Background: Folate is essential for DNA synthesis, repair, and methylation. Polymorphisms in genes associated with folate metabolism may alter these processes and, consequently, modulate cancer development. Aim: We aimed to assess DNMT3B -149C/T (rs2424913), DNMT3B -283T/C (rs6087990), DNMT3B -579G/T (rs2424909), DHFR 19-pb ins/del (rs70991108), SHMT1 1420C/T (rs1979277), and TYMS 28-bp tandem repeat (rs34743033) polymorphisms with risk of head and neck cancer. Methods: A case-control study was conducted in 1,086 Brazilian individuals. Real-time and conventional polymerase chain reactions-PCR were performed for genotyping the polymorphisms. Results: The single nucleotide polymorphism (SNP), DNMT3B -283T/C, revealed a higher risk of head and neck squamous cell carcinoma (HNSCC) when compared with the C group in the codominant (p< 0.001), dominant (p<0.001), and overdominant (p= 0.001) models for T/C and C/C genotypes. DNMT3B -149C/T and DNMT3B -579G/T revealed no association between groups in any model. The DHFR 19-pb ins/del polymorphism protected against HNSCC development compared to the C group by the codominant (p< 0.001), dominant (p< 0.001), and overdominant (p< 0.001) models. In the TYMS, the 3R/3R genotype had a protective effect against HNSCC development compared with the C group by the recessive models (p= 0.009). In contrast, SHMT1 1420 C/T presented no association between the HNSCC and C groups. DHFR 19-pb ins/del polymorphisms protected against oral cavity cancer (p= 0.003), and only TYMS-28 3R/3R decreased the risk of tumor progression (p= 0.023). In the Kaplan–Meier curve, an association was found between DHFR ins/ins and TYMS -28 3R carriers with respect to relapse-free time; further, DNMT3B -579 T and TYMS-28 2R/2R carriers had longer survival times. Conclusion: DNMT3B -283T/C is associated with higher risk, whereas DHFR 19-pb ins/del and TYMS 28 3R/3R protect against head and neck cancer. We also highlighted the association of TYMS 3R/3R genotype carriers with relapse-free cancer protection and survival time.  Presently, head and neck cancer is considered the fifth most common type of cancer worldwide and is associated with a high mortality rate when diagnosed in advanced stages. In 2020, 834,860 new cases of this neoplasm were estimated . This cancer comprises a heterogeneous group of tumors that originate in lining epithelium squamous cells of the upper digestive tract, including the lip, oral cavity, nasal cavity, paranasal sinus, pharynx, and larynx .Elderly males are mostly affected . The main risk factors include tobacco and alcohol consumption, viral infections, particularly with the Epstein-Barr virus and human papillomavirus subtypes 16 and 18, as well as deficiencies or imbalances in vitamins and micronutrients such as folate, vitamins A, C, and E, zinc, and selenium . Smoking and alcohol consumption together increase the risk of this cancer, particularly in the oral cavity and pharynx, as cigarettes have approximately 4,700 chemical substances, of which at least 50 are carcinogenic; frequent alcohol consumption prevents epithelial cells forming the protective barrier against external agents, thus permitting easy entry of carcinogens, thereby forming adducts of DNA that are not recognized during DNA replication and repair . Folate or vitamin B plays a pivotal role in one-carbon metabolism, and generally low levels can be associated with the absence of methylation and DNA repair, thereby promoting carcinogenesis . Thus, several functional gene polymorphisms linked to folate metabolism can cause an imbalance in methylation reactions, as well as free nucleotides for DNA synthesis and repair . In most tumor models, higher intake of folate might decrease the risk of cancer development, as observed in oropharyngeal , esophageal , pancreatic , cervical , and breast cancer ; however, an inverse association can be seen in some tumor models, such as head and neck  and prostate cancers .DNMT3B -149C/T (rs2424913), DNMT3B -283T/C (rs6087990), DNMT3B -579G/T (rs2424909), DHFR 19-pb ins/del (rs70991108), SHMT1 1420C/T (rs1979277), and TYMS 28-bp tandem repeat (rs34743033) with the risk of head and neck cancer. Thus, considering the contradictory results and genetic heterogeneity of the Brazilian population, it is important to evaluate the role of certain folate polymorphisms for susceptibility to head and neck carcinogens. Here, we aimed to investigate the association of polymorphisms Ethics StatementThe Research Ethics Committee of São José do Rio Preto Medical School (FAMERP) in São José do Rio Preto, São Paulo, Brazil, approved this study (Registration Number 013/2012), and written informed consent for the collection of biological material was obtained from all individuals.Subjects and SamplesDNMT3B -149C>T (rs2424913), DNMT3B -283T>C (rs6087990), DNMT3B -579G>T (rs2424909), DHFR 19-pb ins/del (rs70991108), SHMT1 1420C>T (rs1979277), and TYMS 28-bp tandem repeat (rs34743033)].This was a case-control study. The case groups included 378 patients  with a confirmed histopathological diagnosis of cancer. The disease-free control group (C) included 708 Brazilian blood donors , without cancer diagnoses according to government guidelines for donated blood . DNA was obtained from the leukocytes from peripheral blood samples of all participants , and was genotyped for folate polymorphisms [Individuals were classified as having early stage  and advanced stage tumors  based on their tumor, node, and metastasis (TNM) staging. The case and control group participants were interviewed to obtain their demographic and lifestyle data. Those who smoked more than 100 cigarettes in their lifetime were considered as tobacco consumers and those who consumed four doses of alcohol per week were considered as alcohol consumers.Polymorphism GenotypingDNMT3B -149C/T (rs2424913), DNMT3B -283T/C (rs6087990), and SHMT1 1420C/T (rs1979277) polymorphisms were detected via a real-time polymerase chain reaction (PCR) for allelic discrimination using the Step One PlusTM Real-Time PCR System equipment , whereas DNMT3B -579G/T (rs2424909) was detected via PCR-RFLP (PCR-Restriction Fragment Length Polymorphism), and allele-specific PCR was used to assess the DHFR 19-pb ins/del (rs70991108) and TYMS 28-bp tandem repeat (rs34743033). For both PCR techniques, the reaction solution contained the following: 1 × buffer, 15.3 μL of ultrapure H2O, 2.0 μL (0.10 μmol/L) of dNTPs, 0.5 μL (25 mmol/L) of MgCl2, 1.25 μL of each primer (25 mmol/L), 0.2 μL (1 U) of Taq DNA polymerase, and 200 ng of genomic DNA. The PCR amplification products were visualized on a 3% agarose gel (Invitrogen®) with ethidium bromide in the presence of a 100 bp molecular marker. DNA was extracted from the peripheral blood following a previously published protocol  with certain modifications (using Ficoll-Paque™ PLUS to separate blood components). The Statistical AnalysisSNPStats software was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) for the risk associations between polymorphisms and head and neck cancer . The multiple logistic regression models were adjusted for age, gender, drinking, and smoking habits. The effect of the polymorphisms was evaluated in the models as (1) codominant (heterozygous vs. wild-type homozygous and polymorphic homozygous vs. wild-type homozygous); (2) dominant (heterozygous + polymorphic homozygous vs. wild-type homozygous); (3) recessive (polymorphic homozygous vs. wild-type homozygous + heterozygous); (4) overdominant (heterozygous vs. wild-type homozygous + polymorphic homozygous); or (5) log-additive (polymorphic homozygous with 2 + heterozygous vs. wild-type homozygous). Statistical analyses were performed using GraphPad Prism version 5.0  and SNPstats (https://www.snpstats.net/start.htm) . The haplotype frequencies of DNMT3B were inferred using the Haploview program, version 4.0 . A probability level (p) of < 0.05 was considered to indicate statistical significance. DNMT3B -149C>T (rs2424913) was not in Hardy–Weinberg equilibrium (HWE) in the case group (p < 0.001); DNMT3B -579G>T (rs2424909), DHFR 19-pb ins/del (rs70991108), and TYMS 28-bp tandem repeat (rs34743033) were not in HWE in the case  and control groups . The other polymorphisms complied with HWE in both the case and control groups.The The sample group presented a median age of 51 years, and male gender was prevalent in the case (89.0 %) and control (73.0 %) groups. The multiple logistic regression analysis that evaluated the sociodemographic characteristics and risk factors indicated that the male gender , age ≥ 51 years , smoking habit , and alcohol consumption  were associated with greater susceptibility to the development of this neoplasia when compared to the control group .DNMT3B -283T/C (rs6087990) SNP was associated with a higher risk of head and neck squamous cell carcinoma (HNSCC) on comparison with the C group in the codominant model for both T/C and C/C genotypes  and dominant  and overdominant models . The other two SNPs evaluated in this gene, DNMT3B -149C/T (rs2424913) and DNMT3B -579G/T (rs2424909), presented no association between groups in any model evaluated  was associated with a protective effect against HNSCC development compared to the C group by the codominant , dominant , and overdominant models . Similar results were obtained for TYMS 28-bp tandem repeat (rs34743033), where the 3R/3R genotype was associated with a protective effect against HNSCC development when comparing the C group by the recessive model . In fact, SHMT1 1420C/T (rs1979277) revealed no association when compared to the HNSCC and C groups , pharynx , and larynx ; however, DHFR 19-pb ins/del polymorphisms were associated with a protective effect against oral cavity cancer   . With regards to the association of polymorphisms with the TNM staging system, only TYMS-28 3R/3R SNP was associated with decreased risk of tumor progression  .  The DNMT3B haplotypes analyses revealed a higher frequency of the alleles DNMT3B -149C, 283C, and 579T in the case group than in the control group , as well as that of DNMT3B -149T, 283T, and 579G ; however, contradictory results were observed for DNMT3B -149T, 283C, and 579T, , for DNMT3B -149T, 283T, and 579T haplotype , and for 149T, 283C, and 579G haplotypes , which were more frequent in the control group than in the case group.TYMS -28 3R carriers (median = 69%) and TYMS-28 2R/2R (median = 43%), p = 0.050. For the survival time, an association was observed between the DNMT3B -579 G/G (median = 61%) and DNMT3B -579 T carriers (median= 96%), p = 0.040, as well as between TYMS-28 2R/2R (median = 48%) and TYMS-28 3R carriers (median= 96%), p = 0.009. For the other polymorphisms analyzed, no relationship was observed between the relapse-free and survival times.In another statistical analysis, the Kaplan–Meier curve indicated the relapse-free and survival time in allele carriers and noncarriers . Moreover, the consumption of two or more cigarette packs along with four or more alcoholic drinks per day increases the risk of developing this neoplasia by 35-fold . Furthermore, the elderly are prone to cancer risk, with a median diagnosis age of approximately 60–70, which is higher than the median age considered at risk in this study . DNMT3B polymorphisms and cancer risk but yielded conflicting results. One meta-analysis suggests that DNMT3B -283T/C and DNMT3B -579G/T may play a protective role against different types of cancers. Moreover, in the subgroup analysis, DNMT3B -579G/T appeared to contribute to decreased risk of lung and colorectal cancer, whereas DNMT3B -149C/T was associated with a decreased risk of head and neck cancer . Another systematic evaluation of cancer risk demonstrated that DNMT3B -149C/T, DNMT3B -283T/C, and DNMT3B -579G/T polymorphisms were observed as protective factors against cancer in the Asian population ; however, in our study, the DNMT3B -149C/T and -579G/T SNPs did not contribute to the risk