Considering the lack of effective treatments against COVID-19, wastewater-based epidemiology (WBE) is emerging as a cost-effective approach for real-time population-wide SARS-CoV-2 monitoring. Here, we report novel molecular assays for sensitive detection and mutational/variant analysis of SARS-CoV-2 in wastewater. Highly stable regions of SARS-CoV-2 RNA were identified by RNA stability analysis and targeted for the development of novel nested PCR assays. Targeted DNA sequencing (DNA-seq) was applied for the analysis and quantification of SARS-CoV-2 mutations/variants, following hexamers-based reverse transcription and nested PCR-based amplification of targeted regions. Three-dimensional (3D) structure models were generated to examine the predicted structural modification caused by genomic variants. WBE of SARS-CoV-2 revealed to be assay dependent, and significantly improved sensitivity achieved by assay combination (94%) vs. single-assay screening (30%–60%). Targeted DNA-seq allowed the quantification of SARS-CoV-2 mutations/variants in wastewater, which agreed with COVID-19 patients’ sequencing data. A mutational analysis indicated the prevalence of D614G (S) and P323L (RdRP) variants, as well as of the Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.1.1.7/alpha variant in clinical samples of the same period of the third pandemic wave at the national level. Our assays provide an innovative cost-effective platform for real-time monitoring and early-identification of SARS-CoV-2 variants at community/population levels. A cluster of severe pneumonia cases of unknown origin, linked to the Huanan seafood wholesale market in Wuhan, Hubei Providence, China, were reported by the Chinese Health Authorities on 31 December 2019. Sequencing-based analysis of lower respiratory tract samples  identified a novel beta-coronavirus sharing >85% sequence similarity with a bat severe acute respiratory syndrome (SARS)-like coronavirus (CoV), provisionally indicated as 2019-nCoV, to be the causative pathogen of the coronavirus disease 2019 (COVID-19) [CoVs constitute a family of enveloped positive-strand RNA viruses of 26–32 kilobases (kb) . ThereafDue to the lack of established herd immunity and effective therapeutic treatments , the ideWastewater-based epidemiology (WBE) has been successfully applied in numerous case studies worldwide, with the most extensive so far being the estimation of drug consumption ,12. AlthNonetheless, the development and optimization of the analytical protocols needed for SARS-CoV-2 WBE are very challenging due to the nature of the sample , the moderate efficiency of SARS-CoV-2 concentration, and the quality/degradation of the RNA template, for example, due to excessive amounts of detergents in wastewater samples . To overBioinformatic analysis with the ScanFold server provided an informative overview regarding the highly predicted stable regions of SARS-CoV-2 RNA. Specifically, the analyses led to the identification of 300 nt sequences bearing negative z-scores, which were predicted to possess low MFE native values, thus, being more stable. Sequences with notable negative z-scores were found to distribute across the SARS-CoV-2 genome .On the basis of the results from the in silico stability analysis, we designed four different nested PCR/real-time PCR assays on: (a) N gene, nucleocapsid phosphoprotein (N assay); (b) ORF1ab gene, helicase (helicase assay); (c) ORF1ab gene, nsp3 (NSP3 assay); (d) ORF3a gene, ORF3a protein (ORF3a assay).3 to 2.5 RNA copies/reverse transcription) were analyzed by the novel nested PCR/real-time PCR assays, and a newly in-house developed assay using the CDC proposed “2019-nCoV_N1” set of primers and probe (CDC/2019-nCoV_N1-based assay) (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html accessed on 1 July 2020).To investigate the impact of our novel assays on SARS-CoV-2 detection sensitivity, serial dilutions of SARS-CoV-2 complete genome RNA control, covering nine orders of magnitude . The application of the nested PCR/real-time PCR assays improved the detection performance in up to two cDNA copies/PCR reaction for N, helicase, and ORF3a assays. Furthermore, the NSP3 assay detected SARS-CoV-2 RNA in up to five cDNA copies/PCR reaction . SimilarT) values of the positive samples per assay are presented in Our novel nested PCR/real-time PCR assays along with the CDC/2019-nCoV_N1-based assay were applied to 30 wastewater samples (S1–S30) obtained from Wastewater Treatment Plant (WWTP) of Athens from August (n = 2), September (n = 16), and October (n = 12) 2020. The cycle threshold , while 13 samples were negative . Among the 17 positive samples, the CDC/2019-nCoV_N1-based assay was positive in five samples (sensitivity 29.4%). In this study, the developed novel nested real-time PCR assays resulted in the following detection of SARS-CoV-2 RNA: (a) N assay, 10 samples (sensitivity 58.8%); (b) NSP3 assay, 9 samples (sensitivity 52.9%); (c) helicase assay, 7 samples (sensitivity 41.2%); and (d) ORF3a assay, 5 samples (sensitivity 29.4%). As expected, the application of the novel nested real-time PCR assays resulted in significantly lower CT values vs. the CDC/2019-nCoV_N1-based assay , thus, hMost importantly, in the majority of the positive samples , SARS-CoV-2 RNA was detected by a single assay, while five  and one  samples were scored positive by three or four assays, respectively. In this regard, a significantly improved detection specificity was achieved by the following combinations: (a) N and NSP3 assays, 14/17, sensitivity 82.4%; (b) N, NSP3, and helicase assays, 16/17, sensitivity 94.1%. Our results clearly highlight the significantly improved sensitivity by the combination of more than one assays, and that the detection of SARS-CoV-2 in wastewater is genomic region dependent, as different results are being obtained by targeting different regions of SARS-CoV-2 genome.Five well-characterized missense mutations, D614G (23403A>G)-S gene, Q57H (25563G>T)-ORF3a gene, P323L (14408C>T)—ORF1ab/RdRP gene, R203K (28881G>A)—N gene, and G204R (28883G>C)—N gene, were initially targeted as proof-of-principle of our methodology to perform the analysis and quantification of SARS-CoV-2 mutations/strains in wastewater samples. To perform DNA-seq, novel nested PCR assays, against the above-mentioned point mutations, were designed and applied in September (n = 5) and October/November (n = 8) 2020 samples. The PCR products were used to generate DNA-seq barcoded libraries corresponding to September and October/November 2020 samples. The genomic variation profiling (list of existing SNVs or indels) of SARS-CoV-2 is summarized in https://www.gisaid.org/ accessed on 1 May 2021) and Nexstrain (https://nextstrain.org/sars-cov-2 accessed on 1 May 2021) databases, highlighting the significant prevalence of the G164 strain in percentage of >98% and ~100% worldwide and in Europe, respectively. Similarly, the P323L (14408C>T) substitution in the ORF1ab/RdPR gene was also prevalent, ~99.9%, in both time periods of sampling, also in agreement with the genomic epidemiology data (~99% worldwide and in Europe). Interestingly, the Q57H (25563G>T) mutation in the ORF3a gene was solely found observed in October/November samples at a percentage of ~47%, which is in accordance with the growing trend observed worldwide during the last months .The analysis highlighted that the G614 strain of SARS-CoV-2, originating from the D614G (23403A>G) point mutation in S gene, was exclusively detected (>99.9%) over the D614 strain. This finding is in line with the genomic data of the GISAID (https://www.rcsb.org/ accessed on 1 May 2021). Moreover, a novel point substitution, A54V (25553C>T), was also detected at a percentage rate of ~9% of September samples, resulting in the change of alanine to valine at position 54 of the ORF3a polypeptide. The amino acids both represent aliphatic, nonpolar neutral residues, and thus are not expected to induce critical structural alterations in ORF3a polypeptide functionality.In addition to the characterized D614G mutation, a previously unknown point mutation within S gene, H625R (23436A>G), was found at a frequency of 5.7% in September samples. The 23436A>G substitution results in the change of histidine to arginine at position 625 of the spike protein. Even though two similar amino acids are substituted, based on the in silico protein structure analysis, H625R leads to subtle alterations in the spike protein folding . As a reFocusing on the N gene, a declining trend was observed for the missense point mutations 28881G>A (R203K) and 28883G>C (G204R), as well as the synonymous substitution 28882G>A, from ~90% in September to ~70% in October/November samples. Our data agree with the declining trend of the above-mentioned substitutions worldwide , although their absolute percentage in Greek samples remain significantly higher. Interestingly, a point substitution 28884G>T, which has been observed in ~1% worldwide, was overrepresented in our datasets, 09/2020, ~70% and 10–11/2020, ~35%. More importantly, our analysis highlighted the significant correlation of 28884G>T and 28883G>C point mutations, resulting in a novel amino acid substitution from glycine to leucine in position 204 (G204L) as compared with the single 28883G>C (G204R) or 28884G>T (G204V) substitutions. Moreover, the sequencing data revealed the existence of a simultaneous 4nt deletion following position 28880 (28881_28884del) and a 4 nt insertion at position 28885 (28885_28886insACAT). These two simultaneous indels lead to the R203K and G204H missense mutations of the nucleocapsid protein. The findings obviously indicate that R203K co-exists with G204R, G204L, or G204H variations. Although these mutations are located on the linker region (LKR) of the nucleocapsid phosphoprotein, which spans from position 175–254aa, only R203K belongs to the LKR’s crucial Ser/Arg (SR)-rich motif that contains putative phosphorylation sites [Finally, a significant growing trend from 7% in September to 27% in October/November samples was revealed for the missense mutation S194L (28854C>T), in line with a similar trend observed worldwide . Similar to R203K, the S194L mutation is also located on the SR-rich motif of the N protein and involves substitution of the hydroxylic neutral serine with the aliphatic neutral leucine. Since this region regulates the N protein oligomerization upon phosphorylation, the S194L could have a significant impact on protein function; this notion is in accordance with the dramatic changes in the predicted protein structure, and therefore merits further study.To further validate our methodology and to support the need for in-depth epidemiological analysis of existing and/or new emerging variants of SARS-CoV-2, we targeted the mutational analysis of the Spike (S) gene of SARS-CoV-2. In this regard, novel nested PCR assays, against the whole S gene (~4 Kb), were designed and applied in wastewater samples obtained daily in March 2021. The PCR products were used to generate a DNA-seq barcoded library corresponding to March 2021. The analysis led to the conduct of 3.13 million sequencing reads , with a https://covariants.org/per-variant accessed on 20 July 2021) (According to