Studies of the doublesex (dsx) gene in insects, in particular Drosophila melanogaster, reveal that alternative splicing of dsx generates sex-specific Dsx isoforms underlying sexual differentiation. Such a splicing-based mechanism underlying sex-specific Dmrt function is thought to be evolved from a transcription-based mechanism used in non-insect species, but how such transition occurs during evolution is not known. Here we identified a male-specific dsx transcript (dsxM2) through intron retention (IR), in addition to previously identified dsxM and dsxF transcripts through alternative polyadenylation (APA) with mutually exclusive exons. We found that DsxM2 had similarly masculinizing function as DsxM. We also found that the IR-based mechanism generating sex-specific dsx transcripts was conserved from flies to cockroaches. Further analysis of these dsx transcripts suggested an evolutionary pathway from sexually monomorphic to sex-specific dsx via the sequential use of IR-based and APA-based alternative splicing.The  dsxM2, is identified via intron retention, with a masculinizing function weaker than the modern dsxMAn ancestral male-specific doublesex isoform,  Dmrt transcription factors share a common DM (Dsx/Mab-3) domain which is a zinc-finger DNA binding motif, but show little sequence conservation in other parts1. In humans, Dmrt1 is linked to disorders of sex development and testicular germ cell tumors5. Dmrt1 is preferentially expressed in the male gonad and required postnatally to maintain gonadal sex in mammals and other vertebrates8. In the nematode worm Caenorhabditis elegans, mab-3 is mainly expressed in male tails and necessary for proper morphogenesis and differentiation of copulatory structures9. In the fruit fly Drosophila melanogaster, sex-specific Dsx isoforms, DsxM in males and DsxF in females, control sexual differentiation and behaviors in both sexes, and are generated through alternative polyadenylation (APA) with mutually exclusive exons14.Sexually dimorphic traits and behaviors are mediated by sex-specific expression of regulatory genes. Substantial studies reveal that the Dmrt genes in sexual differentiation in metazoans, regulatory mechanisms underlying their sex-specific function are diversified16. The alternative splicing-based regulation of Dmrt genes has only been found in insects and often mediated by the female-specific expression of transformer (tra) with few exceptions19. In vertebrates and nematodes, Dmrt genes are not sex-specifically spliced, but transcribed in a predominantly male-specific manner for the development of male-specific traits2. It has been proposed that the insect-specific mechanism based on alternative splicing of dsx evolved from a more ancient mechanism based on male-specific transcription17, but how such transition occurs during evolution is rarely known.Despite conserved roles of tra-dsx pathway generates sex-specific Dsx isoforms by the selective use of distinct 3′ exons in the two sexes17. In D. melanogaster females, Tra and Tra-2 bind to the female-specific exon of dsx and direct female-specific splicing of dsxF, while in males the absence of Tra function permits the default splicing of dsxM including both common and male-specific exons22. DsxM and DsxF have common DM domain and two oligomerization domains, thus may bind to the same sets of target genes; however, they have been found to oppositely regulate target gene expression to establish male- or female-specific differentiation, possibly through their sex-specific C-terminus26.The canonical insect dsx transcript and propose an evolutionary pathway from sexually monomorphic to sex-specific splicing of dsx. We identify another male-specific dsx transcript (dsxM2), in which the intron linking the last common exon and the female-specific exon is retained. The IR-generated DsxM2 has a masculinizing role like DsxM and is crucial for male courtship robustness in D. melanogaster. We further show that such mechanism generating sex-specific dsx transcripts is deeply conserved in insects from fruit flies to cockroaches. The male-specific intron retention depends on the presence of a weak splicing acceptor sequence inside the intron, as well as the regulation of Tra. Comparison of the structures of dsx transcripts, in light of their difference of conservation, suggests an evolutionary pathway from sexually monomorphic to sex-specific dsx via the sequential use of IR-based and APA-based alternative splicing.In this study, we report that intron retention (IR) generates a male-specific dsx pre-mRNA is sex-specifically spliced to yield the male-specific dsxM and female-specific dsxF mRNAs. In an attempt to determine relative expression of dsx transcripts in the two sexes in wild-type Canton-S (wtcs) and dsx mutant (dsx683-7058/dsx1649-9625) flies, we performed quantitative PCR (qPCR) experiments using two pairs of primers targeting the male-specific or female-specific locus in dsx, respectively  experiments using three different primer pairs targeting different sites of the dsxF transcript ] and found that males with dsxM knocked down were intersexual and displayed little courtship, while males with dsxM2 knocked down had regular male appearance but reduced courtship and mating success with virgin female targets  and validated their efficiency using qPCR experiments and immunostaining with anti-Flag and anti-Myc antibodies , while overexpressing DsxM or DsxM2 masculinized female development  , we next overexpressed these dsx isoforms in a dsx mutant background [dsxGAL4(∆2)/dsx1649-9625] in which both sexes were intersexual, and found that expressing DsxF induced female differentiation in both sexes, while expressing DsxM or DsxM2 strongly masculinized genital and sex comb development in both sexes  Fig. . To avoixes Fig. . These r30. To compare the transcriptional regulation of different Dsx isoforms on target genes, we overexpressed the three Dsx isoforms driven by the dsxGAL4(∆2) and tested relative expression changes of yp2 and yp3. Compared to control females, overexpressing DsxF significantly increased yp2 and yp3 expression, while overexpressing DsxM or DsxM2 significantly reduced their expression in females  have been intensively studied under control of Dsxles Fig. . Note thsxM Fig. . The expion Fig. . Taken tdsxM2 transcript through intron retention, which is a potent mechanism underlying sexual differentiation, we asked if such an alternative splicing form also existed in other animal species. We tested three other Drosophila species including the closely related D. simulans and two other distant species D. mojavensis and D. virilis, by performing RT-PCR experiments using multiple primer pairs targeting sequences including the potentially retained intron and a portion of the female-specific exon, followed by sequencing. We found retention of the 118 bp intron in D. simulans males , but also with the dsx gene structure being previously studied32. We designed a pair of primer targeting the last common exon and the first female-specific exon respectively nNCAG   upstream of the last four nucleotides (NCAG) is at least 9. Indeed, the common (intron 1 and 2) and male-specific (intron 3m and 4m) acceptors have 9–11 pyrimidines upstream of NCAG. As for the retained intron , the acceptor has only 6 pyrimidines upstream of NCAG  Fig. . PreviouCAG Fig. . We nextCAG Fig. . These rDrosophila species. Surprisingly, these intron sequences, despite many differences, share intensive identity in two regions: one in the 3′ end of the intron overlapping the region of the weak acceptor site that may contribute to intron retention as above mentioned, and the other in the middle of these introns containing the core sequence (CAATCAAC) of the tra binding sequence, TC(T/A)(T/A)CAATCAACA, that occurs six times in the female-specific exon   and dsxF (intron spliced out).We next compared full sequences of retained introns in the four xon Fig. . The condsxM2 transcript promoted us to analyze how the two forms of alternative splicing (IR and APA) were evolved to generate the modern day dsx transcripts , and the more conservation of the female-specific exon than the male-specific exons in insect species33, we propose that dsxM2 could serve as an ancient monomorphic transcript in both sexes, followed by two sequential events evolving alternative splicing: the IR-based mechanism generating dsxM2 in males and dsxF in females with the evolution of Tra regulation in females, and later the APA-based mechanism generating the rapid-changing dsxM with the evolution of a stronger splicing acceptor to capture male-specific exons  underlying sexual differentiation in D. melanogaster and many other insects16. Such splicing mechanism involves the selective use of sex-specific 3′ exons, which is under regulation of Tra and Tra-222. Our results identified the male-specific DsxM2 isoform through intron retention, which is a common mechanism generating sex-specific Dsx isoforms in insects across 300 million years.Previous studies have shown that alternative splicing of dsx using a variety of insect species revealed that the tra-mediated female-specific splicing of dsx could be evolved from sexually monomorphic isoforms with male-biased expression17. Indeed, vertebrates, nematodes and crustaceans use male-biased transcription of Dmrt genes to direct male-specific function34. In this regard, the evolution of dsxF may come from dsxM and/or dsxM2 under Tra regulation, possibly through the functional gain of Tra binding sites in the female-specific exon of dsx. Indeed, knocking down Tra function in females induced intron retention-based splicing of dsxM2 instead of dsxF. The coding sequence of the female-specific exon, which is also included as 3′-UTR of dsxM2, is more conserved than the male-specific exon of dsxM that rapidly changes not only across insect orders but also across closely related genera within each insect order33, excluding the possibility of dsxM as the origin of sexually monomorphic dsx transcript. The identification of the intron retention-based dsxM2 transcript fits well into a position as the ancestral isoform of dsx expressed in both sexes before the evolution of dsxM and dsxF. The conserved presence of a weak splicing acceptor upstream of the female-specific exon provides the selection basis for later recruitment of Tra regulation to generate the female-specific dsxF from dsxM2 in females, as well as the evolution of a stronger splicing acceptor to capture male-specific exons and generate dsxM in males. Such an evolutionary pathway of generating sex-specific Dsx isoforms could fill the gap between the splicing-based mechanism in insects and transcription-based mechanism in vertebrates and nematodes.Recent studies on the alternative splicing of M2 isoform contains all common Dsx amino acids and six specific C-terminal residues, thus could act like other Dmrt transcription factors. Our knockdown experiments reveal that DsxM2 still functions in the nervous system to promote courtship robustness in D. melanogaster, but not in the development of male-specific traits such as genitals and sex combs. In addition, the overexpression experiments indicate that