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Can someone link me to resources on the efficiency of sticky end ligation?

Can someone link me to resources on the efficiency of sticky end ligation?


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I really would like to know if sticky end ligation could potentially be performed with very high efficiency, and which factors influence that. However, I can't find any papers on the subject, even though I'm sure they exist. I was hoping someone would know of some literature.


The best resource for troubleshooting ligations I found (and use frequently) is this NEB page. That is assuming you've already referred to the instructions provided with the enzyme you're using. In my case it's T4 Ligase, again from NEB. It's helpful to check the FAQs and the references listed on that page. Also, you can make use of their Molar Calculator tool on Ligation setting (see more from first link).

Good luck with high efficiency!


To measure the frequency of indels at the ligation site you can use a vector with a unique restriction site in the lacZ gene. With a colorimetric assay you can count the number of white cfu. Perfect ligation in-frame yields blue cfu


Promega Connections

One of the easiest methods for cloning blunt-ended DNA fragments including PCR products is T-vector cloning, such as with pGEM®-T or pGEM®-T Easy Vector Systems. This method takes advantage of the “A” overhang added by a PCR enzyme like Taq DNA Polymerase. T vectors are linearized plasmids that have been treated to add 3′ T overhangs to match the A overhangs of the insert. The insert is directly ligated to the T-tailed plasmid vector with T4 DNA ligase. The insert can then be easily transferred from the T vector to other plasmids using the restriction sites present in the multiple cloning region of the T vector.

Proofreading polymerases like Pfu do not add “A” overhangs so PCR products generated with these polymerases are blunt-ended. In a previous blog, we discussed a simple method for adding an A-tail to any blunt-ended DNA fragment to enable T-vector cloning. Below, we think about the next step: Ligation.

Ligation Preparation

You have blunt-ended your DNA insert of interest (from PCR, restriction enzyme digestion, or even sheared and blunted genomic DNA), made sure the fragment is A tailed and are ready to clone into a T vector (e.g., pGEM ® -T Easy Vector). The next step is as simple as mixing a few microliters of your purified product with the cloning vector in the presence of DNA ligase, buffer and ATP.

Estimate the concentration of the prepared insert DNA by spectrophotometric method or compare the staining intensity of your PCR product with that of DNA molecular weight standard of similar size and known concentration on an agarose gel. If the vector DNA concentration is unknown, estimate the vector concentration by the same method you chose for the insert.

It’s a good idea to set up various vector:insert DNA ratios to determine the optimal ratio for a particular vector and insert. Keep in mind, the vector:insert ratio will change depending on the size of the insert. In most cases, a 1:1 or 1:3 molar ratio of vector:insert works well, but you may want to consider 1:5, 5:1 and even a 10:1 ratio.

The equation for calculation of the amount of insert required at a specific molar ratio of vector:insert is:
[(ng of vector × kb size of insert) ÷ kb size of vector] × (molar amount of insert ÷ molar amount of vector) = ng of insert

Example:
How much 500bp insert DNA needs to be added to 100ng of 3.0kb vector in a ligation reaction for a desired vector:insert ratio of 1:3?
Answer: [(100ng vector × 0.5kb insert) ÷ 3.0kb vector] × (3 ÷ 1) = 50ng insert

If the math looks intimidating, don’t worry. Our BioMath Calculators are here to help!

If you’re interested in more details about how to set up a ligation, take a look at this helpful video:

Tips for T-Vector Cloning

Here are a few things to remember while you work:

  1. Nucleases may degrade the T overhangs on the vector. Make sure you use sterile, nuclease-free water in your ligation reactions.
  2. Use high-efficiency competent cells (≥1 × 10 8 cfu/μg DNA) for transformations. Ligation of fragments with a single-base overhang can be inefficient, so it’s essential to use cells with a high transformation efficiency to obtain a reasonable number of colonies.
  3. Limit exposure of your PCR product to shortwave UV light to avoid formation of pyrimidine dimers. Use a glass plate between the gel and UV source. If possible, only visualize the PCR product with a long-wave UV source, or use something like Diamond® Nucleic Acid Dye that allows you to visualize your DNA without the use of UV.

For more information on cloning, consult our Subcloning Technology Guide.

Are you an student or early-career scientist looking for troubleshooting and how-to resources? Or, do you supervise entry-level lab members? Check out our Student Resource Center for more helpful articles, videos and tools!


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Results and discussions

Overview of AFEAP cloning method

The mechanism of AFEAP cloning for assembling multiple fragments is shown in Fig. 1a. AFEAP cloning requires two-round of PCRs to generate overhang adapter sequence at 5′ ends of each DNA molecule that can associate to link DNA segments. All of the nicks between two adjacent fragments are joined by the T4 DNA ligase without the introduction of any scar sequences. The crucial point for successful AFEAP cloning is to assign an “overhang” region. As shown in Fig. 1a and b, the overhang can be a short sequence on the 5′ terminus of the joining sites. AFEAP cloning requires two sets of primers (Fig. 1b). The primers of the first set are designed standard forward and reverse primers that flank the assigned overhang region. The primers of the second set are designed that have additional overhang sequence at their 5′ ends that will then be incorporated into the PCR product. In detail, the assembly of DNA fragments with AFEAP cloning into circular plasmid requires four steps (Fig. 1a): (i) In the first-round PCR, several PCRs are carried out in parallel with forward and reverse primers of the first set, i.e., Fw1–1 and Rv1–1, Fw2–1 and Rv2–1, Fw3–1 and Rv3–1,…, and Fwn-1 and Rvn-1, to produce double-stranded DNA fragments, i.e., dsDNA 1, dsDNA 2, dsDNA 3,…, dsDNA n (ii) In the second-round PCR, two single-primer PCRs run in parallel with each one of the forward and reverse primers of the second set, i.e., Fw1–2 or Rv1–2, Fw2–2 or Rv2–2, Fw3–2 or Rv3–2,…, or Fwn-2 or Rvn-2, using each DNA product generated in the first-round PCR as template. Second-round PCRs yield several pairs of complementary single-stranded DNA products that contain the desired overhang regions at their 5′ ends, i.e., ssDNA1a/ssDNA 1b, ssDNA 2a/ssDNA 2b, ssDNA 3a/ssDNA 3b,…, or ssDNA na/ssDNA nb (iii) the complementary single-stranded DNA products generated in step 2 anneal to form double-stranded DNA fragments with 5′ unpaired overhang (iv) These double-stranded DNA fragments are then subsequently assembled “hand-in-hand”. The nicks in the annealed multi-part DNAs are sealed by ligase to form transformable plasmid. Reconstituted vectors are transformed into competent E.coli cells and the joining sites can be confirmed by DNA sequencing.

AFEAP cloning. a Schematic details show the flow chart of multi-fragment assembly with AFEAP cloning. The first PCR yields linear DNA fragments (step 1), and is followed by a second asymmetric (one primer) PCR (step 2) and subsequent annealing (step 3) that inserts overlapping overhangs at 5′ end of each DNA fragment. These double-stranded DNA fragments are then subsequently assembled “hand-in-hand” (step 4). The nicks in the annealed multi-part DNAs are sealed by DNA ligase to form transformable plasmid (step 4), followed by bacterial transformation to yield the desired plasmids, which is confirmed by DNA sequencing (step 4). b A typical AFEAP cloning showing the region of overhang, and the two set primers of two adjacent fragments. Fw: forward primer, Rv: reverse primer, OH: overhang region, ssDNA: single-stranded DNA

Determination of parameters for effective assembly

To determine the optimum conditions for AFEAP cloning, we evaluated the effects of five key factors, such as overhang length, DNA fragments size, overhang designed as 5′ end of G/C or A/T, ligase treatment, and transformation conditions, which we had hypothesized to be important for AFEAP cloning. A set of DNA fragments with varying sizes (Fig. 2a) were used to evaluate the reaction system and its assembly efficiency. Primers designed for assembling 3′ and 5′ ends of linear DNAs to form the circle were listed in Additional file 2: Table S2. PCR products were subjected to AFEAP cloning protocol as mentioned above (Fig. 1a). The assembly efficiency of AFEAP cloning was characterized as colony-forming units (CFUs) per microgram of ligated DNA after transformation and the percentage of clones containing the desired vectors over total sequenced ones was calculated as fidelity. The join sites of each assembly were confirmed by DNA sequencing (Additional file 3: Figure S1).

Determination of parameters for effective assembly. a Flowsheet of the assembly of 3′ and 5′ ends of linear DNA fragment to form the circle. Effects of the overhang length on the assembly efficiency were characterized as colony-forming units (CFUs) per microgram of ligated DNA (b) and percent of colonies correct (fidelity) (c). d Effects of the overhang designed as 5′ end of G/C or A/T. The relative CFUs produced of overhang designed as 5′ end of G or C are presented as percentages of the CFUs of overhang designed as 5′ end of A or T at the same construct size. e Effects of the ligase treated. The relative CFUs produced of ligase treated are presented as percentages of the CFUs of no ligase treated at the same construct size. f Effects of transformation conditions

We first tested the effects of the overhang length. The tested DNA overhangs length ranges from 0 to 20 bp. The overhang length was showed marked effects on assembly efficiency (Fig. 2b and c). An overhang, which is less than 2 nucleotides in the PCR products is insufficient for assembly, thereby resulting in low positive clones. From 4 nucleotides overhang onwards, a sharp increase of the efficiency of AFEAP cloning is observed up to 10 bp, with the efficiency peak at 9000 CFUs and 98% of colonies correct. From 10 nucleotides overhang onwards, longer overhangs used somehow decrease the efficiency slightly. As a result, 5–8 nucleotides overhang is, therefore, suitable for AFEAP cloning with high efficiency and low cost. And then we investigated the effects of the size of DNA fragments. Five different size points, i.e., 5.5, 8.0, 15, 20, and 30 kb were tested. The CFU did decrease significantly with longer DNA size fragments (Fig. 2b), while the fidelity did not change significantly within the length of overhang range tested (Fig. 2c). Moreover, we evaluated the effects of the overhangs designed as 5′ end of G/C or A/T. The assembly efficiency of DNA fragments, specifically for those of longer DNA size fragments, is benefiting from 5′ end of the overhang as a G or C (Fig. 2d). In addition, we tested the effects of the ligase treatment. As shown in Fig. 2e, the assembly efficiency for different size fragments did increase significantly when treated with ligase. Last, we evaluated the effects of transformation conditions, such as electroporation or chemical transformation, on the assemble efficiency. Electroporation gave higher efficiencies, but lower fidelities (Fig. 2f).

The optimal conditions for effective DNA assembly with AFEAP cloning were summarized and listed in Table 1.

Assembly of multiple fragments

After developing and optimizing the AFEAP cloning method, its efficiency and accuracy in assembling multiple fragments were evaluated. We tested the effects of DNA fragment number, final plasmid size, the molar ratio between longer and shorter DNA fragments, and transformation conditions.

To evaluate the effects of fragment number on efficiency, we built a pET22b-FLAG-T4 L-GGSGGlinker-MCM6 tandem construct, encoding T4 lysozyme (T4 L) [20] and MCM6 protein fused by a peptide linker, from varying number of DNA fragments (8.0 kb, Fig. 3a). PCR products were subjected to AFEAP cloning protocol as mentioned above (Fig. 1a). The join sites of each assembly were confirmed by DNA sequencing (Additional file 4: Figure S2). We first evaluated the effects of the molar ratio between the longer and shorter DNA fragments. It was shown that the molar ratio is critical for obtaining higher assembly efficiency. When increasing the molar ratio of shorter to longer DNA fragments from 1:6 to 20:1, the assembly efficiency increased 6-fold (Fig. 3b). But from the ratio of 10:1 onwards, the assembly efficiency increased slightly. In comparison, the assembly fidelity did not vary significantly for all conditions tested (Fig. 3b). As a result, the molar ratio was determined as 10:1 for high efficiency and low cost. And then we tested the effects of DNA fragment number. As we expected, the AFEAP assembly efficiency was shown a solid negative with increasing number of DNA fragments for assembly (Fig. 3c). The CFU per μg DNA dipped to around 100 when assembling 13 fragments (Fig. 3c). In contrast, the fidelity dropped slightly but remained >76% even for 13-fragment assembly. In comparison with commonly used Gibson method, AFEAP cloning method showed higher assembly efficiency (Fig. 3c), demonstrating the good performance of this new approach. Moreover, we evaluated the effects of transformation conditions on the assemble efficiency of multiple fragments with AFEAP method. Electroporation gave higher efficiencies, but lower fidelities which is similar as we mentioned above (Fig. 3d).