the genetic markers (SNVs) observed , only thly 2021)  regardinConsidering the lack of effective treatments against COVID-19 , accuratΤhe current analytical protocols for WBE of SARS-CoV-2 suffer from reduced sensitivity and false negative results, due to, for example, low viral loads, RNA degradation, and PCR inhibition . To overPreviously, nested PCR approaches have been successfully applied for the detection of SARS-CoV-2 in wastewater ,22,23,24Mutation rates and genetic diversity of RNA viruses are significantly high, providing selective advantage to evolve and adapt to dynamic changes of environments and hosts . DespiteAs the SARS-CoV-2 Spike protein prevails as the main target of COVID-19 vaccines , mutatioIn this regard, we have initially targeted five well-characterized missense mutations spanning different genomic regions of SARS-CoV-2, i.e., D614G (23403A>G), P323L (14408C>T), Q57H (25563G>T), R203K (28881G>A), and G204R (28883G>C), and specific nested PCR amplicons were sequenced using DNA-seq. This approach allowed the quantification of SARS-CoV-2 mutations/variations, and our data highlighted the significant prevalence (>99%) of D614G and P323L mutations in wastewater samples obtained from WWTP of Athens, Greece, during September-November 2020, in line with worldwide data based on COVID-19 patient samples. Additionally, the reported worldwide growing trend of the Q57H mutation was confirmed in our samples, with a percentage of ~47% in samples collected during October/November 2020.Interestingly, a previously unknown missense mutation in the S gene, H625R (23436A>G), was identified in ~6% of September samples. Although H625R involves the substitution of two amino acids with positively charged polar side chains, the in silico structure analysis suggested significant changes in S protein folding. In this regard, specific monitoring of H625R along with other newly emerging mutants in COVID-19 patients and wastewater is necessary to conclude on their potential selective advantage and possible association with SARS-CoV-2 infectivity and effect on existing vaccines.Focusing on N gene, the declining trend of 28881G>A, 28882G>A, and 28883G>C substitutions was confirmed in our samples. Interestingly, the 28884G>A substitution, which reportedly has a ~1% prevalence worldwide, was overrepresented in Athen’s samples  and correlated significantly with 28883G>C, resulting in the G204L substitution of nucleocapsid protein. Moreover, a novel variant originating from a simultaneous 4 nt deletion (28881_28884del) and a 4 nt insertion at position 28885 (28885_28886insACAT), was observed in our samples, leading to the R203K and G204H missense mutations of nucleocapsid protein.Finally, to facilitate the in-depth epidemiological analysis of existing and/or new emerging SARS-CoV-2 variants, we have expanded our methodology towards the mutational profiling of the whole S gene of SARS-CoV-2. The analysis of March 2021 samples as proof-of-principle of the methodology, highlighted that the Β.1.1.7/alpha variant was dominant in wastewater samples from WWTP of Attica, a finding in accordance with the prevalence of Β.1.1.7/alpha variant during the third pandemic wave in Greece.Τhe in silico stability analysis of SARS-CoV-2 RNA was carried out with the ScanFold algorithm . The WuhThe 24 h composite influent wastewater samples were collected from the WWTP of Athens . Influent wastewater samples were collected in precleaned high-density polyethylene (HDPE) bottles, transported on ice to the laboratory and stored at 4 °C. All the collected samples were analyzed immediately after the arrival at the laboratory.g for 30 min at 4 °C to remove debris, bacteria, and large particles. The supernatant was transferred in a clean centrifuge tube, containing 3.5× g PEG and 0.8 g NaCl, mixed at ambient temperature until completely dissolved, and centrifuged at 10,050× g for 2 h, at 4 °C. Most of the supernatant was discarded without disturbing the pellet and the tube was centrifuged at 10,050× g for 5 min, at 4 °C. Finally, the pellet was reconstituted by 500 μL nuclease-free water.Sample concentration was performed immediately after arrival using Polyethylene Glycol 8000  precipitation. In particular, 50 mL of an influent wastewater was centrifuged at 4750× RNA extraction was performed, by 200 μL concentrate using a Water DNA/RNA Magnetic Bead kit  immediately following concentration. The % recovery was calculated by examining three concentrations, 10, 100, and 1000 copies/μL of the EURM-019 SARS-CoV-2 reference material, equal to 78.6%, 82.4%, and 91.3%, respectively ,37. MoreTotal RNA template from wastewater samples was reverse transcribed in a 20 μL reaction containing 5.0 μL RNA, 1.0 μL of 10 mM dNTPs mix , 100 U SuperScript III reverse transcriptase , 50 U RNaseOUT recombinant ribonuclease inhibitor (Invitrogen), and 1.0 μL of 50 μΜ random hexamers (Invitrogen). The mixture of total RNA, dNTPs, and random hexamers was incubated at 65 °C for 5 min, while the reverse transcription took place at 25 °C for 10 min, followed by 50 °C for 50 min. Enzyme inactivation was performed at 70 °C for 15 min. The AMPLIRUN SARS-CoV-2 RNA control  was used as the SARS-CoV-2 complete genome control.Novel nested PCR and nested real-time PCR assays were designed and validated against the identified highly stable regions of SARS-CoV-2 RNA. The Wuhan-Hu-1 reference genome (NC_045512.2) was used for the in silico analysis and design of SARS-CoV-2 specific primers and fluorescent probes .w/v agarose gel, visualized with ethidium bromide staining, and photographed under UV light.A Veriti 96-well fast thermal cycler  was used for the nested PCR assays. The 25 μL of the reaction consisted of 5.0 μL cDNA template (1st PCR) or 2.0 μL PCR product (2nd PCR), 1.0 μL of 10 mM dNTPs mix (Jena Bioscience GmbH), 500 nM of each forward/reverse primer, and 1 U of Kapa Taq polymerase . The thermal protocol consisted of polymerase activation step at 95 °C for 3 min, followed by 15 cycles (1st PCR) or 40 cycles (2nd PCR) of denaturation at 95 °C for 30 s, primer annealing at 60 °C for 30 s and extension at 72 °C for 1 min, followed by a final extension step at 72 °C for 5 min. After the completion of the 2nd reaction, 10 μL of PCR product was electrophoresed on 1.5% The probe fluorescent-based real-time PCR assays were performed in a 7500 Fast Real-Time PCR System (Applied Biosystems). The PCR product of the 1st conventional PCR, as described above, were used as a template for the real-time PCR assay (2nd reaction). The 20 μL reaction consisted of 2.0 μL PCR product, 10 μL Kapa Probe Fast Universal (2X) qPCR Master Mix (Kapa Biosystems), 500 nM of each of the forward, reverse primers, and 125 nM of fluorescent probe. The thermal protocol included an initial polymerase activation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and finally the primer/probe annealing and extension step at 60 °C for 1 min.In-house developed targeted DNA-seq assays, using semi-conductor sequencing technology were performed for the analysis of SARS-CoV-2 variations/mutations in wastewater samples.The five missense mutations, D614G (23403A>G)-S gene, Q57H (25563G>T)-ORF3a gene, P323L (14408C>T)—ORF1ab/RdRP gene, R203K (28881G>A)—N gene, and G204R (28883G>C)—N gene ;The whole Spike (S) gene of SARS-CoV-2 .Nested PCR assays were carried out to amplify the target regions. The Wuhan-Hu-1 reference genome (NC_045512.2) was used for the in silico analysis and the design of specific primers for the analysis of:w/v agarose gel, visualized with ethidium bromide staining, and photographed under UV light.The Veriti 96-well fast thermal cycler (Applied Biosystems) was used for the nested PCR assays. The 25 μL of the reaction consisted of 5.0 μL cDNA template (1st PCR) or 2.0 μL PCR product (2nd PCR), 1.0 μL of 10 mM dNTPs mix (Jena Bioscience), 500 nM of each forward/reverse primer, and 1 U of Kapa Taq polymerase (Kapa Biosystems). The thermal protocol consisted of polymerase activation step at 95 °C for 3 min, followed by 20 cycles (1st PCR) or 40 cycles (2nd PCR) of denaturation at 95 °C for 30 s, primer annealing at 60 °C (most assays) or 57 °C  was employed for the construction of the DNA-seq library, using 1 μg of purified PCR product mix as input. Adapter ligation, nick-repair, and purification of the ligated DNA were carried out, based on the manufacturer’s guidelines. The adapter-ligated library was quantified using the Ion Library TaqMan Quantitation Kit (Ion Torrent) in an ABI 7500 Fast Real-Time PCR System (Applied Biosystems).The sequencing template was generated with emulsion PCR on an Ion OneTouch 2 System (Ion Torrent), using the Ion PGM Hi-Q View OT2 kit (Ion Torrent). Next, the Ion OneTouch ES instrument (Ion Torrent) was used for the downstream template enrichment procedure. Ultimately, semi-conductor sequencing methodology was carried out in the Ion Torrent PGM system for the sequencing of the amplicons.Alignment of the sequencing reads to the reference genome was carried out using the Burrows-Wheeler Aligner (BWA-MEM) . The BAMTo efficiently call variants from the derived NGS datasets, we used the iVar algorithm , which iTo examine the predicted structural modification caused by each detected genomic variant, 3D structure models were generated with the I-TASSER v.5.1 server . For theWe report, here, that SARS-CoV-2 detection in wastewater is assay/genomic region dependent, and that significantly improved detection specificity was achieved by the combination of novel nested PCR/real-time PCR assays against highly stable SARS-CoV-2 genomic regions. Furthermore, we adopted our nested PCR approach for SARS-CoV-2 mutational profiling and variant quantification in wastewater by targeted DNA-seq. Our analysis indicated the high prevalence of the D614G and P323L missense mutations within S and RdRP genes, respectively; the growing trend of Q57H mutation in ORF3a; and the overrepresentation of R203K with G204R or G204L mutations in nucleocapsid phosphoprotein. Moreover, our approach was successfully applied for the mutational profiling of the SARS-CoV-2 S gene in wastewater samples from March 2021, highlighting the prevalence of the Β.1.1.7/alpha variant, in line with the fact that the Β.1.1.7/alpha variant was dominant in clinical samples during the same period of the third pandemic wave in Greece. Further mutational/variant profiling of wastewater samples for a long period of time could clearly shed light on the value of a wastewater-based mutational/variant analysis serving as a leading indicator of SARS-CoV-2 variants spread at community/population levels. We anticipate that our novel methods will further support the global efforts for monitoring SARS-CoV-2 spreading, as well as for detecting the emergence of new viral variants.Provisional patent applications entitled “Detection and mutational analysis of an RNA virus in an environmental sample” were filed by the National and Kapodistrian University of Athens, and the authors M.A., P.G.A., N.V., N.S.T. and A.S. on 11 February 2021 to the Hellenic Industrial Property Organisation (case number 231-0004089064) and on 22 March 2021 to the Patent Cooperation Treaty (PCT application number PCT/EP2021/057316).


Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain  bound to heparan sulfate, while those without a D0 domain  did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed  NK-cell function is dictated by germline-encoded activating and inhibitory receptors that interact with a plethora of ligands expressed on target cells . Major cAn important group of unconventional ligands are carbohydrates expressed on target cells. Indeed, all cells in the body bear a glycocalyx, a term used to refer to the entire array of glycans coating the cell surface . A majorO-sulfated iduronic acid and 6-O- and N-sulfated glucosamine, while NKp44 binds to epitopes containing 2-O-sulfated iduronic acid and N-acetylated glucosamine . We chose KIR3DS1 because it does not bind to HLA class I or class II genes . SuGiven the widespread expression of KIR3DS1 ligands across various human cell lines, we decided to perform a pooled, genome-wide CRISPR/Cas9-based screen [previously described in ] in K562Using high-throughput sequencing, sgRNA barcodes in the cultured post-enrichment cell population were quantified to assess enrichment of specific sgRNA sequences. Remarkably, sequencing showed that the top nine enriched target genes encoded enzymes involved in heparan sulfate biosynthesis Table 1.Given that protein interactions with heparan sulfate are dominated by electrostatic forces between positively-charged basic residues on proteins and negatively-charged sulfate moieties on heparan sulfate , 23, we via electrostatic interactions.To confirm this, we performed surface plasmon resonance on several KIRs to comparatively assess their affinities to heparan sulfate. We included two additional GAGs, hyaluronic acid and chondroitin sulfate, as controls. Remarkably, we found that all Fc constructs of KIRs containing a D0 domain  bound to heparan sulfate but not to chondroitin sulfate or hyaluronic acid, whereas there was no GAG binding observed for KIRs not containing a D0 domain    synthase, an enzyme required to generate PAPS, which is the universal sulfate donor for all cellular sulfotransferase reactions. PAPS synthase can be chemically inhibited by chlorate O-sulfate moieties . To do so, we explored the effects of heparan sulfate elimination on the interaction of KIR3DL1 with its ligand HLA-B*57:01. We performed KIR3DL1-Fc staining of 721.221 cells that were untransduced (null) or stably transduced with HLA-B*08:01 (a non-ligand) or HLA-B*57:01 (a known HLA ligand) and cultured in either normal media or sulfate-free medium containing via surface plasmon resonance and on different cell lines, and predicted to be the dependent on its D0 domain mediating favorable electrostatic interactions. These analyses were extended to other KIRs, resulting in the finding that all tested KIRs containing a D0 domain bound heparan sulfate.Using a genome-wide CRISPR/Cas9 knock-out screen, we identified heparan sulfate proteoglycans as ligands for the activating NK-cell receptor KIR3DS1 on K562 cells. KIR3DS1 binding to heparan sulfate was confirmed via an HLA class I screen that KIR3DS1 binds HLA-F open conformers, a finding that was confirmed by others and was validated biochemically and functionally  exhibit precise compositions of cell-surface heparan sulfate proteoglycans, also referred to as the “heparanosome” signatures. These contain a large variety of interaction motifs for heparan sulfate-binding proteins. Cancer cells of various tissue origins exhibit significantly deregulated expression of heparan sulfate biosynthesis genes, which has been shown to play a role in tumorigenesis affecting various biological processes that are hallmarks of cancer   . Interesgnaling) , 34. TheCis interactions between heparan sulfate and NK-cell receptors like KIR2DL4 have been shown to cause clustering and block trans interactions, allowing for an additional level of tunable control over NK-cell–receptor signaling . It is possible that this form of heparan sulfate may serve as a “decoy” that interrupts intercellular interactions. One study demonstrated that both murine and human NK cells express heparanase upon activation, which was necessary for tumor invasion and prevention of metastases . It is tOverall, we demonstrate the feasibility of performing a genome-wide CRISPR/Cas9 knock-out screen to identify previously unknown ligands of NK-cell receptors, showing the existence of an interaction between several KIRs and heparan sulfate proteoglycans. This interaction may play a wide-ranging role in regulating NK-cell function and immunosurveillance, and studies that further explore the extent and specificity of these interactions may allow for further understanding and manipulating NK-cell biology.2. EBV-transformed B-cell lines (EBV-BCLs) were generated from peripheral blood mononuclear cells from donors bearing specific HLA genotypes; EBV-BCLs were also grown in the same media and conditions. Human samples were used in this study in accordance to protocols approved by Partners Human Research Committee and Institutional Review Board of Massachusetts General Hospital.K562, Jurkat, THP-1, RAJI, and 721.221 cell lines (including HLA transductants) were grown in RPMI-1640 supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 U/mL streptomycin (Gibco) at 37°C/5% COAll KIR-Fc constructs used in this study were produced in mammalian cell lines and purchased from R&D Systems; they included the following: KIR3DS1-Fc, KIR3DL1-Fc, KIR3DL2-Fc, KIR2DL1-Fc, KIR2DL3-Fc, KIR2DL4-Fc, KIR2DS4-Fc, and KIR2DL5-Fc. Cells were stained with KIR-Fc constructs by washing extensively with PBS, incubating for 30–45 min with 25 μg/mL KIR-Fc diluted in PBS at 4°C while shaking, followed by a secondary staining with goat anti-human lgG Fc-PE (Life Technologies) for 30 min at 4°C while shaking. After staining, cells were washed and fixed using 4% paraformaldehyde/PBS (Affymetrix) and flow cytometric analysis was performed on an BD LSR II or LSR Fortessa. Measurement of heparan sulfate expression by flow cytometry was done using anti-HS-FITC .sgRNA Library design. The optimized sgRNA library was designed by first computationally filtering out potential off-target matches (except for sgRNAs that targeted multiple homologs), and also to have high cleavage activity . In total, a library containing 178,896 sgRNAs targeting 18,166 protein-coding genes in the human consensus CDS (CCDS) and 1,004 non-targeting control sgRNAs was constructed. Cell library generation. K562 cells were lentivirally transduced with Cas9/eGFP vector. 2.40 × 108 Cas9-transduced cells were transduced with the viral pool containing the sgRNA library to achieve an average 1000-fold coverage of the library after selection with puromycin for 7 d. Screening procedure. 5 × 107 pooled mutant K562 cells were washed in PBS and stained in 1 mL of 25 μg/mL KIR3DS1-Fc (R&D) for 15 min at 4°C. Cells were then washed with PBS and stained with 1 mL of anti-hIgG(Fc)-PE (Life Technologies) diluted 1:50 in PBS. Fluorescently labeled cells were washed with PBS and stained with anti-PE Microbeads (Miltenyi) following manufacturer’s instructions but using twice the recommended volumes. Magnetically labelled cells were then depleted in an LD column (Miltenyi) following manufacturer’s instructions, and flow-through cells were collected and cultured for 7 d. Screen deconvolution. sgRNA inserts were PCR amplified from 5 million genome equivalents of DNA from flow-through cells. The resultant PCR products were purified and sequenced on a HiSeq 2500 (Illumina); primer sequences are as follows:CRISPR/Cas9-based library of pooled mutant cells was generated similarly to previously described work published in . sgRNA LsgRNA quantification primers:F: AATGATACGGCGACCACCGAGATCTAGAATACTGCCATTTGTCTCAAGR: CAAGCAGAAGACGGCATACGAGATCnnnnnnTTTCTTGGGTAGTTTGCAGTTTT (nnnnn denotes the sample barcode)Illumina sequencing primer:CGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACIllumina indexing primer:TTTCAAGTTACGGTAAGCATATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAAData analysis. Sequencing reads were aligned to the sgRNA library and the abundance of each sgRNA was calculated. Gene-based CRISPR scores (CS) were defined as the average log2 fold change of all sgRNAs targeting a given gene and calculated for the entire screen. To identify enriched genes, the CS distribution was mean-normalized to zero.3-containing media, which consisted of custom-made Advanced RPMI-1640 (Life Technologies) which had 407 μM magnesium sulfate replaced with 407 μM magnesium chloride, 3.03 μM zinc sulfate replaced with 3.03 μM zinc chloride, 5 nM copper (II) sulfate replaced with 5 nM copper (II) chloride, and sodium chloride reduced from 103 mM to 53 mM, and was supplemented with 10% dialyzed FBS (Gibco), 2 mM L-glutamine (Gibco), 100 μg/mL primocin (In vivogen), and 50 mM sodium chlorate  or other NaClO3 concentration if specified, which was balanced with sodium chloride (Sigma-Aldrich).For sulfation inhibition, cells were grown for 48 h (or other time period if specified) in sulfate-free/NaClO5 cells were washed three times with PBS, and then incubated in PBS containing 2 U/mL heparinase II (New England BioLabs), 1 U/mL proteinase K (New England BioLabs), or no enzyme for 1 h at 37°C/5% CO2. Cells were then washed in ice-cold PBS and stained as indicated.For surface enzymatic treatment of cells, 2.5 × 10a values of amino acids  into ExPASy Compute pI/Mw tool, which calculates pI using pKno acids . The KIRno acids , 39 and SPR measurements were conducted in phosphate-buffered saline  containing 0.005% v/v surfactant P20  using a Biacore 3000 system (Biacore AB). To assess binding of various KIR-Fc constructs (R&D) to various GAGs, biotinylated hyaluronate (molecular weight = 29 kDa), biotinylated heparin (molecular weight = 18 kDa) and biotinylated chondroitin sulfate (molecular weight = 50 kDa) , were immobilized onto individual flow cells of an SA (streptavidin) sensor chip  until saturation. A blank flow cell with no immobilized ligand was used as a reference flow cell. Injections of 60 μL of KIR-Fc constructs diluted in PBS to 25 μg/mL were performed at a flow rate of 20 μL/min, with a subsequent 10 min run of buffer to allow sufficient dissociation. Although not presented here, regeneration after each injection was achieved with two pulses of 100 μL of 0.2 M sodium hydroxide (NaOH)  at a flow rate of 100 μL/min. Raw sensograms were corrected by double referencing (subtracting from the reference flow cell response and from PBS injection response). All experiments were done at standard temperature (25°C).Flow cytometry data was acquired on BD LSR II or LSR Fortessa and analyzed using FlowJo software version 10.1 (FlowJo) and analyses were performed using GraphPad Prism 6 (GraphPad Software). Kinetic analyses of SPR data were performed using TraceDrawer 1.9.2 software (Ridgeview Instruments AB) using bivalent analysis model (due to use of dimeric KIR-Fc) to calculate monomeric affinities.https://data.mendeley.com/datasets/3k2rtfv9bx/1.The datasets presented in this study are deposited in the Mendeley Data repository, accessible by KK, WG-B, T-EK, and HD performed experiments and analyzed data. TW provided CRISPR screening tools and analyzed deep sequencing data. AH, SJ, and MA provided significant input regarding the design and execution of this work. KK and WG-B wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by U.S. National Institutes of Health , the National Institute of General Medical Sciences (T32GM007753), Ragon Institute of MGH, MIT and Harvard, Leibniz Institute for Experimental Virology , and German Center for Infection Research (DZIF) .The content is solely the responsibility of the authors and does not necessarily represent the official views or policies of the above-mentioned organizations, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. or German Government.Author HD is employed by Adimab, LLC.