DsxM2 has a potential masculinizing role just like DsxM. However, a direct comparison of DsxM and DsxM2 function could not be faithfully achieved as knockdown and overexpression levels of dsxM and dsxM2 may be different. Another caveat of these results is the lack of direct evidence of DsxM2 expression in males. It is also possible that DsxM2 only serves as an ancient Dsx isoform whose function has largely been replaced during evolution , and now has limited expression and function. Future studies could generate better reagents to testify DsxM2 expression in a temporal and spatial manner in D. melanogaster and other insect species to better understand its function as a possible origin of sex-specific Dsx isoforms underlying sexual development and sexual dimorphism.The DsxCanton-S (wtcs) and w1118 were used as wild-type strains. dsx mutant lines used in Fig. dsx683-7058 and dsx1649-9625, which were used as previously13. Drosophila mojavensis, Drosophila simulans and Drosophila virilis13, R57C10-GAL4 , actin-GAL4 (BDSC_25374), UAS-dsxMRNAi (attP2)14, UAS-flag-dsxF (attP40) and UAS-myc-dsxM (attP40)35, dsxGAL412 and dsxGAL4(Δ2)36 were used as described previously. UAS-dsxM2RNAi (attP2) and UAS-flag-dsxM2 (attP40) were generated in this study and described below in details. Detailed information about fly stocks and other materials used in this study is listed as Table Flies were raised at 22 °C or 25 °C at 12 hr light/12 hr dark cycle at 60% humidity. dsx gene sequence of D. melanogaster from FlyBase (http://flybase.org/). We used NCBI (https://www.ncbi.nlm.nih.gov/) to find the location and gene sequences of dsx in following species: D. simulans (Gene ID: 6727147), D. mojavensis (Gene ID: 6574377), D. virilis (Gene ID: 6633147) and B. germanica32. Gene sequence or amino acid sequences comparisons were performed using Clustal Omega and LaserGene software.We downloaded 27  to analyze splicing events of dsx 14 was digested to be linearized with NheI and EcoRI. We selected a 21 nucleotides sequence (tggctgtgaagtgaaattgta) targeting the 114 bp intron, which was synthesized and generated into hairpin by annealing. The resulting DNA fragment and linearized vector were ligated. The resulting constructs were injected into embryos of attP2 site with PhiC31-mediated transgenesis. The correct insertion was screened by vermillion (vermillion positive eye color) and verified by PCR and followed by DNA sequencing. Oligo primers are as following:To generate the Oligo forward:5′-ctagcagtTGGCTGTGAAGTGAAATTGTAtagttatattcaagcataTACAATTTCACTTCACAGCCAgcg-3′Oligo reverse:5′-aattcgcTCGCACTGTAGCCCAGATCTAtatgcttgaatataactaTACAATTTCACTTCACAGCCAactg-3′.UAS-flag-dsxF and UAS-myc-dsxM lines were generated previously in our lab35. The UAS-flag-dsxM2 line was generated in this study. In brief, the 3×Flag tags (MDYKDHDG-DYKDHDI-DYKDDDDKL) were added to the N terminus of DsxF and DsxM2 protein separately. We simultaneously amplified the flag sequence and the full-length CDS fragment of dsxF and dsxM2 from wild-type cDNAs using the following overhang primers and then cloned into pJFRC2-10XUAS-IVS-mCD8::GFP plasmid (Addgene #26214) to replace the mCD8::GFP sequence.dsxF-forward: CCGAGATCTATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGCTTGTTTCGGAGGAGAACTGGdsxF-reverse: CCATCTAGATCATCCACATTGCCGCGTTG.dsxM2-forward: CCGAGATCTATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGCTTGTTTCGGAGGAGAACTGGdsxM2- reverse: CCATCTAGATCACTGATCAACAGACTTACC.UAS-myc-dsxM transgenic line, the 6×Myc tag (MEQKLISEEDL) was added to the N terminus of DsxM. We first cloned the 6×myc sequence into pJFRC2-10XUAS-IVS-mCD8::GFP between the BglII and the XhoI sites, and then amplified the full-length CDS of dsxM and cloned into C terminus of 6×Myc to replace the mCD8::GFP sequences.For the myc-F: CCGAGATCTTCCCATCGACTTAAAGCTATGmyc-R: CCACTCGAGACTAGTCTCAAGAGGCCTTGAGTTCdsxM-F: CCGACTAGTGTTTCGGAGGAGAACTGGdsxM-R: CCATCTAGACTACGTGGCAGCCGTGGAGmini-white (orange eyes) and verified by PCR and followed by DNA sequencing.The constructed plasmids were injected and integrated into the attP40 sites on the second chromosome through phiC31 integrase mediated transgenesis. The correct strains were screened by Canton-S, w1118 and other species including D. simulans, D. mojavensis, D. virilis and Blattella germanica were used as wild-type strains for RT-PCR. We obtained total RNA from approximate thirty adult females and/or males using a commercial TRizol™ reagent  and purified RNA with DNA-free™ kit  according to manufacturer’s protocol. Purified RNA was finally resuspended in 60μL of DEPC-treated water. First strand cDNA was synthesized for each RNA sample using SuperScript™ IV reverse transcriptase . dsx-specific primers are listed in Table dsx transcripts.To acquire cDNA template, actin was amplified as an internal control for normalization. The primers for qPCR used in this study are listed in Table Total RNA extraction and first strand cDNA template were acquired as described above, except for the data in Supplementary Fig. Canton-S females were group-housed and aged under similar conditions. To measure courtship, a male of each genotype and a Canton-S virgin female were loaded individually into two-layer chambers  which were separated by a plastic transparent barrier until courtship test for 30 min. Courtship index (CI), which is the percentage of observation time a fly displayed any courtship step in 10 min, and percentages mated were counted based on successful copulation in 30 min.Newly enclosed males were collected and group housed for 4–7 days at 25°C and 60% humidity with a 12 hr: 12 hr light/dark cycle. Virgin Adult flies were reared at 25°C and aged for 4–7 days old, and were dissected in Schneider’s insect medium  and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20–30 min at room temperature (RT). Tissues were washed at least 4 times for 15–20 min with PAT3 , and then blocked in 3% normal goat serum (NGS) for 60 min at RT. In Supplementary Fig. For visualizing morphological appearances of flies Fig. , 4–7 dayAll statistical analyses were performed using the Prism 8 (GraphPad software). Experimental flies and genetic controls were tested at the same condition, and data are collected from at least two independent experiments and are reproducible. The Mann-Whitney U test was used for pairwise comparisons. For comparing mating success, Chi-square tests were performed to compare two different groups at 30 min time point.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


PDCD1 gene with breast cancer risk. Similarly, the protein expression data showed that PD-L1 and PD-L2 are associated with an aggressive phenotype, Luminal B, and advanced breast cancer stage. Conclusion: These findings suggest that immune checkpoint polymorphisms are associated with the risk and subtypes of breast cancer.Background: Breast cancer is the most prevalent and second leading cause of cancer-related death in women worldwide. Despite early detection and better treatment therapies, 30% of early-stage breast cancer patients still develop recurrent disease. Breast cancer is a heterogeneous disease comprising several molecular subtypes, commonly classified into clinical subtypes based on the hormone receptor status. These subtypes included luminal A and luminal B, which have different prognoses. Breast cancer development and progression involve many factors. Polymorphisms of PD-1, PD-L1, and PD-L2 genes have been previously associated with high risk and prognosis of cancer. However, no studies have associated PD-1, PD-L1, and PD-L2 polymorphisms with primary breast cancer subtypes. Hence, this study evaluated functional single nucleotide polymorphisms of PD-1, PD-L1, and PD-L2 with primary breast cancer subtypes, luminal A, and luminal B. In addition, we evaluated the PD-L1 protein expression in relation to primary breast cancer subtypes and stages. Results: There were no significant differences in the allele frequencies of PD-1 polymorphisms  and PD-L1 polymorphisms (rs10815225 C>T and rs2282055 T>G) when compared with the general European population. However, a significant difference was detected in one of the PD-L2 polymorphisms (rs1009759 A>G), with the G allele higher in breast cancer patients than in the general European population. A higher prevalence of the T allele of PD-L1 polymorphism rs2282055 T>G was observed in luminal B breast cancer patients compared with luminal A. No significant difference was detected in other polymorphisms. We also observed that the PD-L1 rs2282055 TT genotype was more prevalent in luminal B breast cancer patients compared with luminal A. Our results found no association of the selected SNPs in the  Breast cancer is the most common and second leading cause of cancer-related death in women worldwide ,2. OveraPDCD1 gene, is a cell surface immunosuppressive receptor expressed on immune cells [PDCD-1 gene can influence cancer risk [rs2227981, rs36084323 and rs11568821 polymorphisms are associated with decreased overall cancer risk [rs7421861 significantly augmented the risk of cancer [rs11568821 is associated with increased risk [rs2227981 with the risk of cancer [rs2227982 was significantly correlated with age and tumour size, while PD-1 rs11568821 and rs2227981 were associated with tumour stage and tumour grade, respectively [The interaction of programmed death-1 (PD-1) with programmed death-ligand 1 (PD-L1) plays a vital role in tumour cell evasion from host immune surveillance by downregulating T cell immune response . The binne cells ,13. Studcer risk ,15. Metacer risk ,17,18,19f cancer . Howeversed risk  and no af cancer . PD-1 rsCD274 gene, is a co-inhibitory molecule expressed on activated immune cells, by which cancer cells utilised to evade the host’s immune response [PD-L1 gene by binding to its 3 prime untranslated region (3′UTR) have been reported to play a role in controlling PD-L1 expression [PD-L1 gene enhances PD-L1 mRNA stability in murine and human murine cells [PD-L1 gene [PD-L1, also identified as B7-H1 encoded by the response ,21. Accupression ,23,24. Fne cells . Further-L1 gene .rs7866740 was associated with the risk of non-small lung cell carcinoma (NSCLC) compared to the C allele [rs2890658 C>A and rs822336 G>C polymorphisms were associated with worse overall survival and progression-free survival of NSCLC patients [rs2890658 A>C genotype and A allele increased the risk of breast cancer [rs2890658 significantly reduced the risk of lung and liver cancer in the allele model A>C but increased the risk of hepatocellular carcinoma in the allele model A>C and recessive genetic model AA> AC+CC [PD-L1 s4143815 is associated with a reduced risk of breast cancer (G>C) and hepatocellular carcinoma in homozygote (CC>GG), heterozygote (GC> GG), and dominant (CC+CG >GG) genetic patterns [PD-L1 rs2890658 and rs4143815 were correlated with age [The G allele of PD-L1 C allele . Moreovepatients . Similart cancer . PD-L1 rA> AC+CC . PD-L1 swith age . These fOn the other hand, the role of protein expression of PD-L2 and polymorphism in cancer is not well studied. High expression of PD-L2 was associated with a poor prognostic in patients diagnosed with hepatocellular