of head and neck cancer, whereas DNMT3B -283T/C might be a risk factor for head and neck carcinogenesis. Li et al., (2016) demonstrated that DNMT3B -283T/C (rs6087990) has a potential effect on gastric cancer initiation.Numerous studies have explored the association of DHFR 19-pb ins/del (rs70991108) and TYMS 28-bp tandem repeat (rs34743033) polymorphisms have a protective effect against head and neck cancer. Our study is the first to evaluate the association between a DHFR 19pb deletion polymorphism and the risk of this neoplasia. Evaluation in other cancer types reveals different results. Jokic et al., (2011) and Liu et al., (2013) demonstrated that DHFR deletion was not associated with colon cancer risk. In contrast, Xu et al., (2007) reported an increased breast cancer risk in women with this gene deletion. Corroborating our findings, one study evaluated the DHFR 19-pb ins/del (rs70991108) polymorphism in mother/child dyads in acute lymphoblastic leukemia onset-latency and demonstrated a good prognosis for carrier patients with homozygous deletion .We found that TYMS 28-bp tandem repeat polymorphisms was not associated with acute lymphoblastic leukemia and lung cancer risk development ; however, a study in the Brazilian population revealed increased risk association between the 2R/2R and 2R/3R variants in sporadic and hereditary breast cancer development, which was not in accordance with the results of our study .The role of SHMT1 1420C/T (rs1979277) polymorphism had no association with head and neck cancer risk; however, it was associated with tumor progression. The human SHMT1 gene is located at chromosome 17p11.2; the cytosolic isoform and its coenzyme, vitamin B6, catalyze the reversible conversion of serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate that provide one-carbon units during pyrimidine and purine syntheses . A meta-analysis revealed that no association was found between SHMT1 1420C>T (rs1979277) and the overall risk of cancer; however, in the subgroup analysis, significant associations with a protective effect were found in colorectal cancer and in the Asian population . In our study, the SHMT2 gene that encodes an isoform with the same function. A preliminary study by our research group also found no risk association with head and neck cancer in the Brazilian population , which was in accordance with the results obtained for gastric cancer , colorectal cancer , non-Hodgkin lymphoma , lung cancer , and breast cancer . According to Cheng et al., (2008), mutations in SHMT1 leading to an aberrant protein can be compensated for by the wild-type DNMT3B -579 G/G, have longer survival times in relation to DNMT3B -579 T carriers, and this is also true for TYMS-3R carriers with respect to the TYMS-2R/2R genotype. Moreover, this last polymorphism protects TYMS-3R/3R genotype carriers against cancer recurrence. In vitro and in vivo studies have been carried out to verify the functional consequences of the variable number of tandem repeat polymorphism. In general, the TYMS alleles contain two or three copies of repeats (2R and 3R), and the TS genes with triple sequence have higher expression levels than those with double sequence, and thus have transcriptional activity with the 3R sequence, which can be three to four times greater than that with the 2R carriers . This is the first study to demonstrate an association between survival and relapse-free times. Individuals with wild-type genotype,  DNMT3B 46359C/T and SHMT1 1420C/T polymorphisms, but failed to demonstrate any statistically significant association between the wild-type and polymorphic carriers . In the present study, no association was observed between the SHMT1 1420C/T polymorphism and survival time and relapse-free cancer.A previous study that involved a smaller number of head and neck cancer patients evaluated survival and relapse-free time for theDNMT3B -283T/C is associated with cancer risk, whereas DHFR 19-pb ins/del and TYMS 28-bp tandem repeat polymorphisms have a protective effect against head and neck cancer in relation to that in the control group. Furthermore, we highlight the association of the TYMS 3R/3R genotype with relapse-free cancer protection and the increased survival time of these patients in relation to those with the TYMS 2R allele.Thus, our findings reveal that


Plasmodium vivax malaria is endemic in Mauritania. Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency may develop acute hemolytic anemia when exposed to 8-aminoquinoline antimalarial drugs, which are indispensable for a complete cure. The prevalence of G6PD allelic variants was assessed in different ethno-linguistic groups present in Mauritania. A total of 996 blood samples  were collected from febrile patients in 6 different study sites: Aleg, Atar, Kiffa, Kobeni, Nouakchott, and Rosso. The presence of the African-type G6PD A-  and the Mediterranean-type G6PD B- (C563T) variants was assessed by PCR followed by restriction fragment length polymorphism and/or DNA sequencing. The prevalence of African-type G6PD A- genotype was 3.6% (36/996), with 6.3% (28/447) of hemizygote (A-) males and 1.5% (8/549) of homozygous (A-A-) females. Forty of 549 (7.3%) women were heterozygous (AA-). The following genotypes were observed among hemizygous men and/or homozygous women: A376G/G202A , A376G/T968C Betica-Selma , and A376G/A542T Santamaria . The Mediterranean-type G6PD B- genotype was not observed. The prevalence rates of G6PD A- genotype in male  and heterozygous female  white Moors were lower (p < 0.05) than those of males  and heterozygous females  of black African ancestry. There were only a few homozygous women among both white Moors  and those of black African ancestry . The prevalence of G6PD deficiency in Mauritania was comparable to that of neighboring countries in the Maghreb. Because of the purportedly close ethnic ties between the Mauritanian white Moors and the peoples in the Maghreb, further investigations on the possible existence of the Mediterranean-type allele are required. Moreover, a surveillance system of G6PD phenotype and/or genotype screening is warranted to establish and monitor a population-based prevalence of G6PD deficiency. Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of pentose phosphate pathway, the unique source of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in mature human erythrocytes. NADPH plays a key role in enzyme-based antioxidant system to protect erythrocytes from oxidative damage. When mature erythrocytes are deficient in G6PD and are exposed to various exogenous trigger factors, including infections, a variety of drugs , and vicine and convicine found in fava beans, the antioxidant defense system is unable to neutralize free radicals that accumulate in the erythrocytes, leading to lipid peroxidation and cell lysis, which may be manifested clinically by acute hemolytic anemia ,2.G6PD gene consists of 13 exons and 12 introns encoding a protein with a mass of 59 kDa [G6PD variants, only a few of them result in a functionally deficient G6PD enzyme. On the African continent, the most prevalent G6PD variants of clinical importance are the African-type G6PD A- found in sub-Saharan Africa and the Mediterranean-type G6PD B- variant mainly found in North Africa. Most G6PD mutations are clinically silent unless the G6PD-deficient individual is challenged with a trigger factor. The main clinical manifestation in such individuals is potentially fatal non-immune acute hemolytic anemia.In humans, G6PD deficiency is an X-linked recessive genetic trait which occurs frequently, affecting approximately 400 million people worldwide ,4,5,6. Tf 59 kDa . The genf 59 kDa ,9. DespiPlasmodium vivax malaria is present because 8-aminoquinolines are the only currently available drugs that can kill hypnozoites, the latent liver forms of P. vivax . The hypnozoites remain dormant for weeks or months after the primoinfection and are reactivated due to still unknown mechanisms, leading to new malarial attacks called relapse [P. vivax are not fatal, each attack may worsen anemia. The therapeutic objective in P. vivax-infected patients is complete elimination of both blood and liver stage parasites [P. vivax  malaria attacks from a primoinfection and can also become a source of further transmission to other persons, rendering the objective of malaria elimination difficult to attain.G6PD deficiency is a major issue in areas where  relapse ,11. Altharasites . HoweverP. vivax malaria has been firmly established over the past two decades in the Saharan zone of Mauritania, including Nouakchott, the capital, and Atar, an oasis city [P. vivax-infected patients with normal G6PD activity [The endemicity of sis city ,14,15,16activity . HoweverG6PD genotypes (or phenotypes) in Mauritania is limited to two studies conducted in Nouakchott [G6PD polymorphisms are associated with ethnicity [At present, the amount of data available on uakchott ,19. It ithnicity ,20. Maurthnicity . There aG6PD B- genotype [G6PD A- genotype [We hypothesized that some white Moors, who are of North African Arab-Berber descent, may have inherited the Mediterranean-type genotype ,23. All genotype ,25,26,27G6PD genotypes in the general Mauritanian population, the present study was conducted to determine the prevalence of African-type and Mediterranean-type G6PD genotypes in both men and women of all ethnic groups present in Mauritania and establish a baseline genetic database as the first step to the full implementation of primaquine therapy in P. vivax-infected Mauritanian patients in the near future.Given the scarcity of data on G6PD polymorphisms. The following numbers of samples collected from 6 study sites were genotyped: 101 from Atar, 226 from Nouakchott, 79 from Aleg, 20 from Kiffa, 323 from Kobeni, and 247 from Rosso  were analyzed for om Rosso . Based oG6PD genotypes A (n = 52) or B (n = 367), and 28 ) were A- ) were heterozygous (AA- or BA-), and 8 ) were African-type homozygous A-A- , 91.1–95.8%) men had normal  were A- . Among wous A-A- . The gloG6PD A- genotypes, G6PD A-(202) or A376G/G202A double mutation was observed in 2.2% ; 19 hemizygous males and 3 homozygous females) ; 7 males and 5 homozygous females). Santamaria G6PD A- or A376G/A542T was observed in 2 males, i.e., with a frequency of 0.2% ); there was no homozygous Mauritanian females with this African-type G6PD genotype. Among heterozygous females, the most common genotype was 376A/G-202G/A ), followed by 376A/G-T968T/C ) and 376A/G-542A/T ). We did not find any individuals carrying the A376G/G680T African-type G6PD genotype. Likewise, none of the patients included in the present study had the Mediterranean-type G6PD B- genotype.Among the individuals with females) . The secn) and percentage in each locality. G6PD genotype: African-type genotype in hemizygous male, A-; African-type normal male with a single A376G mutation, A; normal male, B. There were no male Pulars in Kiffa and no male Wolofs in Kobeni.Data are expressed as the number of individual males belonging to one of the ethnic groups (n) and percentage in each locality. G6PD genotype: African-type genotype in homozygous female, A-A-; African-type genotype in female heterozygous carrier, AA- or BA-; normal female with a single African-type A376G mutation with either AA or BA and normal female without any mutation, BB. There were no female Black Moors in Atar, no female Wolofs in Aleg, and no female Soninkes in Rosso in the present study.Data are expressed as the number of individual females belonging to one of the ethnic groups ), 10 males were hemizygous, 3 females were homozygous, and 6 females were heterozygous. Among black Moors with African-type G6PD variant ), 15 males were hemizygous, 3 females were homozygous, and 21 females were heterozygous. Among the individuals belonging to the minority ethnic groups composed of Pulars, Wolofs, and Soninkes ), 3 males were hemizygous, 2 females were homozygous, and 13 