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, and there is a critical need to develop efficacious and tolerable anticancer therapies against this aggressive disease. To uncover druggable RMS-associated tumor antigens, we analyzed the cell surface protein repertoire in RMS tumor cells and normal tissue. We identified several surface proteins highly enriched in RMS, including the immune checkpoint molecule B7-H3. A further analysis using patient specimens showed that B7-H3 is overexpressed in most of RMS tumors and weakly or not detected in normal organs. Interestingly, we found that B7-H3 depletion was associated with higher immune cell killing activity against tumor cells. In line with this, high B7-H3 tumor expression was associated with lower CD8 T-cell density. Our study reveals novel RMS-associated proteins for the development of targeted therapies. In addition, we demonstrate that targeting B7-H3 function can pave the way for the design of new immunotherapies in the treatment of RMS.+-T cells. Furthermore, in vitro functional assays showed that B7-H3 knockout in RMS tumor cells increases T-cell mediated cytotoxicity. Altogether, our study uncovers new potential targets for the treatment of RMS and provides the first biological insights into the role of B7-H3 in RMS biology, paving the way for the development of next-generation immunotherapies.Novel therapeutic strategies are needed for the treatment of rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. By using a combination of cell surface proteomics and transcriptomic profiling of RMS and normal muscle, we generated a catalog of targetable cell surface proteins enriched in RMS tumors. Among the top candidates, we identified B7-H3 as the major immunoregulatory molecule expressed by RMS tumors. By using a large cohort of tissue specimens, we demonstrated that B7-H3 is expressed in a majority of RMS tumors while not detected in normal human tissues. Through a deconvolution analysis of the RMS tumor RNA-seq data, we showed that B7-H3-rich tumors are enriched in macrophages M1, NK cells, and depleted in CD8 PAX3/7 FOXO1 [PAX3/7-FOXO1 fusion protein drives an oncogenic transcriptional program, including the upregulation of genes involved in invasion, proliferation, and survival [PAX3/7-FOXO1 fusion but a variety of mutations on oncogenes, including RAS, PIK3CA, FGFR4, and TP53 [Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma, with nearly 20% of patients presenting with locally aggressive and/or metastatic disease . RMS is survival . In contand TP53 ,7,8. In and TP53 ,10. ThisAntibody-based therapies, including immune checkpoint inhibitors, bispecific antibodies, antibody–drug conjugates, and CAR-T therapy, have transformed the therapeutic landscape of adult cancers . AntibodIn this study, we employed cell surface proteomics (surfaceomics) to establish a comprehensive map of cell surface proteins expressed in RMS. By integrating the transcriptomic data and proteomic data of RMS tumors and normal tissue, we revealed an RMS-specific cell surface protein signature. Among the top upregulated proteins, we identified B7-H3 as the major immunoregulatory molecule expressed in RMS. We found that B7-H3 mediates tumor immune evasion through functional assays and transcriptomic characterization of the tumor immune landscape of RMS tumors. Our findings support B7-H3 as a therapeutic target for antibody-based therapies and provide new biological insights on its immunomodulatory role in RMS. Finally, this study demonstrates the potential of surfaceomics for cancer research and drug development.2 at 37 °C.Human pediatric rhabdomyosarcoma cell lines SJCRH30 (RH30)  fusion-positive, cat# CRL-2061) and RD , fusion-negative, cat# CCL-136) were purchased from ATCC . Two normal human skeletal muscle cell lines were purchased from Lonza (Basel Switzerland) (cat#PCS-950-010) and ATCC (cat#CC-2561). The RMS cell lines RH36 (fusion-negative) and RH18 (fusion-positive) were obtained from Dr. Peter Houghton. The RH30 cell line was grown in RPMI-1640 with 10% FBS  and 1-mg/mL Pen–Strep (Gibco), the RD cell line was grown in DMEM with 10% FBS (Gibco) and 1-mg/mL Pen–Strep (Gibco), and the normal muscle cell lines were cultured in HSkMC Growth Medium . The RMS-MC02 primary cells were isolated from a resected embryonal (fusion-negative) RMS tumor (IRB #16-006956) and cultured in DMEM-F12 (Gibco) with 10% FBS (Gibco) and 1-mg/mL Pen–Strep (Gibco). The B7-H3 knockout RH30 cell line was generated using CRISPR/Cas-9 , and negative selection was performed by flow cytometry using anti-B7H3 PE . All cell lines were maintained in an incubator with a humidified atmosphere and 5% CO7 cells from each cell line were subjected to cell surface biotinylation. EZ-Link-Sulfo-NHS-SS-biotin  was added to cultured cells for 30 min at 4 °C. The cells were washed with 50 mM of glycine to quench the unbound biotin. The cells were lysed in NP-40 lysis buffer, and the biotinylated cell surface proteins were affinity-purified on streptavidin magnetic beads . After stripping off the nonspecifically bound proteins by several rounds of washing with the lysis buffer, the labeled proteins were reduced with 10-mM TCEP  for 30 min at 50 °C and followed by alkylation with iodoacetamide  for 30 min in the dark at room temperature. The proteins were run in a SDS-PAGE electrophoresis gel and submitted for mass spectrometry  and UniProt using the keywords “homo sapiens” and “single-pass transmembrane domain” and “multi-pass transmembrane domain”.n = 101) and normal muscles (n = 5), fusion-negative (n = 66) and normal muscles, and fusion-positive (n = 35) and normal muscles with a fold change ≥ two and adjusted p-value < 0.05 were undertaken for further analysis. To analyze the normal tissue expression of the RMS-enriched cell surface proteins, tissue-specific transcriptomes and proteomes were obtained from Jiang, L et al. that consisted of transcriptomic and proteomic analyses of 201 samples from 32 tissue types of 14 normal individuals [The RNA sequencing data was processed and analyzed as previously reported   were identified and estimated using the QuanTIseq pipeline . The RMS2. The total number of calcein-positive cells and fluorescence intensities of intracellular calcein were calculated for each condition. The tumor cell viability was calculated at 5 h post-incubation. The survival index was obtained by the mean calcein fluorescence in treated cells divided by the mean fluorescence in the control (tumor cells only). An image analysis was performed using Fiji software .Adherent RH30 wild-type and B7-H3KO cells were plated in a 96-well plate and incubated 30 min at 37 °C with calcein-AM . The cells were then washed twice with Live Cell Imaging Solution  and kept in RPMI-1640 (no phenol red) supplemented with 10% HI-FBS. PBMC were isolated from the normal donor blood apheresis cones using a Ficoll gradient and activated overnight with 5 μg/mL of phytohemagglutinin . PBMC were added to each well at a ratio 1:10 and 1:20 (Target:Effector). Each condition was performed in triplicate. The calcein fluorescence was recorded every 10 min for 16 h using the EVOS FL Auto (Thermo) system equipped with the onstage incubator set at 37 °C and 5% COt-test (parametric) and Mann–Whitney U test (nonparametric) were employed to compare the two groups. A paired Wilcoxon test (Wilcoxon’s signed rank test) was performed to analyze the data presented in Figure 6C. The results were considered significant for p-values < 0.05. The p-values were either specified in the figure or denoted as asterisk: * p < 0.05, ** p < 0.01, and *** p < 0.001. All data were analyzed and plotted in GraphPad Prism 9.0.1 .The normality of distribution was assessed using the Shapiro-Wilk normality test. The Student’s Additional materials and methods for the Western blot, immunohistochemistry, and flow cytometry can be found in the In order to define a set of cell surface proteins with an extracellular domain that can serve as potential therapeutic targets in RMS, we used a combination of cell surface capture and proteomic profiling on five RMS cell lines (three fusion-negative and two fusion-positive) and two normal skeletal muscle cell lines A. The prUpon an analysis of the cell surface proteome by label-free mass spectrometry, significant differences in the number of peptides  were detected between individual samples B. To norr = 0.7994 p < 0.0001). Among the fusion-negative and fusion-positive cell lines, positive associations were also observed, but the correlation coefficients varied from 0.2829 (fusion-positive) to 0.4736 (fusion-negative)  of the targets overexpressed in all RMS cell lines at the protein level were also increased at the transcriptional level  . Among tegative) . Similaregative) . To idenal level . When thal level .One major barrier to the development of effective antibody-based therapies is the expression of tumor targets within normal tissue causing on-target, off-tumor toxicity . To imprBased on the RNA and protein expression on the tumor and normal tissues of cell surface antigens, we generated a composite rank by assigning equal weights to transcriptomic and proteomic ranks of overexpressed genes and proteins in RMS . For tumUsing combined proteomic and transcriptomic analyses of RMS tumors and normal tissue, we identified several cell surface proteins enriched in RMS and with limited expression in normal tissue, suggesting they are potential RMS target candidates for antibody-based therapies.Among the cell surface proteins that were upregulated at the RNA and protein levels in RMS compared to the normal muscle and limited expression in normal tissue, we identified the immune checkpoint molecule B7-H3 D. B7-H3 p = 0.06). Strikingly, both PD-L1 (B7-H1) and PD-L2 (B7-DC) were significantly downregulated in RMS compared to normal muscle. The cell surface protein expression of the B7 molecules was also assessed by flow cytometry in normal skeletal muscle cells, fusion-negative (RD), and fusion-positive RMS (RH30) (ρ = 0.84), and no tissue specificity was observed  and fusion-positive RMS (7.03-fold) A. The B7S (RH30) B. SimilaS (RH30) . A WesteS (RH30) C. We anaobserved D.To validate the differential expression of B7-H3 in normal tissue and RMS tumors, we first evaluated the sensitivity and specificity of four commercial antibodies using tumor xenografts of RMS wild-type and knockout cells for B7-H3 . While aN = 97 patients) obtained from our institutional tissue registry . Regardless of the fusion status, B7-H3-high tumors were enriched in M1 macrophages and NK cells and depleted in CD8-T cells and B cells C,D. In fTo confirm a potential association between B7-H3 and CD8-T cells, we performed a T-cell cytotoxicity assay by coculturing activated PBMC