Assembly efficiency of multiple DNA fragments with AFEAP cloning. a Schematic details show the mechanism for the number of fragments characterization. The DNA sequence encoding T4 L and MCM6 proteins is split up into a number of fragments as shown by double-headed arrows. The number of fragments for each assembly is shown on the left of the double-headed arrows and the join sites for assembly is indicated on the below of dash lines. T4 L: T4 lysozyme. V: vector (backbone). b Effects of molar ratio between longer and shorter DNA fragments. c CFU and fidelity as a function of fragment number. d Effects of transformation conditions

Next, we evaluated the effects of the final plasmid size. We assembled four different plasmids of increasing sizes while keeping the number of fragments at six (Fig. 4a). The four chosen plasmids were an 11.5 kb plasmid pET22b harboring avermectin biosynthetic gene cluster (GenBank: AB032524.1) [21], a 19.6 kb pET22b harboring cosmomycin gene cluster (GenBank: DQ280500.1) [22], a 28 kb pET22b harboring the enterocin biosynthetic gene cluster (GenBank: AF254925.1) [23], and a 35.6 kb plasmid pET22b harboring the aureothin biosynthesis gene cluster aurABCDEFGHI (GenBank: AJ575648.1) [24]. The join sites of each assembly were confirmed by DNA sequencing (Additional file 5: Figure S3). As shown in Fig. 4b, the assembly efficiency showed a negative correlation with the size of plasmid assembled. When assembling plasmid size 8 kb, more than 1490 CFUs/μg ligated DNA were generated with an accuracy of 92%. In contrast, the assembly efficiency decreased to 1402, 1329, 1206, and 921 CFUs/μg when 11.5, 19.6, 28, and 35.6 kb plasmids were assembled, respectively. Even so, the accuracy is still more than 82% when assembling 35.6 kb plasmid, which confirms the high capacity of this sequence-independent assembly method.

Effects of plasmid size on assembly efficiency. a The chosen 6 kb, 14.1 kb, 22.5 kb, and 29.1 kb gene clusters used for the size of construct characterization. b Efficiency as a function of construct size

Construction of larger plasmid

As AFEAP cloning shows the ability for large fragment assembly, we tested the feasibility of AFEAP cloning to construct a bacterial artificial chromosome (BAC), which contains 200 kb DNA sequence insert. Accordingly, we proceeded to assemble the salinomycin biosynthesis cluster (200 kb GenBank: HE586118.1) [25] from the Streptomyces albus subsp. albus (ATCC ® 55,161 ™ ) into pCC1BAC™ vector (Epicentre ® ) between the BamH I site (353–358) and the Hind III site (383–388) to form a BAC. As regular DNA polymerases only can amplify up to 40 kb with high fidelity, we plan to divide this 200 kb DNA sequence into 8 consecutive short ones, which are then assembled into BAC with AFEAP cloning. Figure 5a shows the strategy to construct BAC with AFEAP cloning method. Detailed cloning procedure can be found in Additional file 6: Supporting Information. Figure 5b shows the resulting DNA products by AFEAP cloning evaluated by 1% agarose gel electrophoresis, and the 8 consecutive DNA parts and the linear vector backbone were joined to one another and shifted to a higher molecular weight (Fig. 5b, lane 11). The presence of nine join sites was confirmed by DNA sequencing (Fig. 5b). 34 ± 13 CFUs/μg were obtained on transformation with an accuracy of 46.7 ± 4.7%. These results demonstrate that AFEAP cloning could be a powerful DNA assembly tool for multiple fragments, especially for large DNA up to 200 kb.

Construction of larger plasmid. a Schematic diagram of the assembly of 200 kb BAC with AFEAP cloning method. (b, upper panel) Agarose electrophoresis shows the PCR amplification using the primers as listed at Additional file 2: Table S2. Lane 1: 1 kb DNA ladder Lane 2–9: PCR products from first-round PCRs Lane 10: Annealing of PCR products from second-round PCRs before ligation Lane 11: after ligation. DNA samples were electrophoresed in 1% agarose gel. (b, below panel) Sequencing validation of the re-joining junction sites. The overhang regions are marked by red boxes


Rapid urine test

What is a rapid urine test?

A rapid urine test is the quickest way to test urine. This involves dipping a test strip with small square colored fields on it into the urine sample for a few seconds. After that you have to wait a little for the result to appear. Depending on the concentration of the particular substance you are testing for, the fields on the test strip change color. Then the resulting colors of the fields are compared with a color table. The color table can be found on the urine test package. It shows which colors indicate normal and abnormal values.

In a rapid urine test, a test strip is dipped into the urine and then compared with the colored fields on the packaging.

Rapid urine tests are usually done as part of routine examinations – for example at a family doctor’s office, during antenatal visits, when being admitted to the hospital, or before surgery. They are also used in people who have acute symptoms like lower abdominal pain, stomach ache or back pain, frequent painful urination, or blood in their urine. Some people who have diabetes use this test to check their sugar levels.

Rapid urine tests can be done at doctor’s offices, in hospital, or at home. The test strips are available without a prescription at the pharmacy or on the internet. But they are not intended for self-diagnosis purposes, and should be used in consultation with a doctor. 

What substances can a rapid urine test detect? 

Many substances are usually found only in certain amounts in urine, so higher or lower levels indicate a deviation from the norm.


Results

Fusion of FnCas12a and 5′-3′ ssDNA exonuclease can enhance editing efficiency in HEK293T cells

Cas12a generates staggered DSB ends, which can be easily combined by Watson–Crick base pairing and thus will favor the accurate NHEJ repair pathway that does not contribute to the overall editing efficiency (Fig. 1A). To increase the gene editing efficiency of FnCas12a, we assume to bias the DSB repair pathway into imprecise NHEJ by fusing an ssDNA exonuclease with FnCas12a that will degrade the perfectly compatible sticky ends (Fig. 1A). To test our hypothesis, we selected six ssDNA exonuclease candidates, including Artemis from human, RecJ and polA-exo from Escherichia coli, and exonuclease from phage T5, phage T7, and phage λ. Each of them was fused to either the N- or C-terminus of FnCas12a as candidate fusions for comparison tests of the editing efficiency (Fig. 1B). T7E1 assay showed that T5 EXO had the most significant effect on improving the indel efficiency of FnCas12a, increasing by 216% at the N-terminus of FnCas12a and 141% at the C-terminus of FnCas12a (Fig. 1C). Although FnCas12a-Artemis and ecRecJ-FnCas12a showed slightly higher indel efficiency than FnCas12a, the rest of the candidates did not improve the editing efficiency of FnCas12a. Considering that N-terminus-fused T5 EXO-FnCas12a exhibited higher indel efficiency and a smaller size than all other candidate fusions, we chose it for subsequent genome editing tests, and hereafter we refer to it as TEXT.

FIG. 1. FnCas12a, EXO-FnCas12a, and FnCas12a-EXO mediated gene editing in HEK293T cells. (A) Illustration of the FnCas12a and EXO-FnCas12a/FnCas12a-EXO system and corresponding DNA repair pathways. FnCas12a generates fully compatible sticky ends, which are mainly repaired by the precise non-homologous end joining (NHEJ) pathway, and thus will not contribute to insertion and deletion (indel) efficiency. Exonuclease fused FnCas12a can degrade the single-strand DNA overhang after cleavage, and could bias the cellular repair pathway mainly into imprecise NHEJ, which will result in the enhancement of indel efficiency. (B) Schematic illustration of the FnCas12a/EXO-FnCas12a/FnCas12a-EXO expression cassette. (C) Relative indel frequencies induced by FnCas12a, EXO-FnCas12a, and FnCas12a-EXO. Gene editing efficiency was analyzed using T7E1 assay. (Gel images are shown in Supplementary Figure S1.) Color images are available online.

TEXT genome editing at multiple loci of different cell lines

To profile the TEXT system further, we designed FnCas12a crRNAs for targeting three loci—DNMT1, CCR5, and GAPDH—in different cell lines, including HEK293T cells, Hela cells, and human lens epithelial B3 (HLEB3) cells. Compared to FnCas12a at DNMT1 locus, the TEXT system improved gene editing efficiency by up to 172% in Hela cells and 330% in HLEB3 cells (Fig. 2A). At CCR5 locus, TEXT showed two- to threefold higher editing efficiency than FnCas12a in all three cell lines (Fig. 2B). Notably, at GAPDH locus, FnCas12a only showed around 0.3% editing efficiency in HEK293T cells, and undetectable levels of editing in both Hela and HLEB3 cells, while TEXT dramatically improved gene editing efficiency, with a 10-fold increase in HEK293T cells and with significant editing efficiency in Hela and HLEB3 cells (Fig. 2C). These results indicate that the TEXT system can generally increase gene editing efficiency of FnCas12a in human cells, ranging from 2- to 10-fold higher efficiency in our tests, and can significantly edit locus that previously could not be edited by FnCas12a.

FIG. 2. Comparison of gene editing efficiency of the FnCas12a system and the TEXT system in HEK293T, Hela, and HLEB3 cell lines. (A) Gene editing efficiency of the FnCas12a system and the TEXT system for the human DNMT1 gene in Hela and HLEB3 cells. (B) Gene editing efficiency of the FnCas12a system and the TEXT system for the human CCR5 gene in HEK293T, Hela, and HLEB3 cells. (C) Gene editing efficiency of the FnCas12a system and the TEXT system for the human GAPDH gene in HEK293T, Hela, and HLEB3 cells. Indel percentage at each locus was determined using the T7E1 assay and is expressed as the mean ± standard deviation from three biological replicates (*p < 0.05 **p < 0.01 ***p < 0.001 two-tailed t-test). (Gel images are shown in Supplementary Figures S2–S4). Color images are available online.

Varying spacer length of crRNA to optimize editing efficiency of TEXT

The spacer region of crRNA uses Watson–Crick base paring to locate the Cas12a–crRNA complex or TEXT system at specific genomic loci. It has been reported that by optimizing the spacer length of crRNA ranging from 18 to 23 nt, the cutting efficiency of CRISPR-Cas12a can be increased. Typically, 21 nt can achieve higher editing efficiency than other lengths when using FnCas12a. 16 In a similar fashion, in order to investigate the optimal spacer length of crRNA for TEXT-mediated gene editing in human cells, we constructed a series of crRNAs with different spacer lengths ranging from 18 to 23 nt, and we tested the gene editing efficiency of TEXT at DNMT1 locus. Consistent with a previous report, 16 we found that crRNA with a 20–21 nt spacer length can enable higher gene editing efficiency when using FnCas12a (Fig. 3B), while the TEXT system requires a 21–23 nt spacer length to achieve maximum cutting efficiency (Fig. 3B). In addition, it is worth noting that when the spacer length of crRNA was 18 nt, the editing efficiency of FnCas12a sharply decreased to 1.2%. However, the TEXT system retained more than 10% editing efficiency. Overall, these results confirmed that the TEXT system is compatible with a wide range of guide lengths.