carcinoma, while no association was observed in patients diagnosed with gastric, renal cell carcinoma, and esophageal cancer . SimilarAlthough polymorphisms of PD-1, PD-L1, and PD-L2 genes have been reported to associate with a high risk of cancer, to the best of our knowledge, no studies have reported PD-1, PD-L1, and PD-L2 polymorphisms in relation to primary breast cancer subtypes. Hence, this study evaluated functional single nucleotide polymorphisms of PD-1, PD-L1, and PD-L2 in relation to primary breast cancer subtypes, luminal A, and luminal B. In addition, we evaluated the PD-L1 protein expression in relation to primary breast cancer subtypes and stages.n = 41/subtypes) were required to achieve a significantly different. In this study, 123 patients with breast cancer  were analysed for SNP, and 74  for immunofluorescences. Immunofluorescence images were quantified blindly. Samples were fresh-frozen and stored at −80 °C.Human breast cancer samples were collected at the Prolipsis Medical Centre, Athens, Greece between 2007–2012 (ethics approved 12 June 2006). The human samples collection used in this study complied with the guidelines of the National Health and Medical Research Council (NHMRC) Australian Code of Practice for the Care under the approval of the Victoria University Human Ethics Committee (ethics number HREC15-299). All patients signed written informed consent to use their tissues for research purposes. None of the breast cancer patients had a second neoplastic disease or had previously undergone chemo- or radiotherapy. All samples were coded. To achieve a significantly different, a power of 0.8, two-tails, and alpha = 0.05 with two subtypes, medium effect size, F = 0.30 was used to calculate sample size using GPOWER. A minimum of 82 samples . Briefly, samples were incubated overnight with tissue lysis buffer and proteinase K at 55 °C with a rotary shaker. Samples were centrifuged at 10,000 revolutions per minute for 3 min (mins), and the supernatant was transferred to a new microtube. Samples were treated with RNase A, vortexed for 5 s (sec), and flash spun before incubation for 2 min at room temperature. Then, samples were incubated with lysis buffer at 70 °C for 10 min with a rotary shaker. Samples were flash spun, 99% ethanol was added, they were vortexed for 15 sec and flash spun. Lysates were transferred into the cartridge of QuickGene, and washes were performed using wash buffer. Cartridges were incubated with elution buffer for 90 sec at room temperature, and genomic DNA was collected into a new tube. Genomic DNA was run through electrophoresis gel to check purity and qubit to quantify genomic DNA concentration. Genomic DNA was then sent off to the Australian Genome Research Facility  for SNPs analysis using MassARRAYwww.ncbi.nlm.nih.gov/snp/docs/gsr/alfa/ (accessed on 10 March 2020)).Three SNPs  in PD-1, two (rs10815225 and rs2282055) in PD-L1, and two (rs1009759 and rs6476985) in PD-L2 genes were selected based on previously published literature associated with cancer or other diseases. The control data were obtained from the European subgroup of ALFA: Allele Frequency Aggregator  , then placed on a pre-heated hot plate set at 100 °C for 20 min and left to cool at room temperature for another 20 min. Slides were washed with phosphate-buffered saline 3 × 10 min. To reduce the volume, samples were outlined using a liquid blocker super pap pen (ProSciTech). The endogenous activity was blocked by incubating slides with 10% donkey serum for 1 h at room temperature. Slides were then incubated overnight with target primary antibodies against PD-L1  ,31 and P2) was conducted by GraphPad Prism for the relationship of PD-1, PD-L1, and PD-L2 morphisms genotypes with luminal A and B. Statistical analysis of immunohistochemical data was performed by Student’s t-test and one-way ANOVA. Statistical significance was defined as p < 0.05 (two-tailed).SNP analysis: Hardy–Weinberg equilibrium (HWE) was calculated by AGRF. Fisher’s exact test was conducted by GraphPad Prism for the relationship of PD-1, PD-L1, and PD-L2 morphisms allele frequencies with luminal A and B. Chi-square test . Image J software  was employed to convert RGB images to greyscale 8-bit binary; particles were then analysed to obtain the percentage area of immunoreactivity . All sliThe clinicopathological and demographic parameters of breast cancer patients are presented in The distribution of genotype frequencies in controls accorded with Hardy-Weinberg equilibrium (HWE) . For PD-p > 0.05). Similarly, no significant differences were detected for the PD-L1 polymorphisms (rs10815225 C>T and rs2282055 T>G) (p > 0.05). A significant difference was detected in one of the PD-L2 polymorphisms (rs1009759 A>G), with the G allele higher in the breast cancer patients when compared with the general European population (p = 0.038).The allele frequencies of PD-1, PD-L1, and PD-L2 polymorphisms for breast cancer patients are summarized in p = 0.022). Since luminal B is more aggressive, our results demonstrated that rs2282055 T>G is associated with breast cancer severity. However, no significant differences were detected in other polymorphisms.We then compared the allele frequencies of those polymorphisms in two common breast cancer subtypes, luminal A and luminal B. We observed a higher prevalence of the T allele of PD-L1 polymorphism rs2282055 T>G in luminal B breast cancer patients (p = 0.049).We then compared the genotype frequencies of those polymorphisms in the two common breast cancer subtypes. Due to the relatively small sample size, our statistical analyses were not valid for PD-1 polymorphism rs11568821 C>T or PD-L1 polymorphisms (rs10815225 C>T . Howeverp = 0.044). When we combined the TT genotype with the TG genotype, we observed a higher presence in luminal B breast cancer patients when compared with the GG genotype (p = 0.051). We also found a significant difference when PD-1 polymorphism rs7421861 A>G AA+AG genotype frequencies were compared with GG between luminal B and luminal A breast cancer patients (p = 0.042). The GG genotype appears more prevalent in luminal B breast cancer patients.We then applied different genetic models to our analyses. We observed a significant difference in PD-L1 rs2282055 TT vs. GG genotype frequencies between luminal B and luminal A breast cancer patients, with the TT genotype more likely to develop luminal B breast cancer (The fact that the results of gene expression studies are not always identical and should not be directly translated into protein expression studies is well-known. Post-transcriptional mechanisms can shape protein levels independently of mRNA abundance . FurtherOur SNPs data showed that PD-L1 polymorphisms are associated with an aggressive subtype, luminal B. To determine whether, at the protein level in the tumour samples, PD-L1 expression is more abundant in luminal B, immunofluorescence was used. Due to the nature of this study being a blinded pilot study, tumour samples analysed for the subtype and stage were not the same “n” number. All samples were coded, and immunohistochemistry images were quantified blindly. The results showed that overexpression of PD-L1 was associated with luminal B compared with luminal A A–C. In aTaken together, our protein expression data were consistent with SNP data, suggesting that both PD-L1 SNPs and protein expression hold prognostic and predicted values for the breast cancer subtype.The ability of cancer cells to evade T cell responses and avoid immune recognition by disabling effector T cells is dependent on the multiple immunosuppressive mechanisms controlled by immune checkpoints of inhibitory pathways, including but not limited to PD-L1 and PD-L2. These immune checkpoints are initiated by ligand-receptor interactions, some of which have been FDA approved to enhance anti-tumour immunity ,39. The To the best of our knowledge, this study is the first to determine immune checkpoint polymorphisms and their correlation to the stage and subtype of breast cancer. To assess the polymorphism of PD-1, PD-L1, and PD-L2 and their correlation to breast cancer subtype, primary tumour samples were obtained from breast cancer patients, and tumours were defined into subtypes based on the expression of clinically used markers ER, PR, and HER2; and stage I, II, and III. For protein expression, tumour tissues were immunolabelled with PD-L1 and PD-L2 antibodies described under materials and methods.PD-1, PD-L1, and PD-L2 polymorphisms are selected based on previous publications, indicating their association with cancer risk. Out of the seven polymorphisms, only the PD-L2→rs1009759 A>G polymorphism showed a significant difference between the breast cancer patients and the European population. PD-L2 rs1009759 A>G polymorphism has not been studied extensively in the literature. Only one study has checked its association with ankylosing spondylitis and reported no association with the disease . When chOn the other hand, several polymorphisms showing no difference between breast cancer patients and the general European population, including PD-1 rs7421861 A>G and PD-L1 rs2282055 T>G, have been correlated with breast cancer subtypes. Out of 40 luminal B breast cancer patients, the T allele of rs2282055 is more prevalent with no GG phenotype. A 2017 study reported that the G allele of PD-L1 rs2282055 was associated with better clinical response compared with the T allele in non-small-cell lung cancer patients . SimilarCancer cells use multiple mechanisms to avoid being recognized by the immune system and downregulate the expression of PD-L1 on their surface, interacting with PD-1 on tumour-infiltrating lymphocytes . PD-L1 oThe role of PD-L2 and its prognostic significance in cancer remains understudied. Some studies have associated high expression of PD-L2 with poor patient survival ,54,55,56There are a couple of limitations to be acknowledged. First, the control group’s data were obtained from the European population of the ALFA study, which might not represent the exact genetic makeup of the PD-1, PD-L1, and PD-L2 polymorphisms in the Greek population. Secondly, the control population includes males and females; while the present study patients are exclusively females, there might be different allele frequencies between males and females. Lastly, our sample size is relatively small among cancer risk studies. Nevertheless, the fact that the PD-1 rs7421861 A>G and PD-L1 rs2282055 T>G polymorphisms correlate with the severity of cancer probably suggests that these polymorphisms are associated with clinical outcomes among breast cancer patients.The presence of PD-L1 and PD-L2 on the surface of cancer cells functions as an immune resistance mechanism, allowing them to go undetected and leading to cancer cell proliferation and progression of cancer growth. Studies have reported the expression of these markers at protein levels and their relevance to prognostic outcomes. However, there are limited studies on these markers’ polymorphisms. Therefore, revealing the significance of these markers’ polymorphisms in cancer is imperative for understanding breast cancer development and progression.