females were heterozygous. Taken together, there were no statistically significant difference between the number of males of black African ancestry ) and the male white Moors ),) who carried African-type G6PD genotype associated with G6PD deficiency (p = 0.0772). Likewise, there were significantly more heterozygous females among those of black African origin ) than heterozygous white Moor women ) (p < 0.0001). There was no statistically significant difference between the number of homozygous females of black African ancestry ) and that of homozygous female white Moors ) (p = 0.73), but the numbers of homozygous females were too low for a meaningful statistical comparison.n) of affected males with African-type genotype among the total number (N) of males belonging to each ethnic group and percentage of affected males in each ethnic group in parentheses. The proportion of A- allelic variants refers to the type of A- variants in relation to 28 males  with the African-type G6PD variant. There were a total of 447 men in the study.Data expressed as the number (n) of affected females with African-type genotype among the total number (N) of females belonging to each ethnic group and percentage of affected females in each ethnic group in parentheses. The proportion of A- allelic variants refers to the type of A- variants in relation to 48 heterozygous or homozygous females  with the African-type G6PD variant. There were a total of 549 females in the study. Heterozygous 680G/T and homozygous 542T/T and 680T/T genotypes were not observed.Data expressed as the number (G6PD genotypes in six study sites in Mauritania is depicted in The distribution of G6PD genotype by three methods (data not shown). We can therefore tentatively conclude that, based on more than 500 samples (499 from the present study + 8 from the earlier study) from white Moors in six different study sites in Mauritania, the Mediterranean-type G6PD B- variant was not found in our study.One of the hypotheses of the present study that some white Moors may carry the Mediterranean-type G6PD deficiency seems to be invalidated. The results of the present study are in contradiction with those of our earlier study based on multiplex allele-specific PCR . In thatG6PD genotype is rare  or possibly absent in white Moors despite the supposed ethnic and historical ties with peoples from North Africa. It has been estimated that the prevalence rates of G6PD deficiency in the general population in Algeria and Morocco, two neighboring Maghreb countries north of Mauritania, are 2.8% and 0.6%, respectively [G6PD genotype, while 46% had the African-type A- genotype [G6PD genotype was not found in Mauritanians in our study.It can be hypothesized that Mediterranean-type ectively . In Algeectively . A latergenotype . In a stgenotype . By contgenotype , which cgenotype ,23,29. TThe data presented here support the other hypothesis of this study that some Mauritanians of black African origin may be affected by the African-type G6PD deficiency. Among men belonging to black African ancestry , 18 men ) had the A- genotype. This prevalence rate in hemizygous Mauritanian men of black African origin is comparable to that reported for Senegalese males belonging to Sereer ethnic group (10%) and Malian males belonging to the Bambara ethnic group (10.5%), but higher than that of Dogon (2.2%) and Malinké (4.0%) ethnic groups in Mali ,31,32.P. vivax infections [P. vivax infections have been reported in recent years in Duffy-negative individuals in Africa, including in Mauritania [P. vivax infection in Duffy-negative individuals is generally thought to be more of an exception rather than the rule, and the underlying biochemical and molecular mechanisms are poorly understood [P. vivax malaria are slim [P. vivax is endemic, and some migrate to the northern Saharan zone, often for economic reasons, especially to Nouakchott, which has become a melting pot of various ethnic groups. Mauritanians of all ethnic backgrounds are therefore exposed to the risk of P. vivax infection, albeit to a lesser extent in those with black African ancestry due to the relative protection provided by the absence of Duffy antigen on the surface of red blood cells which serves as the principal known receptor for entry of P. vivax merozoites into the cell [Although we found a much higher prevalence of G6PD deficiency in black Moors and three minority ethnic groups than in white Moors in Mauritania, in practice, it may be argued that many of these individuals of black African ancestry may not be confronted with the potential problem of anti-hypnozoite therapy with 8-aminoquinolines because they tend to be Duffy-negative, as in the majority of black Africans elsewhere in sub-Saharan Africa, and may be naturally “resistant” to fections ,34,35,36uritania . At presderstood ,39. Furtare slim ,43,44,45- is generally associated with mild to moderate deficiency  and G6PD B- with severe deficiency (class II) [G6PD genotype are likely to present phenotypically mild to moderate deficiency. However, this assumption is not supported by solid evidence, and WHO experts have recommended against the use of this old classification to make clinical decisions [G6PD−202 is most frequently observed [G6PD Alass II) ,20,46. Becisions . The resobserved . In seveobserved ,27. In MP. vivax-infected patient belongs, G6PD screening test or standard biochemical test is mandatory before treatment with a standard 14-day regimen of primaquine can be considered. If the person is G6PD normal male, the standard dose of primaquine can be given if a qualitative or quantitative G6PD test displays normal enzymatic activity . If the person is G6PD normal female, the standard dose of primaquine can be given only if a reliable quantitative G6PD test is available and confirms a normal G6PD activity [G6PD genotype. The clinician would have to decide on a case-by-case basis whether a modified scheme of anti-hypnozoite therapy with primaquine (once weekly dose for 8 weeks) would be beneficial and outweigh possible risks [Regardless of the A- variant and the ethnic group to which an individual activity . If a Male risks . In someG6PD genotypes in the country. In addition, in some study sites, especially in the oasis city of Atar, none of the black African ethnic groups were seen at the health center. These considerations probably led to sampling bias in our study, with white Moors  over-representing the general population. The actual proportions of the ethnic groups of black African ancestry in Mauritania today are not known. Nevertheless, their proportion  observed in the present study remains most likely less than what is expected. These confounding factors may have considerably modified the representativity of different ethnic groups in the samples, which may, at least in part, explain the lower-than-expected prevalence of African-type G6PD deficiency observed in the present study. This limitation of the study is counterbalanced by the fact that samples were collected from febrile patients spontaneously presenting themselves to health centers and hospitals. A subset of these patients may have been infected by P. vivax and may have required primaquine therapy.It has been estimated that the worldwide prevalence of G6PD deficiency is 5% and that the prevalence is highest in Africa (8.5%) ,4,5,6. IG6PD 202, 376, 542, 680, and 968 polymorphisms associated with African-type G6PD A- and G6PD 563 associated with the Mediterranean-type G6PD B-. Other less common mutations were not analyzed. Although one of our initial hypotheses about the possible presence of the Mediterranean variant associated with moderate G6PD deficiency among white Moors turned out to be unconfirmed among the study participants, preconceived ideas should not preclude investigators and clinicians to rule out G6PD deficiency. An unexpected variant with severe phenotypic deficiency may sometimes be detected in Africa [G6PD genotype and G6PD phenotype is being planned to address this gap in the current knowledge of G6PD epidemiology in the country.Our study has other limitations. The present study was focused on the well-known n Africa . MoreoveField studies were conducted in 2015–2018 in the following cities in Mauritania, from the north to the south, located in six of 12 provinces  of Mauritania : Atar  in Nouakchott; health center of Aleg; Department of Internal Medicine and Infectious Diseases, Kiffa Regional Hospital, Kiffa; health center of Kobeni; and health center of Rosso. Except for Kiffa and Aleg (which is located midway between Nouakchott and Kiffa), these study sites were selected on the basis of previous series of field surveys carried out by our research teams to assess malaria epidemiology in three different settings defined by geoclimatic conditions, namely, the northern Saharan zone (Atar and Nouakchott), southern and southeastern Sahelo-Saharan transition zone , and southwestern Sahelian zone (Rosso) ,42,45,50Fingerprick capillary blood samples were collected as part of the series of epidemiological studies conducted in the country between 2015 and 2018 ,45,50. PA 1 mm-diameter disc of filter paper imbibed with dried blood was punched out and placed in 96-well plates. Human DNA was extracted using an automated MagMAX™-Express system  according to the manufacturer’s instructions. At the end of the DNA extraction protocol, approximately 120 µL of supernatant containing DNA were obtained from each sample.G6PD nucleotide positions, where mutations have been reported to occur most frequently in Africa, giving rise to G6PD A- variant, were amplified by PCR and analyzed by restriction fragment length polymorphism (RFLP) and/or DNA sequencing in a stepwise manner: 376, 202, 542, 680, and 968 [In the present study, DNA fragments including the following  and 968    and the following thermal cycling program: initial step of 95 °C for 5 min, 35 cycles of 95 °C for 1 min, 53 °C  for 1 min, and 72 °C for 1 min, then an extension at 72 °C for 10 min. PCR products (308 base pairs (bp)) were incubated at 37 °C overnight (at least 18 h) with Fok I  endonuclease .Genomic human DNA was first amplified using 2.5–5 µl of eluted DNA template and primer pairs A376G_F (5′-CCCAGGCCACCCCAGAGGAGA-3′) and A376G_R  in a PCR master mix containing DreamTaq DNA polymerase, buffer, 2 mM MgClMbo II endonuclease .PCR amplification was also performed for all samples to determine nucleotide 563 using the primer pairs C563T_F (5′-ACTCCCCGAAGAGGGGTTCAAGG-3′) and C563T_R (5′-CCAGCCTCCCAGGAGAGAGGAAG-3′) (0.5 µM each) . The PCRNla III  .Samples with A376G nucleotide substitution were further analyzed to detect the presence of an additional mutation in 202, 542, 680, or 968. The 216-bp fragment spanning nucleotide 202 was amplified using the primer pairs G202A_F (5′-CCACCACTGCCCCTGTGACCT-3′) and G202A_R (5′-GGCCCTGACACCACCCACCTT-3′) in DreamTaq™ Green PCR Master mix . The theNci I . After enzymatic digestion, digested amplificons were analyzed by 2% agarose gel electrophoresis. The PCR conditions and primers are summarized in If nucleotide 202 was wild-type, PCR was performed to amplify a 283-bp fragment spanning nucleotide 968 using the primer pairs T968C_F (5′-TCCCTGCACCCCAACTCAAC-3′) and T968C_R (5′-CCAGTTCTGCCTTGCTGGGC-3′) . PCR proMbo II to determine the nucleotide 563 in all samples, was sequenced if samples with A376G mutation had neither G202A nor T968C mutation. Sequencing was outsourced .The 547-bp fragment spanning nucleotides 542, 563, and 680, which was initially amplified for RFLP using G6PD gene sequence (GenBank accession number X55448.1) [F for forward primer; R for reverse primer; Tm, annealing temperature. The size in base pairs (bp) refers to the expected fragment size of the PCR product. Three restriction sites were analyzed in the fragment spanning exons 6 and 7. The primer sequences of the fragments amplified from exons 4, 5, and 9  were published by Carter et al. , and thoIn all study