In recent years, H2S has been found to have a variety of important biological functions, and plays an important role in a variety of physiological and pathological processes. In this review, we review the recent role and mechanism of H2S in regulating autophagy in liver disorders, in order to provide a basis for further research in the future.Autophagy is a complex process of degradation of senescent or dysfunctional organelles in cells. Dysfunctional autophagy is associated with many diseases such as cancers, immune dysfunction, and aging. Hydrogen sulfide (H Autophagy is a stable mechanism in eukaryotic cells. In this process, the abnormal proteins, pathogens, and organelles are wrapped by double membranes to form autophagosomes and then transferred to lysosomes for subsequent degradation ,2,3,4. I2S) has been regarded as a toxic gas. However, since the 1990s, more and more studies have demonstrated that H2S, together with NO and CO, is gas transmission signal molecules [2S: cystine sulfide β-synthase (CBS), cystine sulfide γ-lyase (CSE), and 3-mercaptopyruvate sulfur transferase (3-MST). CBS catalyzes the β-substitution reaction of homocysteine with serine to produce L-cystathionine. The elimination of α, γ-cysteine of L-cystathionine is catalyzed by CSE to produce L-cystenine. On the one hand, L-cystine can be catalyzed by CSE/CBS through β elimination reaction to produce H2S; on the other hand, it can also be catalyzed by cysteine aminotransferase (CAT) to transfer amine to α- ketoglutarate to form 3-mercaptopyruvate (3-MP). 3-MP is catalyzed by 3-MST to convert into H2S [2S has been reported to contribute to many physiological processes, such as blood pressure reduction [2S [2S has been reported to act on autophagy through autophagy-related genes  [2S regulates autophagy in many physiological and pathological processes. In this review, we summarize the role and mechanism of H2S regulating autophagy in liver disorders, hoping to provide theoretical reference for further related research in the future.For decades, hydrogen sulfide . It has 2S level in serum of patients with HTG and HTG model of C57BL/6 mice fed by high-fat diet (HFD) decreased. Sodium hydrosulfide  could notably decrease the level of serum TG, liver weight, and liver free fatty acids (FFA) of HTG model of mice, which was counteracted by chloroquine . Further research showed that NaHS promoted autophagy by upregulating LC3BII/LC3BI ratio and downregulating p62 protein level in HFD-fed mice. Electron microscopy imaging revealed that NaHS treatment decreased the number of autophagosomes and lipid droplets, and some lipid droplets were obviously swallowed by autophagosomes in hepatocytes of HFD-fed mice, suggesting that autophagy might reduce lipid droplets in hepatocytes. Additionally, CQ abolished NaHS promotion of autophagy. It could be seen that exogenous H2S decreased serum TG level to improve NAFLD through promotion of autophagy. Furthermore, the p-AMPK level significantly decreased in liver of HFD-fed mice, which was abolished by NaHS treatment. AMPK siRNA in L02 cells abolished NaHS promotion of autophagy; moreover, similar results were obtained in AMPK2−/−mice, indicating that exogenous H2S induced autophagy by activating AMPK/mTOR pathway. Summarizing, exogenous H2S reduced serum TG level to improve NAFLD through autophagy promotion by activating AMPK/mTOR pathway [2S promoted lipophagy to reduce plasma triglyceride levels [2S donor) to treat primary mouse hepatocytes stimulated by oleic acid. The results showed that H2S promoted lipid autophagy [2S inhibited NLRP3-mediated inflammation by upregulating autophagy through activating the AMPK/mTOR pathway in L02 cells [2S in autophagy in liver. Another study of Dongdong Wu et al. showed that H2S improved HFD-induced NAFLD by inhibiting apoptosis and promoting autophagy. In vitro experiments showed that H2S suppressed apoptosis and promoted autophagy by inhibiting reactive oxygen species (ROS)-mediated phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) cascade in L02 cells induced by OA [Nonalcoholic fatty liver disease (NAFLD), affecting 25% of the adult population, is currently considered to be the most common liver disease in the world ,34. It i pathway . Previou pathway . Lipopha pathway . The aboe levels . Similarutophagy , which f02 cells ,48, whiced by OA .2S level and the subsequent sulfhydration-dependent activation of Unc-51-like autophagy-activating kinase 1 (ULK1). Moreover, ULK1 Cys951 Ser sulfhydration by H2S promoted ULK1 kinase activity and autophagy. Whereas the mutation of Cys951Ser in ULK1 inhibited autolysosome formation and exacerbated hepatic lipid accumulation in HFD-fed mice. Summarizing, it indicated that ULK1 sulfhydration upregulated autophagy and improved liver lipid accumulation. Further research showed that CSE silence in HFD-fed mice with SREBP-1c deficiency increased liver lipids accumulation. Collectively, HFD-induced SREBP-1c reduced the level of CSE-dependent H2S in liver through miR-216a, which led to the reduction of sulfur hydration-dependent autophagy of ULK1, thus resulting in excessive accumulation of liver lipids. It can be concluded from above that H2S improved hepatic steatosis by enhancing ULK1 sulfhydration-mediated autophagy. While HFD promoted hepatic steatosis by inhibiting CSE /H2S/autophagy pathway, which is one of the mechanisms by which HFD induces NAFLD. Additionally, in the above study, H2S regulated autophagy by sulfurizing ULK1 mainly through promoting the fusion of autophagosome and lysosome via inducing the formation of UVRAG/Rubicon association and increasing ATG14 phosphorylation [2S can regulate autophagy through other mechanisms in liver remains to be further studied.Sterol regulatory element-binding proteins (SREBP-1c) is a transcription factor responsible for activating genes involved in the synthesis of fatty acids and triglycerides . It is hrylation . Whether2S in plasma and liver, as well as the mRNA expression of CBS, CES, and 3-MST in the liver of HFD-fed mice. Exercise reduced p62 protein expression, but had no notable influence on LC3-II/LC3-I ratio in the liver of HFD-fed mice. The above indicated that exercise upregulated H2S bioavailability and autophagy in the liver of HFD-fed mice, which may help to improve HFD-induced NAFLD. In addition, exercise inhibited malondialdehyde formation, increased GSH/GSSG ratio, and reduced the expression of TNF-α and IL-6 in the liver of HFD-fed mice, indicating that exercise mitigated oxidative stress and inflammation [2S regulation of autophagy remains to be studied.Lifestyle changes, including exercise training, have been reported to be an effective method for NAFLD treatment ,58,59. Eammation . Whether2S could ameliorate hepatic I/R injury by improving the serum levels of ALT and AST and pathological changes induced by hepatic I/R. H2S also attenuated hepatocyte apoptosis and autophagy induced by hepatic I/R in vivo and in vitro. During hepatic I/R injury, the JNK signal pathway was overactivated, which was inhibited by exogenous H2S. JNK1 inhibition with its inhibitor SP600125 potentiated H2S improvement of hepatic I/R injury. Overall, exogenous H2S alleviated hepatic I/R injury by suppressing autophagy and apoptosis through JNK pathway inhibition, which needed to be further confirmed. Further research revealed that further reducing autophagy with 3-MA (autophagy inhibitor) would mitigate H2S protective effects of hepatic I/R injury, while rapamycin (autophagy enhancer) potentiated H2S improvement of hepatic I/R injury [2S can inhibit autophagy to protect against liver ischemia-reperfusion injury. However, rapamycin can enhance this liver protection by reversing the autophagy inhibition of H2S. This shows that the protection mechanism of H2S is multifaceted. Moderate suppression of autophagy by H2S protects cells, whereas excessive suppression of autophagy by H2S has the opposite result. Moreover, the reason for this contradiction may be related to the time of liver ischemia.Tissue ischemia is a significant cause of death and disability worldwide. After ischemia for a period of time, the restoration of blood supply further exacerbates the damage of tissues and organs, which is called ischemia-reperfusion(I/R) injury. Studies have shown that free radical induced cell injury plays an important role in I/R injury ,69,70. HR injury . This se2S ameliorated fatty liver I/R injury by alleviating the pathological changes of liver tissue and downregulating the levels of LDH, ALT, and AST. In-depth research revealed that exogenous H2S enhanced autophagy in peritoneal macrophages through upregulating the level of LC3B and LC3-II/LC3-I ratio in fatty liver I/R injury. Additionally, exogenous H2S also suppressed apoptosis, inflammation, and oxidative stress, and downregulated SRA protein expression in fatty liver I/R injury, indicating that exogenous H2S improved fatty liver I/R injury through promoting autophagy by inhibiting SRA pathway, which required further research through the use of related inhibitors [2S to improve hepatic I/R injury by regulating autophagy. Activation of SRA has previously been shown to inhibit ERS-induced macrophage autophagy [2S promotes autophagy by inhibiting the SRA pathway. In fatty liver I/R injury, exogenous H2S also downregulated ERS [2S regulates the improvement of ERS-mediated autophagy on substance metabolism.Scavenger receptor A (SRA) is a receptor contributing to the macrophage-mediated inflammation, and involved in I/R injury ,80. The hibitors . SRA mayutophagy , suggestated ERS , hence, 2S in regulating autophagy in hepatocellular carcinoma has not been clarified. The results of Shanshan S Wang et al. showed that NaHS suppressed hepatoma cell proliferation, migration, and cell cycle progression to improve hepatocellular carcinoma. The in-depth research revealed that NaHS promoted autophagy by upregulating the expression of LC3-II and Atg5 and downregulated p62 expression in HepG2 and HLE cells. The combination of H2S and rapamycin (an autophagy inducer) further notably upregulated LC3-II expression, suppressed the proliferation, migration, and cell cycle of hepatoma cells. Moreover, both rapamycin and NaHS notably suppressed the protein expression of p-PI3K, p-Akt, and mTOR in hepatoma cells, suggesting that H2S promoted autophagy through the PI3K/AKT/mTOR signaling pathway inhibition. Overall, exogenous H2S ameliorated hepatocellular carcinoma through promoting autophagy by inhibiting PI3K/AKT/mTOR pathway  and increased the level of malondialdehyde. The in-depth studies showed that compared with the control group, high concentrations of H2S inhibited expression of genes related to PI3/Akt/mTOR pathway and increased the expression of other autophagy-related genes , indicating that high concentrations of H2S caused oxidative stress and induced autophagy in chicken hepatocytes by inhibiting PI3K/Akt/TOR pathway and leading liver injury. Moreover, high concentrations of H2S aggravated oxidative stress and autophagy injury induced by LPS [2S can inhibit autophagy and play a cytoprotective role by inhibiting oxidative stress [2S and the different types of tissues and cells. Low concentrations of H2S may often have a protective effect, while high concentrations of H2S have the opposite effects.Oxidative stress plays an important regulatory role in autophagy in liver ,96,97. Se stress . Thus, id by LPS . It has e stress ,100, whi2S regulation of autophagy in different types of liver disorders in recent years as follows:(1) H2S improved NAFLD via reducing serum TG level through autophagy promotion by activating AMPK/mTOR pathway; (2) H2S inhibited apoptosis and promoted