Klebsiella spp. isolated from mastitis samples in Vietnam.This study aimed to determine the antibiotic-resistant profile and to identify molecular characterization of some virulence genes of Klebsiella spp. was identified through biochemical reactions and confirmed by Polymerase chain reaction (PCR). Antimicrobial resistance was tested by disk diffusion method, and virulence and resistance genes were tested by PCR.A total of 468 samples from clinical mastitis cases were collected and submitted to the Laboratory. All samples were cultured, and magA, fimH, and entB, responsible for hypermucoviscosity production, adherence, and enterobactin production, were confirmed in isolates. Multidrug resistance and virulence potential in Klebsiella spp. are changing this mastitis pathogen into a superbug and making its management harder.An antibiogram study showed that a high proportion of isolates are multidrug-resistant (94%). All isolates were resistant to lincomycin and sulfamethoxazole, followed by ampicillin (94%), sulphonamide (66%), amoxicillin (56%), streptomycin (52%), polymyxin B (28%), colistin sulfate (12%), tetracycline (6%), ciprofloxacin (4%), florfenicol (4%), enrofloxacin (4%), piperacillin (2%), trimethoprim (2%), nalidixic acid (2%), imipenem (2%), and sulfamethoxazole/trimethoprim (2%). In contrast, all isolates showed sensitivity to gentamicin and ceftiofur. The appearance of an efflux pump system, extended-spectrum beta-lactamase (ESBL), tetracycline, and sulphonamides-resistant genes was reconfirmed using different specific primers. Capsular serotype K1 and virulence genes Klebsiella spp. associated with bovine mastitis in Nghe An province were mostly multidrug-resistant and carried virulence genes including fimH, entB, and antimicrobials resistant genes , but these isolates were not ESBL producers. Klebsiella was 21.0% (unpublished data).Mastitis is a considerable and recurring economic loss in the dairy industry worldwide. It differs from other animal diseases because several kinds of bacteria can cause the disease; these pathogens colonize the udder, multiply, and produce harmful substances that result in inflammation, decreased milk production, and affected milk quality . MastitiKlebsiella is a genus of Gram-negative, oxidase-negative, rod-shaped bacteria with a prominent polysaccharide-based capsule , submitted, and cultured on the day of arrival.Klebsiella include indole production, citrate utilization, motility [A loopful of mastitis sample (10 μl) was the first culture onto blood agar (incubated at 37°C for 24–48 h); the sample was considered culture-positive if 1 or more colonies were observed (≥100 CFU/ml) , Gram-nemotility , other cAntimicrobial susceptibilities were conducted by using the disc diffusion test (DDT) with the Mueller-Hinton agar (MHA) technique which is described in Clinical and Laboratory Standards Institute (CLSI) M100-30th . The bacESBL production tests followed CLSI M100-30th  using thDNA of each isolate was extracted using Chelex 100 Resin protocol : colonieKlebsiella isolates were first identified by biochemical reactions and then confirmed by using Klebsiella-specific gene gyrA (441 bp) [Klebsiella oxytoca American Type Culture Collection (ATCC) 49131 and Staphylococcus aureus ATCC 25923 were used as the positive and negative control, respectively. PCR products were then purified and identified by agar electrophoresis (Mupid-Exu) and transillumination (Major Science - MBE 200A).The (441 bp) ; the tes(441 bp) : PolymerKlebsiella isolates were confirmed again some specific genes corresponding resistance to ESBL production by blaSHV, blaTEM, blaKPC, blaNDM, blaCTX-M-3 and blaIMP, to Sulphonamides by sul1, sul2, to Tetracycline by tetA, tetB, to Trimethoprim by DHFR-I and to Quinolone by qnrA. The reaction mixture was prepared, and the tests followed the protocol described earlier with primers, sequences, predicted sizes, and annealing temperatures described in Klebsiella spp. were determined by targeting specific gene fimH encoding for type 1 fimbriae, rmpA gene for the regulator of the mucoid phenotype A, magA for hypermucoviscosity production (mucoviscosity-associated gene A), K1 & K2 for encoding capsule serotype, iroN, entB, and iutA genes for encoding siderophore. The reaction mixture was prepared, and the tests followed the earlier protocol with primers, sequences, predicted