sites, samples were randomly selected for analysis, with the exception of patients enrolled in Kobeni where, due to a large number of available samples (more than 2300) , 327 samGACGT) and AA or AB in female. The genotypes denoted A and B in males and AA, AB, and BB in females are considered G6PD normal. The African-type deficient enzyme is designated G6PD A-; there are two mutations, one of which is A376G. The corresponding genotypes are “A-” in hemizygous male  and “A-A-” in homozygous deficient female. The genotypes of heterozygous females are either “AA-” or “BA-.” The Mediterranean variant with C563T (haplotype GAATGT) is designated Mediterranean G6PD B-.The wild-type phenotype of normal enzyme is designated G6PD B. The corresponding genotype in hemizygous male is “B” for haplotype GAACGT (based on nucleotides G202/A376/A542/C563/G680/T968) and “BB” for homozygous normal female. The African variant genotype with a single A376G nucleotide change is associated with the phenotype G6PD A, which is not associated with any change in enzyme activity compared to the normal enzyme. The corresponding genotypes are denoted “A” in hemizygous male (haplotype GG6PD A or G6PD A-. Fok I yields two bands (116 bp + 192 bp) in the presence of the mutant 376G allele, but it has no restriction site in the PCR fragment with the wild-type A376 allele (a single 308-bp band). A heterozygote female with G6PD A or A- genotype is characterized by the presence of three bands (116 bp + 192 bp + 308 bp).If A376G nucleotide change  occurs, the genotype is either Nla III yields two fragments (81 bp + 135 bp) if it carries the mutant allele 202A, one uncut 216-bp fragment if it has the wild-type allele G202, or three fragments (81 bp + 135 bp + 216 bp) in heterozygous females. The genotype with double mutations A376G and G202A is designated “G6PD A−(202).”Enzymatic digestion of the fragment spanning nucleotide 202 with Nci I restriction enzyme digests the 283-bp fragment into two (121 bp + 162 bp) fragments in the presence of mutation T968C or into three fragments (121 bp + 162 bp + 283 bp) in heterozygous females. The genotype characterized by double mutations A376G and T968C is called “G6PD A- Betica-Selma.”Mbo II to determine nucleotide 563. The genotypes with double mutations A376G and A542T or A376G and G680T are called “G6PD A- Santamaria” and “G6PD A−(680),” respectively [Sequencing of some PCR products of samples with A376G mutation, but without G202A or T968C, allowed not only to determine A542T and G680T mutations but also to confirm PCR-RFLP results using Mbo II enzyme digests the 547-bp fragment into 4 fragments (25 + 26 + 199 + 377 bp) if there is a wild-type C563 allele and 5 fragments (25 + 26 + 100 + 199 + 277 bp) in the presence of mutant 563T (Mediterranean-type G6PD B-).G6PD genotype” is synonymous with the wild-type G6PD B and denotes the absence of mutation in one of the following nucleotide positions: 202, 376, 542, 563, 680, and/or 968.In the present paper, the term “normal p-value of < 0.05 was used to test for significance.Proportions were compared using a two-tailed Fisher’s exact test. The G6PD genotyping that addresses the relation between ethnic origin and G6PD deficiency in Mauritania. The African-type G6PD mutations were found in all ethnic groups, more frequently in those of black African ancestry than in white Moors. From a practical viewpoint, every P. vivax-infected Mauritanian patient would require a reliable G6PD screening test and, for females, a reliable quantitative G6PD measurement, before a combination of blood schizonticidal and anti-hypnozoite antimalarial drugs can be administered to ensure that radical cure is achieved to completely eliminate the parasites from the patient. A phenotypic and/or genotypic surveillance of G6PD deficiency is warranted as the country attempts to move on to implement anti-hypnozoite therapy as part of the malaria elimination program.This is the first nationwide survey of


COVID-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. Over the past 12 months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has already infected over 160 million (>20% located in United States) and killed more than 3.3 million people around the world (>20% deaths in USA). As we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack SARS-CoV-2 on multiple fronts. We have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential SARS-CoV-2 viral proteins and their host protein interactors—S/Ace2, Tmprss2, Cathepsins L and K, and Mpro—to prevent binding, membrane fusion and replication of the virus, respectively. All together, we generated an ensemble of structural conformations that increase high-quality docking outcomes to screen over >6 million compounds including all FDA-approved drugs, drugs under clinical trial (>3000) and an additional >30 million selected chemotypes from fragment libraries. Our results yielded an initial set of 350 high-value compounds from both new and FDA-approved compounds that can now be tested experimentally in appropriate biological model systems. We anticipate that our results will initiate screening campaigns and accelerate the discovery of COVID-19 treatments. COVID-19 is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was identified in Wuhan city, in the Hubei province of China in December 2019 . The virhttp://virological.org/ (accessed on 22 May 2021)) .Until February 2021, there was no treatment or vaccine available to prevent or treat COVID-19 ,14. WhilIn addition to vaccine strategies, one of the most efficient ways to attack a virus is to use drug cocktails to target multiple enzymes/pathways used by the virus. Combination therapy has the advantage of being less likely to select for treatment-resistant viral mutants. Such a strategy has been successfully used to treat HCV and human-immunodeficiency virus (HIV) infections. In the case of HCV, the treatment, Epclusa, combines sofosbuvir, which inhibits the viral RNA-dependent RNA polymerase (NS5B), and velpatasvir, a defective substrate that inhibits NS5A. Antiretroviral therapy (ART) against HIV combines drugs from different drug classes to target disparate aspects of the HIV replication cycle. These drug classes include nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, CCR5 antagonists, post-attachment inhibitors, and integrase inhibitors. One example from the HIV-AIDS literature is the randomized comparison of four groups of patients comparing monotherapy to combination therapies: zidovudine (ZDV) monotherapy; ZDV zidovudine and didanosine; ZDV plus zalcitabine; or didanosine monotherapy. This randomized trial showed positive results when ZDT was combined with didanosine or zalcitabine, and for didanosine compared to ZDT monotherapy in raising CD4 counts greater than 50% . Combinapro that are known to control both viral binding or entry and virus replication . We aim to target the SARS-CoV-2 replication cycle at multiple levels to synergistically inhibit viral spread and dissemination. Using a computational pipeline that aimed to expeditiously identify lead compounds against COVID-19, we combined compound library preparation, molecular modeling, and structure simulations to generate an ensemble of conformations and increase high-quality docking outcomes against two essential SARS-CoV-2 viral proteins and their host protein interactions; S/ACE2, Tmprss2, Cathepsin L and K, and Mlication A. Our aplication B will moHit identification in VS also requires careful selection of the methods used based on the goal of the project  . ZINC isAdvances in computing power have increased the utility of in silico screening capabilities and balanced the need for accuracy with virtual high-throughput screening approximations and assumptions ,51,52,53Here, we present in silico screening of both the approved FDA compound library and >30 million compounds representing new chemical entities (NCEs) ,67,68,69For ACE2–S protein, PDB code 6VW1 was used to construct the model . While tA homology model was constructed on the basis of a prothrombin crystal structure in complex with the ligand analog (PDB code 3F68) . We modeMALNSGSPPAIGPYYENHGYQPENPYPAQPTVVPTVYEVHPAQYYPSPVPQYAPRVLTQASNPVVCTQPKSPSGTVCTSKTKKALCITLTLGTFLVGAALAAGLLWKFMGSKCSNSGIECDSSGTCINPSNWCDGVSHCPGGEDENRCVRLYGPNFILQVYSSQRKSWHPVCQDDWNENYGRAACRDMGYKNNFYSSQGIVDDSGSTSFMKLNTSAGNVDIYKKLYHSDACSSKAVVSLRCIACGVNLNSSRQSRIVGGESALPGAWPWQVSLHVQNVHVCGGSIITPEWIVTAAHCVEKPLNNPWHWTAFAGILRQSFMFYGAGYQVEKVISHPNYDSKTKNNDIALMKLQKPLTFNDLVKPVCLPNPGMMLQPEQLCWISGWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADG. Topological domains have the following characteristics: residues 1–84 form the cytoplasmic sequence; residues 85–105 form the transmembrane domain region ; and residues 106–492 form the signal-anchor for type II membrane protein (extracellular), where the protein has two main chains: a non-catalytic chain (Met1-Arg225) and a catalytic chain (Ile256-Gly492), where each domain is modeled as a separate unit built together in composite. Disulfide bonds exist between several residues (113 ↔ 126), (120 ↔ 139), (133 ↔ 148), (172 ↔ 231), (185 ↔ 241), (244 ↔ 365), (281 ↔ 297), (410 ↔ 426), (437 ↔ 465), which can be informative for building the structure. Glycosylation sites are also possible at residues N213 and N249. A cleavage site exists between Arg255 and Ile256 to produce the mature S1 peptidase domain. The second method, homology modeling, was performed using the SwissModel server (2020) after performing a BLAST search on available protein structures in the RCSB database. pro co-crystallized with tert-butyl (1-((S)-1-(((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-3-cyclopropyl-1-oxopropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl)carbamate  PDB 6Y2F was used. This structure was also mostly complete. Residues E47 and D48 were built in COOT. To build the missing residues, the coordinates and structural factors were downloaded, 2mFo-DFc and FEM maps were generated, and real space refine zone/regularize zone was used to manually fit added residues to electron density as well as optimize local geometry. The ligand  was left for usage as a cognate ligand for virtual screening.The structure for MMolecular dynamics simulations (MDS) and Monte Carlo (MC) simulations were performed on the protein to allow local regional changes for all acids in each structure. The X-ray refinement for MC was built using the YASARA SSP/PSSM method ,83,84,85Refinement of the finalized models was completed using either Schrodinger’s LC-MOD MC-based module or NAMD2 protocols. These refinements started with YASARA-generated initial refinement of the proteins ,81,82,84MDS and MC searching were completed on each model for conformational sampling using methods previously described in the literature ,58,64,65+ and Cl− ions to a concentration of 150 mM. All simulations were performed under periodic boundary conditions using the V-Rescale Thermostat algorithm to maintain temperature (310 K) and the Parrinello–Rahman Barostat algorithm for constant pressure (1 bar) pyrrolidine-2-Carboxamide, contained within the ChemblDB repository (CHEMBL1229259) and active against TMPRSS2, prothrombin, and Factor XI. Likewise, another docked model was recovered with macrocyclic ligand (CHEMBL3699198), called:We acquired the dimer protein sequence from the UniProt database. BLAST search showed the highest identification values against factor XI, prothrombin, kallikrein proteases (~41–42%). However, we focused on ligands that could be active against the active form of TMPRSS2 protein. Thus, we found the ligand: (2s)-1-[(2r)-2-(Benzylsulfonylamino)-5-Guanidino-Pentanoyl]-ethyl14-[[(E)-3-[5-chloro-2-(tetrazol-1-yl)phenyl]prop-2-enoyl]amino]-5-(methoxycarbonylamino)-17-oxo-8,16 diazatricyclononadeca-1(18),2(7),3,5,15(19)-pentaene-9-carboxylate.We launched several molecular dynamics simulations (up to 75 ns of duration) to understand the interaction with the target protein-binding