isolated from 10 healthy donors with RH30 wild-type or knockout cells for B7-H3 and monitored the tumor cell survival over time. No significant difference was observed in B7-H3 knockout RH30 cells compared to the wild type A. For eiAltogether, these data show that B7-H3 tumor expression is associated with the distinct immune composition of RMS tumors rich in M1 macrophages, M2 macrophages, and neutrophils and depleted in T cells. In addition, B7-H3 expression in RMS is associated with the inhibition of T-cell cytotoxic functions.In this study, we conducted the first comprehensive characterization of the cell surface proteome (surfaceome) of RMS tumors using cell surface capture and mass spectrometry-based proteomics to identify new therapeutic targets. As a result of the technological advances and reduced costs, conventional proteomics has become an attractive tool for target discovery and therapeutic development. However, it suffers from low sensitivity for cell surface proteins, which are significantly less abundant and soluble than cytosolic proteins . AlthougPAX3/7-FOXO1 target genes were also reported among the top cell surface proteins in FP-RMS, including MET, IL4R, FMR1, and NRCAM [PAX3/7-FOXO1 activity or mutation-associated amplification in FN-RMS may explain the high similarities in the cell surface protein repertoire of both molecular subtypes. This is of utmost interest, as it suggests that therapeutic strategies can be designed to target proteins commonly enriched in both RMS subtypes.Among the cell surface proteins enriched in FN-RMS and FP-RMS, we rediscovered several molecules previously reported by other groups . For insnd NRCAM . Interesnd NRCAM . The hieOur surfaceomic analysis also revealed new targetable RMS-enriched cell surface proteins. Among the commonly overexpressed proteins in both RMS subtypes, we identified the cell adhesion molecule CDH4 and ephrin receptors EFNA5 and EPHA7. CDH4 (R-Cadherin) has been previously found amplified in 43.6% of osteosarcomas, and its overexpression was associated with metastasis and a poor prognosis . Similar+ macrophages and the polarization of M1 to M2 macrophages [Another interesting target identified in our study is the immune checkpoint molecule B7-H3. B7-H3 is a member of the B7 family that contains ten members, including the well-known immune checkpoint PD-L1, the target of FDA-approved immunotherapies . We idenrophages ,48. Whilrophages ,50,51,52While clinical trials evaluating PD-L1/PD-1 inhibitors in pediatric sarcoma have been unsuccessful, B7-H3 has become a popular target for new, targeted therapies . AntibodWhile clinical trials evaluating PD-L1/PD-1 inhibitors in pediatric sarcoma have been unsuccessful, B7-H3 has become a popular target for new, targeted therapies . Antibod


Staphylococcus are zoonotic pathogens and pose a serious threat to public health. Tigecycline resistance in this species appears to be a rare phenomenon, and the mechanisms underlying tigecycline resistance have not been fully elucidated. Here, we report two novel variants of the tet(L) gene in Staphylococcus spp. from swine in China, designed as tet(L)F58L and tet(L)A117V. The tet(L)F58L was located within a 18,720 bp chromosomal multidrug resistance gene cluster flanked by two copies of IS257 in Staphylococcus cohnii 11-B-312, while the tet(L)A117V was located on a 6,292 bp plasmid in S. haemolyticus 11-B-93, which could be transferred to S. aureus by electrotransformation. Cloning of each of the two tet(L) variants into S. aureus RN4220 showed 16- or 8-fold increases in the minimal inhibition concentrations (MICs), which can fully confer the resistance to tigecycline (MICs from 0.125 to 2 mg/liter) and eravacycline (MICs from 0.125 to 1 or 2 mg/liter), but no increase in the MICs of omadacycline, compared with the MICs of the recipient strain S. aureus RN4220. In the in vivo murine sepsis and in the murine pneumonia models, an increase in CFU of S. aureus 29213_pT93 carrying the tet(L)A117V was seen despite tigecycline treatment. This observation suggests that the tet(L)A117V and its associated gene product compromise the efficacy of tigecycline treatment in vivo and may lead to clinical treatment failure. Our finding, that novel Tet(L) efflux pump variants which confer tigecycline and eravacycline resistance have been identified in Staphylococcus spp., requires urgent attention.Tigecycline is regarded as one of the few important last-resort antibiotics to treat complicated skin and intra-abdominal infections. Members of the genus IMPORTANCE Tigecycline and eravacycline are both important last-resort broad spectrum antimicrobial agents. The presence of novel Tet(L) efflux pump variants conferring the resistance to tigecycline and eravacycline in Staphylococcus spp. and its potential transmission to S. aureus will compromise the efficacy of tigecycline and eravacycline treatment for S. aureus associated infection in vivo and may lead to clinical treatment failure. The emergence and wide dissemination of multidrug-resistant bacteria pose serious threats to public health worldwide by compromising the efficacy of antimicrobial treatments in human and veterinary medicine . TigecycAcinetobacter isolates (––tet[X] variants) (tet(A) variant in Klebsiella pneumoniae and a tet(L) variant in Escherichia coli  , 6. In ohia coli , 15.Staphylococcus spp., which can cause a variety of diseases in both animals and humans , which can export tetracycline and doxycycline, but not minocycline and tigecycline, was first described in the genus Staphylococcus in 1992 (tet(L) gene was commonly located on plasmids that differed in size, structure, and co-located antimicrobial resistance genes  gene variants conferring resistance to tigecycline and eravacycline were identified in Staphylococcus spp. of swine origin. In addition, the effect on the efficacy of tigecycline treatment in the presence of one of these tet(L) variants in murine sepsis and pneumonia models was evaluated in vivo.In this study, two novel Staphylococcus spp. isolates were subjected to antimicrobial susceptibility testing (AST) during routine antibiotic resistance surveillance. According to the interpretation criteria of minimal inhibition concentrations (MICs) in the version 11.0 issued by EUCAST in 2021, the breakpoint of tigecycline for Staphylococcus spp. is 0.5 mg/liter, which means that a MIC greater than 0.5 mg/liter is considered resistant. Two isolates (Staphylococcus cohnii 11-B-312 and S. haemolyticus 11-B-93) displayed resistance to tigecycline (4 mg/liter) in addition to tetracycline, erythromycin, chloramphenicol, and ampicillin resistance , tet(L) and tet(X) genes] by PCR and sequencing revealed that only tet(L) mutations, but no other acquired tet genes or mutations were identified in these two tigecycline-resistant Staphylococcus isolates. Further analysis revealed that compared with the reference staphylococcal tet(L) gene from plasmid pG38 (GenBank accession number RCDF01000030.1), the amino acid substitution F58L was present in the deduced Tet(L) sequence of S. cohnii 11-B-312, while the substitution A117V was detected in S. haemolyticus 11-B-93. A BLASTp search for these two Tet(L) variants in GenBank database retrieved no same mutations in Tet(L). Consequently, the two Tet(L) proteins were designed Tet(L)F58L and Tet(L)A117V, respectively.A total of 362 nonduplicate porcine sistance . Screenitet(L)F58L- and tet(L)A117V-carrying strains were performed. Sequence analysis revealed that the tet(L)F58L, along with the pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E), the lincosamide resistance gene lnu(B), and the aminoglycoside resistance genes aadD, spw, and aadE, was located within a 18,720 bp chromosomal multidrug-resistance gene cluster flanked by two copies of IS257 in the same orientation in S. cohnii 11-B-312 (tet(L)-carrying chromosomal DNA of S. aureus NX-T55 (GenBank accession number CP031839) (Whole gene sequencing for 11-B-312 . The BLAP031839) .tet(L)A117V, along with the macrolide-lincosamide-streptogramin B (MLSB) resistance gene erm(T), was located on a small plasmid of 6,292 bp in S. haemolyticus 11-B-93 (tet(L)-carrying plasmids with similar sequences matched the query sequence, namely, pRIVM1183 , pUR1902 , pUR2940  and pUR2941 . Of them, the tet(L)-carrying plasmid pRIVM1183 with a size of 6,183 bp was from a methicillin-resistant S. aureus of human origin A117V could be transferred into S. aureus, where it expressed tigecycline and MLSB resistance.The  11-B-93 . This pln origin  27). Th. Thtet(Ltet(L) variants in conferring tigecycline resistance in Staphylococcus spp., the intact copies of tet(L)F58L and tet(L)A117V including their respective putative promoters were cloned into the pLI50 vector, and then introduced into S. aureus RN4220. The constructs, both RN4220+pLI50_tet(L)F58L and RN4220+pLI50_tet(L)A117V showed 16-fold increases in the MICs of tigecycline from 0.125 mg/liter to 2 mg/liter, compared with those of S. aureus RN4220 and S. aureus RN4220+pLI50_reference tet(L) (tet(L) variants tet(L)F58L and tet(L)A117V can fully confer resistance to tigecycline as the MICs breakpoint of tigecycline for Staphylococcus spp. is 0.5 mg/liter according to the interpretation criteria of MICs in the version 11.0 issued by EUCAST in 2021. In addition, RN4220+pLI50_tet(L)F58L and RN4220+pLI50_tet(L)A117V displayed 16-fold and 8-fold increases in the MICs of eravacycline, reaching 2 mg/liter and 1 mg/liter, respectively  variants tet(L)F58L and tet(L)A117V can also fully confer resistance to eravacycline. However, RN4220+pLI50_tet(L)F58L and RN4220+pLI50_tet(L)A117V displayed no increases in the MICs of omadacycline, a unique first-in class aminomethylcycline (tet(L) variants was also evaluated in Gram-negative bacteria E. coli. DH5α+pLI50_tet(L)F58L and DH5α+pLI50_tet(L)A117V displayed no increases in the MICs of tigecycline, eravacycline and omadacycline, compared with DH5α+pLI50_reference tet(L) (tet(L) variants tet(L)F58L and tet(L)A117V were responsible for the significantly elevated MICs to tigecycline and eravacycline in Staphylococcus spp.To confirm the role of e tet(L) . Therefoectively . Eravacylcycline . In addie tet(L) . These rBacillus in 1988 (tet(L)-carrying plasmid pSTE1 from a porcine Staphylococcus hyicus isolate was identified in 1992 (tet(L) that reported in plasmid pG38 in S. aureus (GenBank accession number RCDF 01000030.1) could confer resistance to tetracycline but not to tigecycline (tet(L)F58L and tet(L)A117V for tigecycline resistance in Staphylococcus spp. in this study, though the comparison of the Tet(L) (RCDF 01000030.1) and the earlier reported Tet(L) (M11036) in Bacillus revealed an L363S residue substitution.Tet(L), composed of 14 transmembrane segments, belongs to the major facilitator superfamily efflux pump that exports tetracycline but neither minocycline nor glycylcyclines from the bacterial cell. Tet(L) was first found in  in 1988  and stap in 1992 . The tetecycline . It was Campylobacter but showed a 4-fold increased tigecycline MICs in E. coli  variant was identified, in which multiple amino acid substitutions/deletions, such as the loss of the first codon, N2M, T3K, S4C, and Y5N were detected. This variant exhibited no elevated tigecycline MICs in  E. coli . The two E. coli .3WDO), which exhibits the most greatest identity