Implementation of a Regional Training Program on African Swine Fever As Part of the Cooperative Biological Engagement Program across the Caucasus Region.

A training and outreach program to increase public awareness of African swine fever (ASF) was implemented by Defense Threat Reduction Agency and the Ministries of Agriculture in Armenia, Georgia, Kazakhstan, and Ukraine. The implementing agency was the company SAFOSO (Switzerland). Integration of this regional effort was administered by subject matter experts for each country. The main teaching effort of this project was to develop a comprehensive regional public outreach campaign through a network of expertise and knowledge for the control and prevention of ASF in four neighboring countries that experience similar issues with this disease. Gaps in disease knowledge, legislation, and outbreak preparedness in each country were all addressed. Because ASF is a pathogen with bioterrorism potential and of great veterinary health importance that is responsible for major economic instability, the project team developed public outreach programs to train veterinarians in the partner countries to accurately and rapidly identify ASF activity and report it to international veterinary health agencies. The project implementers facilitated four regional meetings to develop this outreach program, which was later disseminated in each partner country. Partner country participants were trained as trainers to implement the outreach program in their respective countries. In this paper, we describe the development, execution, and evaluation of the ASF training and outreach program that reached more than 13,000 veterinarians, farmers, and hunters in the partner countries. Additionally, more than 120,000 booklets, flyers, leaflets, guidelines, and posters were distributed during the outreach campaign. Pre- and post-ASF knowledge exams were developed. The overall success of the project was demonstrated in that the principles of developing and conducting a public outreach program were established, and these foundational teachings can be applied within a single country or expanded regionally to disseminate disease information across borders; overall, this method can be modified to raise awareness about many other diseases.

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing.

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

DNA polymerase zeta is essential for hexavalent chromium-induced mutagenesis.

Translesion synthesis (TLS) is a unique DNA damage tolerance mechanism involved in the replicative bypass of genetic lesions in favor of uninterrupted DNA replication. TLS is critical for the generation of mutations by many different chemical and physical agents, however, there is no information available regarding the role of TLS in carcinogenic metal-induced mutagenesis. Hexavalent chromium (Cr(VI))-containing compounds are highly complex genotoxins possessing both mutagenic and clastogenic activities. The focus of this work was to determine the impact that TLS has on Cr(VI)-induced mutagenesis in Saccharomyces cerevisiae. Wild-type yeast and strains deficient in TLS polymerases (i.e. Polzeta (rev3), Poleta (rad30)) were exposed to Cr(VI) and monitored for cell survival and forward mutagenesis at the CAN1 locus. In general, TLS deficiency had little impact on Cr(VI)-induced clonogenic lethality or cell growth. rad30 yeast exhibited higher levels of basal and induced mutagenesis compared to Wt and rev3 yeast. In contrast, rev3 yeast displayed attenuated Cr(VI)-induced mutagenesis. Moreover, deletion of REV3 in rad30 yeast (rad30 rev3) resulted in a significant decrease in basal and Cr(VI) mutagenesis relative to Wt and rad30 single mutants indicating that mutagenesis primarily depended upon Polzeta. Interestingly, rev3 yeast were similar to Wt yeast in susceptibility to Cr(VI)-induced frameshift mutations. Mutational analysis of the CAN1 gene revealed that Cr(VI)-induced base substitution mutations accounted for 83.9% and 100.0% of the total mutations in Wt and rev3 yeast, respectively. Insertions and deletions comprised 16.1% of the total mutations in Cr(VI) treated Wt yeast but were not observed rev3 yeast. This work provides novel information regarding the molecular mechanisms of Cr(VI)-induced mutagenesis and is the first report demonstrating a role for TLS in the fixation of mutations induced by a carcinogenic metal.

Excision repair is required for genotoxin-induced mutagenesis in mammalian cells.

Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of Cr(VI) mutagenesis and the role that DNA repair plays in this process. Our goal was to investigate the role of excision repair (ER) pathways in Cr(VI)-mediated mutagenesis in mammalian cells. Repair-proficient Chinese hamster ovary cells (AA8), nucleotide excision repair (NER)-deficient (UV-5) and base excision repair (BER)-inhibited cells were treated with Cr(VI) and monitored for forward mutation frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. BER was inhibited using methoxyamine hydrochloride (Mx), which binds to apurinic/apyrimidinic sites generated during BER. Notably, we found that both NER-deficient (UV-5 and UV-41) and BER-inhibited (AA8 + Mx) cells displayed attenuated Cr(VI) mutagenesis. To determine whether this was unique to Cr(VI), we included the alkylating agent, methylmethane sulfonate (MMS) and ultraviolet (UV) radiation (260 nm) in our studies. Similar to Cr(VI), UV-5 cells exhibited a marked attenuation of MMS mutagenesis, but were hypermutagenic following UV exposure. Moreover, UV-5 cells expressing human xeroderma pigmentosum complementation group D displayed similar sensitivity to Cr(VI) and MMS-induced mutagenesis as AA8 controls, indicating that the genetic loss of NER was responsible for attenuated mutagenesis. Interestingly, Cr(VI)-induced clastogenesis was also attenuated in NER-deficient and BER-inhibited cells. Taken together, our results suggest that NER and BER are required for Cr(VI) and MMS-induced genomic instability. We postulate that, in the absence of ER, DNA damage is channeled into an error-free system of DNA repair or damage tolerance.

Nucleotide excision repair functions in the removal of chromium-induced DNA damage in mammalian cells.

Some hexavalent chromium (Cr(VI))-containing compounds are human lung carcinogens. While ample information is available on the genetic lesions produced by Cr, surprisingly little is known regarding the cellular mechanisms involved in the removal of Cr-DNA adducts. Nucleotide excision repair (NER) is a highly versatile pathway that is responsive to a variety of DNA helix-distorting lesions. Binary Cr-DNA monoadducts do not produce a significant degree of helical distortion. However, these lesions are unstable due to the propensity of Cr(III) to form DNA adducts (DNA interstrand crosslinks, DNA-protein/amino acid ternary adducts) which may serve as substrates for NER. Therefore, the focus of this study was to determine the role of NER in the processing of Cr-DNA damage using normal (CHO-AA8) and NER-deficient [UV-5 (XP-D); UV-41 (ERCC4/XP-F)] hamster cells. We found that both UV-5 and UV-41 cells exhibited an increased sensitivity towards Cr(VI)-induced clonogenic lethality relative to AA8 cells and were completely deficient in the removal of Cr-DNA adducts. In contrast, repair-complemented UV-5 (expressing hamster XPD) and UV-41 (expressing human ERCC4) cells exhibited similar clonogenic survival and removed Cr-DNA adducts to a similar extent as AA8 cells. In order to extend these findings to the molecular level, we examined the ability of Cr(III)-damaged DNA to induce DNA repair synthesis in cell extracts. Repair synthesis was observed in reactions using extracts derived from AA8, or repair-complemented, but not NER-deficient cells. Cr(III)-induced repair resynthesis was sensitive to inhibition by the DNA polymerase delta/epsilon inhibitor, aphidicolin, but not 2',3'-dideoxythymidine triphosphate (ddTTP), a polymerase beta inhibitor. These results collectively suggest that NER functions in the protection of cells from Cr(VI) lethality and is essential for the removal of Cr(III)-DNA adducts. Consequently, NER may represent an important mechanism for preventing Cr(VI)-induced mutagenesis and neoplastic transformation.