site. pro, a key enzyme for coronavirus replication (SARS-CoV-2), and a potential target for anti-SARS drug development, several peptidomimetics synthetized in early 2012 against SARS-CoV-1 proteases were identified as selective. There is a high degree of sequence identity between the SARS-CoV-1 and SARS-CoV-2 Mpro. This means that SARS-focused ligands could form similar interaction maps with Mpro protein and offers good launching points for 3D-QSAR/machine learning-driven drug design for future iterations. To perform the virtual screening, protein structure was taken from the PDB code 7BQY complex and significant attention was paid to the interaction between the crystallized ligand from the complex and protein-binding site [https://www.diamond.ac.uk/ (accessed on 22 May 2021)) to narrow the source of possible conformations. The binding of the compounds inserted into this region demonstrated a very canonic and recurring interacting motif, represented with α-Keto amide group flanked with aliphatic or saturated rings. We then performed molecular dynamics of 75 ns for the ligand-free dimer structure of the Mpro to evaluate and “catch” the most flexible elements of the binding site. Our simulation revealed that the extended binding pocket was not very stable, unlike its individual subpocket, which contains the catalytic dyad cysteine (C145) and histidine (H41), among other residues .As an example, when examining some prototype compounds from our selected dataset of >300 NCEs screened from >10 million total compounds, we find the predicted interactions between drug and protein  have sompro. Among these are FDA-approved drugs that could be repurposed: 21 against ACE2, 11 against TMPRSS2, and 8 against Mpro  used with levodopa for the treatment of Parkinson’s disease. Glucosamine hydrochloride is a constituent of cartilage and used t treat osteoarthritis joint pain. S4701 or 2-deoxy-d-glucose (2D-DG) compound can induce ketogenic state, a powerful pathway involved in reducing systemic inflammation. Inulin is a natural prebiotic agent that enhances GI function and digestion by increasing prebiotic GI homeostasis critical to stabilize downstream anti-inflammatory effects and prevent overgrowth of harmful bacteria. Metaproterenol is a bronchodilator (beta-2 receptor agonist) that is commonly used to treat a variety of respiratory disorders including asthma, COPD, bronchitis and wheezing associated with viral pneumonias in clinical practice. The novelty of this drug is that is aerosolized and can be given as a breathing treatment to reach the lungs, which have a tremendous surface area, and enter the blood rapidly. By inhalation this drug acts rapidly and potentially alone or in combination with other aerosolized drugs or oral or IV combination drugs. Its inhalational route of delivery also can reach alveolar type II cells which express ACE2 for dual synergism. Metaraminol bitartrate, a stereoisomer of meta-hydroxynorephedrine, is a potent sympathomimetic amine. This drug is used in patients with hypotension or low blood pressure. COVID-19 hospitalized patients in the intensive care unit (ICU) setting often need vasopressor agents to raise blood pressure in a condition called shock (dangerously low blood pressure) from COVID-19 disease or sepsis. Therefore, metaraminol has a dual purpose of antiviral function at ACE2 docking site/entry as well as helping with systemic blood pressure in those acutely ill COVID-19 patients. This drug has immediate repurposing use in this patient population.Isoprenaline hydrochloride (isoprotenerol) is an adrenoreceptor agonist that can be repurposed as a vasopressor to augment cardiovascular function with a beta-receptor side benefit of bronchodilation to improve breathing function. Metaraminol bitartrate, a stereoisomer of meta-hydroxynorephedrine, is a potent sympathomimetic amine to raise blood pressure. Atenolol and nadolol are beta-receptor blocking agents used in chronic hypertension, a comorbid risk factor in COVID-19 patients. Propafenone is an anti-arrhythmic agent approved for patients with life-threatening ventricular tachycardia. Levosulpiride is an atypical antipsychotic medication with prokinetic function that can be used in patients with agitated delirium and gut immotility. Valganciclovir hydrochloride is an antiviral agent used for cytomegalovirus (CMV), varicella zoster virus (VZV), and preventative medication in HIV patients . Recent  T cells . AmikaciAtorvastatin is a statin drug with anti-inflammatory, immunomodulatory  and endoBumetanide is a loop-diuretic used to treat edema. Aloin is an anthraquinone glycoside found naturally in aloe vera plants, a natural cathartic, and decreases 16S rRNA sequencing of dysbiosis-producing butyrate producing bacterial species via an emodin breakdown product . Emodin The liver is the largest metabolic organ, and plays an important role in drug detoxification and metabolism . Hepatocw/v) GelMA with the Ru/SPS visible light photoinitiator as the bioink. This bioprinting system utilized a visible light source from a projector to bioprint the hepatic microtissue constructs based on the digital pattern input in the system , respectively. Calcein AM is a non-fluorescent cell-permeable compound that is hydrolyzed into the green fluorescent anion calcein by intracellular esterase in living cells, whereas EthD-1 is a dead cell-specific red fluorescent dye that binds to DNA. The viability of cells within the hepatic microtissue constructs were found to be more than 96% at day 7.® 594-phalloidin. The cells were counterstained with DAPI for nuclei and imaged using fluorescence microscopy. Fluorescence micrographs showed that these cells were homogenously distributed within the hepatic lobules, suggesting good functions of the cells as well as the resulting microtissues  ,152,153.50 assay identified some new compounds with efficacy similar to the positive control Remdesivir , with over 20% of cases occurring within the United States as of mid-May 2021. In order to accelerate drug discovery, a design funnel using high-powered artificial intelligence was used to screen millions of compounds against macromolecular mechanistic viral targets. At the back end of this funnel, 40 drug candidates emerged, many of which may represent repurposing candidates for use in humans due to known safety and tolerability profiles. However, the approach with the highest probability of overall clinical therapeutic success may be not a single drug therapy for this viral RNA disease but rather a multi-pronged drug approach gleaned from decades of HIV research. A multi-drug approach for HIV has improved survival, markedly reduced viral loads, and vastly improved management of the disease by preventing AIDS end-stage fatal complications. We therefore suggest that a multi-faceted drug approach for SARS-Cov-2 may prove superior by attacking three viral entry and replication cycle sites simultaneously: ACE2 receptor docking site and entry, TMPRSS2 endosomal packaging, and ME. cuniculi, a antimicrobial agents used as an adjuvant treatment for opportunistic AIDS-associated infections. Overall, these compounds are Cathepsin-centric or antibiotic in nature, with little to no effect on our intended targets . Additional top hits identified by Riva et al. include: AMG-2674, an AMGEN compound inhibitor of TRPV-1 (Vanilloid Receptor); SB-616234-A that possesses high affinity for human 5-HT1B receptors; SDZ 62-434 that strongly inhibited various inflammatory responses induced by lipopolysaccharide (LPS) or function-activating antibody to CD29; Hafangchin A , a bis-benzylisoquinoline alkaloid, which acts as a calcium channel blocker; Elopiprazole an antipsychotic drug of the phenylpiperazine class (antagonist at dopamine D2 and D3 receptors and an agonist at serotonin1A receptors) that was never marketed; YH-1238, which inhibits dipeptidyl peptidase IV (DPP-IV) enzyme prolonging the action of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP); KW-8232, an anti-osteoporotic agent that can reduce the biosynthesis of PGE2; Astemizole, an antihistamine; N-tert-butyl Isoquine , an antimalarial drug candidate; and Remdesivir, a broad-spectrum antiviral medication developed by the biopharmaceutical company Gilead Sciences. Again, none of these compounds were geared toward targeting TMPRSS2 or Mpro, and are also not specific to ACE2. While the lack of overlap may be surprising, results generated by Riva and colleagues are not in opposition to our findings and both approaches can complement each other. Most importantly, these FDA-approved compounds can be combined with our set 310 compounds that have been demonstrated to have low toxicity issues based on our chemoinformatics filtering  identified interesting compounds for alternative targets that complement our results. These FDA-approved compounds included MDL-28170, a selective Cathepsin B inhibitor; VBY-825, a non-specific Cathepsin B, L, S, V inhibitor; Apilimod, an inhibitor of production of the interleukins IL-12 and IL-23; Z-LVG-CHN2, a tri-peptide derivative inhibitor for cysteine proteinases; ONO 5334, a selective Cathepsin K inhibitor; and SL-11128, a polyamine analog designed against iltering B. All NCAmong the millions of potential COVID-19 drugs screened, the majority of the final 40 drug candidates have known clinical uses and/or FDA approval for a primary indication  with well-established patient safety and tolerability profiles from large Phase III human trials and post-market (Phase IV) analyses. These large human datasets provide both a clinically significant and scientifically innovative window of opportunity to test 40 compounds on the multi-drug platform, and, in conjunction, observe longitudinal human survival outcomes of COVID-19 patients on these drugs for comparative effectiveness within established and ongoing patient registries. An emerging example of this important parallel is ACE2 pathway drugs (ACE inhibitors and angiotensin receptor blocking drugs), which are increasingly observed in humans with COVID-19 to be associated with improved survival advantage ,161,162.pro. The compounds identified by our large-scale in silico platform can be experimentally validated as binders for intended targets and for efficacy in models of the disease, evaluated for EC50/safety-toxicity data, and carried into hit-to-lead and lead optimization in a drug development pipeline. Structural studies such as X-ray crystallography will also be important to generate structural SAR data for these efforts.A flurry of activity to identify compounds for SARS CoV-2 targets has been underway globally. In our approach, we introduce our novel Maxwell’s demon molecular dynamics method for screening flexibility required to obtain rare and essential conformational transitions and pathways to find the most likely druggable state. We also used our quantum docking technique (QM-driven adaptive molecular dynamics scanning docking)  to identIn sum, our leading edge in silico methods incorporating structural dynamics have produced a set of 350 candidate compounds suitable for screening in biological disease models. Among these, 40 FDA-approved compounds are eligible for rapid clinical trial testing. Additionally, our results bring forward 310 NCEs predicted to possess potency and specificity for viral or host accessory proteins to lower the viral load. Moreover, this resource offers the community a set of chemical tools to probe the behavior of these enzymes essential for SARS-CoV-2 progression, namely, binding, entry and replication. As SARS-CoV-2 is already endemic, the rapid identification of effective antivirals remains a paramount focus until we have generated herd immunity via global vaccination campaigns. We have expectation for >35 effective compounds based on our current 11% hit rate that meets expectations similar to that found in Remdesivir as an antiviral agent, which can then be further tested in our BSL-3 lab.