sizes, and annealing temperatures described in The virulence factors associated with Spreadsheets were used as software tools for data entry, storage, analysis, and visualization in this study.Klebsiella strains by biochemical reactions. Because we did not investigate the prevalence and the sources of this pathogen in the farm  but were interested in the virulence genes and antimicrobial resistance and its related genes. Thus, 50 isolates were randomly selected from 103 strains for further study and were confirmed as Klebsiella spp. by the support of PCR through the gyrA gene  belong to 9 classes, and resistance levels varied between antibiotics  or 6 (14/50) drugs. The 50 isolates resistant to 2–10 different drugs were presented in 23 patterns . All stracrAKp, which might relate to the MDR found. Resistance towards Beta-lactam antibiotics was found with 94% of isolates carrying blaSHV, and 8% blaNDM, and no strains were found positive with blaTEM, blaKPC, blaCTX-M-3, and blaIMP. We found that the proportion of isolates that harbored sul1 and sul2 was 4% and 2%, respectively. Tetracycline-resistant encoded genes were found in 100% of isolates with tetA, but none carried tetB genes. qnrA gene encoding quinolone-resistant was not found in all Klebsiella spp. strains in this study. Analyzed gene patterns, we found 1 isolate with 5 genes (tetA- blaSHV - acrAKp -sul1-sul2), 1 isolate with the pattern (tetA- blaNDM - acrAKp -sul1), another isolate with (tetA- blaSHV - blaNDM - acrAKp), 45 isolates with 3 genes (tetA- blaSHV - acrAKp) and 2 isolates with (tetA- blaNDM - acrAKp) patterns. The results of some antimicrobial resistance genes are summarized in Klebsiella spp. isolates were investigated for some virulence genes; we found that 2 isolates harbored K1 (4%), all strains carried fimH (100%), and 49 Klebsiella spp. positive with entB (98%) genes. None of these isolates harbored K2, magA, rmpA, and iutA genes  is a significant problem in human and veterinary medicine. Fortunately, all isolates in our study were not ESBL producers, similar to a report from Brazil 98.2% ESBL negative [Klebsiella spp. from dairy mastitis might not often be ESBL producers, and attention should be paid to β-lactams as it is an option for mastitis treatment.The worldwide spread of negative  and 98.8negative . This reacrAKp gene coding for a multidrug efflux pump (AcrAb) belonging to the resistance-nodulation-cell-division family [acrAKp, which might relate to the MDR found. A study on the role of this gene revealed that AcrAB was involved in resistance to quinolones, other antimicrobial agents, and host antimicrobial peptides. It was revealed that there is a plasmid that carries a part of the n family . Our resblaSHV, blaTEM, blaNDM, blaCTX-M-3, [blaSHV gene might explain the resistance to AMP and AMO in our study, as this gene often relates to the ability to hydrolyze penicillin and cephalosporins. A study in Iran reported that the proportion of blaCTX, blaSHV, and blaTEM genes were 62.5%, 42.5%, and 87.5%, respectively [blaSHV gene, whereas the blaTEM-1 gene was detected in 2.2% (3/137) isolates [K. pneumoniae strains harbored blaSHV and blaCTX-M, respectively [E. coli and Klebsiella spp. [sul1 and sul2 genes encoding dihydropteroate synthetase with a low affinity for sulphonamides [sul1 was 39% and sul2 was 44% [The development of resistance to β-lactam antibiotics is attributed to the production of β-lactamases coded by different genes, including CTX-M-3, . The preectively . Another 2.2% 3/1 isolatesectively ; these glla spp. . Sulphononamides . Sulphontet genes, which are involved in either the active efflux of the drug, ribosomal protection, or enzymatic drug modification. Among the various tet genes, tetA and tetB are reported in Gram-negative bacteria [K. pneumoniae carried tetA gene [tetA prevalence of 5% and no tetB in the dairy farm [tetA and similar in tetB proportion. Although all isolates harbored tetA gene, the resistance to tetracycline in the current study was only 6%. It may be due to the other resistance mechanisms and need future studies. One of the three main mechanisms of quinolone resistance has been described as the acquisition of plasmid-mediated quinolone resistance genes leads to the protection of quinolone’s targets by qnr proteins [Klebsiella spp. strains, two isolates carried qnrA gene, which