No dolutegravir-associated HIVDRMs were detected, regardless of previous exposure to ART. However, we found E92G in one ART-naïve patient specimen and accessory mutations in 20/460 (4.3%) of the specimens. Moreover, among the 288 integrase amino acid positions of the subtype C, 187/288 (64.9%) were conserved (<1.0% variability). Analysis of the genetic barrier showed that the Q148H/K/R dolutegravir resistance pathway was less selected in subtype C. Docking analysis of the dolutegravir showed that protease- and reverse-transcriptase-associated HIVDRMs did not affect the native structure of the HIV-1 integrase. Our results support the implementation of a wide scale-up of dolutegravir-based regimes. However, the detection of polymorphisms contributing to INSTI warrants the continuous surveillance of INSTI resistance.Dolutegravir-based antiretroviral therapy (ART) has been scaled up in many developing countries, including Ethiopia. However, subtype-dependent polymorphic differences might influence the occurrence of HIV-drug-resistance mutations (HIVDRMs). We analyzed the prevalence of pre-treatment integrase strand transfer inhibitor (INSTI) HIVDRMs and naturally occurring polymorphisms (NOPs) of the integrase gene, using plasma samples collected as part of the national HIVDR survey in Ethiopia in 2017. We included a total of 460 HIV-1 integrase gene sequences from INSTI-naïve ( Following the global increase of pre-treatment drug resistance (PDR) to non-nucleoside reverse transcriptase inhibitors (NNRTIs), the World Health Organization (WHO) recommended the transition from NNRTI to integrase strand transfer inhibitor (INSTI)-based regimens in both treatment-naïve and treatment-experienced patients ,2,3. Sevpol (polymerase) gene, plays a vital role in HIV-1 replication by catalyzing two distinct reactions: 3′-end processing and strand transfer [12H16C40C43) motif that coordinates zinc binding and favors multimerization of the IN subunit [64D116E152 (known as the DDE motif) that plays an essential role in IN enzymatic activity; and the C-terminal domain (CTD) (aa: 213–288), which is involved in binding to viral and cellular DNA, and in protein oligomerization and interactions with the reverse transcriptase [HIV-1 integrase (IN), which comprises 288 amino acids encoded by the 5′-end of the HIV transfer ,7. IN cocriptase .INSTIs inhibit the HIV-1 integrase strand transfer steps to block the integration of HIV viral DNA into the host cell chromosomal DNA through competitive binding to the enzyme’s active site ,10. TherAlthough non-B subtypes dominate the global HIV epidemic, most clinical and virological studies on DTG were based on subtype B. However, subtype-dependent differences in naturally occurring polymorphisms (NOPs) have been implicated in the development of different mutational pathways, leading to varying levels of drug resistance against INSTIs among different HIV-1 subtypes ,32,33,34HIV-1 sequences and structure-based analyses also showed that subtype-specific NOPs, especially at the active site of IN, can affect the genetic barrier to drug resistance by influencing the selection of resistance mutations, native protein structure, and the function of the drug-mediated inhibition of the enzyme ,32,33,37In 2019, an estimated 669,236 people were living with HIV in Ethiopia, and the epidemic was dominated by subtype C ,39. SimiIn this study, we used plasma samples collected from HIV-1-infected patients as part of a national HIVDR survey conducted in Ethiopia. A cross-sectional survey was conducted in 2017 among treatment-naïve patients and patients on first- and second-line regimens in selected health facilities from different parts of the country according to the WHO-recommended HIVDR survey . After ohttps://sequenceqc-dev.bccfe.ca/who_qc (accessed on 28 June 2021)) and the Quality Control program of the Los Alamos HIV sequence database (https://www.hiv.lanl.gov (accessed on 28 June 2021)).Genotyping of the integrase region was performed using an in-house-developed and -validated protocol for IN . Amplicohttp://www.hiv.lanl.gov (accessed on 3 July 2021)). Multiple sequence alignment was conducted using MAFFT version 7 [The HIV-1 subtyping was performed using the online automated subtyping tools REGA v3.0 , COMET , and theersion 7  and was ersion 7 ,50 untilersion 7  with theersion 7 . Clusterhttps://hivdb.stanford.edu/hivdb/by-mutations (accessed on 7 July 2021)). INSTI DRMs were categorized as major resistance mutations, accessory resistance mutations, and other mutations according to the Stanford HIV Drug Resistance Database. Major resistance mutations were primarily nonpolymorphic DRMs that caused a significant reduction in INSTI susceptibility, even when they occurred alone. Accessory mutations were nonpolymorphic or minimally polymorphic mutations that caused only low-level reduction of INSTI susceptibility when they occurred alone, but may have augmented resistance and/or restored the fitness of viral mutants with major resistance mutations. The other mutations included highly polymorphic and/or rare nonpolymorphic mutations that may have been weakly associated (uncertain role) with drug resistance. We further extensively investigated all amino acid positions associated with decreased INSTI susceptibility. Samples harboring resistant and/or a mixture of wild-type and resistant amino acids were considered resistant.INSTI-associated mutations were identified using the Stanford HIV Drug Resistance Database (HIVdB v9.0) ). To avoid the overestimation of variant calling and ensure the sequences included in the analysis were from INSTI-naïve patients, only sequences before 2007 (before the FDA approved INSTIs) were used. The quality of all HIV-1 sequences was verified using the online Quality Control program (http://www.hiv.lanl.gov (accessed on 14 July 2021)). Sequences with stop codons and/or frameshifts and/or poor quality were removed from the analysis. Only one sequence per patient was retained. For a patient with multiple sequences, the earliest sequence was selected and used. The consensus amino acid sequence for IN was generated for Ethiopian HIV-1 subtype C, the global HIV-1 subtype B, and the global subtype C sequence using BioEdit V7.0.9.0 [To comprehensively describe the variability (polymorphism) in the IN sequences, we downloaded global subtype B and C IN sequences that matched the region (HXB2: 4230- 5093 relative to HXB2 clone) from the HIV Los Alamos National Library (LANL) database , NRTI, and/or NNRTIs (HIVDR group) with those with no major HIVDRMs (no-HIVDR groups) in their corresponding protease/reverse transcriptase (PR/RT) gene. To assess differences in the genetic barrier for evolution of drug-resistance substitutions between subtypes C and subtype B, we compared Ethiopian HIV-1 subtype C IN sequences obtained from INSTI-naïve patients and global HIV-1 subtype B sequences obtained from LANL . We calculated the genetic barrier to INSTI resistance for 10 major INSTI resistance amino acid positions (19 substitutions) using a previously published method . Brieflyhttps://www.genome.jp/tools-bin/clustalw (accessed on 1 November 2021)). An amino acid identity matrix was created with Clustal 12.1 ) and visualized using GraphPad Prism 8. For in silico predictions, 20 randomly selected (10 from each ART-naïve (PDR) and ART-experienced (ADR)) sequences were used. The ART-naïve IN sequences used in our analysis had no HIVDRMs against NRTI, NNRTI, and/or PI in their corresponding PR/RT gene, while the ART-experienced group had one or more HIVDRMs against NRTI, NNRTI, and/or PI. A multiple-sequence alignment of amino acid sequences (without any gap) was made using ClustalW (www.rcsb.org (accessed on 2 November 2021)) [The crystallographic structure of full-length HIV-1 IN (accession number: 6u8q.pdb) was obtained from the Protein Data Bank (r 2021)) . To visur 2021)) ,56. Ligap-values ≤ 0.05 were considered statistically significant. Fisher’s exact test, the Chi-squared test, and the Mann–Whitney U-test were used to evaluate the statistical differences between groups. n = 41) or second-line (n = 46) regimen. A total of 460 IN sequences obtained from INSTI-naïve patients were included in the analysis. Among these, 373 sequences were from patients who did not report exposure to any antiretroviral drug at the time of specimen collection (ART-naive), while 87 sequences were from ART-experienced (NNRT-based or PI-based regimens) patients, with virological failure  while on a first-line  of the sequences were subtype C, while 0.43% (2/460), 0.22% (1/460), 0.22% (1/460), 0.22% (1/460), 0.22% (1/460), and 0.22% (1/460) were subtype B, subtype A1, CRF10_CD, CRF02_AG, CRF49_cpx and CRF_A2D, respectively .n = 460) and (n = 414) integrase reference sequences for HIV-1 subtypes (A–K) and circulating recombinant forms downloaded from the HIV-1 LANL database. An ML tree was constructed using the online version of PhyML v 3.0. The reference sequences from the Los Alamos National Laboratory are in black in the figure. All the Ethiopian sequence’s clusters with the HIV-1 subtype C reference sequence are in green, while the non-subtype C Ethiopian sequences are in pink.The phylogenetic tree in No major