autophagy by inhibiting ROS-mediated PI3K/AKT/mTOR to improve NAFLD; (3) H2S improved hepatic steatosis through promoting ULK1 sulfhydration-mediated autophagy; (4) exercise promoted H2S bioavailability and autophagy in the liver of HFD-fed mice to improve HFD-induced NAFLD; (5) H2S improved hepatic I/R injury by reducing autophagy and apoptosis through inhibiting JNK pathway, which needed to be further confirmed; (6) H2S improved fatty liver I/R injury by promoting autophagy through the inhibition of SRA pathway, which required to be further confirmed; (7) H2S improved hepatocellular carcinoma by promoting autophagy through the inhibition of PI3K/AKT/mTOR pathway; (8) high concentrations of H2S promoted autophagy in chicken hepatocytes by inhibiting PI3K/Akt/TOR pathway to induce liver injury (2S regulates autophagy, which can sometimes play a protective role in different liver disorders, and sometimes on the contrary. The reason may be related to the concentration of H2S. In addition, sometimes H2S can promote autophagy, while sometimes it can inhibit autophagy, which may be related to the different stages of the pathological processes and the basic level of autophagy. In addition, in liver disorder, the signaling pathway involved in H2S regulating autophagy needs to be further studied.In this review, we summarized the role of Hr injury . It can 2S regulation in many diseases [2S can improve NAFLD by regulating autophagy/NLRP3 inflammasome is a topic worthy of study. With the deepening of the research, the role of H2S in regulating autophagy may become an important therapeutic strategy for liver disorders.In recent years, the interaction between autophagy and NLRP3 inflammasome has been reported to be involved in many metabolic disorder-related diseases, including NAFLD . Moreovediseases . Therefo


The Integrase (IN) strand transfer inhibitor (INSTI), Dolutegravir (DTG), has been given the green light to form part of first-line combination antiretroviral therapy (cART) by the World Health Organization (WHO). DTG containing regimens have shown a high genetic barrier against HIV-1 isolates carrying specific resistance mutations when compared with other class of regimens.n = 20) and (n = 287) sequences data derived from HIV Los Alamos National Laboratory database. The possible impact of NOPs on protein structure caused by HIV-1 CRF02_AG variations was addressed within the context of a 3D model of the HIV-1 IN complex and interaction analysis was performed using PyMol to validate DTG binding to the Wild type and seven mutant structures.We evaluated the HIV-1 CRF02_AG IN gene sequences from Cameroon for the presence of resistance-associated mutations (RAMs) against INSTIs and naturally occurring polymorphisms (NOPs), using study sequences (We observed 12.8% (37/287) sequences to contain RAMs, with only 1.0% (3/287) of the sequences having major INSTI RAMs: T66A, Q148H, R263K and N155H. Of these,11.8% (34/287) of the sequences contained five different IN accessory mutations; namely Q95K, T97A, G149A, E157Q and D232N. NOPs occurred at a frequency of 66% on the central core domain (CCD) position, 44% on the C-terminal domain (CTD) position and 35% of the N-terminal domain (NTD) position. The interaction analysis revealed that DTG bound to DNA, 2MG ions and DDE motif residues for T66A, T97A, Q148H, N155H and R263K comparable to the WT structure. Except for accessory mutant structure E157Q, only one MG contact was made with DTG, while DTG had no MG ion contacts and no DDE motif residue contacts for structure D232N.Our analysis indicated that all RAM’s that resulted in a change in the number of interactions with encompassing residues does not affect DTG binding, while accessory mutations E157Q and D232N could affect DTG binding leading to possible DTG resistance. However, further experimental validation is required to validate the in silico findings of our study. Sub-Saharan Africa (SSA) remains one of the regions highly burdened by HIV infection at 70% of the global epidemic. SSA has a particularly high HIV-1 genetic diversity and it is documented that diverse subtypes may affect the clinical treatment outcome in patient management –4. Furthhttp://www.hiv.lanl.gov/) HIV-1 database to assess the occurrence of mutations and natural occurring polymorphisms (NOPs). NOPs are categorized under secondary mutations which on their own play a limited role in resistance [The United States of America (USA) Food and Drug Administration (FDA) has approved four HIV-1 INSTIs, including raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG) and bictegravir (BIC) . Howeversistance . Howeversistance . In thisn = 20) available in GenBank under the following accession numbers: MN816445- MN816488 [n = 287) INSTI treatment Naïve patients’ sequences (https://www.hiv.lanl.gov/components/sequence/HIVsearch.com), completed on 01 February 2020. All Cameroonian HIV-1 subtype CRF02_AG IN sequences for treatment naïve patients, were included in our search criteria [n = 287) and CRF02_AG cohort sequences from our previous study (n = 20) [https://www.hiv.lanl.gov/content/sequence/QC/index.htm) was employed to screen nucleotide sequences for quality and to verify for stop codons, insertions and/or deletions. The MAFFT version 7 sequence alignment tool, was used to perform a multiple sequence alignment from which the consensus sequence was derived [To compare our study CRF02_AG treatment naïve sequences (criteria . We sele derived . In an ehttp://www.bioafrica.net/subtypetool/html/subtypinghiv.html) and COMET-HIV (https://comet.lih.lu/).HIV-1 subtyping based on IN sequences was performed using two online available programs; REGA version 3 (http://hivdb.stanford.edu/). All drug resistance mutations results were classified as either major or minor mutations, last accessed 01 April 2020.Mutations associated with resistance to INSTIs were identified using the Stanford University genotypic resistance interpretation algorithm, HIVdb version 8.3 (https://servicesn.mbi.ucla.edu/SAVES/) namely; ERRAT [The crystal structure of the HIV-1B intasome  (PDBID: 5U1C) was used as a homologous template to generate a three-dimensional tetrameric structure of HIV-1CRF02_AG IN using the consensus sequence of recombinant subtype CRF02_AG sequence that we generated. The SWISSMODEL webserver was used for model construction by first constructing a pairwise sequence-structure alignment between HIV-1C wild-type (WT) amino acid sequence and template 5U1C . The quay; ERRAT  VERIFY3Dy; ERRAT  and PROCy; ERRAT  to assesThe predicted 3D structure of HIV-1 CRF02_AG IN was energy minimized using GROMACS software  and the HIV-1 subtyping was done using HIV-1 subtyping online-automated tools and all sequences were verified using phylogenetic tree as HIV-1 subtype CRF02_AG  sequences to contain RAMs, with only 1.0% (3/287) having major INSTI RAMs: T66A, Q148H, R263K and N155H. Two mutations, Q148H and R263K, occurred together in one sequence (0.3%), whereas T66A and N155H were present individually in one sequence each. A total of 11.8% (34/287) of the sequences contained five different IN accessory mutations, namely Q95K, T97A, G149A, E157Q and D232N. Mutations G149A and D232N occurred together in one sequence (0.3%). Notably, one sequence dating back from 2010 had two major mutations; Q148H and R263K in combination with two other minor mutations G149A and D232N.n = 287) and cohort sequences (n = 20), identified 20 naturally occurring polymorphisms (NOPS): E11D,K14R, V31I, M50I, I72V, L74MVI, L101I, T112V, T124A, G134N, I135V, K136K/Q, V201I, T206S, T218I, L234I, A265V, R269K, S283G , i.e. the M50I, T66A, L74I, L74M, T97A, G118S, S119R, P145S, Q148H, G149A, N155H, E157Q, D232N and R263K substitutions resulted in destabilizing effects of − 0.582, − 0.703 -1.069, − 0.93, − 1.051, − 0.492, − 0.091, − 0.485, − 0.133, − 0.421, − 0.975, − 1.111, − 0.512 and − 0.455 Kcal/Mol each, respectively. Only substitution Q95K resulted in a slightly stabilizing effect of 0.146Kcal/Mol. Interaction analysis of the single amino acid changes indicated differences in the number and type of interaction between neighbouring residues and the DNA. The T97A showed four polar contacts for T97 compared to the three of A97  and/or transmitted DRM, leading to reduced susceptibility to INSTIS and possible treatment failure , 28. TheIn a recent study on CRF02_AG IN, we reported that accessory mutations can impact the binding of DTG with or without combination of primary resistance mutations , 36. In In our study, we observed five IN accessory RAMs; namely Q95K, T97A, G149A, E157Q and D232N. T97A mutation can reduce EVG susceptibility by 3-fold  and combWe further analysed the effect of NOPs on the stability of the structures and neighbouring residues. Most of the variants noted in our study were shown to destabilise the protein structure, except for one mutation Q95K, that showed to exert a slightly stabilising effect on the protein structure and no changes in the number of polar contacts with neighbouring residues making it unlikely to affect the IN protein structure. It is known that destabilising effects of mutations on the protein structure might reduce drug binding. This was further explored by performing interaction analysis between the drug DTG and energy minimized structures of the WT and mutants T66A, T97A, Q148H, N155H, E157Q, R263K and D232N. The findings revealed accessory mutations E157Q and D232N had the potential to reduce and or prevent DTG binding to HIV-1 CRF02_AG IN structure based on the loss of MG ion interactions, while known RAM’s does not seem to influence DTG drug binding. However, the effect of RAM’s on DTG drug binding needs to be validated using molecular dynamic simulations to calculate the change in free energy of binding between DTG and HIV-1 CRF02_AG IN. Interestingly, the mutation E157Q occurred within the stable alpha-helix secondary structure element and made more contacts with DNA , while the D232N mutation occurred within the stable Beta-sheet secondary structure element and in close proximity to the flexible G140’s loop region suggesting that these changes can affect the protein conformation and thereby interfere with drug binding leading to resistance.A limitation of the study is the use of online database sequences that may contain contaminated and truncated sequences leading to spurious phylogenetic tree results and also these databases are not regularly updated. Furthermore, gaps in the aligned regions between the homologous template and target sequence may result in unresolved loop regions within the protein model which is one of the limitations of 3D protein modelling that can result in inaccurate interaction prediction. Furthermore, the interaction analysis was done for only a single static structure of the protein structure and does not consider the dynamic behaviour of the protein structure that might result in the loss and under estimation of crucial interaction partners. An important finding in this study is the fact that sequence diversity amongst different subtypes may affect different folding conformations of the HIV-1 IN subtypes thereby allowing not only RAM’s but accessory mutations to result in less efficacious INSTI binding to HIV-1 IN structures.Molecular modelling and interaction analysis provided novel insights into the effect of accessory mutations (E157Q and D232N) on HIV-1 CRF02_AG IN drug resistance. This emphasise the need to screen for the presence of INSTIs major RAM’s and accessory mutations in patients on INSTI treatment. This would help identify pathways that contribute to drug resistance and help tailor more effective treatment regimens in INSTI naïve patients.