with Tet(L)  is currently not available in the Protein Data Bank database. Therefore, we established the predicted structure of Tet(L) by homology modeling, based on the crystal structure of YajR (PDB ID code h Tet(L) . The restet(L), the variants tet(L)F58L and tet(L)A117V into this same vector pLI50, respectively, and then transform them into the same recipient to eliminate the influence of the copy number of these resistance genes on gene expression to some extent. As both tigecycline and eravacycline are important options for the treatment of S. aureus infections, the emergence of the tet(L) variants in Staphylococcus spp. might impair the efficacy of treatment with these two antibiotics.In addition, the copy number of the resistance genes affects gene expression, which leads to the elevated MICs , 31. In tet(L)A117V-mediated tigecycline resistance on the efficacy of tigecycline treatment in vivo. The tet(L)A117V-carrying plasmid pT93 was introduced into S. aureus ATCC 29213, designed 29213_pT93, and the S. aureus ATCC 29213 was used as control  indicated that the alveolar structure had disappeared, and a large number of bacterial clumps were scattered, accompanied by inflammatory cell infiltration, which remained even after tigecycline treatment in 29213_pT93 infection group A117V compromises the efficacy of tigecycline treatment in vivo and may lead to tigecycline treatment failure in humans.The tet(L) variants in Staphylococcus spp. from swine, which are able to confer full resistance to tigecycline and eravacycline whether in native host or in the S. aureus host. The presence of these tet(L) variants may compromise the efficacy of tigecycline treatment as shown in the murine sepsis and pneumonia model, which eventually may lead to clinical treatment failure. From the perspective of “One Health,” measures to monitor and control the dissemination of the novel tet(L) variants in both animal and human clinical strains are needed.In conclusion, we report the presence of two novel Staphylococcus spp. isolates were investigated in this study. These isolates were collected in 2019 from nasal swabs of swine in Henan province/China and were cultured on Staphylococcus chromogenic medium. After incubation at 37°C for 24–36 h, colonies were selected for 16S rRNA sequencing as described previously  issued by the Clinical and Laboratory Standards Institute (CLSI)   was used.AST was performed by broth microdilution according to the recommendations given in document M100 (30e (CLSI) . S. aureStaphylococcus cohnii 11-B-312 and Staphylococcus haemolyticus 11-B-93 with tigecycline MICs of 4 mg/liter were screened for the presence of the tet(A), tet(K), tet(Y), tet(M), tet(L) and tet(X) genes by PCR using the primers listed in Table S1. The PCR mixture was composed of 12.5 μl of Ex Taq , 0.5 μl of each primer, 0.5 μl of chromosomal DNA template, and 11 μl of sterile distilled water. All PCR products were subjected to Sanger sequencing.S. cohnii 11-B-312 and plasmid DNA of S. haemolyticus 11-B-93 were extracted using the Gentra Puregene Yeast/Bact. kit or the Qiagen plasmid extraction midi kit . The plasmid DNA of strain 11-B-93 was introduced into the recipient strain S. aureus RN4220 and the QC strain S. aureus 29213 by electrotransformation as previously described (Whole cell DNA of escribed . Brain hS. cohnii 11-B-312 and the transfomant of S. haemolyticus 11-B-93 (transformant S. aureus T93) were sequenced by the PacBio RS and Illumina MiSeq platforms. The PacBio sequence reads were assembled with HGAP4 and CANU (version 1.6) and corrected by Illumina MiSeq with pilon (version 1.22). The prediction of ORFs and their annotations were performed using Glimmer 3.0. Insertion sequences were predicted by using ISfinder (www-is.biotoul.fr). Resistance genes were identified with ResFinder (version 4.0) (Whole genome DNA of ion 4.0) .tet(L) variants confer tigecycline resistance, tet(L)F58L and tet(L)A117V were separately cloned into the E. coli-S. aureus shuttle vector pLI50 (tet(L) variants and their putative promoter from S. cohnii 11-B-312 and the transformant S. aureus T93, respectively, using the online assembly tool NEBuilder . The same cloning procedure was conducted for the reference tet(L) gene from the S. aureus plasmid pG38 (26). Then, the amplicons were ligated into the linearized plasmid pLI50 by using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). Each of the recombinant plasmids, pLI50-tet(L)F58L and -tet(L)A117V, but also pLI50-reference tet(L), were subsequently electrotransformed into S. aureus RN4220, respectively, as described previously , Group 29213_TGC (infection with S. aureus ATCC 29213 and treated with tigecycline), Group 29213_pT93 (infection with S. aureus 29213_pT93), Group 29213_pT93_TGC (infection with 29213_pT93 and treated with tigecycline), and Group NC, which represented the negative control . Each group contained six mice.All animal experiments were conducted in accordance with the approved guidelines of the Institutional Animal Care and Use Committee. Seven- to 9-week-old BALB/c female mice weighing 15–18 g served for the sepsis model and SPF level KM female mice for the pneumonia model according to the methods described previously , 37. The8 CFU of the S. aureus 29213_pT93) and KM female mice were infected by bacteria though the nasal cavity (5 × 107 CFU of S. aureus 29213_pT93). The mice were treated with tigecycline by subcutaneous injection at an initial dose of 100 mg kg−1 1 h after the infection and then subsequently with 50 mg kg−1 tigecycline every 12 h, which is consistent with the treatment levels of tigecycline used in humans. The mice were euthanized and were dissected after 48 h (Prior to tigecycline injection, BALB/c female mice were intraperitoneally injected with bacterial suspension (1 × 10ter 48 h . The livS. cohnii 11-B-312 and the plasmid sequence of S. aureus T93 plasmid, pT93, determined in this study have been deposited in GenBank under accession numbers CP068241 and CP068248, respectively.The genomic sequence of


Furthermore, through PGRMC1-EGFR/GLP1R-PI3K-Akt pathway, knockdown or inhibition (by AG205) of PGRMC1 mimics, whereas its overexpression moderately alleviates CLZ-induced glucose disturbances. Potentially, the PGRMC1 target may be regarded as a novel therapeutic strategy for AAPD-induced hepatic glucose metabolism disorder.Newly emerging evidence has implicated that progesterone receptor component 1 (PGRMC1) plays a novel role not only in the lipid disturbance induced by atypical antipsychotic drugs (AAPD) but also in the deterioration of glucose homoeostasis induced by clozapine (CLZ) treatment. The present study aimed to investigate the role of PGRMC1 signaling on hepatic gluconeogenesis and glycogenesis in male rats following CLZ treatment (20 mg/kg daily for 4 weeks). Recombinant adeno-associated viruses (AAV) were constructed for the knockdown or overexpression of hepatic PGRMC1. Meanwhile, AG205, the specific inhibitor of PGRMC1 was also used for functional validation of PGRMC1. Hepatic protein expressions were measured by western blotting. Meanwhile, plasma glucose, insulin and glucagon, HbA1c and hepatic glycogen were also determined by assay kits. Additionally, concentrations of progesterone (PROG) in plasma, liver and adrenal gland were measured by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Our study demonstrated that CLZ promoted the process of gluconeogenesis and repressed glycogenesis, respectively mediated by PI3K-Akt-FOXO1 and GSK3β signaling  It is well recognized that abnormal glucose and lipid metabolism is a high-risk factor of cardiovascular and cerebrovascular events, contributing to the premature death of schizophrenic patients from physical diseases greatly . CLZ, thPGRMC1, as a type of adaptor protein, is directly involved in cell signaling by trafficking and interacting other transmembrane receptors to the plasma membrane. A recent translational study demonstrated decreased levels of PGRMC1 protein and RNA in patients with insulin resistance, suggesting a role of PGRMC1 in insulin signaling , 12. PGRThe liver plays a central role in the maintenance of blood glucose by balancing new synthesis (gluconeogenesis) and glycogen synthesis . Under pvia the interaction with GLP1R and EGFR.Intriguingly, new evidence provided support for the coprecipitation and colocalization between PGRMC1 and epidermal growth factor receptor (EGFR) in cytoplasmic vesicles in cells . FurtherThe present study aims to evaluate the effects of 4-week CLZ treatment on glucose homeostasis and hormone secretion in male rats. We also examined whether the PGRMC1 signaling is involved in the CLZ-induced disruption of glucose homeostasis. On the basis of above, the dual regulatory effects on knockdown and overexpression of PGRMC1 on EGFR/GLP1R-PI3K-Akt pathway were further validated in CLZ-induced hepatic glucose dysregulation.To avoid possible influence of cyclic, systemic PROG fluctuation caused by menstruation cycle , only maCLZ was purchased from Wuhan Yuancheng Co-Creation Technology Co., Ltd. . AG205 (purity≥97.5%) was synthesized by Jining Drug Research and Development Center. CLZ  and AG205  were initially dissolved in 5% (v/v) ethanol and then further diluted in 0.9% saline water containing 22.5% 2-hydroxypropyl β–cyclodextrin to obtain the final concentration. All the solutions were injected intraperitoneally. The CLZ dose used in our experiments was selected on the basis of our previous research , which w12 vg/ml) for PGRMC1 overexpression and AAV/BBB 2.0-r-PGRMC1 shRNA3-GFP  for PGRMC1 knockdown. The shRNA sequence targeting rat PGRMC1 is AATTCGATTGTGTACTCGGATGATGAAGAATTCAAGAGATTCTT-CATCATCCGAGTACACAATCTTTTTTG (Top strand); GATCCAAAAAAGATTG-TGTACTCGGATGATGAAGAATCTCTTGAATTCTTCATCATCCGAGTACACAATCG (Bottom strand). Detailed information about the genetic tools has been provided in supplementary material.The AAV tools targeted at PGRMC1 as well as the control vectors were designed and provided by Hanbio Biotechnology Co., Ltd. . Specifically, there are two types of AAV used in our study including AAV/BBB 2.0-CMV-r-PGRMC1-3xflag-GFP : the vehicle group (NC), down-regulated AAV group (PGRMC1-KD) and overexpression AAV group (PGRMC1-OE). All kinds of AAV were injected via tail vein only once with a volume at 300 μl. In 28 days, anatomy was exerted to observe the GFP immunofluorescence in liver sections and the rat liver tissues were collected for western blot analysis. The second batch of rats were divided into five groups (n=9/group): the vehicle group (NC), down-regulated AAV group (PGRMC1-KD), and blank AAV + CLZ group , CLZ + overexpression AAV group (CLZ+PGRMC1-OE), AG205 group , the rat liver tissue samples were collected and frozen immediately in liquid nitrogen. Whole blood was collected from the truncal vessel in EDTA anticoagulation vacuum tubes. The plasma was obtained by centrifugation of blood at 4°C, 3000 rpm for 15 minutes and stored at −80°C before analysis  and Fluorescein (FITC)-conjugated Affinipure Donkey Anti-Goat IgG (H+L) secondary antibody (SA00003-3). Liver samples were fixed in 4% paraformaldehyde overnight at 4°C, embedded in paraffin and sectioned at 4-μm thickness. Tissue sections were stained with hematoxylin and eosin (H&E). Then, the histopathological examination was performed under light microscopy. Periodic