accounted for 14.3% [qnrA and DHFR-I genes. Resistance to tetracycline is governed by bacteria . A similetA gene , whereasiry farm ; compareproteins . A studyor 14.3% . ConnectKlebsiella spp. will depend on many factors, including the virulence profile of each isolate. The polysaccharide capsule is one of the most important virulence factors [magA (mucoviscosity-associated gene A and specific to K1 capsule serotype), k2A (specific to K2 capsule serotype), and rmpA [entB, iutA, iroN genes [fimH [The severity of infections caused by  factors , which iand rmpA . AdditiooN genes . Type 1 es [fimH .fimH was 100% isolates, iroN 4.4%, rmpA 4.4% [entB (78%), fimH1 (55%), kfu (31%), and mrkD (24%) were the prevalence of virulence genes among K. pneumoniae, whereas entB (50%), fimH1 (30%), and mrkD (22%) were prevalent in K. oxytoca [entB, 12% iutA, 94% fimH, and 3% rmpA [fimH and entB, which may be the main virulence factors of Klebsiella spp. from dairy animals in Vietnam and China. Significant differences in the frequency of antibiotic-resistant and virulence genes in different studies may be due to different sources of isolation in different regions, their methods of investigation and sensitivity, and the number and types of samples.Similar studies in China reported that the prevalence of mpA 4.4% ; entB , siderophore (entB), and antimicrobials resistant genes . Results from this study provided information about the distribution and characteristics of pathogenic Klebsiella spp.-infected bovine mastitis that could support dairy farmers/ owners and veterinarians to improve the usage and management of antimicrobial in mastitis treatment, especially with Klebsiella mastitis in large-scale herds.In conclusion, this is the first study to report the occurrence and molecular characterization of


We compared 2 human papillomavirus (HPV) assays to detect the 14 high-risk HPV (hrHPV) genotypes in self-collected anal samples. We found a good agreement and similar performance to detect HPV-16, HPV-18, and the 12 other hrHPV genotypes. The global performance to detect the 14 hrHPV genotypes was not significantly different between the 2 assays. Around 30 000 new cases of anal squamous cell carcinoma (ASCC) are diagnosed each year worldwide . Key popIn this study, we aim to compare the agreement and performance of the Cobas 4800 HPV test (Roche) and the Anyplex II HPV28 Detection assay (Seegene Inc) to detect hrHPV in self-sampling anal swabs from asymptomatic MSM and transgender women (TGW) in Phnom Penh, Cambodia, especially for HPV-16 detection, which was the most prevalent HPV genotype found in this population [A cross-sectional retrospective study was conducted between 1 October 2021 and 30 November 2021 in different clinics in Phnom Penh, Cambodia, and under the scope of activities of Men's Health Cambodia, a nongovernmental and nonprofit organization working with men, MSM, and TGW in Cambodia. During consultation, a questionnaire was used to collect patient data, and anal collection  was self-performed using anal swabs discharged in viral transport media (VTM). They were then stored at −80°C at the Institut Pasteur du Cambodge (IPC) until further analysis. A comprehensive explanation of eligibility criteria and procedures has been published previously [First analysis was performed at IPC on 400 μL of sample with the Cobas 4800 HPV test (Roche), which allows the detection of 14 hrHPV types by polymerase chain reaction (PCR) and nucleic acid hybridization. This test specifically identifies HPV-16, HPV-18, and 12 other hrHPV types in the same channel including HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Then, 1 mL of frozen VTM was sent to the Virology Department of the Pitié-Salpêtrière Hospital, Paris, France. DNA extraction (from 200 μL of sample to 100 μL) was performed with the E-mag device (bioMérieux), and both samples and extracted DNA were stored at −80°C. Then, HPV testing was performed with the Anyplex II HPV28 Detection assay (Seegene Inc), which detects and differentiates 28 distinct HPV types including the 14 hrHPV types detected by the Cobas 4800 HPV test and 14 other HPV types . Both HPV tests also detect a human gene as internal control to assess the quality of the sampling and the efficiency of the PCR amplification. If internal control was not amplified, samples were referred as invalid.rstatix