Cunninghamellaelegans and Cunninghamella echinulata fungal strains. A sulphate-depleted medium was designed for the preferred production of ZEN glycosides. Both Cunninghamella strains were able to produce zearalenone-14-β-D-glucopyranoside (Z14G), zearalenone-16-β-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S). In a rich medium, Cunninghamellaelegans preferably produced Z14S, while Cunninghamellaechinulata preferably produced Z14G. In the sulphate-depleted medium a dramatic change was observed for Cunninghamellaelegans, showing preferred production of Z14G and Z16G. From 2 mg of ZEN in sulphate-depleted medium, 1.94 mg of Z14G and 0.45 mg of Z16G were produced. Following preparative Liquid Chromatography-Mass Spectrometry (LC-MS) purification, both fractions were submitted to 1H and 13C NMR and High-Resolution Mass Spectrometry (HRMS). These analyses confirmed that the purified fractions were indeed Z14G and Z16G. In conclusion, the presented research shows that a single Cunninghamella strain can be an effective and efficient tool for the controlled biotransformation of ZEN glycosides and other ZEN metabolites. Additionally, the biotransformation method was extended to zearalanone, β-zearalenol and other mycotoxins.In recent years, conjugated mycotoxins have gained increasing interest in food safety, as their hydrolysis in human and animal intestines leads to an increase in toxicity. For the production of zearalenone (ZEN) glycosides reference standards, we applied  Fusarium spp. It occurs in grain commodities, and it can cause reproductive disorders in farm animals and lead to hypoestrogenic syndromes in humans − m/z 525.19720 +/− 2 Da was isolated and fragmented with 50 NCE.For the separation of ZEN metabolites in the fungal extract, after filtration, 10 µL was injected on the analytical column, which was kept at 40 °C. The ZEN metabolites were separated with a gradient elution  using ru


Scientific Reports 10.1038/s41598-021-00661-4, published online 01 November 2021Correction to: The Acknowledgements section in the original version of this Article was omitted. The Acknowledgements section now reads:“This study has been funded by Instituto de Salud Carlos III through the project PI18/00462 to M.V.G.G., co‐funded by European Regional Development Fund "A way to make Europe,  and the Spanish Ministerio de Ciencia (RTI2018-096961-B-I00 to E.L-P. and RTI2018-096918-B-C41 to R.J.J.R.). This study was also supported by the Plan Estatal de I+D+I 2013-2016, with funding from the European Regional Development Fund (ERDF) “A way to build Europe” initiative. The CNIC is supported by Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).”The original Article has been corrected.


Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus. Chronic hepatitis (CHB) caused by the Hepatitis B virus (HBV) is a serious liver disease. CHB is associated with severe liver conditions ranging from fibrosis and cirrhosis to hepatocellular carcinoma or acute liver failure ,2. The WThe Hepatitis B virus is a small, enveloped virus with a 3.2 kb long, partially double-stranded circular DNA genome. It encodes only four overlapping open reading frames that are transcribed into 6 RNAs and translated to seven proteins, e.g., pre-core (HBeAg), core , viral polymerase (P), envelope/surface proteins  and the X protein (HBx) ,5.The HBV core protein is a phospho-protein consisting of 183 or 185 amino acids (aa) with an approximate molecular mass of 21.5 kDa. As demonstrated by numerous reports, the HBV core protein (HBc), which is the main component of viral nucleocapsid, plays multiple roles in the HBV life cycle . In the In terms of its structure, the HBc protein could be divided into N- and C-terminal domains that are connected with a 9 amino acids linker region. The N-terminal domain (NTD), also known as the assembly domain, encompasses amino acids from 1 to 140, and is necessary for capsid assembly . The C-tThe major phosphorylation of the CTD occurs on each of three serine-proline (SP) sites  ,20,21. ARecently, there is growing evidence that the ATM (Ataxia-telangiectasia-mutated) and ATR (ataxia telangiectasia and Rad3-related) pathways play an important role in the positive regulation of HBV replication. ATM and ATR belong to the class-IV phosphoinositide 3-kinase (PI3K)-related kinase (PIKK) family, along with the mammalian target of rapamycin (mTOR) and DNA-dependent protein kinase (DNA-PK) ,27. TheySeveral studies have already documented the importance of the DDR pathway in HBV infection and replication. Zhao et al. ,32 showe2O2, and subsequent mutational analysis revealed that HBc is phosphorylated in response to ATM-Chk2 activation predominantly on serine residue 170 (S168 in a 183-aa HBc variant). Importantly, etoposide treatment of HBV-infected hepatocytes led to increased levels of intracellular HBc and HBe antigen in cell supernatants as well as higher levels of HBc phosphorylation at SQ sites.In the present study, we addressed the potential role of ATM/ATR kinases in phosphorylation of the HBc protein. The HBc protein contains three SQ motifs, all located in its C-terminal domain. Treatment of HBc-expressing human hepatocytes with DNA damaging agents, etoposide and HHepG2-NTCP  was obtained from Dr. Stephan Urban ). HepG2.2.15  was obtained from Dr. David Durantel ). They were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and puromycin (0.05 mg/mL) or G418 (0.4 mg/mL), respectively.CACCATCACCACCATCACGCGAAAGAAAACCTGTATTTTCAGGGTAGCTACCCATACGATGTTCCAGATTACGCTGACATTGACCCGTATAAAGAATTTGG-3′ and HBc-R: 5′-gccgctcgagtctagTTAACATTGAGATTCCCGAGATTGAGATCTTCTG-3′ and Q5 polymerase  from plasmid pHY92CMV as a template. The His6x tag sequence is underlined, the HA sequence is in italics, the sequence in small capitals represents homologous regions to the XhoI linearized pcDNA3.1 vector. PCR product was inserted into XhoI linearized pcDNA3.1(-)  by an In-Fusion approach . The HBx expression plasmid  was generated by PCR amplification of HBx ORF with primers X-Nhe-F 5′-ATATACGCTAGCACCATGGCTGCTAGGCTGTGCTGCCAAC-3′ and X-TAA-Xho-R 5′-CTCTAGACTCGAGTTAGGCAGAGGTGAAAAAG-3′ ), followed by subcloning into the pcDNA4-V5-HisA vector. The C-terminal truncation of HBc  was described previously [The full-length HBc  expression plasmid was generated by PCR amplification of HBc ORF ), followed by subcloning into pcDNA3.1 . The full-length His-HA-HBc  expression plasmid was constructed as follows: HBc coding sequence was amplified by PCR using primer pair HBc-F: 5′-aaacgggccctctagATGeviously . The S-tTM West Femto Maximum Sensitivity Substrate  and LAS-4000 imager. As secondary antibodies for the LI-COR system, we used goat anti-rabbit IgG (H + L) IRDye 800CW  and goat anti-mouse IgG (H + L) IRDye 680RD . The Western blots were visualized using an LI-COR Odyssey CLx system and the Image Studio Lite Software was used for densitometric analysis.The primary antibodies and reagents used for the Western blot and immunoprecipitations include antibodies against β-actin , HA , alpha-tubulin , lamin A/C  and anti-HA magnetic beads . Antibodies against HBc  and HBx  were obtained from Dr. Rudolf K. Beran . The following antibodies were purchased from Cell Signaling : anti-phospho-ATM/ATR Substrate (S*Q) detecting phosphorylated SQ motifs (p-SQ), anti-ATM, anti-phospho-ATM , anti-ATR, anti-phospho-ATR , anti-Chk1, anti-phospho-Chk1 , anti-Chk2 and anti-phospho-Chk2 . Secondary antibodies include goat polyclonal anti-mouse horseradish peroxidase (HRP)  and goat polyclonal anti-rabbit-HRP . For visualization, we used a SuperSignalAll siRNAs were purchased from Thermo Fisher Scientific . The siRNAs were s42813 and s57219 for ATM, and s536 for ATR. Two different Select Negative Control siRNAs 1 and 2 were used as the control. The siRNAs were transfected using Lipofectamine RNAiMAX , according to the manufacturer’s recommendations. The specificity and inhibitory potential of ATM- and ATR-siRNAs was estimated by RT-qPCR using the following primer pairs: ATM-F 5′-AGTGGGACCATTGCACTTCC-3′, ATM-R 5′-TCTTCCACTTCTTTTACTCTGGCA-3′; ATR-F 5′-ACTGCAGCTATCTTCCACTACC-3′, ATR-R 5′-AGTGCTCTCAGAGGTCTCCTTT-3′. RNA was isolated from transfected HepG2-NTCP cells using RNeasy Plus kit  and reverse-transcribed using a Maxima First Strand cDNA Synthesis kit , according to the manufacturer’s recommendations. qPCR was performed with gb Elite PCR Master Mix  and SYBR Green dye . The 20 μL qPCR mixture consisted of 5 μL cDNA, primers (0.2 μM each), SYBR Green dye diluted 1:2500 and 1 × gb Elite Master Mix. Thermal cycling conditions for qPCR run were as follows: denaturation (95 °C for 10 min), denaturation and annealing/elongation steps repeated 40 times with fluorescence measurement at the end of the elongation step . The levels of ATM and ATR mRNAs were normalized to housekeeping gene Hypoxanthine phosphoribosyl-transferase 1 (HPRT1). For amplification of HPRT1 cDNA, the following primers were used: HPRT1-F 5′-TGGTCAGGCAGTATAATCCAAAG-3′, HPRT1-R 5′-TTTCAAATCCAACAAAGTCTGGC-3′.2O2) or 4-Hydroxynonenal (4-HNE), all from Merck Millipore . After treatment, the cells were washed twice and cultured in complete DMEM medium supplemented with 10% FBS for an additional 4 h. Dimethyl-sulfoxide (DMSO) was used as the vehicle for etoposide and 4-HNE. Both the ATM inhibitor KU55933  and ATR inhibitor IV VE821  were used at a concentration of 10 μM for two hours before etoposide treatment, and at a concentration of 5 μM after treatment.For the induction of DNA damage, the cells were exposed to a 1 h treatment of etoposide (Etp), hydrogen peroxide  per cell) in the presence of 4% PEG8000 overnight. Sixteen hours later, the cells were washed 3 times with PBS followed by treatment with etoposide for 1 h. After treatment, the cells were washed twice and maintained in DMEM supplemented with 2.5% DMSO. At different time-points after the addition of Etp, the cells were harvested and protein extracts were isolated and analyzed by Western blotting. The levels of HBe antigens were