FIG. 3. Different spacer lengths of crRNA result in distinct cleavage pattern and gene editing efficiency. (A) Cleavage pattern of FnCas12a under different crRNA spacer lengths (18–23 nt). With a 18 nt spacer (crRNA-18 nt), FnCas12a cleavage sites were mainly after the 13th and 14th bases on the non-target strand and from the 22nd bases on the target strand. With a 19 nt spacer, FnCas12a cleavage sites were mainly after the 14th, 13th, and 17th bases on the non-target strand and from the 22nd bases on the target strand. With a 20 nt spacer, FnCas12a cleavage sites were mainly after the 18th, 17th, and 16th bases on the non-target strand and from the 18th and 19th bases on the target strand. With a 21–22 nt spacer, FnCas12a cleavage sites were mainly after the 18th bases on the non-target strand and from the 21st and 22nd bases on the target strand. With a 23 nt spacer, FnCas12a cleavage sites were mainly after the 18th bases on the non-target strand and from the 20th, 21st, and 22nd bases on the target strand. The cleavage sites are indicated by red arrows. (B) Gene editing efficiency of the FnCas12a system and the TEXT system for the human DNMT1 locus with different spacer lengths of crRNA in HEK293T cells. Gel images are shown in Supplementary Figure S5. (C) Editing efficiency fold increase of the TEXT system compared to FnCas12a at different spacer lengths. The ratio was determined by the formula A/B, where A and B represent the gene editing efficiency of TEXT and FnCas12a, respectively. (D) The relative ratio of deletions to insertions induced by FnCas12a or the TEXT system with 18–23 nt spacer lengths of crRNA. (E) The relative proportions of different size deletions among total deletions that were induced by FnCas12a or the TEXT system with 18–23 nt spacer lengths of crRNA. (F) Ratio of deletions size (1–30 nt) induced by the TEXT system with a 18 nt spacer length of crRNA. (Gel images are shown in Supplementary Figure S5. Ratio of deletions size (1–30 nt) induced by FnCas12a and the TEXT system with other spacer lengths of crRNA are shown in Supplementary Figures S6 and S7). Color images are available online.

TEXT improves gene editing under different lengths of crRNA

Previously, it was commonly believed that Cas12a would cut DNA at the 18th base downstream of the PAM site on the non-target strand and the 23rd base on the target strand. 9 However, recently, it has been reported that FnCas12a cleavage sites are located after the 13th, 14th, 18th, and 19th bases on the non-target strand and from the 21st to 24th bases on the target strand, 29 which supports the scheme that FnCas12a has a different cleavage pattern when using different spacer lengths of crRNA (Fig. 3A). When the spacer length of crRNA is 18 nt, which typically directs Cas12a to generate 8–9 nt sticky end, the gene editing efficiency of the TEXT system is 11 times higher than FnCas12a (Fig. 3A and C). When the spacer length is 19 nt, which directs Cas12a to generate a 5–9 nt sticky end, TEXT can increase the editing efficiency of FnCas12a by five times (Fig. 3A and C). When the spacer length is 20–23 bp, with 3–5 nt sticky end, the gene editing efficiency of TEXT is around twofold higher than FnCas12a (Fig. 3A and C).

Deep sequencing analysis of TEXT indel patterns

Furthermore, we analyzed the indel pattern of TEXT and FnCas12a with different spacer lengths of crRNA by deep sequencing (Fig. 3D–F). In general, TEXT induced a higher indel efficiency than FnCas12a (Fig. 3B and C). In addition, the ratio of deletion frequency to insertion frequency (del/in) is significantly higher in TEXT than it is in FnCas12a (Fig. 3D). When using 18 nt spacer crRNA, TEXT increased the del/in of FnCas12a from 20.17 to 92.86, while it only increased from 56.78 to 80.84 when using the 23 nt spacer crRNA (Fig. 3D). Then, we measured the lengths of the deletions generated by TEXT and FnCas12a. When using FnCas12a, under different crRNA spacer lengths from 18 to 23 nt, the proportion of deletion sizes <3 bp and <6 bp are about 20% and 30–40%, respectively, while when using TEXT, the deletion sizes that are <3 bp and <6 bp account for 10% and 20%, respectively (Fig. 3E). It is worth noting that when the spacer length is 18 nt, the deletion size that is <6 bp is 42.6% by FnCas12a and 14.3% by TEXT (Fig. 3E). In addition, we analyzed the deletion pattern and found that the proportion of 8–9 nt deletion of TEXT is up to 41.2% with a 18 nt spacer length crRNA, while that of FnCas12a is 9.8% (Fig. 3F). Next, to understand further the role of T5-EXO in TEXT editing, we analyzed next-generation sequencing data to generate detailed indels pattern of TEXT, in which most of the deletion alleles do not contain the sequence of the 5′ overhangs (Supplementary File S5). The results further confirm that the TEXT system improves the gene editing efficiency of FnCas12a by resecting the 5′ single-strand overhang at the DSB ends. Collectively, the TEXT system substantially increased the deletion frequency and deletion size at the targeted locus.

TEXT performs efficient on-target editing with minimal off-target effects

Further characterization of the TEXT system showed substantial editing activities in human cells. We compared the editing efficiency of FnCas12a, TEXT, LbCas12a, and AsCas12a, with NTTV PAMs in human cells. In all cases, we observed higher editing efficiency achieved by TEXT than by FnCas12, LbCas12a, and AsCas12a (Fig. 4A). In particular, TEXT showed robust editing activities on multiple endogenous target sites with ATTV and GTTV PAMs compared to AsCas12a and LbCas12a (Fig. 4A). These results confirm that TEXT recognizes TTV PAM, which will help target genomic sites that are not accessible to LbCas12a and AsCas12a. Next, we determined the off-target effects of TEXT compared to FnCas12a, LbCas12a, and AsCas12a. We used CRISPR RGEN prediction tools to generate a set of potential off-target sites and found that only one off-target site had detectable efficiency by TEXT and other Cas12a orthologs, while most predicted off-target sites showed undetectable off-target effects (Fig. 4B). The results indicated that the TEXT system does not significantly increase off-target effects when enhancing on-target editing efficiency.

FIG. 4. Comparison of editing efficiencies and off-target effects between TEXT and Cas12a orthologs over a diverse range of endogenous targets. (A) Comparison of indel efficiency of TEXT, FnCas12a, LbCas12a, and AsCas12a at various protospacer adjacent motif (PAM) sites in HEK293T cells. Four types of PAM (ATTV, CTTV, GTTV, and TTTV) were analyzed, in which three to four different sites were selected to represent each type of PAM. Indel efficiency was detected by T7E1 assay. (B) Off-target profile of TEXT, FnCas12a, LbCas12a, and AsCas12a at selected loci in HEK293T cells. (Off-target sites and their amplification primers are listed in the Supplementary File S4. Gel images are shown in Supplementary Figures S8–S15). Color images are available online.


Here are some questions to consider when selecting a birth control method:

  • How well does the method prevent pregnancy? To tell how well a method works, look at the number of pregnancies in 100 women using that method over a period of 1 year.
  • What are your feelings about getting pregnant? Would an unplanned pregnancy create hardship or distress to a woman or her partner? Or would a pregnancy be welcomed if it occurred earlier than planned?
  • How much does a method of birth control cost? Does your insurance plan pay for it?
  • What are the health risks? Talk about these risks with your health care provider before believing what you hear from others.
  • Is your partner willing to accept and use a given method of birth control?
  • Do you want a method that you only need to use when you have sex? Or do you want something that is in place and always working?
  • Is preventing infections spread by sexual contact important? Many methods do not protect you from sexually transmitted infections (STIs). Condoms are the best choice for preventing STIs. They work best when combined with spermicides.
  • Availability: Can the method be used without a prescription, a provider visit, or, in the case of minors, parental consent?

BARRIER METHODS OF BIRTH CONTROL

  • A condom is a thin latex or polyurethane sheath. The male condom is placed around the erect penis. The female condom is placed inside the vagina before intercourse.
  • A condom must be worn at all times during intercourse to prevent pregnancy.
  • Condoms can be bought in most drug and grocery stores. Some family planning clinics offer free condoms. You do not need a prescription to get condoms.

DIAPHRAGM AND CERVICAL CAP:

  • A diaphragm is a flexible rubber cup that is filled with spermicidal cream or jelly.
  • It is placed into the vagina over the cervix before intercourse, to prevent sperm from reaching the uterus.
  • It should be left in place for 6 to 8 hours after intercourse.
  • Diaphragms must be prescribed by a woman's provider. The provider will determine the correct type and size of diaphragm for the woman.
  • About 5 to 20 pregnancies occur over 1 year in 100 women using this method, depending on proper use.
  • A similar, smaller device is called a cervical cap.
  • Risks include irritation and allergic reactions to the diaphragm or spermicide, and increased frequency of urinary tract infection and vaginal yeast infection. In rare cases, toxic shock syndrome may develop in women who leave the diaphragm in too long. A cervical cap may cause an abnormal Pap test.
  • Vaginal contraceptive sponges are soft, and contain a chemical that kills or "disables" sperm.
  • The sponge is moistened and inserted into the vagina, to cover over the cervix before intercourse.
  • The vaginal sponge can be bought at your pharmacy without a prescription.

HORMONAL METHODS OF BIRTH CONTROL

Some birth control methods use hormones. They will have either both an estrogen and a progestin, or a progestin alone. You need a prescription for most hormonal birth control methods.

  • Both hormones prevent a woman's ovary from releasing an egg during her cycle. They do this by affecting the levels of other hormones the body makes.
  • Progestins help prevent sperm from making their way to the egg by making mucus around a woman's cervix thick and sticky.

Types of hormonal birth control methods include:

    : These may contain both estrogen and progestin, or only progestin. : These are small rods implanted beneath the skin. They release a continuous dose of hormone to prevent ovulation. , such as Depo-Provera, that are given into the muscles of the upper arm or buttocks once every 3 months.
  • The skin patch, such as Ortho Evra, is placed on your shoulder, buttocks, or other place on the body. It releases a continuous dose of hormones.
  • The vaginal ring, such as NuvaRing, is a flexible ring about 2 inches (5 centimeters) wide. It is placed into the vagina. It releases the hormones progestin and estrogen. : This medicine can be bought without a prescription at your drugstore.
  • The IUD is a small plastic or copper device placed inside the woman's uterus by her provider. Some IUDs release small amounts of progestin. IUDs may be left in place for 3 to 10 years, depending on the device used.
  • IUDs can be placed at almost any time.
  • IUDs are safe and work well. Fewer than 1 out of 100 women per year will get pregnant using an IUD.
  • IUDs that release progestin may be for treating heavy menstrual bleeding and reducing cramps. They may also cause periods to stop completely.

PERMANENT METHODS OF BIRTH CONTROL

These methods are best for men, women, and couples who feel certain they do not want to have children in the future. They include vasectomy and tubal ligation. These procedures can sometimes be reversed if a pregnancy is desired at a later time. However, the success rate for reversal is not high.


What to Expect After Cardiac Ablation

What is recovery like?

It depends on the type of procedure you have:

Catheter ablation. You may need to spend a night in the hospital, but most people can go home the same day. If so, you'll rest in a recovery room for a few hours while a nurse closely watches your heart rate and blood pressure. You need to lie flat and still to prevent bleeding from where your skin was cut. Plan to have someone drive you home.

The doctor will prescribe a medication to prevent blood clots and another to prevent AFib. You’ll probably take them for 2 months. A shower is OK once you’re home, but keep the water on the cooler side. Don’t take a bath, swim, or soak for 5 days or until the cuts have healed.

Continued

  • Don’t lift more than 10 pounds.
  • Skip activities that make you push or pull heavy things, like shoveling or mowing the lawn.
  • If you get tired, stop and rest.
  • Don’t exercise. You can go back to normal in week two.

Continued

Maze procedure. You’ll probably be in the hospital about a week. You’ll spend the first couple of days in an intensive care unit (ICU) and then move to a regular room before you go home. Full recovery takes about 6 to 8 weeks, but you should be able to return to normal activities within 2 or 3 weeks. You should start to feel better in about 4 weeks. You’ll probably take a blood thinner for about 3 months.