acid–Schiff (PAS) staining was used to visualize the glycogen deposits in liver tissues. Liver samples were soaked in 4% paraformaldehyde solution and then mounted in paraffin and sectioned. Sections were oxidized in periodic acid for 20 min at room temperature and rinsed for three times in deionized water. The liver sections were counterstained with haematoxylin and eosin for 1 min, rinsed in deionized water and observed for the red-staining parts as the presence of glycogen using a light microscope.Plasma glucose and hepatic glycogen levels were tested using Biochemical Assay Kits  following the manufacturer’s instructions. Insulin, HbA1c and glucagon levels were measured by immunoassays using commercial ELISA Kits .g for 10 min at 4°C. Then the resulting supernatant was evaporated to dryness by a centrifugal vacuum concentrator at 4°C. The residue was next resuspended with 100 μL of 2-propanol/acetonitrile/water  solvent mixture. Finally, the concentrations of PROG in the tissues and plasma were measured by an LC-MS/MS method as previously reported  was transferred and mixed with 1500 μL of methyl tert-butyl ether/methanol  extraction solvent, vortexed for 3 min and centrifuged at 20627reported , 25. The473), GSK-3β, FOXO1, β-actin, PCNA , phospho-EGFR, phospho-GSK-3β (Affinity) overnight at 4°C. The membrane was then incubated with appropriate secondary horseradish peroxidase-conjugated antibodies  at room temperature for 1 h and developed color rendering by the ECL kit. The film signal was digitally scanned and then quantified using ImageJ software .Proteins from liver tissues were extracted and quantified using a BCA kit as described previously , 24. Appp<0.05.The data are presented as the mean ± SD and were analyzed using GraphPad Prism 8.0 . For parameters including weight gain, food intake and feeding efficiency, due to the effects of treatment and time, two-way repeated ANOVA was selected for data analysis. Owing to small samples and non-normally distributed variance, nonparametric Kruskal–Wallis one-way analysis of variance was used for data analysis followed by pairwise multiple comparisons. Statistical significance was considered at In order to evaluate the effects of 4-week CLZ treatment on glucose homeostasis, we analyzed the body weight gain (BWG), cumulative food intake (CFI) and feeding efficiency of rats. As shown in H=28.96, p<0.0001), HbA1c , insulin , glucagon  and hepatic glycogen  among groups. Notably, CLZ-treated rats exhibited increased fasting plasma glucose and HbA1c levels, while reduced the insulin level and hepatic glycogen content, compared with NC group. Although there was an elevated tendency of glucagon levels in CLZ group, the differences did not approach significance.In addition, we also measured relevant biochemical parameters related with glucose metabolism. As shown in H=12.53, p=0.0138)    and the ratio of p-Akt/Akt  was down-regulated in CLZ-treated rats in comparison with NC group  and total FOXO1 (H=23.30. p<0.0001) were upregulated in CLZ-treated rats  via inhibition of the PI3K-Akt signaling, thereby activating hepatic gluconeogenesis and inhibiting glycogenesis.We also examined whether the downstream PI3K-Akt- FOXO1/GSK3β signaling are involved in the clozapine-induced disruption of glucose homeostasis. We found that PI3K  compared to NC group  and their phosphorylated forms  in the liver was compared among groups. CLZ treatment significantly downregulated the protein expression of the three key factors -ShRNA-PGRMC1 and (AAV)-CMV-PGRMC1 to knockdown or upregulate hepatic PGRMC1 expression. Moreover, the specific inhibitor of PGRMC1, AG205 was also used for functional validation.H=24.71, p<0.0001; GLP1R: H=26.02, p<0.0001; p-EGFR/EGFR: H=24.15, p<0.0001) compared to NC group  and the ratio of p-Akt/Akt  was down-regulated in PGRMC1-KD, AG205 in comparison with NC group, but PGRMC1-OE restored the CLZ-induced downregulation of PI3K and p-Akt/Akt  and total FOXO1 (H=23.30. p<0.0001) were upregulated in PGRMC1-KD and AG205, which were eliminated by PGRMC1-OE  clozapine may directly damage the secretory function of β-pancreatic islets, leading to the decrease of insulin secretion . Howeverl muscle . Therefo studies . We alsoIn addition, hormonal levels might play an important role in CLZ-induced metabolic disturbance especially in glucose dysregulation. It could be hypothesized that CLZ as an inducive factor, lead to hormonal excretion disturbance. As mentioned before, PROG has strong affinity with PGRMC1 among all hormones. Previously, Christine et, al  reportedvia an increase in glucagon secretion and subsequent stimulation of hepatic glucose production .All authors contributed to and have approved the final manuscript. HC: Conceptualization, Reviewing and Editing. TC: Methodology, Data curation, Writing- Original draft preparation. QC: Methodology, Investigation. BZ, XW, CZ, and SZ: Visualization, Investigation, Validation.This work was supported in part by the grants from Hunan Provincial Natural Science Foundation of China [2021JJ30922], Hunan Provincial Health Commission Research Project [202113010595], Wu Jieping Medical Foundation Funded Special Clinical Research Project [320.6750.2020-04-2], Changsha Municipal Natural Science Foundation [kq2007045], the Fundamental Research Funds for the Central Universities of Central South University  and New Clinical Medical Technology Project of the Second Xiangya Hospital of Central South University ([2021]94).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Multidrug resistance gene 1 (MDR-1) encodes for P-glycoprotein (P-gp) recognized for removing cytostatic drugs from tumor cells. MDR1 gene polymorphisms change function of P-gp. In this study, we are interested in investigating whether MDR1 C1236T single nucleotide polymorphisms (SNPs) affect the susceptibility and treatment-related toxicities in B-cell non-Hodgkin lymphoma (B-NHL) in the population of eastern China.Materials and methods: A group of 107 B-NHL patients and 150 healthy donors, unrelated ethnic Han Chinese and residents of eastern China, were included in this study. The MDR1 C1236T polymorphisms were determined using polymerase chain reaction-allele specific primers after extraction of genomic DNA. Analyses were performed using SPSS and Arlequin software.Results: MDR1 C1236T polymorphisms were not significantly related to the risk and treatment-related toxicities of B-NHL. A significant association between extranodal sites and C1236T allele was observed (C vs T: P=0.01).Conclusion: Our findings could expand our understanding of MDR1 in B-NHL and provide references for further research in multidrug resistance. B-cell non-Hodgkin lymphoma (B-NHL) comprises a heterogeneous group of cancers and accounts for 85-90% of non-Hodgkin's lymphoma (NHL) Multidrug resistance gene 1 (MDR-1), also known as the adenosine triphosphate binding cassette B1 gene (ABCB1), is located on chromosomal region 7q21.1 In this study, we are interested in investigating whether MDR1 C1236T SNPs affect the susceptibility and treatment-related toxicities in B-NHL in the population of eastern China. Our findings could expand our understanding of MDR1 in B-NHL and provide references for further research in multidrug resistance.A group of 107 B-NHL patients and 150 healthy donors were included in this study. All Subjects were unrelated ethnic Han Chinese, residents of Shanghai, Jiangsu and the surrounding regions. Patients were dignosed based on pathology according to 2008 and 2016 World Health Organization (WHO) classification for tumors of hematopoietic and lymphoid tissues Genomic DNA was extracted manually from peripheral blood. The MDR1 C1236T polymorphism was determined using the polymerase chain reaction-allele specific primers assay. Each polymerase chain reaction (PCR) reaction mixture contained: a PCR Master Mix reaction buffer (10 μl), DNA template (1 μl), primer (2 μl) and double distilled water (7 μl). For allele T, forward primer is 5-GTCACTTCAGTTACCCATCTCG-3 and reverse primer is 5-CTGCACCTTCAGGTTCGGA-3. For allele C, forward primer is 5-GTCACTTCAGTTACCCATCTCG-3 and reverse primer is 5- CTGCACCTTCAGGTTCTGG-3. For T/C, forward primer is 5-GTCACTTCAGTTACCCATCTCG-3 and reverse primer is 5- GGTCATAGAGCCTCTGCATCA-3. The PCR conditions were 10 cycles of denaturation at 96 °C for 10 min, annealing at 58 °C for 30 min, and extension at 60 °C for 75 min, and 25 cycles of denaturation at 96 °C for 10 min, annealing at 55 °C for 30 min, and extension at 60 °C for 75 min. DNA fragments generated after restriction enzyme digestion were separated in a 2% agarose gel. The C1236T polymorphism of MDR1 was genotyped homozygous CC genotype, homozygous TT genotype and heterozygous CT genotype.The Hardy-Weinberg equilibrium test was performed by Arlequin software 3.5. Associations between allele, genotype frequencies of MDR1 polymorphism and risk of B-NHL patients were determined using Binary logistic regression analysis. Pearson Chi-squared and Fisher's exact test were used to analyze association between patients' MDR1 polymorphism and clinical characteristics, treatment-related toxicities. All tests were two-sided and differences were considered significantly when p-value was <0.05. All analyses were performed using SPSS for Windows .The study consisted of 107 B-NHL patients. 56 were men and 51 were women. 52.3% patients were ≤60 years old. Clinical characteristics, including sex, age, staging, grouping, extranodal sites, ECOG score, LDH and IPI score, are shown in Table Table To estimate the association between MDR1 polymorphisms and the risk of developing B-NHL, Binary logistic regression analysis was used. For MDR1 1236T allele, 70.1% were in patients and 64.7% in controls . For MDR1 1236CT genotype, 46.7% were in patients and 45.3% in controls , and for MDR1 1236TT allele, 46.7% were in patients and 42% in controls . Analysis did not show any significant differences. Details are described in Table To evaluate the association between MDR1 polymorphisms and clinical characteristics of B-NHL, 107 B-NHL patients and 150 healthy people were enrolled. As shown in table We evaluated the association between polymorphisms and treatment-related toxicities among 107 B-NHL patients. The frequencies of major hematological toxicities after chemotheraphy were compared between different alleles and genotypes of MDR1 C1236T. None of the MDR1 alleles and genotypes was associated with treatment-related toxicities Table .Even to the same treatment, clinical courses and responses of different B-NHL patients are variable Up to now, relationship between MDR1 polymorphisms and a variety of diseases have been studied Niveditha Muralidharan et al. showed that MDR1 3435C>T gene polymorphism influences adverse events to MTX in the South Indian cohort of patients with RA In conclusion, we have investigated association between MDR1 C1236T polymorphisms and B-NHL in Han population of eastern China. Extranodal sites and C1236T allele is highly correlative, but no relationship was found between MDR1 SNPs and incidence or chemotheraphy-related toxicities of B-NHL. Unfortunately, our results are lack of complete follow-up records. Thus, no survival analysis was performed. Furthermore, we may complete data of follow-up. Besides, we might pay more attention on connection between MDR1 and relevant mRNA.