Flavonoids give plants their rich colors and play roles in a number of physiological processes. In this study, we identified a novel colorless maize mutant showing reduced pigmentation throughout the whole life cycle by EMS mutagenesis. E183K mutation in maize chalcone synthase C2 (ZmC2) was mapped using MutMap strategy as the causal for colorless, which was further validated by transformation in Arabidopsis. We evaluated transcriptomic and metabolic changes in maize first sheaths caused by the mutation. The downstream biosynthesis was blocked while very few genes changed their expression pattern. ZmC2-E183 site is highly conserved in chalcone synthase among Plantae kingdom and within species’ different varieties. Through prokaryotic expression, transient expression in maize leaf protoplasts and stable expression in Arabidopsis, we observed that E183K and other mutations on E183 could cause almost complete protein aggregation of chalcone synthase. Our findings will benefit the characterization of flavonoid biosynthesis and contribute to the body of knowledge on protein aggregation in plants. Flavonoids, which mainly consist of such compounds as chalcones, flavones, flavonols, anthocyanins, and proanthocyanins , are a gDue to the success of isolating mutants with alterations in pigmentation , the metArtificial mutants serve as essential materials for forward and reverse genetics. In Arabidopsis and rice, T-DNA insertion mutagenesis has been widely used . The sucTraditional map-based cloning of mutants relies on repeatedly screening putative markers and evaluating the entire population, which is time-consuming and labor-intensive. Recently, a significant cost reduction was achieved by combining the bulked segregant analysis (BSA) strategy  with higIn the process of gene expression, from DNA to a functional protein or RNA, modification or regulation frequently occurs and plays indispensable roles. Posttranslational regulatory steps include posttranslational modifications, protein degradation, and aggregation. Intracellular protein degradation is the breakdown of abnormal or superfluous proteins into smaller polypeptides or amino acids. Some misfolded proteins may escape from degradation, instead accumulating and clumping together to form aggregates . One of In this study, combing ethyl methane sulfonate (EMS) mutagenesis and MutMap strategy, we identified and did gene mapping for a novel colorless maize mutant. The causal mutation proved to cause protein aggregation and dysfunction. Also, the effect of causal mutation on transcriptomic and metabolic levels were investigated.For whole lifetime phenotypic observation, maize materials were grown in the field with regular management. In all other cases, materials were grown to three leaf stages in a greenhouse. For anthocyanin measurement, fresh tissues were used. For DNA, RNA, and metabolome extraction, samples were immediately frozen in liquid nitrogen and stored at −80°C.v/m, where m represents the weight of tissues (g), v is the volume (1 ml) of extraction buffer.Anthocyanin was extracted with a methanol–HCl method . BrieflyMutant mapping was taken with a modified MutMap strategy . An F2 p1 using BWA  in mutant-type pool (MP). Lowess fitness was performed variant-index for smoothing using the “lowess” function in R .3 were used for the visualization of sequencing peaks. Sequence alignment was performed using pairwise sequence alignment tools available at EMBL-EBI4.For variant validation, the PCR products were directly sequenced using the corresponding PCR primers (5 (Oryza sativa Japonica Group)  with 92.15% alignment identity to ZmC2 was selected as a modeling template. Visualization of protein structure was performed using PyMOL  and 2x SYBR Green qPCR Master Mix . The Real-Time qRT-PCR program had three steps: step 1 (Hot-Start DNA Polymerase Activation), 95°C for 30 s; step2 (PCR), 40 cycles of 95.0°C for 15 s and 60.0°C for 30 s; step 3 (Melt Curve), 95.0°C for 15 s, 60.0°C for 60 s, increment of 0.3°C increase/cycle to 95.0 and 95.0°C for 15 s. The primers for ZmC2  were CAGAAGGCGATCAAGGAGTG and GGTACATCATGAGGCGGTTC (product size: 151 bp) (ZmTUB2  was used as internal control for normalization with the primers CTACCTCACGGCATCTGCTATGT and GTCACACACACTCGACTTCACG (product size: 297 bp) (–ΔΔCT method (RNA was extracted from maize first sheaths or leaves using an Ultrapure RNA Kit (DNase I) . cDNA was generated using UEIris II RT-PCR System for First-Strand cDNA Synthesis Kit . qRT-PCR was performed with three biological replicates using the Eco 151 bp) . ZmTUB2  297 bp) . The relT method .2FoldChange— ≥1 and q-value ≤ 0.05. Gene annotation was achieved from Ensembl-Plants BioMart6.RNA-seq was performed on a pool of B73-type F2 progenies  and a pool of mutant-type F2 progenies . Each pool consists of 20 individuals’ first sheaths at the three-leaf stage. cDNA library preparation and sequencing were performed by Sangon  using Illumina HiseqXTen platform following a standard protocol. Bioinformatics analysis followed a common approach. Quality control was performed on raw reads using Trimmomatic . Clean r7 ; MS, Applied Biosystems 4500 Q TRAP8). OPLS-DA was performed using R package “MetaboAnalystR”  and a pool of mutant-type F2 progenies . Each pool consists of 50 individuals’ first sheaths at the three-leaf stage. Sample preparation and extraction, UPLC-MS/MS analysis was performed by Beijing Guoke Biotechnology  following a widely targeted metabolomics method . In brienalystR” . SignifiystR”  was performed using Clustal-Omega11  database in NCBI-Omega11 . The MSAlview v2 . For con14 (The expression pattern for ZmC2 and ZmWHP1 in 23 tissues spanning multiple stages of maize development  were ach14 .CHS (ZmC2 or ZmWHP1) was cloned using cDNA as template (NdeI and XhoI restriction enzymes) to generate the fusion protein His6-tag:CHS  with a PVDF membrane  following the instructions of the manufacturer. Proteins were transferred from gel to PVDF membrane using a semidry method. The first antibody used was an anti-His mouse monoclonal antibody, and the second antibody used was HRP-conjugated goat anti-mouse IgG. ECL luminescence reagent was used as the substrate of HRP, and X-film was used in the darkroom to capture the luminescence.A. thaliana .Maize protoplast isolation and transformation were carried out as previously described in thaliana  with somAgrobacterium tumefaciens strain GV3101. Transformation of Arabidopsis was done following the vacuum infiltration method ; TAIR accession At3g18780] was used as internal control (primer #3). Leaves of positive lines were subjected to protoplasts extraction and YFP observation following a similar procedure as “maize leaf protoplasts extraction and GFP observation.”Constructed plasmids were transformed into n method . TransfoA novel colorless mutant was screened from an EMS-induced mutant library for the maize B73 inbred line. Throughout the life cycle, this mutant showed reduced level of pigmentation, especially in sheath, aerial roots, cobs, etc., which are tissues expected to contain notably abundant pigment .The mutant was crossed with wild-type B73. Compared to B73 with red/purple leaf sheaths and high anthocyanin content, the mutant line showed green leaf sheaths with almost zero anthocyanin content, and the F1 hybrids showed intermediate color type and anthocyanin content . InvestiTo identify the causal mutation, we used MutMap pipeline and an F2 population derived from B73 and the colorless mutant . Over 20In total, 176,899 variants  were identified for the three samples . Over 90ZmC2)  . To valiZmC2) .ZmC2 is known to encode a naringenin-chalcone synthase ; the enzyme catalyzes the reaction 4-coumaroyl-CoA + malonyl-CoA → naringenin chalcone, which is the first dedicated step in flavonoid biosynthesis. Three-dimensional (3D) protein structure was built for ZmC2 based on OsCHS1 ; NCBI accession: XP_015618054.1) (ZmC2 in the sheath or leaf (18054.1) . ZmC2 is18054.1) . Further or leaf .A. thaliana, that is At5g13930 , and the alignment identity for them is 82.7% (tt4) mutant contains a T-DNA insertion in the second exon of AtCHS  and widely targeted metabolome  were performed on the first sheaths of B73-type F2 progeny (BP) and those of mutant-type F2 progeny (MP).q-value ≤ 0.05), where 12 genes were up-regulated, and 5 were down-regulated. Zm00001d017186 (Shikimate O-hydroxycinnamoyltransferase) is the only DEG with slight down-regulation related to flavonoid biosynthesis , it is obvious that most of the detected metabolites in the downstream of ZmC2 involved reaction (EC: 2.3.1.74) showed significant decrease . Also, dZ. mays); NCBI accession: NP_001149022.1] with 97.25% identity. These two maize chalcone synthase (CHS) was in the same branch with CHS from Setaria italica, Panicum hallii, and seven CHS from Sorghum bicolor. Furthermore, we explored the conservation of chalcone synthase within species but among different varieties. Publicly available variation information was used for ZmC2, ZmWHP1, OsCHS1, and AtCHS , 1 belongs to Metazoa (Kingdom), 167 belong to Plantae (Kingdom)-Magnoliopsida (Class)-Poales (Order), and the rest belong to the other orders or classes in Plantae (Kingdom). Principal components analysis  and phylnd AtCHS . There wZmC2 had much higher (about 100 times) transcription level than ZmWHP1. Besides, ZmC2-E183K mutation did not cause a differential regulation of ZmWHP1 -tag was fused at the C-terminus of ZmC2-WT and ZmC2-E183K, and the reconstructed plasmids were transferred into maize leaf protoplasts for transient expression. ZmC2-WT proteins existed as evenly distributed in the cytoplasm without aggregation (type I) or with some aggregation (type II). Notably, ZmC2-E183K fused proteins existed as totally aggregated (type III) .Arabidopsis tt4 mutant. ZmC2-WT fused proteins were evenly distributed in the cytoplasm (type I) in independent transgenic lines, while ZmC2-E183K significantly aggregated into some points (type III) (Another convincing evidence for the aggregation of ZmC2-E183K was gained through Arabidopsis transformation. ZmC2-WT and ZmC2-E183K were fused with YFP (yellow fluorescent protein)-tag at their C-terminus and stably expressed in ype III) .2)2-COOH and -CH2-COOH respectively, the E183D mutation leading to protein aggregation may indicate a restrictive structural requirement, i.e., chain-length of the acidic side chain. In summary, E183K and other mutations on E183 could cause protein aggregation of chalcone synthase, leading to dysfunction.Similar phenomena were observed for ZmWHP1, the most identical protein of ZmC2. In prokaryotic expression, ZmWHP1-WT proteins were detected as soluble and precipitated, while ZmWHP1-E183K proteins completely aggregated/precipitated ; when trStructure modeling of ZmC2 indicated the wild-type proteins exist as homodimers, and the side chain of E183 has polar contacts with A21, K179 in the same molecule