Mini maze. You'll be in the ICU for a few hours to a day. You’ll probably stay for 2 to 4 days, total.

Open-heart maze. This is major surgery. You'll spend a day or two in intensive care, and you may be in the hospital for up to a week. At first, you'll feel very tired and have some chest pain. You can probably go back to work in about 3 months, but it may take 6 months to get back to normal. Once you’re home:

  • You may need someone to drive you for a while. The doctor will tell you when you can drive again.
  • You’ll probably need help at home.
  • You’ll need to go back in about 10 days to get the stitches out.
  • Don’t lift anything heavy for several weeks.

Continued

Convergent procedure. This usually requires a 2- to 3-day hospital stay. Recovery is similar to catheter ablation.

Life after cardiac ablation

Catheter ablation may not cure your AFib, but it will often ease your symptoms. You could still have AFib episodes during the first 3 months because it takes that long for the scars to form.

If you've had AFib a long time, you'll probably need another treatment to keep your heartbeat regular. You may also need medicine to control your heart rhythm for a few months after the procedure.

Continued

Most people who have the maze procedure get long-term relief from their symptoms. And many don't need to take heart rhythm medicine afterward.

Your doctor may recommend lifestyle changes to keep your heart healthy, including:

  • Eat a good diet with less salt and alcohol.
  • Quit smoking.
  • Keep a healthy weight.
  • Get more physical activity.
  • Try to manage stress and strong emotions.

Sources

Stop Afib.org: “Maze Procedure: (Surgical Ablation),” “What to Expect After Cardiac Ablation,” “What to Expect After a Maze Procedure,” “What to Expect After Mini Maze Surgery.”

Keck School of Medicine of USC: “Robotic-Assisted Maze Surgery.”

Frankel Cardiovascular Center, Michigan Medicine: “Frequently Asked Questions: Catheter Ablation.”

Inova Heart and Vascular Institute: “Frequently Asked Questions.”

Johns Hopkins Medicine: “Atrial Fibrillation Surgery.”

Lahey Hospital & Medical Center: “Convergent Procedure.”

National Heart, Lung, and Blood Institute: “Catheter Ablation.”

American Heart Association: “What is Atrial Fibrillation (AFib or AF)?” “Non-surgical Procedures for Atrial Fibrillation (AFib or AF),” “Treatment Guidelines of Atrial Fibrillation (AFib or AF),” “Why Atrial Fibrillation (AF or AFib) Matters,” “Ablation for Arrhythmias,” “Surgical Procedures for Atrial Fibrillation (AFib or AF).”

Cleveland Clinic: “Surgical Procedures for Atrial Fibrillation (MAZE),” “After Catheter Ablation,” “Heart Surgery for Atrial Fibrillation (MAZE): After the Procedure.”

Heart Rhythm Society: “Symptoms of Atrial Fibrillation (AFib),” “Types of Ablations.”

University of Southern California Keck School of Medicine: “MAZE Procedure for Treatment of Atrial Fibrillation.”

Verma, A. Circulation, published August 2005.

Mayo Clinic: “Atrial Fibrillation: Symptoms,” “Atrial fibrillation ablation,” “Cardiac ablation.”


Background

Long INterspersed Element-1 (LINE-1, L1) is one of the most abundant mobile DNAs in humans. With roughly 500,000 copies, LINE-1 sequences comprise about 17% of our DNA [1]. Although most of these exist in an invariant (fixed) state and are no longer active, about 500 insertions of the Homo sapiens specific L1 sequences (L1Hs) are more variable and derive from a few ‘hot’ L1Hs that remain transcriptionally and transpositionally active [2,3,4,5,6,7]. The activity of LINE-1 results in transposable element insertions that are a significant source of structural variation in our genomes [8,9,10,11]. They are responsible for new germline L1 insertion events as well as the retrotransposition of other mobile DNA sequences including Alu Short INterspersed Elements (SINEs) [12,13,14,15] and SVA (SINE/VNTR/Alu) retrotransposons [16]. Additionally, LINE-1 can propagate in somatic tissues, and somatically-acquired insertions are frequently found in human cancers [17,18,19,20,21,22,23].

Characterizations of transposable element sequences remain incomplete in part because their highly repetitive nature poses technical challenges. Using these high copy number repeats as probes or primer sequences can create signals or products in hybridization-based assays and PCR amplifications that do not correspond to discrete genomic loci. Moreover, both the absence of many common insertion variants from the reference genome assembly as well as the presence of hundreds of thousands of similar sequences together complicate sequencing read mappability. Detecting insertions that occur as low frequency alleles in a mixed sample presents an additional challenge, such as occurs with somatically-acquired insertions. Nevertheless, several recent studies describe strategies for mapping these elements and highlight LINE-1 continued activity in humans today. These methods include hybridization-based enrichment [24,25,26,27,28,29] selective PCR amplification [6, 17, 30,31,32,33,34,35,36,37,38,39] and tailored analyses of whole genome sequencing reads [10, 11, 18, 19, 40, 41].

Here we present a detailed protocol to amplify and sequence human LINE-1 retrotransposon insertion loci developed in the Burns and Boeke laboratories, Transposon Insertion Profiling by sequencing (TIPseq) [22, 23, 42,43,44]. This method uses ligation-mediated, vectorette PCR [45] to selectively amplify regions of genomic DNA directly 3′ of L1Hs elements. This is followed by library preparation and Illumina deep sequencing (see Fig. 1a). TIPseq locates fixed, polymorphic, and somatic L1Hs insertions with base pair precision and determines orientation of the insertion (i.e., if it is on the plus (+) or minus (−) strand with respect to the reference genome). It detects, though does not distinguish between, both full length and 5′ truncated insertions as short as 150 bp. TIPseq is highly accurate in identifying somatic L1 insertions in tumor versus matched normal tissues, and allows sequencing coverage to be efficiently targeted to LINE-1 insertion sites so it is an economical way to process samples for this purpose. We have used TIPseq to demonstrate LINE-1 retrotransposition in pancreatic [22] and ovarian [23] cancers, and to show that somatically-acquired insertions are not common in glioblastomas [44]. Together with the machine learning-based computational pipeline developed in the Fenyӧ Lab for processing TIPseq data, TIPseqHunter [23], this protocol allows researchers to map LINE-1 insertion sites in human genomic DNA samples and compare insertion sites across samples.

Steps in the TIPseq protocol. a Steps in TIPseq are shown from top to bottom in a vertical flow chart. These include (i.) vectorette adapter annealing, (ii.) genomic DNA (gDNA) digestion, (iii.) vectorette adapter ligation, (iv.) vectorette touchdown PCR, (v.) PCR amplicon shearing, (vi.) sequencing library preparation, (vii.) Illumina sequencing, and, (viii.) data analysis. The first seven of these steps are shown adjacent to schematic representations in part b., to the right. b Vectorette adapter annealing is shown first. Mismatched sequences within the hybridized vectorette oligonucleotides are illustrated in red and blue, and create a duplex structure with imperfect base pairing. The sticky end overhang on one strand of the vectorette (here, a 5′ overhang on the bottom strand) is drawn in gray. This overhang in the annealed vectorette complements sticky ends left by genomic DNA digest, and the digest and vectorette ligations are shown in the subsequent two steps. The black box within the gDNA fragment illustrate a LINE-1 element of interest (i.e., a species-specific L1Hs). Most gDNA fragments will not have a transposable element of interest, and thus cannot be amplified efficiently by the vectorette PCR. In vectorette PCR, the L1Hs primer begins first strand synthesis (1) and extends this strand through the ligated vectorette sequence. The reverse primer complements this first-strand copy of the vectorette (2) and the two primers participate in exponential amplification (3) of these fragments in subsequent cycles. c Amplicons are sheared, and conventional Illumina sequencing library preparation steps complete the protocol. Paired-end sequencing reads are required to perform data analysis with TIPseqHunter. d A diagram of read pile-ups demonstrate how there is deep coverage of the 3′ end of L1Hs elements. For elements on the plus (+) strand with respect to the reference genome, the amplified sequences are downstream of the insertion site (i.e., covering genomic coordinates ascending from the transposon insertion). For minus (−) stranded insertions, sequences are recovered in the opposite direction


Chemical and Biological Weapons: Use in Warfare, Impact on Society and Environment

Since the end of World War II there has been a number of treaties dealing with the limitations, reductions, and elimination of so-called weapons of mass destruction and/or their transport systems (generally called delivery systems). Some of the treaties are bilateral, others multilateral, or in rare cases universal. In the present paper only the chemical and biological weapons will be discussed, with emphasis on the Convention to eliminate them (CBWC).

The term “Weapons of Mass Destruction” (WMD), used to encompass nuclear (NW), biological (BW), and chemical weapons (CW), is misleading, politically dangerous, and cannot be justified on grounds of military efficiency. This had been pointed out previously by the author [1] and discussed in considerable detail in ref. [2]. Whereas protection with various degrees of efficiency is possible against chemical and biological weapons, however inconvenient it might be for military forces on the battlefield and for civilians at home, it is not feasible at all against nuclear weapons. Chemical weapons have shown to be largely ineffective in warfare, biological weapons have never been deployed on any significant scale. Both types should be better designated as weapons of terror against civilians and weapons of intimidation for soldiers. Requirements on their transport system differ vastly from those for nuclear warheads. They are able to cause considerable anxiety, panic, and psychosis without borders within large parts of the population. Stockpiling of biological weapons is not possible over a long time scale [3, 4]. Only nuclear weapons are completely indiscriminate by their explosive power, heat radiation and radioactivity, and only they should therefore be called a weapon of mass destruction.

However, if one wants to maintain the term “Weapons of Mass Destruction (WMD)“, it is a defendable view to exclude chemical and biological weapons, but put together with nuclear weapons all those that actually has killed millions of people in civil wars since World War II. These are mainly assault rifles, like AK47s, handguns, and land mines, to a lesser extent mortars, fragmentation bombs, and hand grenades.

This paper gives in Chapter 2 an overview on the history of chemical warfare, addresses in Chapter 3 the inventory of chemical weapons, discusses in Chapter 4 the elimination of chemical weapons and possible problems resulting for the environment (CW), reviews in Chapter 5 some non-lethal chemical weapons and chemical weapons which may be on the borderline to conventional explosives, and describes in Chapter 6 some of the old and new biological weapons (BW). Chapter 7 evaluates and compares the use of biological and chemical weapons by terrorists and by military in combat. The present status and verification procedures for the Chemical and Biological Weapons Convention (CBWC) are addressed in the conclusions in Chapter 8.

2. Chemical Warfare, Its History [5]

The Greeks first used sulfur mixtures with pitch resin for producing suffocating fumes in 431 BC during the Trojan War. Attempts to control chemical weapons date back to a 1675 Franco-German accord signed in Strasbourg. Then came the Brussels Convention in 1874 to prohibit the use of poison or poisoned weapons. During the First Hague Peace Appeal in 1899, the Hague Convention elaborated on the Brussels accord by prohibiting the use of projectiles that would diffuse “asphyxiating or deleterious” gases (Laws and Customs of Wars on Land). This Convention was reinforced during the second Hague conference in 1907, but prohibitions were largely ignored during World War I. At the battle of Ypres/Belgium, canisters of chlorine gas were exploded in April 1915 by Germany, which killed 5,000 French troops and injured 15,000. Fritz Haber, a Nobel price winner in 1919 for invention of ammonium fixation, had convinced the German Kaiser to use chlorine gas to end the war quickly. History taught us about a different outcome. During World War I all parties used an estimated 124,000 tons of chemicals in warfare. Mustard gas – “the king of battle gases” – then used on both sides in 1917 killed 91,000 and injured 1.2 million, accounting for 80% of the chemical casualties (death or injury). Chemical weapons caused about 3 percent of the estimated 15 million casualties on the Western Front [3, 6]. To put these numbers into perspective, the total loss of Allied lives was ³ 5 million, of the Central Powers 3.4 million, and the total of all wounded soldiers 21 million. Despite of its intensive use, gas was a military failure in WW I. The inhuman aspect and suffering was soon recognized and the year 1922 saw the establishment of the Washington Treaty, signed by the United States, Japan, France, Italy and Britain. In 1925 the Geneva Protocol for the Prohibition of the use in war of Asphyxiating, Poisonous or Other Gases and Bacteriological Methods of Warfare was signed, and it had been a cornerstone of chemical and biological arms control since then. The Geneva Protocol did neither forbid the stockpiling or the research on chemical weapons.