determined from cell supernatants by ELISA at each time-point.All transfections of expression plasmids were carried out using Lipofectamine 3000 transfection reagent , according to the manufacturer’s recommendations. The HepG2-NTCP cells were transfected  with the HBc, HBx expression plasmids, or an empty vector, pcDNA.2O2-treatment, the cells were lysed in lysis buffer (20 mM HEPES (pH 7.9), 50 mM NaCl, 5 mM EDTA, 1% (v/v) Igepal CA-630, 10% (v/v) glycerol, 1 mM dithiothreitol, 1 mM phenyl-methyl-sulfonyl fluoride (PMSF), 0.2 mM protease inhibitor cocktail ) and phosphatase-inhibitor-mix I . Protein extracts were analyzed either by Western blotting or immunoprecipitation (IP). For IP, the protein extracts (800 μg or 1 mg) were incubated with anti-HA  magnetic beads at 4 °C overnight. Immune complexes were extensively washed with lysis buffer, and co-precipitated complexes were analyzed by Western blotting using phospho-ATM/ATR substrate (S*Q) antibody  detecting phosphorylated SQ motifs (p-SQ).At different time-points after etoposide- or H2, 1 mM PMSF supplemented with 0.2 mM protease inhibitor cocktail  and phosphatase-inhibitor-mix I . Cells were incubated for 10 min on ice. Thereafter, CHAPS  was added at a final concentration of 0.6% (v/v), and lysates were homogenized by being passed through a 20 G needle for three times. Nuclei were pelleted by centrifugation at 600× g for 5 min at 4 °C, and supernatant containing cytoplasmic proteins was collected and stored at −80 °C. The remaining nuclei were washed three times in buffer A containing 0.6% CHAPS. The nucleic pellets were lysed in buffer B containing 20 mM HEPES, pH 7.9, 0.4 M NaCl, 2.5% glycerol, 1 mM EDTA, 1 mM PMSF, 0.5 mM DTT, supplemented with 0.2 mM protease inhibitor cocktail  and phosphatase-inhibitor-mix I , by repeated freezing and thawing. Supernatants containing soluble nucleic proteins were collected by centrifugation at 20,000× g for 20 min and stored at −80 °C.For nuclear and cytoplasmic extract preparation, the cells were resuspended in five packed cell volume of buffer A containing 20 mM Tris, pH 7.6, 0.1 mM EDTA, 2 mM MgClThe intensity of the HBc, ATM, p-HBc or p-ATM signal was calculated as the mean of two to three independent experiments, as specified in figure legends, using Image Studio Lite Software for densitometric analysis, and normalized to the corresponding level of β-actin, total HBc or total-ATM expression, respectively. The levels of p-HBc, p-ATM, HBc or ATM in control cells (untreated cells or at time-point 0 h) were arbitrarily set to 1. Each independent experiment was performed in one replicate.g for 10 min to remove cellular debris, transferred to clean tubes and stored at −80 °C until the antigen measurement. The titers of HBeAg were measured using a commercial ELISA kit  according to the manufacturer’s instructions.The titers of secreted HBeAg were quantified by ELISA. HepG2-NTCP cell culture supernatants were collected and centrifuged at 120× t test, n = 3.Statistical analyses were performed in GraphPad Prism version 9.1.2 . Results in graphs are expressed as mean and standard deviations. The differences in the mean of p-HBc, p-ATM, HBc or HBeAg between different Etp treatments and the control (ctrl) at various time points were analyzed with a two-tailed paired Several recent studies identified the ATM/ATR pathway as an activator of HBV replication and cccDNA formation. These findings led us to investigate whether activation of ATM and/or ATR kinases may result in phosphorylation of the HBc protein. ATM and ATR share substrate specificity, recognizing Ser-Gln (SQ) and Thr-Gln (TQ) motifs. While there is no TQ motif present in the HBc protein sequence, the C-terminal domain contains three SQ sites involving serine residues at positions 170, 178 and 183  or 4-hydroxynonenal (4-HNE)  D. The im (4-HNE) E. The us 8000 µM F, the le 8000 µM G and pho 8000 µM H gradual µM  production that led to the accumulation of γ-H2AX foci and increased p-ATM and p-Chk2 levels . Based o2O2 or doxorubicin, resulted in upregulation of HBV replication that was assayed on the level of HBV RNAs, intracellular or secreted HBV DNA and the HBs antigen. Therefore, we decided to determine whether etoposide treatment may have any effect on the HBc protein and its phosphorylation levels and/or secretion of the HBe antigen (HBeAg) in HBV-infected cells. To test this, HepG2-NTCP cells were infected with HBV  per cell) and subsequently treated with 100 µM etoposide for 1 h  and subsequently treated them with different concentrations of etoposide ranging from 10 to 300 µM A. The leTo analyze ATM-regulated phosphorylation of HBc in HBV-infected hepatocytes, HepG2-NTCP cells were infected with HBV (MOI 500 VGE per cell), followed by 1-h etoposide treatment 6 days after infection A. The leMany DNA viruses rely on the host DDR pathway for their efficient propagation. Viruses take advantage of the DDR to modulate the cell cycle and hijack cellular proteins to support viral replication ,39,40. I2O2 treatment. Importantly, the down-regulation of ATM using ATM-specific siRNAs led to substantially reduced levels of HBc phosphorylation. Thus, this study revealed that ATM is likely to be the major kinase responsible for the etoposide-induced HBc phosphorylation. Surprisingly, none of the genotoxic drugs used in this study could activate the ATR-Chk1 pathway. Although H2O2 was previously shown to trigger the activation of Chk1 independently of ATM, and H2O2 treatment was successfully used in other HBV study using HepG2 cell line [Growing evidence that the Hepatitis B virus relies on activated DDR for its effective replication prompted us to study the interaction between the host ATM/ATR pathway and viral proteins. In this study, we investigated the role of activated ATM/ATR in phosphorylation of the HBc protein. Our presented data are the first to identify HBc as the phosphorylation target regulated by ATM-Chk2 after the induction of the DNA damage response pathway by etoposide or Hell line , we wereThe mutation analysis identified the serine residues that were modified by ATM-mediated phosphorylation. All three serines, S170, S178 and S183 , that were part of SQ motifs located in the CTD were phosphorylated. Notably, the S-to-A mutation involving S170 displayed the highest reduction in phosphorylation, suggesting that this serine is predominantly modified by activated ATM kinase. As reported previously, the majority of wt HBc expressed in human cells is hyperphosphorylated that could be detected by phos-tag SDS PAGE as two slowly migrating species ,42. SuchFurthermore, we showed that efficient ATM-mediated phosphorylation of HBc requires prior phosphorylation at S157 and S164. Similar phenomenon was described previously for Plk1 and HBc , indicatDynamic phosphorylation and dephosphorylation of HBc CTD and cross-talk between phosphorylation sites are important mechanisms involved in various steps of HBV replication, including pgRNA packaging, reverse transcription, nucleocapsid (NC) formation and virion maturation ,44,45,46Plk1 was shown to phosphorylate two out of three HBc SQ sites involving S170 and S178 that were also identified as substrate motifs targeted by ATM in the presented study . InteresAlthough the ATM protein was shown to be localized mainly in the nucleus of mitotic cells, a number of studies indicated that ATM was also present in the cytoplasm, including peroxisomes and cytoplasmic vesicles ,58,59,602O2 led to significant upregulation of pgRNA, S-RNA, intracellular and secreted HBV DNA and HBsAg. Here, we extended their findings by showing that etoposide treatment may provide growth advantage to the replicating virus as demonstrated by elevated levels of intracellular HBc protein and secreted HBe antigen. Especially at later time-points after etoposide-treatment, the differences in levels of HBc and HBe became clearly apparent. The possible mechanism of ATM action on HBe and HBc expression could act via the induction of cell cycle arrest leading to increased gene expression from accumulated cccDNA in non-dividing cells. This could be enough to cause relatively weak effect on HBeAg secretion in the infection studies.The effects of DNA damaging agents on HBV replication were well documented by Kostyusheva et al. . The autFrom a diagnostic perspective, HBeAg is an important marker of HBV replication. The differences in HBeAg secretion in our study appear to be relatively low (less than 2-fold changes) compared to chronically infected patients where viral reactivation often leads to dramatic upregulation of antigen production. In patients, HBV reactivation and liver disease progression depends on many different factors, that include patient’s age and immunosuppression status, co-infection with other viruses and additional anti-viral or anti-cancer therapies. Therefore, we conclude that even relatively low differences observed in infection studies may have biological significance and become clinically important.Consistent with our in vitro results, frequent HBV reactivation has been observed in patients with chronic hepatitis after the use of etoposide during different anti-cancer chemotherapies ,68,69. IThe importance of the ATM signaling pathway in HBV infection and HBV-associated carcinogenesis was also recently supported by the study of Dansako et al. , who demIn summary, the activation of the ATM pathway leads to upregulation of HBc phosphorylation that occurs on SQ motifs located in the C-terminal domain. Further in-depth analysis will be needed to explore the interaction between HBV and the host ATM pathway and to define the role of ATM-mediated HBc phosphorylation in HBV replication or reactivation.The importance of the host DDR pathway in HBV replication and associated pathogenesis has been emerging recently. The detailed understanding of the molecular mechanism and functional outcome of the interaction between HBV and host ATM/ATR will enable future studies that may lead to the design of new therapeutic approaches.