from R software. McNemar and kappa indices were calculated to compare the proportion of positive samples and the degree of agreement between the 2 methods to detect the 14 hrHPV types, respectively. A 2-tailed P value of <.05 was considered significant.Statistical analysis was performed with the package Overall, 162 participants were included with a median (IQR) age of 28.1 (24.4–31.8) years, of whom 56% were MSM and 8% were HIV infected. None of them received any HPV vaccine.P = .52), 16% versus 17% (P = .86), and 50% versus 59% (P = .18), respectively  and 41 (25%) samples, respectively. The prevalence of HPV-16, HPV-18, and other hrHPV types was not significantly different between the 2 methods: 20% versus 25% (ectively .i) 3 for HPV-16 positivity, of which 2 HPV-16 were missed by Roche and 1 missed by Seegene; and (ii) 12 for other hrHPV positivity, of which 8 hrHPV types were missed by Roche and 4 by Seegene  (P = .44).The agreement (Cohen kappa) for detection of HPV-16, HPV-18, and other hrHPV types was 0.91  (.81–1.00), 1.00 (1.00–1.00), and 0.75 (.62–.88), respectively. The performance to detect HPV-16, HPV-18, and other hrHPV positivity were not significantly different between the 2 tests  . In totaTo our knowledge, this is the first study conducted in self-collected anal samples that compared the Cobas 4800 HPV test (Roche) and the Anyplex II HPV28 Detection (Seegene) tests for hrHPV detection.+/GP6+ PCR testing followed by genotyping with Luminex [First, comparing the 2 methods on 97 samples revealed a good agreement between them and comparable worldwide ability to identify any of the 14 hrHPV genotypes currently recommended for cervical cancer screening. This result was also consistent for HPV-16 alone, HPV-18 alone, and the other hrHPV genotypes detected with the 2 methods. However, compared to the Seegene approach, the Cobas test missed more anal hrHPV infections. In term of hrHPV prevalence in normal cervical tissue, a difference was also identified between the Cobas method and GP5The prevalence of HPV-16 was the highest in our high-risk population, as expected, and the 2 methods showed high agreement and performance to detect the most oncogenic genotype in the anal site. This is of importance as HPV-16 testing could be one of the biomarkers used as anal cancer screening in key populations in the near future. For instance in France, new guidelines propose to use HPV-16 testing in anal cytology specimen collected by physicians as an anal cancer screening marker in MSM living with HIV . If posiSimilarly to self-collected vaginal swabs for cervical cancer screening , self-coIn conclusion, both the Anyplex II HPV28 Detection assay and the Cobas 4800 HPV test are suitable to detect the 14 hrHPV genotypes, in particular HPV-16, in self-collected anal samples.ofad540_Supplementary_DataClick here for additional data file.


Cervical cancer being one of the primary causes of high mortality rates among women is an area of concern, especially with ineffective treatment strategies. Extensive studies are carried out to understand various aspects of cervical cancer initiation, development and progression; however, invasive cervical squamous cell carcinoma has poor outcomes. Moreover, the advanced stages of cervical cancer may involve lymphatic circulation with a high risk of tumor recurrence at distant metastatic sites. Dysregulation of the cervical microbiome by human papillomavirus (HPV) together with immune response modulation and the occurrence of novel mutations that trigger genomic instability causes malignant transformation at the cervix. In this review, we focus on the major risk factors as well as the functionally altered signaling pathways promoting the transformation of cervical intraepithelial neoplasia into invasive squamous cell carcinoma. We further elucidate genetic and epigenetic variations to highlight the complexity of causal factors of cervical cancer as well as the metastatic potential due to the changes in immune response, epigenetic regulation, DNA repair capacity, and cell cycle progression. Our bioinformatics analysis on metastatic and non-metastatic cervical cancer datasets identified various significantly and differentially expressed genes as well as the downregulation of potential tumor suppressor microRNA miR-28-5p. Thus, a comprehensive understanding of the genomic landscape in