Despite the conventions, banning chemical weapons, Italians used them during the war 1935-36 in Ethiopia, the Japanese in China during World War II (1938-42), and they were used also in Yemen (1966-67). Various new chemicals were developed for use in weapons. Sarin, Soman, and VX followed Tabun, the first nerve gas, discovered in 1936.

During the Vietnam War (1961-1973), the US was accused of using lachrymatory agents and heavy doses of herbicides (defoliants) in much the same manner as chemical weapons. Some international organizations consider Napalm, its trade name, to be a chemical weapon, others put it on equal level with flame throwers, and consequently not falling under any of the articles of the CWC.

Saddam Hussein used chemical weapons against Iraqi civilians as well as against Iran soldiers between 1980 and 1988. It is estimated that of the approximately 27,000 Iranians exposed to Iraqi mustard gas in that war through March 1987, only 265 died. Over the entire war, Iraqi chemical weapons killed 5,000 Iranians. This constituted less than one percent of the 600,000 Iranians who died from all causes during the war [6].

The Convention on the Prohibition of the Development, Production, Stockpiling, and Use of Chemical Weapons And on Their Destruction (CWC) [7], entered into force in 1997 after deposit of 65 ratification documents, and is signed as of May 1999 by 122 states-parties. There are 46 non-ratifying signatories, and 22 non-states parties [8, 9].

3. The Inventory of Chemical Weapons

Chemical weapons have been produced during the twentieth century by many countries and in large quantities. They are still kept in the military arsenals as weapons of in kind or flexible response. Old ammunition is partially discarded in an environmental irresponsible way.

3.1 Military value of chemical weapons

By their nature, chemical arms have a relatively limited range: they create regional rather than global security problems, and slow the tempo of operations. In this, they are militarily more akin to conventional arms than to nuclear or biological weapons.

Even extended use of chemical weapons had no decisive impact on outcome of wars, had only local success, and made wars uncomfortable, to no purpose. For this and other reasons it is difficult to see why they are around in the first place. However, they had been produced in enormous quantities and mankind has to deal with their very costly elimination.

Should scientists be held responsible for their invention, production, use, and also for the elimination of chemical weapons? Certainly not entirely, since military and politicians demanded their production. However, we need the help of scientists for the difficult job of neutralising or eliminating them.

3.2 Classification of chemical weapons

Binary munitions contain two separated non-lethal chemicals that react to produce a lethal chemical when mixed during battlefield delivery. Unitary weapons, representing the by far largest quantity of the stockpile, contain a single lethal chemical in munitions. Other unitary agents are stored in bulk containers. The characteristics of chemical warfare agents and toxic armament wastes are described in detail in ref. [10]. The reader is referred to this article, which summarises the chemical and physical characteristics of blister, blood, choking, nerve, riot control, and vomiting agents, as well as their effects on the human body.

The easiest – say cheapest – way to eliminate (?) chemical weapons in the aftermath of World War II appeared to dump them into ocean [11]. There had been a worry that, after their defeat in 1945, Germans could be tempted to use part of their arsenal, which totaled 296,103 tons. Therefore, the weapons were captured and dumped into the sea. There are more than 100 sea dumping of chemical weapons that took place from 1945 to 1970 in every ocean except the Arctic. 46,000 tons were dumped in the Baltic areas known as the Gotland Deep, Bornholm Deep, and the Little Belt. According to The Continental Committee on Dumping the total was shared by 93,995 tons from the US, 9,250 tons from France, 122,508 tons from Britain, and 70,500 tons from Russia.

The US dumped German chemical weapons in the Scandinavian region, totaling between 30,000 and 40,000 tons, nine ships in the Skagerrak Strait and two more in the North Sea at depth of 650 to 1,180 meters.

The Russians alone have dumped 30,000 tons in an area, 2,000 square kilometers in size, near the Gotland and Bornholm Islands.

Between 1945 and 1949, the British dumped 34 shiploads carrying 127,000 tons of chemical (containing 40,000 tons mustard gas) and conventional weapons in the Norwegian Trench at 700 meters depth.

The chemical weapons at the bottom of the Baltic Sea (mean depth of the Baltic Sea is 51 meters) and the North Sea represent a serious danger for the aquatic life. The shells of the grenades corrode and will eventually start to leak. The corrosion of these weapons is already so advanced that identification of the former owners is virtually impossible. Consequently, nobody can be made nowadays responsible for the ultimate elimination.

The US is responsible for 60 sea dumping totaling about 100,000 tons (equal to 39 filled railroad box cars), of chemical weapons filled with toxic materials in the Gulf of Mexico, off the coast of New Jersey, California, Florida, and South Carolina, and near India, Italy, Norway, Denmark, Japan, and Australia.

Some of the above figures appear to be not entirely coherent and do not add up well to the total, demonstrating among other things that no careful bookkeeping had been done during this inadmissible actions.

During the 1950s, the US conducted an ambitious nerve gas program, manufacturing what would eventually total 400,000 M-55 rockets, each of which was capable of delivering a 5-kg payload of Sarin [11, 12]. Many of those rockets had manufacturing defaults, their propellant breaking down in a manner that could lead to auto ignition. For this reason in 1967 and 1968 51,180 nerve gas rockets were dropped 240 km off the coast of New York State in depths 1� to 2,190 meters, and off the coast of Florida.

The CWC does not cover sea-dumped chemical weapons in fact it makes a clear exception for them (CWC, Article III, § 2). The CWC does not provide the legal basis to cover chemical weapons that were dumped before 1985. They remain an uncontrollable time bomb.

3.4 The existing arsenal

The arsenal of chemical weapons has to be subdivided into two categories: (i) The “stockpile” of unitary chemical warfare (CW) agents and ammunitions, comprising the material inside weapons and chemicals in bulk storage, and (ii) The “non-stockpile” material, including buried chemical material, binary chemical weapons, recovered chemical weapons, former facilities for chemical weapons production, and other miscellaneous chemical warfare material.

3.4.1 The stockpile of unitary chemical warfare agents and ammunition

The Defence Intelligence Agency (DIA) in the US reports [13, 14]:

Egypt: First country in the Middle East to obtain chemical weapons training, indoctrination, and material. It employed phosgene and mustard agent against Yemeni Royalist forces in the mid-1960s, and some reports claim that it also used an organophosphate nerve agent.
Israel: Developed its own offensive weapons program. The 1990 DIA study reports that Israel maintains a chemical warfare testing facility. Newspaper reports suggest the facility be in the Negev desert.
Syria: It began developing chemical weapons in the 1970s. It received chemical weapons from Egypt in the 1970s, and indigenous production began in the 1980s. It allegedly has two means of delivery: a 500-kilogram aerial bomb, and chemical warheads for Scud-B missiles. Two chemical munitions storage depots, at Khna Abu Shamat and Furqlus. Centre D’Etude et Recherche Scientifique, near Damascus, was the primary research facility. It is building a new chemical-weapons factory near the city of Aleppo.
Iran: Initiated a chemical and warfare program in response to Iraq’s use of mustard gas against Iranian troops. At end of war military had been able to field mustard and phosgene. Had artillery shells and bombs filled with chemical agents. Was developing ballistic missiles. Has a chemical-agent warhead for their surface-to-surface missiles.
Iraq: Used chemical weapons repeatedly during the Iraq-Iran war. Later it attacked Kurdish villagers in northern Iraq with mustard and nerve gas. Since end of Gulf War UN destroyed more than 480,0000 liters of Iraq’s chemical agents and 1.8 million liters of precursor chemicals.
Libya: Obtained its first chemical agents from Iran, using them against Chad in 1987. Opened its own production facility in Rabta in 1988. May have produced as much as 100 tons of blister and nerve agents before a fire broke out in 1990. Is building a second facility in an underground location at Tarhunah.
Saudi Arabia: May have limited chemical warfare capability in part because it acquired 50 CSS-2 ballistic missiles from China. These highly inaccurate missiles are thought to be suitable only for delivering chemical agents.
Yugoslavia: The former Yugoslavia has a CW production capability. Produced and weaponized Sarin, sulphur mustard, BZ (a psychochemical incapacitant), and irritants CS and CN. The Bosnians produced crude chemical weapons during the 1992-1995 war.
Romania: Has research and production facilities and chemical weapons stockpiles and storage facilities. Has large chemical warfare program, and had developed a cheaper method for synthesizing Sarin.
Czechoslovakia: Pilot-plant chemical capabilities that probably included Sarin, Soman, and possibly VX.
France: Has stockpile of chemical weapons, including aerosol bombs.
Bulgaria: Has stockpile of chemical munitions of Soviet origin.

Has the second largest arsenal of chemical weapons in the world, consisting of

31,000 tons of chemicals, and 3.6 million grenades [15]. The chemical weapons contain about 12,000 tons of agents, and 19,000 tons are in bulk storage. Details on composition and location are given in Table 1.

An estimate of the Russian stockpile in 1993 puts it at

40,000 agent tons, of which one-fourth is of pre-World War II vintage. A larger portion seems to be in bulk storage [16]. Out of the officially declared quantity 30,000 tons are phosphoric organic agents (Sarin, Soman, VX), the remaining 10,000 tons are composed of 7,000 tons lewisite (in containers ?), 1,500 tons of mixture of mustard gas and Lewisite (GB, GD, VX), and 1,500 tons mustard gas. Slightly different numbers on the composition of the arsenal are given in ref. [17]. Some independent analysts believe that the 40,000 tons formally declared by Russia is only a fraction of a total of 100,000 to 200,000 tons, the rest of which were probably disposed of in some manner [18].

Locations of the US Unitary Chemical Stockpile
Site Agent Agent Tons Percent of Stockpile
Anniston Army Depot (ADAD), Anniston, AL GB, HD, HT, VX 2,253.63 7.4
Aberdeen Proving Ground (APG), Edgewood, MD HD 1,624.87 5.3
Blue Grass Army Depot (BGAD), Richmont, KY GB, HD, VX 523.41 1.7
Johnston Island (JI), Pacific Ocean GB, HD, VX 1,134.17 3.7
Newport Chemical Activity (NECA), Newport, IN VX 1,269.33 4.2
Pine Bluff Arsenal (PBA), Pine Bluff, AR GB, HD, HT, VX 3,849.71 12.6
Pueblo Depot Activity (PUDA), Pueblo, CO HD, HT 2,611.05 8.5
Tooele Army Depot (TEAD), Tooele, UT H, HD, HT, GA, GB, L, TGA, TGB, VX 13,616.00 44.5
Umatilla Depot Activity (UMDA), Herminston, OR GB, HD, VX 3,717.38 12.2
Total 30,599.55 100.0

Non-persistent nerve gas agents: Tabun (GA) and Sarin (GB) and their thickened products (TGA and TGB) Mustard agents (H, HD and HT) Lewisite (L) Persistent nerve agent (VX)

Agents of the US Unitary Chemical Stockpile
Agent Site Agent Tons Percent of Stockpile Total
GA TEAD 1.41 0.005 1.41
GB ANAD 436.51
BGAD 305.64
JI 617.48
PBA 483.69
TEAD 6,045.26
UMDA 1,041.01 29.1 8,902.59
H TEAD 319.77 1.5 319.77
HD ANAD 456.08
APG 1,624.87
BGAD 90.63
JI 164.86
PBA 94.20
PUDA 2,551.94
TEAD 5,694.64
UMDA 2,339.52 42.5 13,016.74
HT ANAD 532.30
PBA 3,124.55
PUDA 59.11
TEAD 181.51 12.7 3,897.47
L TEAD 12.96 0.004 12.96
TGA TEAD 0.64 0.002 0.64
TGB TEAD 3.48 0.01 3.48
VX ANAD 828.74
BGAD 127.15
JI 351.83
NECA 1,269.33
PBA 147.27
TEAD 1,356.33
UMDA 363.86 14.5 4,444.51
TOTAL 100.0 30,599.55

US Binary Chemical Stockpile
Site Type Fill Component Total Tons
APG QL 0.73
DF 0.57 1.30
PBA QL 48.21
DF 126.51 174.72
TEAD OPA 33.58 33.58
UMDA OPA 470.59 470.59
TOTAL 680.19

Methylphosphonic difluoride (DF) Isopropyl alcohol and isopropylamine (OPA) Ethyl 2-diisoprpylaminoethyl methylphosphonite (QL)

Tables 1. US Unitary and Binary Chemical Stockpiles

The above tables give the location of the nine depots and the variety of chemical weapons stored, which is an indication for the complexity for their elimination or transport problems.