DRMs known to be associated with DTG resistance  were detected among INSTI- naïve individuals, regardless of previous exposure to ART. However, one (0.22%) sequence from a person without previous ART exposure was found to harbor E92G, a mutation that moderately reduces EVG susceptibility but does not reduce susceptibility to RAL and DTG. p = 0.9)  of the sequences contained five different IN accessory mutations: −E157Q (2.39%), G163R/K (0.65%), Q95K (0.65%), T97A (0.43%), and G149A (0.22%). There was no significant difference in the prevalence of accessory mutations among ART-naïve and ART-experienced patients (p = 0.9) . Only onTo assess the impact of ART exposure to NRTI, NNRTI, and/or PI on the selection of INSTI-resistance mutations, we further compared the INSTI HIVDRMs from patients with one or more major HIVDR mutations to NRTI, NNRTI, and/or PI (HIVDR group) with those with no HIVDRMs in their corresponding PR/RT genes (no-HIVDR group) .p = 0.8); while 4.29% (15/373) and 5.75% (5/87) accessory mutations were detected among ART-naïve and ART-experienced groups, respectively (p = 0.6).Briefly, among the total 460 IN sequences used in our analysis, 327 had a corresponding PR/RT gene sequence, of which 234 had no major HIVDRMs (no-HIVDR group), while 93 of the sequences (HIVDR group) had one or more HIVDRMs against the NRTI, NNRTI, and/or PI see . No majoHigh similarity was also observed when comparing the consensus sequence from ART-naïve and ART-experienced patients, as shown in An alignment of the 453 HIV-1 subtype C IN sequences from the INSTI-naïve Ethiopian patients was extensively analyzed and compared to the HIV-1 subtype B reference sequence (GenBank accession number: K03455). Based on our definition of polymorphism (≥1.0% variability), an overall 64.9% (187/288) amino acid positions of the IN were conserved. The conservations of the NTD, CCD, and CTD were 60% (30/60), 66.1% (107/162), and 66.8% (50/76), respectively. The distribution of polymorphisms in 453 HIV-1 subtype C IN sequences is shown in 12H16C40C43) involved in the multimerization of the IN subunit, stabilization of folding, and interaction with LEDGF/p75 were highly conserved [Within the NTD, the Zn-binding motif H12H1C40C43 in64D116E152 was highly conserved, and was found within the conserved regions 61–70, 114–118, and 152–155, respectively. The critical positions for DNA-binding HIV-1 integration and replication  [186, R187, K188), which is vital for the integrase:integrase:oligomerization at the dimer:dimer interface [In the CCD, the catalytic triad Dnd N155)  and the nd N155) , and thenterface , showed Among the INSTI-mutation positions in the CCD residues that directly reduced the INTSI susceptibility, H51, T66, E92, F121, G140, Y143, Q146, S147, Q148, S153, N155, and E157Q were highly conserved, except for codon position E157Q, which was a polymorphic position (>1.0% variability). However, a highly polymorphic residue in the CCD including V72I, I84M, F100Y, L101I, T112V, T124A, T125A, R127K, K136Q, D167E, K188R, and V201I was observed. Within CTD, the two large consecutive residues, L241-Q252 and I257-K264, which are involved in the binding of viral and cellular DNA, were found to be highly conserved, except for positions I251 and V257, which were mutated to I251L and V259I in 3.5% and 0.7% of the sequences, respectively. However, the important positions for DNA binding and integrase multimerization  [Our analysis also showed that 24 amino acid positions were highly polymorphic (>20.0% variability): D10E, K14R, S24N, D25E, V31I, M50I, V72I, I84M, F100Y, L101I, T112V, T124A, T125A, R127K, K136Q, D167E, K188R, V201I, K215N, T218I, A265V, R269K, D278A, and S283G. Six of these  belonged to the NTD, whereas 12  belonged to the CCD, and the other 6  belonged to the CTD. Our comparison of the NOPs’ distribution with the global subtype B and global subtype C sequences downloaded from LANL showed that the Ethiopian HIV-1 subtype C IN sequences had a high similarity to the global subtype C sequence, but were quite different from the global subtype B, as shown in The consensus IN sequence for the global HIV-1 subtype B and global subtype C were generated using 1884 and 1410 sequences, respectively. Our comparison of the 288 amino acid sequence alignment of the consensus Ethiopian HIV-1 subtype C with the global HIV-1 subtype C showed high similarity, except for the mixture of amino acid sequences at positions 25E/D, 100Y/F,124T/A, 136K/Q, 167E/D, 215K/N, and 218I/L in the Ethiopian consensus; and 50M/I, 72I/V, and 265A/V in the global HIV-1 subtype C consensus sequence. However, it differed from the global subtype B consensus at eight positions with complete amino acid replacement , while a mixture of amino acids was detected at positions 11E/D, 72I/V, and 101I/L in the global subtype B consensus sequences and at 24N/S, 25E/D, 100Y/F, 124T/A, 136K/Q, 167E/D, 215K/N, and 269K/R in the Ethiopian subtype C consensus sequence .In this study,19 substitutions conferring major resistance to DTG at 10 amino acid positions in the IN  were assessed to explore the genetic barrier to DTG. For each codon, the number of transitions and/or transversions required for a IN drug resistance associated substitution were calculated. A total of 1884 global HIV-1 subtype B sequences and 453 Ethiopian subtype C sequences from INSTI-naïve patients were compared for differences in the genetic barrier to INSTI resistance .Overall, the sequence analysis of the two subtypes showed similar predominant codon use at the selected amino acid positions, resulting in a similar minimum score for the genetic barrier to DTG. However, at position 140, the predominant codons in subtype C were GGG (53.6%) and GGA (45.9%). In contrast, in subtype B, GGC (85.0%) was the predominant codon resulting in a difference in the calculated genetic barrier at this position. For subtype C, two transversions (minimum score of 5) were required to mutate to G140C (GGG/A to ATG/C); while for subtype B, one transversion and transition (minimum score: 3.5) were required to mutate to G140C (GGC to TGT). Similarly, a two-point mutation (one transversion and one transition) (minimum score of 3.5) was required to mutate to G140S (GGG/A to AGT/C) for subtype C; while subtype B required a one-step transition (minimum score of 1) (GGC to AGC). The effects of HIVDRMs in HIV-1 PR and/or RT on the secondary structure of HIV-1 IN were investigated on 20 sequences: 10 from ART-naïve (PDR) and 10 from ART-experienced (ADR) individuals representative of randomly selected HIV-1 IN sequences. The sequence identity matrix a showed Overall, our results revealed no major DTG associated HIVDRM mutations among INSTI-naïve individuals, regardless of previous exposure to ART. In one individual, the E92EG mutation was found, which moderately reduced EVG susceptibility, but had no effect on DTG. However, INSTI accessory mutations and NOPs, which could influence INSTI susceptibility and the genetic barrier to INSTI resistance, were detected. Our polymorphism analysis showed that 64.9% (187/288) of amino acid positions of the HIV-1 subtype C IN sequences from INSTI-naïve individuals were conserved (<1.0% variability). The majority of amino acids involved in key functions of the enzyme (the HHCC motif and the DDE motifs ) were fuThe absence of major INSTI DRMs among INSTI-naïve patients in our study was consistent with other studies from Africa ,62,63,64When present alone, accessory mutations have a minimal effect on INSTI susceptibility, but may serve to augment resistance and/or restore the fitness of viral mutants with major resistance mutations ,30. INSTE157Q was the most common nonpolymorphic accessory mutation detected in our analysis. It is a natural polymorphism present in 1–10% of untreated individuals, depending on the subtype. It has no effect on the susceptibility of INSTI. However, it may act as a compensatory substitution for R263K-induced resistance to DTG . Q95K waL74M/I (2.9%) and M50I (18.8%) were the other polymorphic mutations detected in our study. L74M/I has been reported at levels between 0.5–20% in the untreated population, with a high prevalence in subtypes A, G, and A/G recombinants. It does not decrease INSTI susceptibility alone, but it can contribute to a high-level resistance when occurring with major INSTI-resistance mutations, mainly the Q148H/K/R mutation ,58,78,79The other nonpolymorphic and polymorphic accessory mutations detected were G163R and T97A, which can contribute to a high-level resistance when occurring with Y143 and N155H major INSTI-resistance mutations . In this study, we characterized the distribution of amino acid variants among the 453 HIV-1 subtype C IN sequences from INSTI-naïve individuals. Our results revealed that 64.9% of HIV-1 IN amino acid positions were conserved (<1.0% variability). The conserved position in the NTD, CCD, and CTD were 60%, 66.0%, and 65.8%, respectively. This was comparable to the study by Rhee et al. (2008) that showed 70% (202/288) of IN amino acid positions of the 1500 sequences obtained from INSTI-naïve (ART-naive or ART-experienced) individuals with different subtypes (<1.0% variability) . In general, our results showed that the majority