Brassica species possesses sporophytic self-incompatibility (SSI), which is controlled by the pollen- and stigma-determinant factors SP11/SCR and SRK. However, the mysterious molecular mechanism of SI remains largely unknown. Here, a new class II S haplotype, named BrS-325, was identified in a pak choi line ‘325’, which was responsible for the completely self-compatible phenotype. To obtain the entire S locus sequences, a complete pak choi genome was gained through Nanopore sequencing and de novo assembly, which provided a good reference genome for breeding and molecular research in B. rapa. S locus comparative analysis showed that the closest relatives to BrS-325 was BrS-60, and high sequence polymorphism existed in the S locus. Meanwhile, two duplicated SRKs (BrSRK-325a and BrSRK-325b) were distributed in the BrS-325 locus with opposite transcription directions. BrSRK-325b and BrSCR-325 were expressed normally at the transcriptional level. The multiple sequence alignment of SCRs and SRKs in class II S haplotypes showed that a number of amino acid variations were present in the contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I and HV II) of BrSRK-325s, which may influence the binding and interaction between the ligand and the receptor. Thus, these results suggested that amino acid variations in contact sites may lead to the SI destruction of a new class II S haplotype BrS-325 in B. rapa. The complete SC phenotype of ‘325’ showed the potential for practical breeding application value in B. rapa.Most flowering plants have evolved a self-incompatibility (SI) system to maintain genetic diversity by preventing self-pollination. The  Brassica species, the SI system is characterized by sporophytic self-incompatibility (SSI), in which the diploid parental genotype determines the pollen grain self-incompatible phenotype, and is controlled by a single Mendelian locus (named S locus) [S locus contains multiple physically linked genes and segregates with the SI phenotype [S locus genes have been identified, including S locus Glycoprotein (SLG), S locus receptor kinase (SRK), and S locus cysteine-rich protein (SCR)/S-locus protein 11 (SP11). SLG was the first identified S locus gene, encoding a protein secreted in stigmatic papilla cells [Brassica [SRK was the second identified gene, and encodes a transmembrane serine/threonine receptor kinase located at the stigmatic papilla cell plasma membrane, which had been testified as being the female determinant factor [SP11/SCR was the third identified gene in the S locus, and encodes a peptide protein located in anther tapetum cells and/or pollen grains, which had been testified as being the male determinant factor [S haplotype, SRK is activated and a signaling cascade is elicited within the stigmatic papilla cell to reject self-pollen [In flowering plants, self-incompatibility (SI) evolved to maintain genetic diversity and prevent self-pollination, in which pistils inhibit pollen germination or pollen tube growth after self-pollination . In BrasS locus) . The S lhenotype . The S lla cells  and may Brassica ,6,7. SRKt factor ,9,10,11.t factor ,13,14. Wf-pollen ,16. S haplotypes were classified into two groups, class I and class II [S haplotypes are dominant over class II in pollen and the dominance relationship of S haplotypes is involved in SCR/SP11 expression suppression, which is controlled by the SCR methylation inducer (Smi) [S haplotypes is observed in pistils [S haplotypes have been identified in B. rapa [S haplotypes have been reported in B. oleracea [S haplotypes have been identified in B. napus, including BnS-1 to BnS-7 and BnS-1300 [S haplotypes in B. rapa, including BrS-40, BrS-60, BrS-29, and BrS-44 and three class II S haplotypes in B. oleracea, including BoS-2b, BoS-5, and BoS-15 [S haplotypes has been observed through reciprocal test pollinations in B. rapa (BrS-44 > BrS-60 > BrS-40 > BrS-29) [SCR methylation inducer 2 (Smi2) [SCR and SRK genes in the recessive class II S haplotypes have been characterized in B. rapa [S locus organization of the class II S haplotypes was limited to the BrS-60 haplotype [S locus.Based on the nucleotide sequence similarity, class II . Class Ier (Smi) ,20,21,22 pistils ,24. More B. rapa , and abooleracea , while sBnS-1300 ,28. Howe BrS-29) ,23, and  B. rapa . Howeveraplotype ,33, whicS locus genes, including SCR genes [SRK genes [S locus gene disruption also changes the SI phenotypes, such as M-locus protein kinase (MLPK) [EXO70A1), Phospholipase D α1(PLDα1), and Glyoxalase1(GLO1), were identified as self-compatible factors working downstream of ARC1 [In the last few decades, researchers have concentrated on identifying the SI reaction factors; however, the self-incompatible mechanism and self-compatible pathway remain unexplained. Mutant screening and research are very valuable to identify new factors involved in the SI system ,36,37,38CR genes ,39,40 anRK genes ,42. On te (MLPK) ,44 and Ae (MLPK) ,46,47,48e (MLPK) ,50,51. S of ARC1 ,53,54,55 of ARC1 . Thus, tBrassica. Some SC lines have been identified from SI populations [Brassica rapa subspecies, including heading Chinese cabbage, yellow sarson, and no-heading pak choi [Brassica genomic variation research. In this study, a completely self-compatible line with a single S locus mutation was identified, which had a new class II S haplotype, named BrS-325. Complete genome assembly was gained by means of Nanopore sequencing strategy and the S-intergenic region was determined. The S locus genes were identified, and BrSCR-325 and BrSRK-325b had normal expression levels. Previous reports investigated leaf development, flowering, and LTR-RT expansion based on the assembled genomes in B. rapa [S locus of two class II S haplotypes, BrS-60 and BrS-325, which are each other’s closest relatives. The S locus of BrS-60 and BrS-325 showed high sequence polymorphism with few genes’ distribution. Meanwhile, multiple sequence alignment of class II SCRs and SRKs was carried out to reveal amino acid variations in the contact regions (CR II and CR III) of BrSCR-325 and the hypervariable regions (HV I and HV II) of BrSRK-325s, which may influence the interaction between the ligand and the receptor. Here, a complete pak choi genome was obtained and many amino acid variations were identified in the contact regions of BrSCR-325 and the hypervariable regions of BrSRK-325s, which may lead to self-incompatibility destruction.The screening and establishment of new self-compatible (SC) lines are critical for SI system research in ulations ,35,38,57ulations ,59,60,61ulations ,63,64,65pak choi ,33,66, aS haplotype by cloning and analyzing S locus genes. The line ‘325’ could be amplified by universal primer pairs of class II, but not class I, indicating that a class II S haplotype existed  were obtained by the means of PCR and sequenced (SCR and SRK E1 sequences in ‘325’ were different from the reported class II S locus genes and showed the highest sequence similarity with that of BrS-60 (79% sequence similarity of SCR and 91% sequence similarity of SRK E1)  . Phylogef BrS-60 D,E. Thus1 hybrids were created from the SI parent ‘326’ (BrS-12) and SC parent ‘325’ (BrS-325), and pollination behavior displayed self-incompatibility (1 hybrids (1 hybrids (1.29 and 1.48 cm per silique)  , indicating that the SC trait in ‘325’ was controlled by a single genetic locus. S haplotypes in the F2 population were segregated into S-12S-12: S-12S-325: S-325S-325 =20:50:22 , and all the pollination phenotypes were consistent with the genotypes  B,C. Aversilique) B,D. Baseenotypes . All theBrS-325 and discover the underlying mechanism of SC in ‘325’, we carried out Nanopore sequencing and next de novo assembly to obtain the pak choi genome [B. rapa genome assemblies , retrotransposons (42.41%), and unclassified elements (3.41%). The annotated retrotransposons mainly consisted of the long terminal repeat retrotransposon (LTR-TE), which covered 120 Mb and occupied 31.96% of the assembled genome . In totaSCR and SRK from ‘325’, homologous genes were blasted in our assembled genome. Finally, we obtained a 145 kb sequence from contig, named ctg00061, which covered the whole BrS-325 locus and boundary regions   . BrS-325 showed the highest sequence similarity with those of BrS-60 , and their F1 hybrid. BrSCR-325 showed a higher expression level in the anther of the SC line ‘325’ than that of BrSCR-12 in the SI line ‘326’, while in the F1 hybrid, the expression of BrSCR-325 was hardly detected  and amino acid deletion . As we know, the hypervariable region of SRK and contact regions were necessary for the interaction between SCRs and SRKs with the same plotypes ,74,75. BS domain of BrSRK-325  and three class II S haplotypes in B. oleracea  [S haplotype, named BrS-325, was identified in a pak choi line‘325’   A. The sine ‘326’ . All theS haplotype BrS-325  and two duplicated SRK genes (BrSRK-325a and BrSRK-325b) exist in the BrS-325 locus; however, expression analysis results showed that the expression of BrSCR-325 and BrSRK-325b are normal, which may not be responsible for the SC phenotype  was performed in a 10 μL reaction system, including 1 μL of DNA, 0.5 μL of each primer at 10 μM, 5 μL of 2× Taq Reaction Buffer  , and 3 μL of distilled water. The amplification program involved one cycle of 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min. PCR products were electrophoretically-fractionated in a 1% agarose gel, stained with ethidium bromide, and then viewed under a UV illuminator. The SCR sequence was determined using the II SCR-1Land II SCR-1R primer pair, and the partial sequence (included first exon and four to seven exons) of SRK was determined using SRK II E1L and E1R and SRK II E4L&E7R in the self-compatible line “325”. PCR products were sequenced to determine the S haplotype. To explain the mutation locus of the self-compatible inbred line ‘325’, the F1 hybrid was created by crossing the SI parent ‘326’ (BrS-12) and the SC parent ‘325’, and self-pollinated to produce the F2 segregation population. S haplotypes were determined with the specific primer pairs: PK1and PK4 for detecting class I S haplotypes and SRK II E4L and SRK II E7R for detecting class II S haplotypes. PCR was performed under the same conditions mentioned above. Primer information for detecting S haplotypes is listed in Genomic DNA from all individuals was extracted from young leaves as described by Murray and Thompson . The S hand PS15  and PK1aand PS15  to identand PS21  and SRK Genomic DNA was extracted from the first young leaves of the self-compatible inbred line ‘325’, according to the procedure described by Belser et al . Genomichttp://www.girinst.org/repbase; accessed on 21 March 2020) (software:RepeatMasker [http://www.repeatmasker.org/RepeatModeler/ accessed on 21 March 2020), Piler [http://bix.ucsd.edu/repeatscout/ accessed on 21 March 2020) and repetitive sequence features (software: TRF (https://tandem.bu.edu/trf/trf.html accessed on 21 March 2020) and LTR-Finder [In our assembled genome, we carried out genomic annotation, including the prediction of repeated sequences, the prediction of non-coding RNA, and the prediction of gene structure and functional annotation. Repetitive sequence annotation is combined with the homologous prediction method and the de novo ab initio prediction method . The homatMasker  and Repe), Piler , RepeatSR-Finder ).http://rfam.xfam.org/ accessed on 21 March 2020) [The structure prediction of coding genes was usually combined with a variety of prediction methods, such as homolog prediction , de novoch 2020) .3PO4) for 3 h. The stained pistils were placed on the glass slide in distilled water to detect the pollen germination and pollen tube growth using the blue channel (UV 340–380 nm) of a fluorescence microscope .The aniline blue staining assay was performed according to the established procedure with minor modifications . The antTM RT reagent kit . The real-time quantitative polymerase chain reaction (RT-qPCR) was performed using 2× SYBR Green master mix  with the 10 μL reaction system, including 5 μL of 2× SYBR Green master mix, 0.4 μL of each primer at 10 μM and 4.2 μL of 50× diluted cDNA. Amplification was performed using the CFX96 Touch Real-Time PCR Detection System . The amplification program involved one cycle of 95 °C for 5 min, followed by 45 cycles at 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s. After each run, a melting curve was performed by heating up the samples from 60 to 95 °C. All analyses were repeated with three biological replicates. Actin gene (GenBank accession No: AF111812) was used as an internal control and actin was used to normalize transcript levels for all expression analyses [t-test. The primer pairs coming from S locus genes (SRK and SCR) are developed. All primers are listed in Anthers were cut from medium-size buds (2 to 3 days before flowering) and stigmas were gained from mature buds (on the day of flowering). Total RNA of stigmas and anthers was isolated from more than 30 stigmas and from six anthers of more than 20 buds using the SV Total RNA Isolation System kit  following the manufacturer’s instructions. One microgram of total RNA was used for the cDNA synthesis using a PrimeScriptanalyses . SignifiBrSRK-60 came from the genome of ‘Chiifu’ [http://nrbsc.org/gfx/genedoc 26 August 2015). Class II SCR drafts were drawn referring to the research results [CDS and amino acid sequences of class II SRK and SCR were obtained from the NCBI and their GenBank or entry name are listed in ‘Chiifu’ . MEGA-X ‘Chiifu’  program  results  and clas results . Phyloge results  using thS haplotypes, BrS-60 was obtained from the genome of ‘Chiifu’ [S haplotype and a contrasting SI phenotype, and BrS-325 was obtained from our assembled genome. Collinearity analysis of S locus sequences was performed using the software: MuMmer [S locus collinearity analysis of BrS-325 and BrS-60 was performed with the MuMmer tool. Then, visualizations of the S locus collinearity analysis were displayed using a JCVI tool.To display the synteny relationships of two class II ‘Chiifu’  and ‘Z1’‘Chiifu’ , which p: MuMmer  and JCVI: MuMmer . The S lt-test.The self-incompatibility phenotype was measured as in . When thS haplotype in Brassica, which displayed completely self-compatibility. It is a good resource for breeding practices due to its recessiveness to all class I S haplotypes [S haplotype in B. rapa.In conclusion, we identified a new class II plotypes . Here, wplotypes . In this