and R12, N184 in the paired molecule. Of them, A21 and R12 may be important for the conformation of N-terminal helix . An aggrAccording to this proposed model, the N-terminal helix is essential for generating stable dimer. We performed artifact damage on the N-terminal helix . ZmC2-A2_P20del proteins were almost totally precipitated/aggregated in prokaryotic expression and transient expression in maize protoplasts . As E183ZmC2-WT genes would generate ZmC2-WT homodimers directly, while ZmC2-E183K genes generate ZmC2-E183K aggregates directly, thus ZmC2-WT/ZmC2-E183K proteins (unknown status) only occupy a tiny proportion or even do not exist. In F1 or F1-type F2 progenies, there are half ZmC2-WT homodimers  and half ZmC2-E183K aggregates , which is completely consistent with the observed mid-colored phenotype. However, there may be other mechanisms for the aggregation instead of our proposed hydrophilic and/or hydrophobic model. Furthermore, in vivo experiments are in need to determine ZmC2-E183R, ZmC2-E183D, and ZmC2-E183R_R12E proteins are functional or not.The protein aggregation model is consistent with the observed phenotype . In B73 Protein aggregation raises strong interest in animal fields due to its involvement in human diseases . PreviouC2-Idf mutant  was crossed with B73. Only 5654 variants homozygous and unique in the mutant were taken as markers, and over 90% of them are C > T or G > A substitution . The EMSPRJNA593329). Raw RNA-seq data are available at NCBI-SRA (PRJNA593349). Other relevant data are within the article and The raw data for whole genome sequencing are available at NCBI-SRA (YY, HD, and HL planned and designed the research. HD, HL, YX, SS, SL, XS, HKL, NJ, XW, and ZZ performed the experiments, analyzed the data, conducted the field work, etc. HD, HL, and YY wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P = 0.002; female, P <0.001) and cyclooxygenase-2 (COX-2) . Collectively, our findings indicate that estrogen ameliorates the immune microenvironment represented by PD-L1 expression and enhances its effect in the absence of Nrf2.Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a dual role in carcinogenesis. We previously reported that Nrf2 deficiency enhances the anti-tumorigenic effect of 17β-estradiol (E2) in an azoxymethane (AOM)/dextran sodium sulfate (DSS) model of colitis-associated cancer (CAC). Herein, we aimed to determine a possible explanation for our recent work and investigated the immune microenvironment represented by programmed death-ligand 1 (PD-L1) expression. One week after the AOM injection, mice were administered with DSS in drinking water for seven days; daily E2 injections were intraperitoneally administered during this period. The mice were sacrificed 16 weeks after AOM injection and analyzed for PD-L1 expression in the distal colon tissues using Western blotting and immunohistochemistry (IHC). Based on Western blotting results, PD-L1 expression was reduced in Nrf2 knockout (KO) female and E2-treated male mice when compared with their wild-type counterparts, following AOM/DSS treatment; this supports the association of PD-L1 expression with tumor progression. Additionally, this finding was in good agreement with the IHC results for PD-L1. Furthermore, we observed that PD-L1 is predominantly expressed in stromal cells rather than on epithelial cells in the colon. Western blotting revealed that PD-L1 expression in the colon positively correlates with expressions of inducible nitric oxide synthase (iNOS) (male,  Helicobacter pylori-induced gastritis have been associated with an increased risk of cancer (Inflammation is known to predispose patients to cancer initiation and development . For insf cancer . Tumor if cancer . For exaf cancer . TherefoA programmed death 1 (PD-1) is an immune checkpoint expressed on activated T cells, and its engagement with programmed death-ligand 1 (PD-L1) attenuates T cell activation . PD-L1 evia ERβ  transcription factor maintains redox homeostasis during oxidative stress and suppresses inflammation . ConsistBased on this background, we hypothesized that estrogen might exert anti-tumorigenic effects and cooperate with Nrf2 deficiency in the immune microenvironment Therefore, this study aimed to determine the sex difference in PD-L1 expression and its correlation with Nrf2 expression in the colon tumor microenvironment of the AOM/DSS-induced CRC mouse model. Also, as part of the sex difference, we tried to confirm the effect of E2 on PD-L1 expression in male mice.AOM (# A5486) and E2 (# E8876) were purchased from Sigma-Aldrich . DSS  was procured from MP Biomedicals . The antibodies used in this study are listed in +/− mice (C57BL/6 background) were kindly provided by Prof. Young-Joon Surh. As shown in −/−, KO) were generated by cross-breeding of heterozygous Nrf2+/− mice  guidelines. All animal experiment protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Seoul National University Bundang Hospital (BA1705-223/043-01).Heterozygous Nrf2+/− mice , 28. Micus study , 28. AllThe AOM/DSS protocol is well established and is commonly used as a mouse model of CAC. Both WT and Nrf2 KO mice were randomly divided into the following five groups: male control group, male AOM/DSS group, male AOM/DSS + E2 group, female control group, and female AOM/DSS group; each group was composed of 9–12 mice. In the AOM/DSS-treated group, mice were intraperitoneally injected with 10 mg/kg of AOM dissolved in phosphate-buffered saline (PBS). Seven days after injection, 2.5% (w/v) DSS was added to the drinking water for 7 days, followed by switching to regular water. E2 was dissolved in olive oil and administered intraperitoneally daily at a dose of 10 mg/kg during 2.5% DSS treatment Figure 1Colon samples were homogenized using a handheld homogenizer in 300 μl of RIPA buffer supplemented with complete protease inhibitors . Then, tissue lysates were denatured and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoresis, separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in TBST  for 1 h. Next, the membranes were incubated with primary antibodies Table 1 Gapdh.Colon tissues were homogenized using a handheld homogenizer in 500 μl of TRIzol reagent , and total RNA was extracted. According to the manufacturer’s instructions, 2 μg of total RNA was reverse transcribed using a Reverse Transcription kit . The cDNA was used to perform qRT-PCR using specific primers  on a Benchmark XT autostainer , with standard antigen retrieval. The UltraView Universal DAB Detection Kit  was used according to the manufacturer’s instructions. PD-L1 expression was evaluated in both the tumor and non-tumor areas. Quantitative assessment of PD-L1 immunoreactivity was performed using the Image-Pro Plus analysis system, with measurements expressed as the proportion of immunoreactive area . The reported values are averages of different images from different mice within each group.P <0.05. The correlation of PD-L1 expression with COX-2 and iNOS was determined using Spearman’s correlation coefficient.Statistical analysis was performed using SPSS version 18.0 . Data are presented in bar graphs as the mean ± standard error of the mean (SEM). The statistical significance of differences between the two groups was evaluated using the Mann–Whitney test, and the graphs were generated using GraphPad Prism 5.0 . The statistical significance of differences between more than two groups was determined using a nonparametric factor Kruskal–Wallis sum-rank test. Statistical significance was set at P = 0.003) and Nrf2 KO (P = 0.004) male mice when compared with the control group. The administration of E2 significantly reduced the expression of AOM/DSS-induced PD-L1 protein in Nrf2 KO male mice (P = 0.009); a marginal decrease was observed in WT male mice when compared with that in the AOM/DSS group (P <0.001), but not in WT males (P = 0.004) and Nrf2 KO (P <0.001) males , but not in Nrf2 KO females. Similar to PD-L1 expression, iNOS and COX-2 protein levels were strongly elevated by AOM/DSS treatment only in WT females (P = 0.002 for iNOS and P = 0.018 for COX-2), not in Nrf2 KO females (P = 0.033) and pro-inflammatory mediators (P = 0.003 for iNOS and P = 0.045 for COX-2) than WT mice after AOM/DSS treatment . In terms of sex, the mRNA expression of Pd-l1 and iNos increased by AOM/DSS treatment was significantly lower in female mice than in males, only in Nrf2 KO groups  and COX-2 (P <0.001) (P <0.001) and COX-2 (P <0.001) in both WT and Nrf2 KO female mice  when compared with WT male mice in the non-tumor area . PD-L1 expression was analyzed in male and female mice, and representative images were obtained from the indicated mice. As shown in P = 0.034 for WT and P = 0.020 for Nrf2 KO; P = 0.034 for WT and P = 0.020 for Nrf2 KO; P = 0.025 for male vs female; Similar to the male control group, PD-L1-positive cells were barely detectable in the controls of WT and Nrf2 KO female mice  remains elusive. Recent studies using meta-analysis have shown that PD-L1 expression is a negative prognostic factor for overall survival and disease-free survival , 31. Altvia its ability to reduce ROS accumulation  of the Seoul National University Bundang Hospital.+/− transgenic mice and revised the manuscript. All authors contributed to the article and approved the submitted version.CK drafted the manuscript and performed molecular experiments. C-HS revised the manuscript and performed molecular experiments. NK designed and supervised the experiments and revised the manuscript. RN, C-HS, SC, JY, and HN performed the animal experiments. JK advised on the concept of PD-L1 in Nrf2 knockout mice. HYN assisted with the analysis of immunohistochemistry of PD-L1. RN performed the immunohistochemical analysis. JC and H-NL revised the manuscript. Y-JS provided Nrf2This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the government of the Republic of Korea (2019R1A2C2085149).