invasive and metastatic cervical cancer will help in stratifying the patient groups and designing potential therapeutic strategies. Cervical cancer (CC) is the most common tumor of the female reproductive system and the fourth most prevalent cancer type overall –3. The iSeveral genetic and epigenetic variations and tumor microenvironment heterogeneity contribute to malignant transformation , 13. TheIt has been established that the cervicovaginal microbiome dysbiosis in concert with HPV infection contributes to CC initiation , 27. TheFusobacterium species ; pathogen gene response ; 2q13 (PAX8), 5p15.33 (TERT-CLPTM1L), and 17q12 (GSDMB). Polymorphisms in ARRDC3, INS-IGF2, SOX9, TTC34, ACACB have also been associated with a risk of developing CC; however, this remains to be validated [A large number of genetic variations are observed in CC, and the burden of variations increases with high grade and severity of the disease . The gentibility . With thalidated , 178. SeEP300, MUC4, MUC16, SYNE1, KMT2C, PIK3CA, FLG, KMT2D, DST, MAPK1, and TTN [NSD1, ARID1A, CTCF, ARID1B, KMT2C, SETBP1, PBRM1, and KMT2D were specific to recurrent/metastatic CC [APOBEC3A was also observed for recurrent/metastatic CC. Also, in some cases of CC, nuclear-encoded sigma factor 6 (SIG6) mutation is associated with defect in mismatch DNA repair [Although mutations are prevalent in CC, only a few can act as driver mutations, which are expected to initiate and promote growth, and these are common mutations identified in cancer invasion and metastasis . Additio and TTN . Also, t and TTN . Integratatic CC . APOBEC-A repair .MCL-1 leading to the progression of CC by evading apoptosis signaling [FOXM1. However, circCLK3 sponges miR-320a, thus resulting in increased FOXM1 expression and significant promotion of cell metastasis in vitro as well as in vivo [p21 expression, regulates expression of MMP-9, VEGF and genes related to EMT, all of which are essential for migration and invasion of CC [Non-coding regions within the genome have a major impact on the progression of cancer. Deregulated expression of small miRNAs as well as lncRNAs and circRNAs has been associated with distinct stages of CIN and development of CC . Specifi in vivo . miR-320 in vivo . Similar in vivo , 197. Ci in vivo . The HOXon of CC , 188. Reon of CC . Additioon of CC , MALAT1 on of CC , and TMPon of CC , therebyHIF-1α, thus promoting proliferation, invasion, and migration as well as suppression of apoptosis by escalating glycolysis through circAGFG1/miR-28-5p/HIF-1α axis as observed in non-small-cell lung cancer (NSCLC) [Our bioinformatic analysis of TCGA data of CC revealed reduced expression of miR-28-5p in CC patient (unpublished data). miR-28-5p is located at 3q27.3, which is frequently gained in CC. The significant downregulation in CC could be due to circRNA ArfGAP with FG repeats 1 (circAGFG1), which binds to miR-28-5p and targets  (NSCLC) . Further (NSCLC) . Similar (NSCLC) , 202.GSE26511 dataset from TCGA including 39 samples (20 CC-lymph node-negative samples and 19 CC-lymph node-positive samples) was analyzed by Ge et al. . ApproxiNR5A2 in metastatic tissue compared to non-metastatic tissue of CC [NR5A2 had been identified to contribute in developing CC during GWAS [CXCL9, SPEG, MUC21, and APOBEC3A were downregulated, POSTN was upregulated in metastatic CC compared to primary tumor [Dataset analysis of 116 non-metastatic and 10 metastatic samples from “TCGA” indicated differential expression of transcription factor ue of CC . Earliering GWAS . NR5A2 ping GWAS  and repring GWAS . The gening GWAS , 209. CSing GWAS . Signifiing GWAS . Vimentiing GWAS . EMT is ing GWAS , 212. Thing GWAS . Transcring GWAS . Matrix ing GWAS , 214. Diry tumor .p-value < 0.05 and logFoldChange > 1.5 for upregulation, logFoldChange < -1.5 for downregulation was set as the standard filter. The genes that fall under the standard filter criteria were selected as DEGs. We have identified 70 upregulated and 46 downregulated genes in at least two datasets. The overlapping DEGs identified in more than two of the selected datasets were extracted and represented in the UpSetR plot Fig. KRT6B, TGM3, ALOX12B, and CRCT1 were found to be upregulated Fig. DDX58, IFI44, OAS2, IFI44L, and IL15 Fig. We performed in silico analysis with the limma R package  for idenp-value cut-off score of 0.05.Further, the