of amino acids involved in key functions of the enzyme, including the zinc-binding HHCC motif, the multimerization of IN subunits, and the binding with the human cellular factor LEDGF/p75 in the catalytic core domain, the catalytic triad DDE ,22 was hIt was also interesting to note that 20.5% 93/453) of our study participants were found to harbor a major HIVDR mutation (transmitted and acquired HIVDR) for NRTI, NNRTI, and/or PI in their corresponding PR/RT gene. However, DRM directed toward sites other than IN did not have a significant effect on INSTI susceptibility. In line with our findings, different studies have shown that previous NRTIs mutations appeared to have no impact on the risk of virological failure in patients switched to DTG with NNRTIs ,84,85,863/453 of p = 0.5 and p = 0.4)), while an increased prevalence of V165I was observed among ART-experienced groups ). Furthermore, our comparison of the HIVDR and no-HIVDR groups showed no differences .However, in our study, no significant difference was found in I84V and M154I prevalence between the ART-naïve and ART-experienced patients  required for the virus to escape drug-selective pressure . It is aOverall, our analysis of the codon distribution of the selected amino acid position of HIV-1 subtype C and subtype B revealed a similar genetic barrier for the development of DTG resistance between subtype C and B, except at codon position 140, where subtype C had a higher genetic barrier to develop the G140C and G140S mutations compared to subtype B, highlighting a higher genetic barrier for the Q148H/R/K resistance pathway in subtype C. The G140S mutation has been shown to rescue the catalytic defect due to the Q148H mutation, enabling the recovery of viral fitness . A similThis study was comprehensive, and included both treatment-naïve and treatment-experienced (first- and second-line regimens) patients, and will be a benchmark for INSTI DRM monitoring in Ethiopia. However, our analysis was based on the Sanger dideoxy sequencing method, which does not detect drug-resistance minority variants below 20% of the virus population, and might have underestimated the prevalence of INSTI DRMs among our study participants .Our results showed no major clinically relevant INSTI-associated mutations among INSTI-naïve patients regardless of exposure to other antiretroviral agents, supporting the implementation of the wide scale-up of DTG-based regimes in Ethiopia. However, the detection of polymorphisms contributing to INSTI resistance and the expected increased use of DTG-based regimens in Ethiopia warrant the need for continuous surveillance of INSTI resistance. The genetic barrier analysis showed that subtype C had a high genetic barrier to acquiring the G140C and G140S mutations, highlighting that the Q148H/K/R mutation DTG resistance pathway was selected less in subtype C. Moreover, the docking analysis of the dolutegravir showed that protease- and reverse-transcriptase-associated HIVDRMs did not affect the native structure of the HIV-1 integrase.


SARS-CoV-2 coronavirus is a recently identified novel coronavirus that is the causative agent of the COVID-19 pandemic that began in 2020. An intense research effort has been undertaken by the research community in order to better understand the molecular etiology of this virus and its mechanisms of host cell subjugation and immune system evasion. To facilitate further research into the SARS-CoV-2 coronavirus we have generated adenovirus 5-based viral vectors that express SARS-CoV-2 proteins—S, N, E, NSP7, NSP8, and NSP12 as hemagglutinin (HA)-tagged and untagged variants. We have also engineered two additional viruses that express the S protein receptor binding domain and a fusion of the receptor binding domain to the N protein. We show that these vectors are expressed in several different cell lines by Western blotting and real-time quantitative reverse transcriptase (qRT-PCR), we evaluate the subcellular localization of these viral proteins, and we show that these coronavirus proteins bind to a variety of cellular targets. The flexibility of adenovirus vectors allows them to be used in a variety of cell models and, importantly, in animal models as well.IMPORTANCE The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus has brought untold personal and economic suffering to the world. Intense research has made tremendous progress in understanding how this virus works, yet much research remains to be done as new variants and continued evolution of the virus keep shifting the rules of engagement on the pandemic battlefield. Therefore, wide availability of resources and reagents to study SARS-CoV-2 is essential in overcoming the pandemic and for the prevention of future outbreaks. Our viral vectors provide additional tools for researchers to use in order to better understand the molecular biology of virus-host interactions and other aspects of SARS-CoV-2. Coronaviridae family within the order Nidovirales, initially identified in the 1930s as the causative agent of bronchitis in chickens, pig gastroenteritis, and hepatitis and neurological disease in mice  was initially identified as the causative agent of cell degeneration during attempts to culture epithelial-like cells from adenoid tissue surgically removed from children . HAdVs oIn the present study, we report on the design, construction, and validation of multiple HAdV5-based viral vectors that express several different SARS-CoV-2 proteins. We decided to make vectors expressing proteins that would be of the most interest from a clinical perspective, including those that are good antigen candidates  or those that can be targeted for drug development (RdRP). We show that these proteins can be immunoprecipitated from infected cells, and we investigate their subcellular localization. These vectors were designed to be freely available to anyone interested in using them for research purposes in animal and cell models.in vivo recombine a transgene of interest into the viral backbone (To design recombinant HAdVs (rHAdVs) we used the method of Hardy et al.  that utibackbone . A previbackbone , 13 isolbackbone  that havExpression testing of recombinant viruses was performed in either 293 or A549 cells . The 293To test the expression of nontagged constructs, qRT-PCR was used after RNA extraction from infected 293 cells . QuantifWe wanted to determine whether the SARS-CoV-2 proteins expressed from rHAdV were able to interact with potential cellular proteins. SARS-CoV-2 proteins were immunoprecipitated from 293 cells infected with rHAdV using anti-HA affinity resin and resolved on SDS-PAGE . We usedWe wanted to determine the subcellular localization of the various SARS-CoV-2 proteins expressed from our rHAdV vectors. To do this, HT1080 cells were infected and stained for the various viral proteins using the anti-HA rat antibody 3F10 , with thin vitro cell culture-based model, but also as tools for animal studies due to the broad range of species that adenoviruses are able to transduce.In the present study, we report the design, construction, and characterization of rHAdV vectors expressing several different SARS-CoV-2 proteins. We show that all of our viruses express SARS-CoV-2 proteins tagged with an HA-tag, and untagged variants express SARS-CoV-2 mRNA. We further demonstrate the subcellular localization of these proteins, and we show that they interact with a variety of unknown cellular proteins. Together, these viruses represent valuable tools for researchers aiming at understanding SARS-CoV-2 and offer the unique ability of using these not only in an The impact of the COVID-19 pandemic on the world has been tremendous, affecting all aspects of society and our way of life . This coWe have designed our rHAdV to express either HA-tagged or untagged versions of SARS-CoV-2 proteins, as antibodies, particularly to the NSPs, are not readily available or are of various quality. Therefore, having consistent antigen detection may be important in certain circumstances. Ultimately, having the choice of using either HA-tagged or untagged versions of our viruses provides added flexibility in an assay-dependent manner. Interestingly, recombination of HA-tagged S construct was unsuccessful despite repeated attempts, while untagged S recombined easily. It is unclear why that was the case. It is possible that the combination of the sequences of HA-tag and S gene somehow interfered with the Cre recombinase, or it may be that the HA-tagged S protein itself was somehow toxic and killed recombined cells before efficient virus production was achieved. Ultimately, we felt it was more important to have S in any form, as there is a wide variety of anti-S antibodies, and not having an HA-tagged variant is of limited consequence overall.Expression of all NSPs and other structural proteins was easily detectable by a Western blot for the HA-tag  using thAs a proof-of-principle, we performed immunoprecipitation of the HA-tagged SARS-CoV-2 proteins to determine whether they interacted with cellular targets . Most ofIn conclusion, we have engineered a number of viral vectors based on HAdV5 that individually express a number of SARS-CoV-2 proteins. Characterization of these vectors shows