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We performed single-genome sequencing of full-length HIV-1 pol in a highly treatment-experienced patient, and determined drug susceptibility of patient-derived HIV-1 genomes using a phenotypic assay encompassing full-length pol gene. We show the genetic linkage of multiple InSTI-resistant haplotypes containing major resistance mutations at Y143, Q148 and N155 to protease inhibitor (PI) and reverse transcriptase inhibitor (RTI) resistance mutations. Phenotypic analysis of viruses expressing patient-derived IN genes with eight different InSTI-resistant haplotypes alone or in combination with coevolved protease (PR) and RT genes exhibited similar levels of InSTI susceptibility, except for three haplotypes that showed up to 3-fold increases in InSTI susceptibility (p ≤ 0.032). The replicative fitness of most viruses expressing patient-derived IN only significantly decreased, ranging from 8% to 56% (p ≤ 0.01). Interestingly, the addition of coevolved PR + RT significantly increased the replicative fitness of some haplotypes by up to 73% (p ≤ 0.024). Coevolved PR + RT contributes to the susceptibility and viral fitness of patient-derived IN viruses. Maintaining patients on failing cART promotes the selection of fitter resistant strains, and thereby limits future therapy options.Integrase strand transfer inhibitors (InSTIs) are recommended agents in first-line combination antiretroviral therapy (cART). We examined the evolution of drug resistance mutations throughout HIV-1  Since the introduction of combination antiretroviral therapy (cART) against HIV-1 in the late 1990s, the number of overall new HIV infections and AIDS-related deaths has decreased ,3. To dapol gene, the target of the four main classes of ARVs used in cART: PIs, NRTIs, non-nucleoside reverse transcriptase inhibitors (NNRTIs) and InSTIs. This is because routine genotypic resistance testing in HIV-infected patients normally involves standard “bulk” population sequencing of viral genes of interest; usually protease (PR) and the N terminal of RT, or the catalytic core domain of integrase (IN) separately, or, alternatively, short read (75 to 300 bp) next-generation sequencing is used [gag-pol fragments [pol genes [pol single-genome sequencing assay to investigate the development, evolution and linkage of PI, RTI and InSTI resistance mutations in a patient undergoing InSTI-containing therapy. We also investigate the effect on InSTI susceptibility and viral fitness of patient-derived IN only or in combination with coevolved PR and RT, using a single-replication-cycle drug susceptibility assay. Originally, RAL was used as part of a second-line treatment or salvage regimen for those failing previous lines of therapy . However is used ,9. In adragments ,11,12,13ol genes ,15,16. CSingle genomes were generated from five out of six samples over a 16-month period . A totalAnalysis of the 16 single genomes before exposure to RAL (preRAL) revealed no RAL resistance mutations, except for the presence of an amino acid substitution (G163E) in a single genome at a position associated with RAL resistance . On the Within four months following the withdrawal of RAL treatment (4 post), 97.4% (38/39) of the single genomes contained no RAL resistance-associated mutations, with only one single genome still harbouring the G163R mutation and a novel substitution at major resistance position 143 (Y to G). This minor variant was not detected in any of the single genomes at 4RAL or 5RAL. Two weeks after RAL therapy was re-initiated (ReRAL), this novel Y143G + G163R mutant dominated the viral population with all of the single genomes (n = 13) containing the double mutation.We investigated the linkage of RAL resistance mutations in IN to drug resistance mutations in PR and RT. We found little variation in the composition of PI and RTI resistance mutations over the sampling period. All 117 amplified single genomes contained numerous PI , NRTI  and NNRTI  resistance mutations . This ispol gene in 100% (n = 64) of single genomes that contained RAL resistance mutations  was equivalent to that of the wild-type control virus (p8.9NSX) at 4.2 ± 0.11 vs. 4.5 ± 0.37 nM . Interestingly, the major RAL-resistant haplotype at 4RAL and 5RAL, Y143R + G163R, had the highest decrease in RAL susceptibility (200-fold), compared to a moderate decrease in RAL susceptibility of 50-fold for the Y143G + G163R haplotype, that emerged during re-initiation of RAL therapy.We investigated the effect of the different drug resistance-associated mutations identified during the development of RAL resistance on RAL susceptibility. We generated nine recombinant virus vectors expressing patient-derived quencing . As expe50 of the virus expressing the patient-derived wild-type IN from pre-RAL time point (ptA_WTIN) was significantly higher compared to the wild-type control (p8.9NSX) at 0.24 ± 0.026 vs. 0.1 ± 0.016 nM . The remaining six viruses expressing patient-derived IN only were found to have significantly higher EVG EC50 values compared to the wild-type control, ranging from 0.48 ± 0.064 to 23.31 ± 0.69 nM (p < 0.05). This includes the virus expressing the Y143R + G163R mutation combination, which exhibited the highest fold change in RAL EC50 (200-fold), but which only resulted in a 5-fold change in EVG susceptibility. The highest fold change in EVG susceptibility (227-fold) was exhibited by the virus expressing the Q148R + G140A mutation combination, which also resulted in a very high fold change in RAL susceptibility (147-fold).Next, we investigated the susceptibility of the viruses expressing patient-derived fragments against a different InSTI, EVG. Interestingly, the EVG ECcontrols ,18. Two PR and RT genes influence the susceptibility to the InSTIs RAL and EVG, we generated recombinant vectors expressing patient-derived full-length pol gene. The virus expressing the patient-derived full-length pol with resistance mutations in PR and RT but wild-type in IN (designated ptA_pol), as well as the virus expressing the patient-derived PR and RT only (designated ptA_PR + RT) had RAL susceptibilities comparable to that of the wild-type control virus (p8.9NSX) with EC50 values of 5.5 ± 1.1 nM, 4.1 ± 0.48 and 4.5 ± 0.37 nM, respectively . This suggests that the coevolved PR and RT can confer a negative effect on resistance to RAL, which is dependent on the combination of resistance mutations in IN.To determine if the coevolved PR and RT also influences EVG susceptibility, we investigated the EVG susceptibilities of viruses expressing patient-derived full-length pol (IN gene only (ptA_WTIN), the virus expressing patient-derived full-length pol with resistant PR and RT genes but wild-type IN gene (ptA_pol) also exhibited a significantly higher EVG EC50 compared to wild-type control virus at 0.18 ± 0.033 vs. 0.1 ± 0.016 nM (p = 0.03). Overall, the EVG EC50 values of viruses expressing patient-derived full-length pol were similar to that of viruses expressing patient-derived IN gene only, with the exception of the virus expressing Y143C + E92Q mutations, which showed a significant decrease in EVG susceptibility for patient-derived IN gene only compared to full-length pol gene (p = 0.0064). Again, this may indicate that the patient coevolved PR and RT genes’ influence on the susceptibility to EVG is dependent on the combination of InSTI resistance mutations; however, this effect is different between EVG and RAL.To determine if the coevolved ngth pol B. Similapol or IN gene only (PR + RT (ptA_PR + RT) and the virus expressing patient-derived full-length pol with wild-type IN showed similar replicative fitness to the virus expressing patient-derived wild-type IN only  at 110.5 ± 7.7%, 109.7 ± 12.3% and 102.9 ± 8%, respectively (p ≥ 0.39). Next, we tested the replicative fitness of the viruses expressing patient-derived full-length ene only . Using aIN was the patient-derived full-length pol or IN only virus, expressing the rare IN Y143G + G163R mutation combination at 86.9 ± 7.6 and 102 ± 6.6%, respectively (p = 0.28 and 0.85). All other viruses expressing patient-derived full-length pol or IN only had significantly lower replicative fitness than ptA_WTIN, ranging from 12.9 ± 0.6 to 72.9 ± 3.4% (p ≤ 0.01). Interestingly, the only other virus that showed replicative fitness comparable to that of ptA_WTpol was greater than or comparable to that of viruses expressing the respective IN gene only. The viruses showing significantly increased replicative fitness upon expression of patient-derived full-length pol relative to IN gene only were those with the following RAL resistance mutation combinations: Q148R + G140A (p = 0.0023), Y143R + G163R (p = 0.024), Y143R + G163K (p ≤ 0.0001) and Y143C + G163R (p = 0.013). Furthermore, viruses that dominated the population during the two RAL treatment phases had both high levels of RAL resistance and replication fitness  during treatment failure, albeit on different genomes, which is also consistent with previous findings ,21,22,23IN [Our findings also shed light on the complexities of the intrapatient evolution of the Y143 resistance pathway, and the effects of accessory mutations linked to this pathway. Previous studies suggest that the accessory mutations linked to the Y143 mutations play a positive role in IN activity and/or RAL resistance ,24,30. IIN ; therefo50 than the Y143C + G163R mutant, and had a significantly higher replicative fitness compared to the Y143R + G163R and Y143C + G163R mutants. The replicative fitness of Y143G + G163R was in fact the highest of all RAL resistant variants identified in this patient, and was comparable to that of wild-type virus. It is therefore likely that the subsequent outgrowth of this viral variant after the re-initiation of RAL therapy may be due to its higher replicative fitness compared to the other RAL resistant viruses present in the patient. The late and rare development of the Y143G substitution could be due to a high genetic barrier. The wild-type codon for position 143 in IN in this patient was TAC. One nucleotide change was required for the Y to C substitution (TAC to TGC), whilst two nucleotide changes were needed to generate the G substitution (TAC to GGC) or R substitution (TAC to CGC). The second change required for the Y to R substitution (T to C) is a transition which occurs at a higher frequency compared to the T to G transversion required for the Y to G substitution. Thus, although more advantageous to the virus in terms of fitness, the Y143G substitution is likely to occur less frequently compared to Y143R/C substitutions. This is supported by analysis of data available from the Stanford HIV Drug Resistance Database, which showed no instances of Y143G mutation in InSTI-experienced subtype B infected patients , linked to the same accessory mutation (G163R). This finding is contrary to another study which showed that the RAL susceptibilities of both the Y143R and Y143C mutants were similar . Howeverpatients B. This aPR and RT genes affect the susceptibility and replicative fitness of an IN gene harbouring InSTI resistance mutations by up to three-fold. This concurs with another study, that showed an effect on viral fitness and susceptibility to EFV and RAL for certain combinations of NNRTI and InSTI resistance mutations [IN gene [pol gene and their effects on susceptibility and viral fitness, including that to the new InSTIs DTG, BIC and CAB. However, taken together, these data suggest that analysis of only part of the HIV-1 genome is probably not sufficient to gauge the true dynamics in the evolution and extent of drug resistance in the era of cART, as shown by recent studies linking InSTI resistance to env and cPPT regions [pol gene or more of the viral genome may provide useful insights into drug resistance mechanisms, and help devise better treatment strategies as well as improve the prediction of the emergence of drug resistance and subsequent