ATP7B (encoding a copper-transporting P-type ATPase) variants that shows various characteristics according to race and geographical region. This study was aimed to provide a comprehensive analysis of ATP7B variants in China and to investigate a plausible role of common variants in WD manifestations.Wilson’s disease (WD) is an autosomal recessive disorder of copper metabolism caused by ATP7B gene sequencing and information of age and symptoms at onset was collected. The genotype–phenotype correlation was assessed in the index patients who were examined with two pathogenic variants and onset with hepatic (n = 276) or neurologic (n = 665) symptoms.A total of 1366 patients (1302 index patients and 64 siblings) clinically diagnosed with WD (Leipzig score ≥ 4) were recruited. They underwent ATP7B variants  in the 1302 index patients, including 116 novel variants. The most frequent variant was c.2333G>T , followed by c.2975C>T , c.2621C>T , c.2755C>G , and c.3646G>A . In 1167 patients, both pathogentic variants were identified, of which 532 different variant combinations were found. By binary logistic regression analysis, the factor associated with neurological presentation was high age-at-onset, but not sex, protein-truncating variant (PTV), or the common missense variants . In the neurological group, low age-at-onset was a factor associated with dystonia, gait abnormality, and salivation; high age-at-onset was a factor associated with tremor; and the sex, low age-at-onset and A874V were independent factors associated with dysarthria. In addition, PTV, R778L, and P992L were predominant in early-onset patients, whereas A874V was predominant in late-onset patients, and patients with R778L/A874V genotype displayed a higher age-at-onset than patients with R778L/R778L or R778L/P992L genotype.We identified 294 potentially pathogenic ATP7B variant spectrum and highlighted the differences among patients with WD in age-at-onset and ATP7B variants, which may provide some valuable insights into the diagnosis, counseling, and treatment of patients with WD.Our work expanded the The online version contains supplementary material available at 10.1186/s40035-022-00287-0. ATP7B) [ATP7B variants [Wilson’s disease  is an autosomal recessive disorder of copper metabolism caused by variants in the gene encoding a copper-transporting P-type ATPase (ATP7B) –3. WD isATP7B) . The clivariants , 6.ATP7B (Ensembl ENST00000242839.10) contains 21 exons and encodes an eight-transmembrane-domain protein consisting of 1465 amino acids that is located on trans-Golgi networks (TGN). The protein is abundantly expressed in the liver and is expressed at lower levels in the kidneys, placenta, brain, lungs, and heart [ATP7B gene [The canonical transcript of P7B gene .ATP7B truncating variants are associated with early onset of WD [ATP7B variants in the aim to extend the variant spectrum and decipher the relationship between the phenotype and the genotypes of the most common variants in China, which are distinct from those in Caucasians.Patients with WD usually present with various phenotypes, with hepatic and/or neurological presentation being the main feature, and their ages of onset range from 8 months to 74 years –16. The et of WD , 18. So et of WD . HoweverATP7B variant analysis , symptoms at presentation , presence of K–F rings, age at diagnosis, and laboratory findings, were reviewed. Clinical diagnosis of WD was based on the Leipzig score , 20, andATP7B were amplified with the primers  [http://www.wilsondisease.tk/) [https://asia.ensembl.org/index.html). The pathogenicity of the variants was identified with InterVar (http://wintervar.wglab.org/) or artificially by applying the American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines [http://provean.jcvi.org/index.php) and PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/index.shtml) [The variants identified in this study were compared with the list of reported pathogenic variations in the Human Gene Mutation Database  , and theidelines , 23, andx.shtml) , 25.ATP7B variants were included  associated with neurologic/hepatic presentation, acute/chronic hepatic disease, or specific neurological symptom . Only factors that showed significant associations in univariate analyses were included in the multivariate analysis. To control type I errors caused by multiple-hypothesis test, the P-value was adjusted by the Benjamini–Hochberg method [P < 0.05.All statistical analyses were performed using SPSS version 23 . Quantitative data (age at onset and diagnosis) are expressed as mean ± SD, and categorical variables are given as absolute (number) and relative frequencies (%). To compare continuous variables, Student’s g method . The criATP7B sequencing  were enrolled for ATP7B, with a majority of variants before mental binding domain 5 (MBD5) being PTV  were characterized with two potential disease-causing variants (204 homozygotes and 963 compound heterozygotes), 115 patients (8.83%) were characterized with one potential disease-causing variant, and 20 patients (1.54%) were characterized with no potential disease-causing variants (Table ATP7B variants and had onset with hepatic (n = 276) or neurologic (n = 665) symptoms , which was different from the previous finding in Caucasians that females were more common in the hepatic group [P = 0.002) and those with a neurologic presentation , implying that liver injury tends to start at an earlier age in males than in females. In fact, males had an earlier age at onset than females in the hepatic group , but not in the neurologic group .Next, we focused our attention on index patients who carry two potential pathogenic oms Fig. . We notiic group . In addivs neurologic: 17.32 ± 10.36 years vs 19.17 ± 7.99 years, P = 0.008). Further analysis was performed in the hepatic or neurologic presentation groups with various symptoms, and the results indicated that the low age-at-onset was a factor associated with acute hepatic disease in patients with hepatic presentation  and neurological presentation, and no association between the presence of PTV or the three common variants  and neurological presentation Fig. a. InsteaFurther analysis was focused on common homozygotes and compound heterozygotes in the hepatic and neurologic groups. As shown in Table Next, we evaluated the distribution frequencies of several common variants  and their percentages in specific ranges of age-at-onset. As shown in Fig. ATP7B in 1302 index patients from 30 provinces of China, which allowed us to delineate the variant spectrum, clinical features, and genotype–phenotype correlations of WD in China. Overall, 294 potential pathogenic variants were identified in this study, among which 178 have been reported to be disease-causing variants in the Wilson Disease Mutation Database [ATP7B variants. Significantly, two of the previously identified variants (I390V and A476T), which were predicted to be ‘tolerated’ and ‘benign’, and two of the novel variants (L510R and L1154R), which were predicted to be ‘damaging’ and ‘probably damaging’, were found to co-exist with two other known pathogenic variants in the patients  [Saccharomyces cerevisiae, the ccc2 mutant can be partially rescued by H1069-ATP7B, whereas only weak complementation was found with R778L-ATP7B and P992L-ATP7B [The different presentations of WD may result from various properties associated with the lum (ER) . The mislum (ER) , 12. Thelum (ER) . In addilum (ER) . In Sacc2L-ATP7B , 39. Rec2L-ATP7B , and theATP7B variants, and demonstrated that PTV, R778L, P992L and A874V were not associated with hepatic or neurologic presentation. Besides, we also noted no correlations of PTV, R778L, P992L and A874V with acute hepatic disease, dystonia, gait abnormality, salivation, tremor and swallowing difficulty, except that A874V was negatively associated with dysarthria. In addition, patients with the R778L/A874V genotype displayed a higher age-at-onset than patients with R778L/R778L or R778L/P992L genotype. Our research highlights the differences among WD patients in age-at-onset and ATP7B variants, which may provide some valuable insights into the diagnosis, counseling, and treatment of patients with WD. Comparing the clinical features of patients with R778L/R778L and R778L/A874V genotypes and studying their underlying mechanisms will be helpful for understanding the progression of WD.In summary, our study identified 116 novel variants that substantially expand the spectrum of pathogenic Additional file 1: Table S1. Primers used for amplification and sequencing of exons and exon–intron boundaries of ATP7B.Additional file 2: Table S2. Details of potential pathogenic variants of ATP7B identified in this study.Additional file 3: Table S3. Non-pathogenic variants of ATP7B identified in this study.Additional file 4: Fig. S1. Sequencing results for 64 novel missense variants of the ATP7B gene. Fig. S2. Sequencing results for novel variants of the ATP7B gene except for missense variants. Fig. S3. Homology comparisons of novel missense variants in the ATP7B protein.Additional file 5: Table S4.ATP7B variant combinations in this cohort.