that they express efficiently and provide new tools in the race to understand the virus. Importantly, the flexibility of adenovirus-based vectors enables these to be used in a variety of cell types and animals. These vectors are freely available to anyone interested in their use for their research.Rat monoclonal anti-HA 3F10 (MilliporeSigma) was used at a dilution of 1:3,500 for Western blotting. Mouse monoclonal anti-S (MilliporeSigma) was used at a dilution of 1:2,000 for Western blotting. For immunofluorescence, 3F10 rat monoclonal anti-HA was used at a dilution of 1:100, and 2B3E5 was used at a dilution of 1:67. Secondary goat anti-rat and goat anti-mouse horseradish peroxidase (HRP)-conjugated antibodies were purchased from Jackson Immunoresearch and were used at a dilution of 1:100,000 for Western blotting, and Alexa Fluor 594 secondary antibody was purchased from Thermo Fisher Scientific and used at a dilution of 1:600 for immunofluorescence.2. Cotransfections for viral vector recombination were carried out in 10-cm plates of 293Cre9 cells. Complete medium was changed on 293Cre9 plates, and then a transfection mixture containing 1 mL of serum free DMEM, 7 μg of extracted Ψ5 viral DNA, 3 μg of SfiI (New England Biolabs) digested pAdLoxP expression plasmid, and 20 μl of polyethylenimine (PEI) were added. Viruses were allowed to recombine until all cells were dead. All virus infections were carried out in serum-free medium for 1 h, after which, complete medium was added without removal of the infection medium. For all infections, virus titers were determined using viral genome quantification via qPCR, and crude freeze-thawed lysates were used. All recombinant viral vectors were purified by picking plaques from plaque assays performed on 293 cells.293Cre9 cells, 293 cells (ATCC CRL-1573), human epithelial lung carcinoma A549 cells (ATCC CRM-CCL-185), and human connective tissue fibrosarcoma HT1080 cells (ATCC CCL-121) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone) and supplemented with either 10% (293Cre9 and 293) or 5% (A549 and HT-1080) fetal bovine serum (VWR Seradigm) and streptomycin and penicillin (HyClone). Cells were incubated at 37°C under 5% COAll genes were amplified using the specific cloning primers described below, using a Phusion PCR kit (New England Biolabs), according to the manufacturer’s specifications. Genes were then cloned into either shuttle vector pAdLoxP and pAdLoxP-WPRE or the expression vector pCAN-HA using standard techniques. pCAN-HA constructs were then subcloned into the pAdLoxP- and pAdLoxP-modified shuttle vectors. Each shuttle vector was then digested using the restriction enzyme SfiI and ethanol extracted, in preparation for cotransfection with Ψ5 viral DNA as previously described.All primers were purchased from Integrated DNA Technologies, and an annealing temperature of 60°C was used.ACTGTACTCGAGACCATGAGCAAGATGAGCGACGTAAAAT and ACTGTATCTAGATTACTGCAATGTCGCCCGGTTGT; NSP8 primers, ACTGTAGGATCCACCATGGCAATCGCATCTGAATTTTCT and ACTGTAGGATCCTTACTGCAGTTTGACTGCGCTGTTAG; NSP12 primers, ACTGTAGGATCCACCATGTCAGCAGACGCACAAAGTTTTC and ACTGTAGGATCCTTACTGCAGGACGGTGTGAGGCG; E primers, ACTGTAGGATCCACCATGTACAGCTTCGTATCAGAAGAAACC and ACTGTAGGATCCTTAAACGAGGAGATCCGGCACCC; N primers, ACTGTAGGATCCACCATGAGCGATAACGGCCCCCA and ACTGTAGGATCCTTACGCCTGAGTAGAATCGGCTGAG; S-RBD primers, untagged, ACTGCCAAGCTTACCATGGTACAACCTACGGAATCTATCGTACGC and with HA-tag, ACTGCCAAGCTTACCATGGGCTACCCCTACGACGTGCCCGACTACGCCGTACAACCTACGGAATCTATCGTACGC and ACTGCCGGATCCTCCAAATGAGCATGGAGTTATATCG; S primers, ACTGTAGGATCCACCATGTTTGTTTTCTTGGTTCTTTTGCCAC and ACTGTAGGATCCTTACGTGTAGTGCAATTTTACGCCTTTA. The real-time gene expression PCR primers were the following: NSP7 primers, TGAGCAAGATGAGCGACGTA and CATTGACAGGAGAACGCTCA; NSP8 primers, CCGCTCAGAGGATAAACGAG and GAACGTCGTACCATCACACG; NSP12 primers, TGTCAATTTGCATTCCTCCA and TACCGGGTTTTACGGTTTGA; E primers, CCGGGACACTGATCGTAAAT and GGCACCCTGCTAGAATTGAG; N primers, GCATCAGAGGTGGAGATGGT and ACGATTGCGGCATTATTAGC; S-RBD primers, ACTGCCAAGCTTACCGTACAACCTACGGAATCTATCGTACGC and ACTGCCGGATCCTCCAAATGAGCATGGAGTTATATCG; S primer, AACTGAATCGGGCACTTACG CCCAAGACAATCGCCATACT; GAPDH primers, GAGTCAACGGATTTGGTCGT and TTGATTTTGGAGGGATCTCG.The cloning PCR primers were the following: NSP7 primers, N-morpholino)ethanesulfonic acid] buffer (Thermo Scientific). Proteins were subsequently transferred to a polyvinylidene difluoride (PVDF) membrane using Genescript’s eBLOT L1 with default settings, and the membranes were blotted using the previously described antibodies. Detection was performed using secondary HRP-conjugated antibodies and exposure to radiographic film.The 293 and A549 cells were plated, allowed to grow until confluent, and then infected with each viral vector at the indicated specific MOI. Cells were subsequently harvested at the indicated time points, and protein was extracted using NP-40 lysis buffer. Cell lysate was resolved on SDS-PAGE using Novex BOLT 4 to 12% (Thermo Scientific) gradient gel using MES [2-. Samples were then washed and run on a 10% SDS-PAGE hand cast gel at 30 V for 16 h and then finished at 200 V for 2 h in a Bio-Rad Protean II cell. The protein was visualized via silver staining as per the manufacturer’s protocol (Invitrogen SilverQuest silver-staining kit) according to the manufacturer’s protocol.Cell staining was performed as previously described . Briefly


Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain  Streptomyces, the largest genus among the actinomycetes, and more than 70% of them relate to the biosynthesis and late-stage tailoring of secondary metabolites  form a superfamily of heme-containing proteins that catalyze the oxidative modification of different organic substrates , unless otherwise indicated. DNA oligonucleotides were synthesized by Eurofins Genomics . The DNA polymerase Prime STAR GXL  and KOD FX Neo  were used for high-fidelity DNA amplification and inverse polymerase chain reaction (PCR). The Diversify PCR Random Mutagenesis kit  was used to perform error-prone PCR. All DNA restriction enzymes were obtained from Takara Bio . M-I, M-II, M-IV, and M-V were isolated and purified from the fermentation broth of i et al. .E. coli strains were cultivated in Luria Bertani (LB) medium . NZCYM medium  was used for whole-cell assays performed with E. coli cells. M. griseorubida strains were cultured in MR0.1S medium  was used for the fermentation of M. griseorubida strains.The strains used in this study are listed in mycG gene was amplified by PCR using primers mycG_NdeI-F/mycG_SpeI-R and total genomic DNA of M. griseorubida A11725 as a template. The PCR product was cloned into the pDrive cloning vector . After confirming the sequences, the 1.2-kb NdeI-SpeI DNA fragment containing mycG was inserted into the P450 expression vector pCYPcamAB carrying the putidaredoxin reductase gene camA and the putidaredoxin gene camB derived from Pseudomonas putida  . Site-directed mutagenesis into the mycG region of pMCGcamAB was performed by inverse PCR using the primer pairs listed in The plasmids and PCR primers used in this study are listed in ycG_RM-R . This PCM. griseorubida were also constructed by inverse PCR using the previously prepared pMG520 as a template  transformants carrying a plasmid expressing the MycG mutant with CamA and CamB were cultivated at 32°C for 20 hr in LB medium containing ampicillin (50 μg/ml) and chloramphenicol (25 μg/ml). The seed culture (250 μl) was transferred into 25 ml of NZCYM medium supplemented with ampicillin (50 μg/ml) and chloramphenicol (25 μg/ml), and cultivated at 37°C until the OD600 reached 0.6–0.8. Later, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 80 mg/ml 5-aminolevulinic acid were added, and the cultivation was continued at 22°C for 20 hr. The cells were harvested by centrifugation, washed with CV buffer  and resuspended in CV buffer at 0.05 g/ml (wet weight). Later, 500-μl cell suspension and 0.1 mM of M-II, M-IV, and M-V were mixed and incubated at 27°C for 20 hr with vigorous shaking. The pH of the reaction mixture was adjusted to 9 using 28% ammonia solution, and mycinamicins were extracted using 2 × 500 μl ethyl acetate. The resulting organic extract was dried and dissolved in 50-μl methanol for high-performance liquid chromatography (HPLC) analysis. HPLC analysis was performed using a Hitachi Chromaster HPLC system  comprising of a 5110 pump and a 5430 diode array detector under the following conditions: column, ODS-80TM ; mobile phase, acetonitrile/0.06% trifluoroacetic acid (30:70); flow rate, 0.8 ml/min; UV wavelength, 220 nm and 280 nm. M-I, M-II, M-IV, and M-V were quantified based on the peak areas from HPLC using peak areas derived from standard solutions. As the retention times of M-II and M-V were almost the same and the peaks overlapped, their concentrations were calculated as follows: the absorption maximum for M-II occurred at 220 nm and that of M-V was at 220 nm and 280 nm. At same concentration, M-II and M-V have equal peak areas at 220 nm. Therefore, the concentration of M-II was calculated by subtracting the concentration of M-V quantified at 280 nm from the total concentration of M-II and M-V quantified at 220 nm. All reactions were performed and analyzed in