treatment failure.A limitation of the study is that the analysis, although in depth, is from one patient. Future studies will focus on investigating this phenomenon in a large group of patients, and include investigation of resistance to second generation InSTIs, such as DTG and use of full-length genome clones. Nonetheless, the data show that the coevolved utations . On the [IN gene . Differe regions ,36,37. TThe plasma samples used in this study were obtained from a patient attending the Mortimer Market Clinic, UCLH, who was infected with subtype B virus and initiated on RAL salvage therapy (600 mg daily) in September 2007. They continued RAL in combination with darunavir/ritonavir (DRV/r) and etravirine (ETR) until February 2008, when the patient experienced virological failure. The patient was then switched onto therapy containing tenofovir (TDF) and lamivudine (3TC), but experienced virological failure again 2 months later (April 2008). RAL treatment was then re-started in combination with TDF/emtricitabine (FTC), DRV/r and ETR in September 2008. Six samples were obtained, and these were: pre-RAL therapy (preRAL); 2, 4 and 5 months on RAL ; 4 months after RAL was stopped (4post); and 0.5 months after RAL was re-started (reRAL) . InformeIN, and used to sequence full-length pol from sequential samples. cDNA synthesis and single genome PCR reactions were carried out as described, but using the antisense primer KVL069 (5′-TTCTTCCTGCCATAGGARATGCCTAAG-3′) [A previously described, single genome sequencing assay  was modiTAAG-3′)  followedTAAG-3′)  or 5222-gag-pol expression vector, which contains a unique ApaI restriction site in gag (upstream of the PR start codon) and an EcoRI restriction site in vif (downstream of the IN stop codon), was modified by introducing a unique ClaI restriction site at the beginning of IN (flanking amino acids 4/5), creating the p8.9NSXClaI+ vector. In parallel, mutagenesis was used to introduce the same ClaI and EcoRI sites in nine of the single genomes amplified from the patient in the single genome assay. The nine single genome variants were selected from different on- and off-RAL treatment time points, which included all the different RAL resistance mutation combinations observed in the patient, with these being: N155H + V151I, Q148R + G140A, Y143R + G163R, Y143R + G163K, Y143C + E92Q, Y143C + T97A, Y143C + G163R, Y143G + G163R as well as a wild-type IN from the pre-RAL time point (used as a control and designated ptA_WTIN).To study phenotypic drug susceptibility, we used a previously described three plasmid-based retroviral vector system, utilising luciferase expression as a measure of infectivity ,40. The ApaI restriction site in gag and the ClaI restriction site at the beginning of IN were introduced into a single genome containing L10F, V32I, L33F, M46I, I47V, I54L, A71T, I84V, L89V and L90M in PR, and M41L, D67N, L74V, M184V, T215Y, K219E, L100I, K103N and N348I in RT. These resistance mutations were present in all single genomes chosen for the analysis. The unique restriction sites were then used for the subcloning of patient-derived PR + RT (ApaI and ClaI) or IN (ClaI and EcoRI) into p8.9NSXClaI+, to generate ptA_PR + RT and patient-derived IN only vectors, respectively , according to the manufacturer’s protocol. The presence of mutations was verified by sequencing.In addition, the ectively . The genIN control, as described previously [The two other vectors used in this assay are the retroviral expression vector, pCSFLW, which encodes the firefly luciferase reporter gene, and pMDG, which encodes the vesicular stomatitis virus G protein. Viruses were generated by cotransfection of 293T cells as previously described, and then used to infect fresh 293T cells in the presence of serially diluted RAL or EVG ,42. The eviously .RAL and EVG  were obtained from the National Institute for Biological Standards and Control.50 values and replicative fitness were calculated using the Student’s t test tool, available on www.graphpad.com (accessed on 10 June 2021). p values which were <0.05 were regarded as significant.Differences in ECThe single-genome sequences generated and used in this study have been submitted to GenBank and assigned the accession numbers MH663797-MH663975.


Aedes aegypti and Aedes albopictus mosquitos and is thus capable of replicating in both human and mosquito cells. CHIKV has a broad tropism in vivo, capable of replicating in various tissues and cell types but largely excluding blood cells. This was reflected in vitro by a broad array of adherent cell lines supporting CHIKV infection. One marked exception to this general rule is the resistance of the lung cancer-derived A549 cell line to CHIKV infection. We verified that A549 cells were restrictive to infection by multiple alphaviruses while being completely permissive to flavivirus infection. The adaptive growth of a primary CHIKV strain through multiple passages allowed the emergence of a CHIKV strain that productively infected A549 cells while causing overt cytopathic effects and without a fitness cost for replication in otherwise CHIKV-susceptible cells. Whole genome sequencing of polyclonal and monoclonal preparations of the adapted virus showed that a limited number of mutations consistently emerged in both structural (2 mutations in E2) and non-structural proteins (1 mutation in nsP1 and 1 mutation in nsP2). The introduction of the adaptive mutations, individually or in combinations, into a wild-type molecular clone of CHIKV allowed us to determine the relative contributions of the mutations to the new phenotype. We found that the mutations in the E2 envelope protein and non-structural proteins contributed significantly to the acquired phenotype. The nsP mutations were introduced in a split-genome trans-replicase assay to monitor their effect on viral genome replication efficiency. Interestingly, neither mutation supported increased viral genomic replication in either Vero or A549 cells.Chikungunya virus (CHIKV) is an emerging arthropod-borne virus that has spread globally during the last two decades. The virus is mainly transmitted by  Aedes mosquito, mainly Aedes aegypti and Aedes albopictus [Aedes aegypti was the primary vector of CHIKV, limiting its main distribution primarily to Africa and Asia. However, mutations in the envelope proteins (E1 and E2) have enabled CHIKV to use Aedes albopictus as an insect vector more efficiently. This allowed CHIKV, in combination with climate change and increased global travel and trade, to spread to previously unaffected regions  [Aedes albopictus was first reported in 2005, allowing an increased midgut infection of Aedes albopictus but not of Aedes aegypti [Aedes albopictus adaptation have been identified in E2  is a single stranded RNA virus transmitted to humans through the bite of an infected bopictus . CHIKV wbopictus , and thebopictus . CHIKV ibopictus ,4,5. Unt Europe) ,7. The E aegypti . Since tnd K252Q ). Interebopictus .Alphavirus genus of the Togaviridae family. It is a small (60–70 nm) enveloped virus carrying a single-stranded positive sense RNA genome containing two open reading frames that respectively encode non-structural and structural proteins. The non-structural proteins nsP1, nsP2, nsP3, and nsP4 are directly translated from the genomic RNA and together form the replication complex. The replication complex is responsible for synthesizing the complementary negative-sense RNA viral genomes and 26S subgenomic (SG) RNAs. This SG mRNA is used to translate the structural proteins capsid, E3, E2, 6k, TF, and E1. Both non-structural and structural proteins are synthesized as precursor polyproteins that are proteolytically cleaved in individual proteins [CHIKV belongs to the To complete its lifecycle, the virus replicates in the cells of both its mammalian and insect host. In humans, CHIKV is introduced in the skin by a mosquito bite and subsequently infects and replicates in various tissues and cell types in the skin, joints, liver, muscle, and the secondary lymphoid organs where fibroblast, epithelial cells, and macrophages were described to be infected ,14. In vIn this study, we applied viral passaging to adapt CHIKV to productive replication in restrictive A549 cells and subsequently characterized the adaptive mutations in the viral genome. The in vitro virus evolution system provides insights into the genetic plasticity of the virus, and the role of mutated viral proteins in overcoming cellular restrictions provides hints into the presence of cellular restriction factors as well. Interestingly, we found four non-synonymous amino acid substitutions, two in E2, one in nsP1, and one in nsP2, while one synonymous mutation was found in E1. The mutations in the ns-proteins were observed after only two passages, while the mutations in the structural proteins were identified in passage P7. The nsP1 mutation contributed most to the increased viral replication observed in both A549 and Vero cells. The adaptive mutations in E2 seemed specific to A549 cells and did not support increased viral replication in Vero cells, although viral attachment was not hampered. Using a trans-replicase assay, we could not identify an effect of the nsP mutations on the efficiency of the viral replication complex. Our results confirm the genomic plasticity of CHIKV that allows a rapid adaptation to cells, which are naturally restrictive to infection by this virus.2 in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine. Vero cells (ATCC CCL-81) were cultured at 37 °C and 7% CO2 in Eagle’s Minimal Essential Medium (EMEM) supplemented with 10% FBS and 1% L-glutamine.A549 cells (ATCC CCL-185) were cultured at 37 °C and 7% COThe following viruses were used: a reporter CHIKV construct with a nanoluciferase gene inserted in nsP3 , CHIKV 6 A549 cells. After every passage, aliquots of the supernatant were stored at −80 °C. Viral RNA present in the cell-free supernatant was quantified by RTqPCR using SYBR Green Master Mix  and primers E3F: AGTCTTGCCATCCCAGTTATGTGC; E3R: GCGTCGCTGGCGGTGGGGAG. The amount of viral envelope proteins present in the supernatant was determined by Western blot. As a primary antibody, anti-E2  was used, followed by incubation with the secondary anti-mouse antibody .A549 cells were infected with the Indian-Ocean lineage (IOL) CHIKV strain. In a blind passage, the supernatant was transferred in a 1/1000 dilution to a new 6-well containing 1 × 10® Luciferase Assay System  on the TriStar LB 941 Multimode Microplate Reader .A549 and Vero cells were infected with a Nanoluc CHIKV. A serial dilution of the reporter virus stock was made in a culture medium containing 2% FBS. 24h after infection, nanoluciferase activity was measured using Nano-GloNear full-length CHIKV genomes of the polyclonal or monoclonal viruses were sequenced using the MiSeq system . Briefly, viral RNA was purified , eluted in DNase/RNase-free water, and the viral