The locations of the Soviet chemical weapons are spread over large parts of the West-European and Asian part of Russia at seven sites (Table 2 [18]). About 80 percent are weaponized and consist mostly of organophosphorus nerve agents. The remainder of the material is stored in bulk at two sites – Kambarka and Gornyi.

Site % of Stockpile Agents
Kambarka 15.9 Lewisite
Gorny 2.9 Mustard
Lewite
Kizner 14.2 Vx
Sarin
Soman
Lewisite
Maradykovsky 17.4 Vx
Sarin
Soman
M/L mix
Pochep 18.8 VX
Sarin
Soman
Leonidovka 17.2 VX
Sarin
Soman
Shchuchye 13.6 VX
Sarin
Soman
Phosgene

Table 2. Russia’s chemical weapons storage sites [18]

3.4.2 The non-stockpile material

Data on non-stockpile material are scarce. Some estimates are available for the US [12]. All the material recovered in the US thus far contains only hundreds of tons of agent and could, in theory, be placed in a single 8-metre-by-25-metre storage building [12]. A considerable amount of money will be required for the destruction of all former facilities for chemical weapons production constructed or used after January 1, 1946.

Abandoned chemical weapons do represent a safety risk. Between 1985 and 1995 Dutch fishermen reported more than 350 cases where chemical weapons, dumped into the Baltic Sea, were caught in fishing nets, some resulting in serious burns.

In China during World War II the Japanese left 678,729 chemical weapons. Recent negotiations resulted in Japan’s agreement to collect and destroy these weapons.

The most persistent agents – mustards and lewisite – can remain dangerous for decades. Even after lewisite breaks down, the resulting arsenic compounds can remain in soil and contaminate ground water [19].

Recovery of ammunitions from World War I still continues. Annual collections by France amount to about 30-50 tons along the old front line, by Belgium to 17 tons (c. 1,500 items) [20].

4. Elimination of Chemical Weapons

The CWC not only prohibits the use, production, acquisition and transfer of chemical weapons, but also requires the states-parties to destroy their existing weapons and production facilities. For the US the deadline is April 29, 2007. The CWC prohibits disposal by dumping into a body of water, land burial or open-pit burning, and requires that the chosen technology destroy the chemical agent in an irreversible manner that also protects the safety of humans and the environment.

4.1 Program, costs and status of the destruction of the existing active arsenal

Since the weight of a typical chemical weapon is roughly ten times that of the agent it contains, and other nations may have as much as 10-15 percent of the combined Russian and US stockpile, the mass of the material to be destroyed comes to roughly 500,000 tons – nearly 100,000 truckloads of material.

In general, the ignition part of ammunition has to be removed or inactivated prior to destruction. Then starts the main part of elimination of the weapon. The US choose high-temperature incineration and chemical neutralization as its preferred destruction technique, which has to destroy the chemicals together with the metal casing. The cost of this procedure can outrun the cost of agent destruction many fold – in some cases by 10-20 times.

The process of elimination is a slow, tedious one, with rising costs as time passes by. A bilateral US – USSR agreement in June 1990 to destroy at least 50 percent of their stockpiles by 1999 and to retain no more than 5,000 tons of agent by 2002 is long outdated [21].

Since 1985, the US Army’s cost estimate for the stockpile disposal program has increased from estimates in 1985 of $1.7 billion to $15.7 billion as of today, and its projected completion date has slipped from 1994 to 2007 [16, 12]. At the end of 1999 about 22 percent of its chemicals had been incinerated [8, 9].

The destruction of the Russian arsenal faces both, financial and technical challenges [17] and is seriously behind schedule. The first deadline imposed by the CWC – destruction of 1 percent of stockpiles by April 29, 2000 – has already been missed. Under the revised program approved by the Russian government in July, this milestone will not be achieved until 2003, while the entire destruction process is scheduled to last until 2012. Russia does not want to copy the well-proven American incineration technology. Its own neutralization-bituminization program has not been developed beyond the laboratory bench, and therefore had destroyed only a few thousand weapons [22]. The idea of incineration of their chemical weapon arsenal by nuclear explosion is studied in Russia’s former weapons laboratories [23]. This procedure, even if it is feasible deep underground, is not compatible with the Comprehensive Test Ban Treaty (CTBT) and will find also serious resistance from environmentalists.

Most estimates for Russia’s costs are in the $6 billion to $8 billion range [18].

4.2 The abandoned weapons

Chemical weapons are buried on land, dumped into the sea and simply lost at many places on our globe [20]. Finding, collecting and destroying them might be as difficult, dangerous and time consuming as those of land mines.

The non-stockpile disposal program is currently projected to cost $15.1 billion – nearly the cost of the stockpile disposal program – and will take until 2033 to complete [12]. There the major cost factor arises from the difficulties of detection of scattered chemical weapons, due to insufficient book-keeping, the necessity to design and built new mobile disposal systems, and last not least overcoming the public opposition of destruction or transporting lethal CW in the vicinity of habitats. The provisions in the CWC will not apply to weapons buried on its territory before 1 January 1977.

4.4 A Comparison of chemical weapons agents with other waste

Our civilization produces a great variety of waste products, with differing degrees of danger for the environment and people. They range from household waste, electronic waste from the information age, to toxic waste from chemical factories, by-products of the mining industry, coal and oil firing, and last not least to those from military and civil use of nuclear energy. Among these waste products is a largely unknown environmental hazard due to the one-to-two-hundred tons of Mercury, that have been discharged into nature during the manufacturing of nuclear weapons in the US (mainly at Oak Ridge, also at Hanford/Washington). Its impact on the food chain can become catastrophic on a regional level [24]. Even the most widely used propellant of weapons, Trinitrotoluol or TNT, is a threat to the environment because of its persistency and its ability to enter easily into ground water.

A crude estimate of the importance of the chemical weapon waste relative to other human waste production can be made taking data from the annual production of waste in kilogram per inhabitant in France:

Waste Kg/person/year
Household (kitchen garbage, diverse domestic scrap) 360
Agriculture (plastic, farming scrap) 7,300
Industrial waste (metal waste, iron, non-iron, powders, technology waste) 3,000
thereof classified as toxic waste 100
Hospital waste 15
Nuclear waste (packaged) 1.2
Total waste 10,776

Table 2 Annual waste production in kilogram per person in France [25]

And by assuming that waste production per person in France (population 58 million) and the United States (population 267 million) is comparable (probably an underestimation of the US figures), the total waste of these categories can be estimated for the US in tons per year:

Waste Tons/year
Household 100· 10 6
Agriculture 2·10 9
Industrial waste 800·10 6
thereof toxic waste 30·10 6
Nuclear waste 320·10 0
Chemical weapons waste 500·10 0
Total waste 3·10 9

Table 3 Crude estimate of annual waste production in the US

It is assumed that the 30,000 tons of US chemical weapons material were accumulated over

60 years, i.e. on the average 500 tons produced per year. The above order of magnitude estimateshows, that nuclear and chemical weapons wastes are in the same ball part, but are hundred thousand times smaller than the other toxic/dangerous waste. Due to the complexity of the toxic items, a qualitative comparison of present and future dangers for mankind and environment by taking only the quantitative aspects into consideration can and should not be made since it may lead to wrong conclusions.

5. Non-lethal chemical weapons

All weapons are made out of chemical elements, be it the metal shell of a grenade, sometimes made of depleted uranium, the explosive agent to propel it or the material filled into its encasing. The dangers of highly toxic, volatile rocket fuel on the delivery systems of nuclear warheads in Russia may be very high [26]. For this simple reason alone it is difficult to come up with an all-encompassing definition for chemical weapons.

Are chemicals still material of weapons if they are used in very low concentrations? The latter point may be illustrated by the double use of Zyklon B (or Cyclon B in English), that is used as fumigant for the purpose of pest and vermin control. It had been applied in low concentration in a beneficial way in the Nazi concentration camp of Dachau, while utilized in high concentration in the gas chambers of Auschwitz, it lead to one of the most criminal acts committed in the twentieth century [27].

Dozens of technologies are being studied or developed under the elastic rubric of “non-lethal weapons” [28]. They include infrasound, supercaustics, irritants like tear gas, and all those that could be aimed at non-human targets – such as combustion inhibitors, chemicals that can immobilize machinery or destroy airplane tires. The text of the CWC does not give always an unambiguous answer or definition what is a chemical weapon agent. It could be asked if the following agents fall into the category of chemical weapons, some of them old as war [10], like (i) Military Smoke Agents, (ii) Incendiaries producing fires and burns of skin? Where do the recently used or newly developed ones belong, like (iii) Sticky Foam, Super Lubricants (“slickums and stickums”), or (iv) Pulsed Chemical Laser Beams? A special case takes (v) Depleted Uranium Ammunition, which can be considered a biological or a radiological weapon.

The preamble to the Convention on Prohibitions or Restrictions on the Use of Certain Conventional Weapons Which May Be Deemed To Be Excessively Injurious or To Have Indiscriminate Effects (CCW), and less formally referred to as the “Inhuman Weapons Convention”, expressed the wish for amendments [30]. Among those was the elimination of laser weapons, which are now banned by the Protocol IV, which was adopted by the Conference of the States Parties to the Convention and entered into force on 30 July 1998 [28, 29].

Other weapons are being negotiated, like submunitions in the form of bomblets assembled in clusters and delivered by aircraft or by artillery, rockets or guided missiles, be equipped with devices making them harmless if they fail to explode. One canister may contain 50 bomblets, or 600, or even as many as 4,700, depending on the model, and may cover a ground area from 100 to 250 meters in diameter. The bomblets, when fitted with delayed action fuses, are effective area-denial weapons. Usually about 30% fail to explode and remain as mines, like many in Kosovo after the 1999 war.

Depleted Uranium (DU) [31], which draw a lot of public attention in the recent decade, is a by-product of enriching natural uranium – increasing the proportion of the U235 atom which is the only form of uranium that can sustain a nuclear reaction and is used in nuclear reactors or nuclear weapons. The remaining depleted uranium has practically no commercial value. The Department of Energy in the US (DoE) has a 560,000-metric-ton stockpile, with very limited civilian use as a coloring matter in pottery or as a steel-alloying constituent [32]. Depleted uranium is chemically toxic like other heavy metals such as lead, but can produce adversary health effects being an alpha particle emitter with radioactive half-life of 4.5 billion years.