duplicates.E. coli cells suspended in CV buffer at 0.05 g/ml (wet weight) during whole-cell assays were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cell suspension was sonicated using Ultrasonic Disruptor UD-201 . The insoluble material was removed by centrifugation at 25,000 × g for 10 min. The protein concentration in the soluble fraction was measured using the Quick Start Bradford Protein Assay   and spread on MR0.1S agar plates. The plates were incubated at 27°C for 20 hr and then overlaid with 1-ml water containing nalidixic acid (0.5 mg) and apramycin (1 mg). The plates were subsequently incubated at 27°C for 2–3 weeks until the exconjugants were observed. The introduction of genes encoding MycG mutants into the resulting exconjugants was confirmed by PCR and sequence analysis.pMG522, pMG523, pMG524, pMG525, and pMG526 were introduced into the previously prepared M. griseorubida, the culture extracts were analyzed by HPLC. Each M. griseorubida strain was cultured in 5 ml of MR0.1 broth at 27°C on a rotary shaker at 220 rpm for 4 days. The seed culture (400 μl) was inoculated in a 500-ml Erlenmeyer flask containing 40 ml of FMM medium, and incubated at 27°C on a rotary shaker at 140 rpm for 4–14 days. One milliliter of the broth was adjusted to pH 9.0 using 28% ammonia solution and extracted with an equal volume of ethyl acetate. The organic layer was mixed with an equal volume of water at pH 2.0 using hydrochloric acid. Next, the pH of the water layer containing mycinamicins was adjusted to 9.0 using 28% ammonia solution, followed by extraction with an equal volume of ethyl acetate. After concentrating the organic solution to dryness in vacuo, the extracts were dissolved in 20 μl of methanol for HPLC analysis. HPLC analysis was performed under the same conditions as the whole-cell assays using E. coli.To compare the levels of mycinamicins produced by each strain of M. griseorubida A11725 strain. To stimulate the efficient production of intermediate products by multifunctional P450, MycG mutants that preferentially catalyze monohydroxylation were screened from a library of random mycG mutations. Screening was performed using previously reported E. coli whole-cell assays utilizing a P450 expression system (Iizaka et al., Pseudomonas putida as surrogate redox partners. The library of E. coli expressing MycG random mutants was constructed using the modified MEGAWHOP method (Miyazaki, Multifunctional P450 MycG catalyzes sequential C-14 allylic hydroxylation and C-12/13 epoxidation to generate M-II from M-IV. The M-V intermediate is generated from initial C-14 hydroxylation by MycG resulting in low-level accumulation in the mycinamicin-producing E. coli expressing MycG mutants with single or double amino acid substitutions derived from RM92, RM96, and RM98 were prepared and subjected to whole-cell assays using individual mycinamicins as substrates (Fig. tes Fig.  and S4. tes Fig. . All thrWhole-cell assays using M-V as a substrate were performed to evaluate the catalytic activity of the R111Q/V358L, W44R, and V135G/E355K mutants for conversion to M-II and display reduced catalytic activity for the terminal epoxidation step (Fig. E. coli by SDS-PAGE and CO-bound reduced difference spectra (E. coli cells, except for the negative control carrying the P450 expression vector pCYPcamAB. As no difference was observed in the signal intensity, the expression level of each mutant was essentially equivalent compared to wild-type strain. However, the concentrations for R111Q/V358L, W44R, and V135G/E355K mutants based on the CO-bound reduced difference spectra were lower than 1/10 of the MycG concentration detected for the wild-type strain. These analyses suggest that R111Q/V358L, W44R, and V135G/E355K mutants induced almost no spectral change owing to CO binding and may not carry correctly folded active forms of P450s. Despite the fact that these mutants catalyze monohydroxylation to generate M-V from M-IV, it remains unclear why the CO-bound reduced difference spectra were not observed.To evaluate the associations between alterations in catalytic activity and expression levels in whole-cell assays, we confirmed the expression of wild-type MycG and its mutants in  spectra . The 44-M. griseorubida A11725 predominantly produces M-II owing to the sequential MycG-mediated hydroxylation and epoxidation of M-IV via M-V. The wild-type accumulation of M-V, an intermediate within multiple reactions catalyzed by MycG, is very low. Whole-cell assays revealed that MycG mutants R111Q/V358L, W44R, and V135G/E355K selectively generate M-V from M-IV. We then examined whether it is possible to efficiently produce M-V by in vivo reconstitution of these mutants in the mycinamicin biosynthetic pathway. Furthermore, MycG mutants, V135G and E355K, which showed a reduced tendency for direct epoxidation of M-IV to M-I, were also investigated in M. griseorubida.mycG-disruption strain M. griseorubida TPMA0025 and the mycG complementation strain M. griseorubida TPMA0047 were prepared as previously described (Anzai et al., mycG gene in strain TPMA0025 was replaced with the disruption cassette FRT-oriT-neo-FRT-attB. TPMA0047 is a strain in which mycG is inserted into the chromosome of TPMA0025 by recombination between the attP and attB sites by the site-specific integration vector pSET152. We obtained M. griseorubida TPMA0073, TPMA0074, TPMA0075, TPMA0076, and TPMA0077 with the genes encoding the MycG mutants R111Q/V358L, W44R, V135G/E355K, V135G, and E355K, respectively (mycG region of the plasmid used to obtain TPMA0047, followed by the integration of the plasmid into TPMA0025.The ectively . These sM. griseorubida A11725 and TPMA0047. The amount of M-V produced by TPMA0073 was almost the same as that produced by TPMA0047. The highest M-V productivity was demonstrated by TPMA0075, which was consistent with the data from whole-cell assays, wherein M-IV was reacted with E. coli expressing the MycG mutant V135G/E355K (Fig. M. griseorubida.To compare the production profiles of mycinamicins by strains reconstituted with the MycG mutants, culture extracts after 10 days of incubation were analyzed by HPLC Fig. ; Table 255K Fig. . Further55K Fig. . TogetheAs for TPMA0075, which showed the best enhancing M-V productivity, the production of M-I, M-II, M-IV, and M-V was compared to the wild-type strain A11725 for 4–14 days to confirm the change to production profiles of mycinamicins toward culture period . The proThe current study reports the identification of amino acid residues in MycG that result in altered catalytic activity and selectivity reflected by product profile changes. Substrate recognition by MycG involves three distinct regions, the hydrophobic pocket that binds the mycinose sugar of M-IV, the active site, and the FG/BC loop that form a salt–bridge interaction with the desosamine sugar of M-IV (Yang et al., in vivo reconstitution in M. griseorubida. The other MycG mutants V135G and E355K display enhanced sequential hydroxylation and epoxidation to generate M-II with corresponding reduced direct epoxidation of M-IV to M-I compared to the wild-type. The five amino acid substitutions derived from these mutants occurred at sites that were structurally different from the above-mentioned three regions involved in substrate recognition by MycG (Table E. coli whole-cell assays and in vivo studies using M. griseorubida. This variant outcome could be due to alternative redox partners in the two expression systems. Previous studies have revealed that the functional characteristics of MycG can vary depending on the type of the redox partner (Zhang et al., M. griseorubida. Interestingly, although the single mutations of V135G and E355K preferred the two-step oxidation pathway of hydroxylation and epoxidation over direct epoxidation of M-IV, the corresponding double mutations result in selective monohydroxylation to generate M-V. The mutation sites of W44R, V135G, and E355K were also different from the previously reported substrate recognition of MycG (Li et al., Bacillus megaterium makes the substrate binding domain (Ravichandran et al., E. coli; however, the active P450 concentration according to the CO-bound reduced difference spectra was significantly reduced.We obtained two types of MycG mutants with altered product profiles using random mutagenesis and subsequent mutational analysis. MycG mutants R111Q/V358L, W44R, and V135G/E355K selectively catalyze monohydroxylation to form M-V from M-IV. The V135G/E355K mutant is especially effective in producing M-V following in vivo genetics is expected to be useful in the field of natural product biosynthesis. Although the multistep reactions by MycG have not been completely controlled, the detailed functional analysis of MycG mutants obtained in this study will lead to further elucidation of the mechanism underlying the multifunctionality exhibited by MycG.Over the past decade, the reactions of several multifunctional P450s derived from bacterial secondary metabolite pathways have been controlled or modified. Bifunctional GfsF catalyzes C-8/9 epoxidation followed by C-10 hydroxylation in FD-891 antibiotic biosynthesis (Kudo et al., kuab069_Supplemental_FileClick here for additional data file.