genome RT-PCR amplified in two overlapping fragments using primers . Dual inAll mutants were introduced separately or together in pBR332-ChikFlic. Seven different constructs were made: E2-T196K, E2-H232R, E2-T196K-H232R, nsP1-M314V, nsP2-H687Y, nsP1-M314V + nsP2-H687Y and E2-T196K-H232R + nsP1-M314V + nsP2-H687Y using primers nsP1-M314V-F: GTGTGCAAGACTACCGACACGGTTG; nsP1-M314V-R: CAGGAATCCGTCTGCGTGGTGGG, nsP2-H687Y-F: TACACACCTTTTCGCATACACCATTACC, nsP2-H687Y-R: GATGTTTATGACCACTAGGTCATACC, E2-T196K-F: AGGTGCGGTACAAGTGTAATTGCGG, E2-T196K-R: TCTGGCCATTGACTGTGATCTTTACGTTGC, E2-H232R-F: GCAAAAAGTGGCAGTATAACTCCCC, E2-H232R-R: GATTGGTGACCGCGGCATGACATTG. Clones were verified by Sanger sequencing. Viral RNA was produced using the SP6 mMessage mMachine transcription kit  following the manufacturer’s instructions. Viral RNA was transfected in Vero cells, and after 48 h, the viral stocks were harvested, filtered (0.45 µm), and stored at −80 °C. Viral stocks were titrated on Vero and A549 cells using a plaque assay, and TCID50 was calculated using the Spearman-Kärber method . Stocks ® Reporter Assay System (Promega). Mutant TRA expressing plasmids were produced by TRA replicase plasmid digestion with EcoRV and PCR fragments carrying the nsP1 and nsP2 mutations insertion after PCR amplification from the corresponding ChikFLic molecular clone using primers nsP1-TRA-M314V-F: GTGTGCAAGACAACCGATACCG, nsp1-TRA-M314V-R: CAGAAAGCCGTCGGCGTGGTGGG, nsP2-TRA-H687Y-F: TACACCCCTTTCCGCATCCACCAC, nsP2TRAH687Y-R: GATGTTGATCACGACCAGGTCG. Clones were verified by Sanger sequencing.The trans-replicase assay as described before  was util2 for 90 to 120 min. The virus dilutions were removed and the cells were washed two times with PBS. One ml of medium containing 10% FBS and 1% carboxy-methyl-cellulose (CMC) high viscosity was added, and the cells were incubated at 37 °C and 7% CO2 for 6 days. Subsequently, cells were washed two times with PBS and fixed with 1 mL of 3.7% formaldehyde for 30 min. Finally, cells were stained with 1 mL of naphthalene black solution for 30 to 60 min, after which the wells were washed with water.80,000 A549 cells or 200,000 Vero cells were plated in a 24 well plate one day before infection. Cells were washed once with PBS and virus dilutions prepared in cell culture medium containing 2% FBS was added, and mixtures were incubated at 37 °C and 7% CO2 for 2 hr. The virus was removed and the cells were washed twice with PBS. Three ml of a 0.3% agarose overlay in DMEM was added, and the cells were incubated at 37 °C and 7% CO2 for 6 days. Subsequently, 1.5 mL of a 0.5% agarose overlay containing Neutral Red was added and incubated for another 20 min. A pipette tip was inserted into the agar overlay to pick up a single plaque. The tip was immersed in cell culture media, and the clonal virus was expanded on A549 cells.To prepare plaque-purified monoclonal viruses, 600,000 A549 cells were seeded in a 6 well plate one day before infection. Cells were washed once with PBS, virus dilutions prepared in cell culture medium containing 2% FBS were added, and mixtures were incubated at 37 °C and 7% CO100,000 A549 or Vero cells were plated in a 24 well plate. The next day, a cold medium was added to the cells for 30 min at 4 °C. Cells were incubated with 10-fold dilutions of WT CHIKV or equal amounts of adapted CHIKV for 90 min at 4 °C. Stocks of WT and adapted CHIKV were quantified by RTqPCR. Cells were washed four times in a cold medium with 2% FBS. After washing, the cells were re-suspended in TRIzol. RNA was purified, and viral genomes were quantified using RT-qPCR using SYBR Green Master Mix  and primers E3F: AGTCTTGCCATCCCAG-TTATGTGC; E3R: GCGTCGCTGGCGGTGGGGAG. A standard curve was produced from in vitro transcribed CHIKV RNA to calculate CHIKV vRNA copies.To verify the restriction of CHIKV replication in A549 cells, the infectivity of CHIKV and related alphaviruses was assessed on both A549 and Vero cells using both a plaque assay and a nanoluciferase reporter virus. For the plaque assay, a clinical isolate of CHIKV (IOL 06-049) was used. We demonstrated that A549 cells were restrictive for CHIKV A. Also, To gain insight into the determinants that govern the restriction of CHIKV replication by A549 cells, the IOL 06-049 CHIKV strain was serially passaged eight times. During the continuous passaging, the observed cell death gradually increased, coinciding with an increasing amount of viral RNA and viral envelope proteins in the supernatant A. To ideTo determine the relative contribution of the adaptive mutations on the acquired phenotype, the mutations were introduced in a CHIKV molecular clone as individual mutations and together. Seven different constructs harboring following mutation(s) were made: E2-T196K, E2-H232R, E2-T196K-H232R, nsP1-M314V, nsP2-H687Y, nsP1-M314V + nsP2-H687Y and E2-T196K-H232R + nsP1-M314V + nsP2-H687Y. Viruses were rescued, obtained viral stocks were titrated on both Vero and A549 cells using a plaque assay, and TCID50 was calculated and normalized for viral input RNA (vRNA).In A549 cells, the viral envelope E2-T196K mutation contributed significantly to the increased viral replication of the adapted virus. The E2-T196K mutant stimulated viral replication 238-fold while a small or no additional effect of the E2-H232R mutation alone or in combination with E2-T196K, respectively, was observed . InteresIn contrast to the A549 cells, when introducing the envelope E2 mutants in Vero cells either separately or in combination, viral replication was impeded . After iIn conclusion, the envelope localized E2-T196K-H232R mutations are required to adapt CHIKV to A549 cells but suppress replication in Vero cells. The nsP1-M314V mutation allows increased viral replication in both A549 and Vero cells. The nsP2-H687Y mutant has minimal effect on viral replication in both cell types. The four mutations acquired by adapting CHIKV to A549 cells not only allowed increased infectivity in A549 cells but also in Vero cells.Firefly luciferase (Fluc) and Gaussia luciferase (Gluc) reporter gene respectively. As such, the FLuc activity reflects the overall activity of the replication complex, and the Gluc expression, controlled by the subgenomic (SG) promotor, correlates with late-stage SG RNA synthesis. Replicase expressing plasmids carrying nsP1-M314V and nsP2-H687Y alone or together were created and tested alongside the WT and nsP4-GAA catalytic dead mutant as controls in both A549 and Vero cells. The different luciferase activities were measured at two time points, 17 h and 27 h (A split-genome trans-replicase assay (TRA)  was usedand 27 h . No cleaand 27 h A. By conand 27 h A. As expand 27 h B. The adand 27 h B,C. Afteand 27 h B. Similaand 27 h C. Overaland 27 h B,C. Whiland 27 h , no effeand 27 h . Neither7 vRNA copies), in three 10-fold dilutions, were allowed to bind for 90 min at 4 °C to both A549 and Vero cells. After extensive washing, the cell-associated viral RNA was determined by RTqPCR , neither of the nsP mutations identified here were found in these regions.Therefore, we also consider that the nsP1 mutation, shown to be critical for increased viral replication, plays a role outside the viral replication complex. This indicates the presence of a so-far unknown restriction mechanism for alphaviral replication in A549 cells. The antiviral factor tetherin is a potential candidate as it is known that nsP1 downregulates its expression . The nsPAedes albopictus mosquitoes. Our findings re-emphasize the genomic plasticity of alphaviruses to overcome restriction barriers at the cellular level.In conclusion, after serial passaging of CHIKV on restrictive A549 cells, four mutations were rapidly acquired to enable CHIKV to replicate in A549 cells. Of these mutations, two were found in the envelope proteins and two in the non-structural proteins. While the mutations in the non-structural proteins also contributed to an increased CHIKV replication in permissive Vero cells, the mutations in the structural proteins impeded CHIKV replication in Vero cells. Previous adaptive mutations in CHIKV E1 have been reported that facilitated transmission by


Asbestosis and silicosis are characterized by diffuse or nodular interstitial lung fibrosis resulting from exposure to asbestos or silica dust, respectively. This study was designed to detect programmed cell death protein (PD-1)/programmed death ligands (PD-Ls) expression in patients with asbestosis and silicosis and to explore the possible clinical significance of PD-1/PD-Ls expression in patients with the two diseases.Thirty patients with asbestosis, 23 patients with silicosis and 25 healthy controls were consecutively recruited and provided informed consent to participate in the study. Clinical data were collected from patients’ clinical charts. PD-1/PD-Ls expression in peripheral blood (PB) was detected using flow cytometry.+ or CD8+ peripheral T cells from patients with asbestosis and silicosis than on cells from healthy controls. Similarly, significantly lower PD-L1 and PD-L2 expression was detected on CD14+ monocytes from patients with asbestosis and silicosis than on cells from healthy controls. In addition, no significant differences in PD-1, PD-L1 and PD-L2 expression were observed between the asbestosis and silicosis groups. Moreover, the proportions of PD-1+ CD4+ T cells and PD-1+ CD8+ T cells in patients with asbestosis were positively correlated with the percentage of forced vital capacity predicted.PD-1 was expressed at significantly lower levels on CD4+ T or CD8+ T cells in PB was positively correlated with the asbestosis severity, implying that pulmonary fibrosis development in patients with asbestosis was positively correlated with the downregulation of the PD-1/PD-Ls pathway.Decreased PD-1 expression on CD4The online version contains supplementary material available at 10.1186/s12890-021-01531-5. Interstitial lung disease (ILD), or diffuse parenchymal lung disease, is a group of diseases that appear in the lung interstitium and alveolar cavity, resulting in alveolar capillary dysfunction . PulmonaAsbestosis and silicosis are two types of pneumoconiosis. Asbestosis is characterized by diffuse interstitial pulmonary fibrosis caused by long-term asbestos exposure. The most common clinical symptom of asbestosis is progressive dyspnoea on exertion. The disease is associated with a restrictive lung impairment and decreased diffusing capacity . Chest hAsbestosis and silicosis are currently incurable and may be progressive even after dust exposure cessation . The patProgrammed cell death protein 1 (PD-1), also known as CD279, is a member of the B7/CD28 immunoglobulin superfamily. PD-1 has two ligands, programmed death ligand-1 (PD-L1 or CD274) and programmed death ligand-2 (PD-L2 or CD273) [+ or CD8+ T cells in peripheral blood (PB) was positively correlated with the disease severity of asbestosis.No existing study has shown whether the