In the 1950’s the US became interested in using depleted uranium metal in weapons because it is extremely dense, pyrophoric, cheap, and available in high quantities. Kinetic energy penetrators do not explode they fragment and burn through armour due to the pyrophoric nature of uranium metal and the extreme flash temperatures generated on impact. They contaminate areas with extremely fine radioactive and toxic dust. This in turn can cause kidney damage, cancers in the lung and bone, non-malignant respiratory decease, skin disorders, neurocognitive disorders, chromosomal damage, and birth defects [33]. Depleted uranium weapons are proliferating and are likely to become commonly used in land warfare. The United States, the United Kingdom, France, Russia, Greece, Turkey, Israel, Saudi Arabia, Kuwait, Bahrain, Egypt, Thailand, Taiwan and Pakistan are possessing or manufacture depleted uranium weapons. Many NATO countries may follow suite. These weapons were used in large quantities first in the 1991 Gulf War [33, 34], and then again during the Kosovo War in 1999 [35]. The question can be asked if DU is mainly a chemical, or a radiological weapon? An immediate answer is not to be expected before classified material becomes available, and the medical reason for the Golf-War Syndrome is identified, which shows up in thousands of American soldiers. It appears that effect of the radioactive by inhalation of small doses will have only a small impact on risk to die of cancer, whereas the heavy metal effect seems to dominate [36]. Be it as it might be, depleted uranium is dangerous, but is pales in comparison with the other direct and indirect effects of war.

Due to their double use properties, some chemical weapons may be masked as pesticides, fertilizers, dyes, herbicides, or defoliants. Between 1962 and 1971 more than 72 million liter herbicides were distributed over South Viet Nam [37], thereof more than 44 million liter were the defoliant agent orange, containing about 170 kg dioxin. American scientists developed a means of thickening gasoline with the aluminum soap of naphtenic and palmitic acids into a sticky syrup that carries further from projectors and burns more slowly but at a higher temperature. This mixture, known as Napalm, can also be used in aircraft or missile-delivered warheads against military or civilian targets. A small, high explosive charge scatters the flaming liquid, which sticks to what it hits until burned out. Is Napalm still only a herbicide even when used in too large a quantity, and then accidentally affecting humans?

White phosphorous is used as a shell and grenade filler in combination with a small high-explosive charge. It is both an incendiary and the best-known producer of vivid white smoke. Small bits of it burn even more intensely than Napalm when they strike personnel.

Herbicides are not covered by the Convention but they are banned under the Prohibition of Military or any other Hostile Use of Environmental Modification Techniques (ENMOD), adopted by the UN General Assembly on the 10th of December 1976 and entered into force the 5th of October 1978 [38].

In order to curb the production of chemical weapons, require their identification, e.g. by trace elements in ammunition!

6. Old and New Biological Weapons

The use of biological agents as weapon has always an even more adverse world opinion than chemical warfare. A SIPRI Monograph describes among other topics the changing view of biological and toxin warfare agents, the new generation of biological weapons, the changing status of toxin weapons, a new generation of vaccines against biological and toxin weapons, and the implications of the BWC [39].

Claims that biological agents have been used as weapons of war can be found in both the written records and the artwork of many early civilizations [40]. As early as 300 BC the Greeks polluted the wells and drinking water supplies of their enemies with the corpses of animals. Later the Romans and Persians used the same tactics. In 1155 at a battle in Tortona, Italy, Barbarossa broadened the scope of biological warfare, using the bodies of dead soldiers as well as animals to pollute wells. In 1863 during the US Civil War, General Johnson used the bodies of sheep and pigs to pollute drinking water at Vicksburg. The use of catapults as weapons was well established by the medieval period, and projecting over the walls dead bodies of those dead of disease was an effective strategy for besieging armies. In 1763 the history of biological warfare took a significant turn from the crude use of diseased corpses to the introduction of specific decease, smallpox (“Black Death”), as a weapon in the North American Indian Wars. This technique continued with cholera or typhus infected corpses. In 1915, during World War I, Germany was accused of using cholera in Italy and plague in St. Petersburg. There is evidence Germany used glanders and anthrax to infect horses (1914) and cattle, respectively, in Bucharest in 1916, and employed similar tactics to infect 4,500 mules in Mesopotamia the next year.

The period 1940 – 1969 can be considered the golden age of biological warfare research and development. Especially the 1940s were the most comprehensive period of biological warfare research and development.

The US had signed the Geneva Protocol, but the Senate voted only in 1974 on it. Detailed information on the history of the US Offensive Biological Warfare Program between 1941 and 1973 can be found in ref. [41].

It has been reported recently that the US tested a Soviet-designed germ bomb and assembled a germ factory in the Nevada desert from commercially available materials, in particular to produce potentially more potent variant of the bacterium that causes anthrax, a deadly disease ideal for germ warfare [42]. It is debatable if such a research is consistent with the treaty banning biological weapons.

The Former Soviet Union had an important biological weapons program, which might have extended well into the period after its dissolution [43].

For a decade after 1972 there was hope that the problem of Biological Warfare was going to be eradicated. However, the last two decades have produced indications that some eight developing nations, in addition to China and Israel, have initiated biological weapon development programs of varying degrees.

Biological warfare (BW) agents, or biological weapons, are ‘living organisms, whatever their nature, or infectious material derived from them, which are intended to cause disease or death in man, animal, and plants, and which depend for their effects on their ability to multiply in the person, the animal, or plant attacked’. BW agents, however, might be used not only in wars, but also by terrorists. One should therefore refer to living organisms ‘used for hostile purposes’.

The Biological Weapons Convention (BWC) prohibits bacteria such as salmonella being used against soldiers. It would permit bacteria, that eat petroleum or rubber for the destruction of equipment for peaceful purposes, but prohibits their use for hostile application.

6.2 Toxic warfare agents and other chemical warfare agents

Toxins are poisonous substances usually produced by living organisms. Toxin warfare (TW) agents, or toxic weapons, are toxins used for hostile purposes. TW agents unequivocally are types of chemical warfare (CW) agent. CW agents, or chemical weapons, are chemical substances whether gaseous, liquid, or solid, which are used for hostile purposes to cause disease or death in humans, animals or plants and which depend on their direct toxicity for their primary effect.

TW agents, like all other CW agents, are inanimate and are incapable of multiplying. They are CW agents irrespective of whether they are produced by a living organism or by chemical synthesis or even whether they are responsible for the qualification of that organism as a BW agent.

Nevertheless, TW agents are often mistakenly considered to be biological weapons, and definitions of biological warfare (BW) occasionally include TW agents. New chemical weapons agents, who are 5 to 10 times more dangerous than VX, the most dangerous toxic gas known today.

The successful control of biological weapons is a daunting task [44]. Ensuring safety from biological and toxin weapons is a more complex issue than totally prohibiting chemical or nuclear weapons. This is due to the character of the relevant technologies. More than those, biotechnology is of dual-use, i.e. the same technology can be used for civilian and permitted military defensive purposes as well as for prohibited offensive or terrorist purposes.

6.3 Biological Warfare against Crops

Intentionally unleashing organisms that kill an enemy’s food crops is a potentially devastating weapon of warfare and terrorism [45]. All major food crops come in a number of varieties, each usually suited to specific climate and soil conditions. These varieties have varying sensitivities to particular diseases. Crop pathogens, in turn, come in different strains or races and can be targeted efficiently against those crop brands. This way it might be possible to attack the enemy’s food stock, but preventing damage to the own. However, such a strategy may not work for neighboring countries, where agricultural conditions are similar to the aggressor. The spread of those organisms holds the risk of worldwide epidemic, and the use of these weapons may very well be counter productive. Any such warfare would be directed primarily against the civilian population. Due to the delays involved it would not affect immediately the outcome of a war.

Nevertheless, many countries developed during the twentieth century anticrop substances.

Iraq manufactured from the 70s onward wheat smut fungus, targeting wheat plants in Iran. France’s biological weapon program by the end of the 1930s included work on two potato killers. During the Second World War the British concentrated on various herbicides. Germany investigated during the same period diseases like late blight of potatoes and leaf-infecting yellow and black wheat rusts, as well as insect pests, such as the Colorado beetle. Japan’s World War II biological weapons program is not too well known, but it contains pathogens and chemical herbicides. The American efforts were substantial. They centered on products attacking crops of soybeans, sugar beets, sweet potatoes and cotton, intended to destroy wheat in the western Soviet Union, and rice in Asia, mainly China. Between 1951 and 1969 the U.S. stockpiled more than 30,000 kilograms of the fungus that causes stem rust of wheat, a quantity probably enough to infect every wheat plant on the planet [45]. According to another source [46] 36,000 kilograms of wheat stem rust, and additional quantity of stem rust of rye, only 900 kilograms of rice blast were produced and stockpiled. The U.S., using the “feather bomb” and free-floating balloons developed ingenious distribution and transport systems.

7. WMD: Warfare, Terrorism, Comparative Perspective

The concept of weapons of mass destruction (WMD) should be revisited, as pointed out in the Introduction of this article. Physical efficiency and psychological effect of these weapons may differ considerably when they are used in warfare on soldiers or in peacetime by terrorists. Industrialized countries can develop reliable and sophisticated technologies, which may not be available to small groups.

7.1 Weapons in Warfare

The efficiency of weapons in warfare is closely related to the time parameter:

  • Number of enemy casualties in a given period,
  • Number of weapons employed to obtain the desired result,
  • Delivery time of weapons,
  • Possibility for stockpiling over extended periods,
  • Infrastructure affected by its use,
  • Avoidance of negative impact upon own troops and civil population,
  • End a war quickly,
  • No efficient defense against weapons on short or long term.

Evidently, nuclear weapons are “superior” to any other weapons on all these points. Is a specific weapon category useful in conflicts between countries and/or in civil war? Can it serve as a deterrent? Does its use have long term effects on the crop area?

The efficiency of chemical and biological weapons depends heavily on its dispersion, upon the weather condition, determining the exposure and lethality for the combatants. A presumptive agent must not only be highly toxic, but also ‘suitably highly toxic’, so that it is not too difficult to handle by the user. It must be possible to store the substance in containers for long periods without degradation and without corroding the packaging material. Such an agent must be relatively resistant to atmospheric water and oxygen so that it does not lose its effect when dispersed. It must also withstand the shearing forces created by the explosion and heat when it is dispersed. Transport of these agents by long-range missiles and efficient distribution will face enormous difficulties, causing their decomposition, mainly due to the heat development of the warhead at re-entry into the atmosphere. A few developed countries may already be capable to overcome these hurdles [47].

Finding an answer to these questions can be facilitated by evaluation of previous wars.

In World War I an average of one ton of agent was necessary to kill just one soldier. Chemical weapons caused 5 percent of the casualties. The use of chemical weapons did not end the war quickly as had been predicted. During the war between Iraq and Iran through March 1997 27,000 Iranians were exposed to chemical grenades, only 265 died. During the entire war between these two countries chemical weapons killed 5,000, out of the total 600,000 from all causes, i.e. less than 1 percent [6].

The efficiency of chemical/biological weapons in future wars is difficult to predict. Estimates cover a wide range, as shown below.

Under ideal conditions 1 ton of Sarin dropped from an airplane could produce 3,000 to 8,000 deaths, however, under breezy conditions only 300 to 800 [6]. To obtain a sensible effect requires that airplanes fly at very low altitude (less than about 100 meters), and consequently the zone of lethality that could be covered remains small. Furthermore, agent particles larger than 10 micrometers do not reach the non-ciliated alveolar region in the lungs, and those, with a size of about 1-micrometer are exhaled. The optimal size is somewhere between 10 to 5 micrometers, which can not be obtained easily. Sunlight kills or denatures most biological agents. Anthrax efficiency may drop by a factor of thousand when the agent is used during a sunny day. Therefore, the agents have to be sprayed during nighttime.

Chemical weapons depend more than other armament upon atmospheric and topographical factors, whilst temperature, weather and terrain are important factors in determining the persistence of a given chemical agent. Chemical attacks can contaminate an area for between several hours and several days. Weight-for-weight, biological weapons are hundreds to thousands of times more potent than the most lethal chemical weapon [47. 48]. Contamination time is between several hours and several weeks.

A Scud missile warhead filled with botulinum could contaminate an area of 3,600 square kilometers, or 16 times greater than the same warhead filled with the nerve agent Sarin [49].