PD-1/PD-Ls signalling pathways play a role in cellular immunology in pulmonary fibrotic diseases caused by asbestos or silica. We explored the expression of members of the PD-1/PD-Ls pathway in patients with asbestosis and silicosis and further elucidated the relationship between the expression of these molecules and clinical indexes. Here, we described the expression of components of the PD-1/PD-Ls pathway in human pulmonary fibrosis, which provided a deeper understanding of the mechanism underlying the development of pulmonary fibrosis and might provide clues for the identification of new drug targets for the treatment of pulmonary fibrosis. Our study found that decreased PD-1 expression on CD4Thirty patients with asbestosis and 23 patients with silicosis were consecutively recruited from the Department of Occupational Medicine and Toxicology, Beijing Chao-Yang Hospital. Clinical data, including age, sex, current and past medical history, occupational history and pulmonary function values, were collected from patients’ clinical charts.2 tetrahedra and Mg(OH)2 octahedra. (2) Patients’ lungs exhibited honeycombing and septal and interlobular fissure thickening, as well as diffuse pleural thickening and/or pleural plaques, on chest HRCT; and (3) patients in whom other known causes of ILD were excluded [Asbestosis was diagnosed in patients based on the criteria listed below. (1) Patients had a definite occupational history of exposure to chrysotile dust through asbestos product manufacturing and a prolonged latency. Chrysotile asbestos is a general term for serpentine asbestos. Serpentine asbestos is a trioctahedral silicate mineral with a double-layer structure composed of SiOexcluded . Silicosexcluded . Twenty-All patients were in a stable clinical state and showed no clinical, radiographic, or electrocardiographic signs of heart failure, acute pulmonary infection, or pulmonary thromboembolism. None of the patients were receiving treatment with corticosteroids and/or immunosuppressants. The study protocol (No. 13-KE-61) was approved by the Ethics Committee of Beijing Chao-Yang Hospital, Capital Medical University. All the participants provided written informed consent before enrolling in the study.HRCT was performed with 1-mm sections and 1-s scan times. The apex-base scans, which included both lungs in the field of view, were performed at 10-mm intervals. The CT images were reviewed independently by two experienced thoracic radiologists, and the CT patterns were obtained and recorded by two observers (MHQ and YQ). Patients who showed evidence of coexisting emphysema  on HRCT were not included in the study.2), forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), FEV1/FVC ratio, total lung capacity (TLC), and diffusing capacity of the lung for carbon monoxide (DLCO)  of each subject.Pulmonary function tests were performed according to American Thoracic Society guidelines . We recoPB samples were collected from each subject, placed in ethylenediaminetetraacetic acid-treated tubes and processed to measure PB mononuclear cell (PBMC) counts for subsequent flow cytometry procedures. The blood samples were layered onto Ficoll-Paque Plus , centrifuged (400 g for 20 min at 21 °C), and then PBMCs were harvested. The cells were washed once with divalent cation-free Hanks balanced salt solution at 300 g for 5 min at 4 °C. The PBMCs were subsequently resuspended, and the number of viable cells was counted.5 cells were acquired for subsequent data analyses.Freshly obtained human PBMC samples were stained with anti-hCD273-FITC, anti-hCD279-PE, anti-hCD4-APC, anti-hCD8-PerCP, anti-hCD14-FITC, anti-hCD274-PE, anti-hCD45-PerCP and matched isotype control antibodies and incubated in a dark room for 30 min at 4 °C. Anti-hCD8-FITC, anti-hCD4-APC, anti-hCD28-PE, anti-hCD69-PerCP, anti-hHLA-DR-PerCP, and anti-hCD38-PE antibodies were used for surface marker staining of effector T cells. All antibodies were purchased from BD Biosciences . Data acquisition and analysis were performed with Canto II Software . Approximately 10t test, Wilcoxon’s rank-sum test, or the chi-square test, as appropriate, and correlations were assessed by calculating Pearson’s correlation coefficient or Spearman’s rank correlation coefficient. P < 0.05 was considered statistically significant. Statistical analyses were performed with SPSS for Windows V17.0  and GraphPad Prism 5 software .Data are presented as the means ± standard deviations (SD) or as medians and interquartile ranges (IQRs) when appropriate. Group comparisons were performed using analysis of variance, Student’s P < 0.01); however, no significant difference in age was observed between the silicosis and healthy control groups. The PaO2 level was significantly lower in the asbestosis group than in the silicosis and control groups (P < 0.05). Pulmonary function tests indicated that restrictive ventilation and/or impaired gas exchange were present in the asbestosis group, while normal lung function or mild airflow limitation and a mildly decreased DLCO were present in the silicosis group.Thirty patients with asbestosis, 23 patients with silicosis and 25 healthy controls were evaluated in this study Table . None of+ or CD8+ T cell fractions among the three groups (P > 0.05). We first investigated PD-1 (CD279) expression in PB. As shown in Fig. + T cells in PB from the asbestosis (mean 7.753%) and silicosis (mean 6.676%) groups than in PB from the healthy control group . Similarly, PD-1 expression on CD8+ T cells in PB was also significantly decreased in the asbestosis (mean 9.556%) and silicosis groups (mean 9.132%) compared to the healthy control group   and silicosis (mean 0.310%) groups compared to the healthy control group (mean 0.705%) (P < 0.05)  and silicosis (mean 0.544%) groups compared to the healthy control group . Similarly, lower percentages of circulating PD-L2+ CD14+ monocytes were observed in the asbestosis (mean 0.541%) and silicosis (mean 0.525%) groups than in the healthy controls   and PD-L2 (CD273) expression on CD14 and PD-L+CD8+ T cells were significantly lower in patients with asbestosis and silicosis than in the healthy controls (P < 0.05)  , and the percentage of CD69+CD8+ T cells tended to be higher in the silicosis group than in the healthy control group  and PD-1+CD8+ T cells  were positively correlated with the percentage of FVC predicted in the asbestosis group . We did not identify any correlations between PD-1 expression and DLCO% predicted, TLC% predicted, PaO2, or CPI  is also a chronic fibrotic lung disease. IPF is characterized by the excessive accumulation of extracellular matrix in the interstitial and alveolar spaces, leading to scarring and the destruction of the normal pulmonary epithelium [In our study, we first observed decreased expression of molecules in the PD-1/PD-Ls pathway in patients with asbestosis and silicosis. More importantly, the proportions of CD4ithelium . IPF, a ithelium . In a prithelium . Howeverithelium , 34. Intithelium .In the present study, PD-1 and PD-L1 expression showed different trends in patients with asbestosis and silicosis compared with patients with IPF. A potential explanation for some of the differences is provided below. Asbestosis and silicosis have different aetiologies than IPF. Asbestosis and silicosis are a group of heterogeneous fibrotic lung diseases that develop through the inhalation of asbestos and small silica crystals. Macrophages, key regulators of fibrosis that reside in the small airways, ingest silica crystals and asbestos and produce numerous profibrotic soluble mediators, chemokines, and matrix metalloproteases, thereby controlling extracellular matrix (ECM) deposition. Monocytes and macrophages function as antigen-presenting cells that send costimulatory and coinhibitory signals to T cells and thus have the ability to promote Th2 responses that induce and activate TGF-β1 in macrophages through an IL-13 and matrix metalloproteinase (MMP)9-dependent mechanism , 37. Alt+ CD8+ T cells was positively correlated with the percentage of CD28+ CD8+ T cells in PB from patients with asbestosis and silicosis. Therefore, we hypothesized that lower PD-1 expression on T cells was correlated with greater T cell activation and greater CD28 depletion.Downregulation of the PD-1/PD-Ls pathway potentially leads to overactivation of T cells. Therefore, we detected the effector T cell activation status in patients with asbestosis and silicosis. Activated T cells were detected in patients with asbestosis and silicosis, and these cells exhibited lower CD28 expression and higher HLA-DR expression. CD28, a marker of early T cell activation, is the second T cell activation signal, while HLA-DR is a marker of late T cell activation , 42. CD2To our knowledge, the present study is the first to explore the PD-1/PD-Ls pathway in occupational pulmonary diseases. We performed a comprehensive analysis of PD-1/PD-Ls expression in PB from patients with asbestosis and silicosis. These findings may facilitate the development of therapies for pulmonary fibrosis. However, this study had some inherent limitations. First, the control population was significantly younger than the asbestosis group, whereas it was not different from the silicosis group. A previous study showed that PD-1 mRNA and protein expression levels increase with ageing . Therefo+ T or CD8+ T cells in PB was positively correlated with the percentage of FVC predicted, implying a link between pulmonary fibrosis development and downregulation of the PD-1/PD-Ls pathway. Based on the results, the PD-1/PD-Ls pathway potentially represents a therapeutic target.In conclusion, the data provided further evidence showing that the PD-1/PD-Ls pathway plays a role in the pathogenesis of asbestosis and silicosis. We first showed the downregulation of the PD-1/PD-Ls pathway on cells in PB from patients with asbestosis and silicosis. Importantly, lower PD-1 expression on CD4Additional file 1. Fig. S1: Analysis of PD-1 expression patients with different stages of asbestosis. PD-1 expression on CD4+ (a) or CD8+ (b) T cells in patients with different stages of asbestosis.Additional file 2. Fig. S2: Correlations between PD-1 expression with DLCO% predicted and TLC% predicted in patients with asbestosis. Correlations between PD-1+CD4+ T cell fractions and DLCO predicted  (a), PD-1+CD8+ T cell fractions and DLCO predicted  (b), PD-1+CD4+ T cell fractions and TLC% predicted  (c); and PD-1+CD8+ T cell fractions and TLC% predicted  (d) are shown. DLCO, diffusing capacity of the lung for carbon monoxide; TLC: total lung capacity.Additional file 3. Fig. S3: Correlations between PD-1 expression with PaO2 and CPI in patients with asbestosis. Correlations between PD-1+CD4+ T cell fractions and PaO2  (a), PD-1+CD8+ T cell fractions and PaO2  (b), PD-1+CD4+ T cell fractions and CPI  (c), and PD-1+CD8+ T cell fractions and CPI  (d) are shown. CPI: composite physiological index.