A United Nations study [50] compared the hypothetical results of an attack carried out by one strategic bomber using either nuclear, chemical or biological weapons. A one-megaton nuclear bomb, the study found, might kill 90 percent of unprotected people over an area of 300 square kilometers. A chemical weapon of 15 tons might kill 50 percent of the people in a 60 square kilometer area. But a 10-ton biological weapon could kill 25 percent of the people, and make 50 percent ill, over an area of 100,000 square kilometers.

If a ballistic missile hits a city delivering 30 kilograms of anthrax spores in a unitary warhead against a city with no civil defense measure could result in lethal inhalation dosage levels over an area of roughly 5 to 25 square kilometers. With no treatment, most of the infected population would die within a week or two. For typical urban population densities this could result in the deaths of tens of thousands or even hundreds of thousands of people [51].

Exaggerated, counterproductive, essentially incorrect, and even dangerous remarks by a US high-ranking official have been made. He claimed that about 2.5 kilograms of anthrax if released in the air over Washington, DC, would kill half of its population, that is, 300,000 people (TV, Nov.1997). In March 1988, four of the most qualified experts on anthrax serving in the US government published a paper in the Archives of Internal Medicine which used a different estimate: 50 kilograms of anthrax released over a city of 500,000 people could kill up to 95,000 people, and possible fewer, depending on urban atmospheric conditions. The first estimate was approximately 100 times higher [46, 52].

These above efficiencies assume, however, that chemical and biological agents can be spread over a large surface and reach the ground level, whereas nuclear weapons can be exploded at any predetermined altitude and on ground level with the desired efficiency.

7.2 Weapons for Terrorists

There is a largely unjustified fear of the public concerning terrorist attacks with chemical or biological agents, their impact on daily life, their frequency, and number of people possibly affected.

Between 1960 and 1980 there have been 40,000 international terror incidents (according to CIA), but only 22 out of them were performed with chemical or biological agents, showing a tiny ratio of 1/2,000. From 1900 till today there occurred 71 terrorist acts worldwide involving the use of biological or chemical agents, resulting in 123 fatalities, among those only one was American, hit by a cyanide-laced bullet. These acts produced 3,774 nonfatal injuries (784 Americans, 751 out of them by salmonella food poisoning by an Oregan-based religious sect). During the first nine decades of the 20th century there have been 70 biological attacks (18 by terrorists), causing 9 deaths [6].

The Aum-Shinrikyo sect in Japan had about $1 billion (another source gives $1.2 to 1.6 billion) at its disposal for development of chemical and biological weapons.

  • Aum had appropriate equipment (even more than it was necessary).
  • Aum had used commercial front companies to buy the equipment.
  • Aum may have spent about $10 million in their effort to produce biological agents.
  • Several of the individuals had post-graduate degrees.
  • Aum had gathered a research library.
  • Aum had sufficient time – four years – for their attempts.
  • Aum had attempted to purchase expertise in Russia and obtain or purchase disease strains in Japan.

However, Aum failed to produce either of two biological agents, Clostridium botulinum, to obtain Botulinum toxin, and anthrax, and also did not manage to “disperse” them. Despite its efforts, spending $10 million on the development of biological agents. Aum sprayed botulinum toxin over Tokyo several times in 1990, and conducted similar activities with anthrax spores in 1993, but without any known effects. Actually, the cult had used a relatively harmless anthrax vaccine strain and the aerosolizer had no sufficient efficiency [53, 54].

There are two well-publicized Aum attacks with chemical agents (Matsumoto, 3 kg of pure Sarin, 1994 Tokyo subway, 6-7 kg 30% pure Sarin, 1995), the latter made in a confined area, limiting a detrimental effect of air current. Nevertheless, the Matsumoto assault killed only seven non-targeted innocents, and in Tokyo only twelve people died from direct contact with the liquid and not from fumes [54].

A more detailed description of risk assessment by terrorism with chemical and biological weapons can be found in [54]. This article provides results from computer simulation for dispersion of chemical and biological agents under various atmospheric conditions and their impact parameters on human health.

7.3 Comparative Perspective

Analysts have defined Mass Casualty as anything between 100 and 1,000 individuals arriving at hospitals. The numbers in the previous section are related to deaths, and a factor of up to about ten has to be applied to encompass individuals suffering non-lethal injuries. Evidently, similar factors have to be used for victims of conventional weapons in war.

In the discussion of biological agent terrorism as a potential mass casualty event it is quite revealing to look at the annual mortality in several public health sectors in the USA [53]:

• Food-borne disease incidence: 76 million cases per year
315,000 hospitalizations per year
5,000 deaths per year
• Medical error mortality: between 44,000 and 98,000 deaths per year
• Hospital contracted infections: 20,000 deaths per year
• The 1993 cryptosporidium outbreak in Milwaukee (water pollution) sickened 400,000 people
• Air pollution in the US results in 50,000 deaths per year
• Firearms result in 35,000 death per year.

Compared with these data, the impact of biological and chemical agents terrorism in the past is negligible and will remain probably (hopefully!) small.

8. Implementation of the Chemical and Biological Weapons Convention and Conclusions

Like most scientific and industrial developments there is the possibility to apply them for the good or for the bad. The responsibility of the scientists, as well as the politicians and military, is challenged. The production of the basic material for military or civilian application is closely intertwined. This makes any inspection and accusation of intended military use extraordinary difficult. In addition manufacturers fear for their patents and are worried about industrial espionage.

Production of biological warfare agents can be done in any hospital or basement rooms in small quantities by qualified personal, for chemicals it requires larger plants. The 121 States Parties and 48 signatory states of the Chemical Weapons Convention have an implementation body, the Organisation for the Prohibition of Chemical Weapons (OPCW), which is operational since two years from The Hague [7]. It performed already more than 500 inspections. The OPCW has about 500 staff members, consisting of 200 inspectors and 300 administrative staff. Out of these 300 administrators most are verification experts and inspection planers. Among the most important old issues are: guidelines for low concentrations, the usability of old and abandoned chemical weapons. As mentioned above the Chemical Weapons Convention (CWC) does not cover sea-dumped chemical weapons.

There has not yet been progress in the establishment of an analogue organization for Biological and Toxin Weapons Convention (BWC). It might be placed in The Hague or in Geneva. Work on the protocol to strengthen the Biological Weapons Convention, as well as the verification protocol is still in its initial state, and a success of the 5 th BWC Review Conference to be held in Geneva in November 2001 is not at all assured [46]. Of the 141 States Parties to the BWC only around 60 send delegations to the Ad Hoc Group (AHG). Not all of the AHG accept the concept of random visits. The establishment of an international organization to oversee the implementation of the BWC protocol is estimated to consist out of a staff of 233 people and an annual cost of approximately $30 million. There might be eventually about 70 inspectors carrying out approximately 100 visits per year. One of the disputed topics is related to new forms of biological weapons, caused by the biotechnology revolution [38]. The delivery system or the efficiency of these new agents has not changed, but their capability to manipulate human life processes themselves. Biological weapons should now be seen as a global threat to the human species, but not as an efficient weapon in warfare.

Inspections of biological agents will hit more resistance by the pharmaceutical and bio-technical industry than the one in the chemical industry.

The dangerous leftovers from the chemical weapons race, like the ones from nuclear weapons construction, not to forget the land mines, will be still with us for a long time. Ethics, politics and international security should be closely interlaced to remove these inhuman weapons from Earth. There is an excellent opportunity for fruitful collaboration between defense conversion sector and the environmental community.

The CBWC has certainly the beneficial effect in reducing the arsenal of old weapons, but will not give a guarantee that new, clandestine developments in various countries will go on unnoticed.

The difficulty to use these weapons efficiently is in general underestimated, but their impact exaggerated. This combination causes unjustifiable fear of the public and leads policy makers to wrong conclusions, among them to designate them as WMDs and keep nuclear weapons as a deterrent.

The critical, comparative assessment of the three types of weapons (one may want to include radiological weapons) presented in this article are not intended to slow down efforts for the elimination of chemical and biological weapons. The CBWC should remain an important treaty and negotiations on enforcement provisions should be accelerated, so that it can be eventually fully implemented. In particular, the arsenal of unused weapons, being in storage or “disposed” in the oceans or elsewhere, presents a considerable danger on short and long term for humans and the environment. Anybody killed by these weapons is one too much. However, we have to put these weapons and the ratified conventions in the right quantitative perspective.

In the view of the author most of the conventional weapons, in particular small arms, are weapons of Mass Killing: According to a Red Cross inquiry [57] Assault Rifles, like AK47s, Handguns, and Land Mines, caused 64%, 10% and 10% of civilian casualties, respectively. The remaining 16% are almost equally shared between Hand Grenades, Artillery (including fragmentation and incinerating bombs), Mortars, and Major Weapons. During the 20th century these weapons had been used to kill 34 million soldiers in combat, 80 million civilians, plus soldiers who died from wounds, accidents or disease. The world was “fortunate” that only two nuclear bombs have been dropped in warfare until now. They killed “only

200,000 people. Nevertheless, the nuclear arsenal has to be on the top of the WMD-category, since it has the potential to erase humans from our planet in almost no time.

Maintaining nuclear weapons by the Nuclear Weapon States (NWSs) to deter production and stockpiling of chemical and biological weapons, mainly in countries of concern, can only be interpreted as an unjustifiable, unreasonable pretext to keep nuclear weapons indefinitely in stock. Is it politically wise to change the unfortunate, misleading definition of weapons of mass destruction (WMD = NW + CW + BW), repeated again and again in the media, and deeply engraved into the mind of people? Will a new definition distract from the importance of the two, universally ratified treaties? Might it be counterproductive to do so in a time, where scientists are under increasing scrutiny and attack?

The author felt that informing the educated public and policy makers on a re-definition of WMD warrants the change and outweighs possible negative repercussions.

I like to thank Professor W.K.H. Panofsky for carefully reading a previous version of this article, and for valuable criticism and useful suggestions. Dr. Milton Leitenberg is thanked for providing a lot of relevant literature and sharing with me his profound knowledge and insight into the problem of biological warfare and terrorism. I profited much from participation in workshops in Como/Italy and Rome, organized by Professor Maurizio Martellini, and thank him for the kind invitation to these events. The opinion expressed in this article is those of the author and under his sole responsibility.

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[27] http://www.altavista.com/cgibin/query?pg=q&sc=on&hl=on&q=Zyklon+B&kl= XX&stype=stext&search.x=24&search.y=7

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[29] Protocol IV on Blinding Laser Weapons, http://www.austlii.edu.au/au/other/dfat/multi/19980730.html

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[45] P. Rogers, S. Whitby and M. Dando, “Biological Warfare against Crops”, Scientific American, June 1999, pp. 62-67.

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[52] Milton Leitenberg, “Terrorism and Weapons of Mass Destruction”, ISPAC, International Scientific and Professional Advisory Council of the United Nations Crime Prevention and Criminal Justice Program, International Conference on Countering Terrorism Through Enhanced International Cooperation, Courmayeur, Mont Blanc, pp. 1-33, 22-24 September 2000.

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[54] Jean Pascal Zanders, Edvard Karlsson, Lena Melin, Erik Näslund and Lennart Thaning, in SIPRI Year book 2000, Oxford University Press, Appendix 9A. “Risk assessment of terrorism with chemical and biological weapons”.

[55] Milton Leitenberg, “Biological Weapons: A Reawakened Concern”, The World & I, pp. 289-305, January 1999.

[56] Milton Leitenberg, “Biological Weapons Arms Control”, Project on Rethinking Arms Control, Center for International and Security Studies at Maryland, PRAC Paper No. 16, May 1996, http://www.puaf.umd.edu/CISSM/publications/bwarmscon.pdf

[57] Jeffrey Boutwell and Michael T. Klare, “A Scourge of Small Arms”, Scientific American, June 2000, pp. 30-35.



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