gene ontology study identified that the upregulated genes were primarily involved in protein binding and serine-type peptidase activity and the downregulated genes were involved in catalytic activity, localization, and immune response Fig. p-value > 0.05 set as the cut-off, suggesting that the upregulated genes were significantly involved in Wnt signaling, chromosomal and microsatellite instability in colorectal cancer, apoptosis modification and signaling Fig. CNKSR3, CSNK1A1, EPHB3, and IL1R2, interacting with multiple microbial protein domains Fig. SPTBN1, CFI, DST, and SYT1 interacting with two microbial domains Fig. IDS, MAP4K4, COL4A2 and KHDRBS1 among the upregulated and, in SPTBN1 and DST among the downregulated genes Fig. When pathway analysis was performed on the DEGs from WikiPathways using EnrichR tool  with p-vIDS encoding iduronate 2-sulfatase (ID2S) was overexpressed in CC epithelial tissue compared to the normal tissue with an unknown mutation status. ID2S is primarily reported in mucopolysaccharidosis, and its association with the regulation of breast cancer metastasis has been reported [MAP4K4 is associated with the activation of NF-κB and JNK signaling pathway and EMT induction in hepatocellular carcinoma (HCC) [MAP4K4 is reported as one of the potential metastatic genes in colorectal cancer metastasis [MAP4K4 in cancer cell lines inhibits cell proliferation and cell growth [Protein expression analysis was performed for DEGs using Human Protein Atlas , revealireported , 227. MAreported  that plareported –232. Ovema (HCC) . Studiesma (HCC) . MAP4K4 tastasis  and CC [tastasis . Downregl growth , 238, apl growth , 240, anl growth .Fig. 6ImCOL4A2 encodes type IV collagen and impacts cancer progression and pathogenesis. In HCC, COL4A1 overexpression promotes metastasis [COL4A1 overexpression is positively correlated with dendritic cells and macrophage expression in CC [COL4A2 inhibits migration and proliferation in triple-negative breast cancer [KHDRBS1/SAM68 detected in CC tissues with pelvic node metastasis and was not found in normal tissues [SAM68 in malignant keratinocytes increases the sensitivity to DNA damage and decreases tumor burden [tastasis . COL4A1 on in CC . Supprest cancer . Elevate tissues . The RNA tissues . SAM68 i tissues . SAM68 e tissues . Furtherr burden .SPTBN1 expression decreased significantly with the progression of epithelial ovarian cancer. The decreased expression resulted in cell proliferation, migration, and increased vimentin expression [SPTBN1 is downregulated in HCC and loss of its expression stimulates Wnt signaling, thereby promoting the acquisition of stem cell-like characteristics, and eventually, contributing to the growth of malignant tumors [SPTBN1 is a member of the β spectrin family and is important for the stability of lateral membrane. It also functions as a TGF-β adaptor protein and modulates Smad complex . SPTBN1 pression . It inhipression . Furtherpression . In primpression . Similart tumors .KLK8, KLK7, KLK10) were found upregulated in metastasized CC. Kallikrein (KLK) proteins are a subgroup of serine proteases. KLKs play an important role in carcinogenesis by altering cell proliferation, EMT, compromising oxygen balance, and degrading ECM. They also allow tumor cell detachment by infiltrating through ECM barriers and promoting metastatic dissemination [Further, analysis from two datasets (GSE6791 and GSE9750), various isoforms of kallikreins (KLKs) -mination . Kallikrmination , 255. KLmination . The woumination . The uprmination . Furthermination . Though mination .KLK7) levels were reported to be higher, both of which are associated with tumor growth, invasion and metastasis [KLK7 was found to be regulated by histone modifications and promoter methylation. Trichostatin A (TSA) enhanced KLK7 expression in cervical and pancreatic cancer cell lines, and the transcription factor specificity protein 1 (SP1) supported the effect of TSA on KLK7 by transcriptional activation [KLK7 in pancreatic cancer was associated with urokinase-type plasminogen activator receptor (uPAR), which inhibited cellular adherence to vitronectin [KLK7 overexpression inhibited cell adhesion by lowering integrin expression and inducing spheroid formation by increasing MCAM/CD146 expression [KLK7 expression, which significantly induced the invasion and metastasis [In CC tissues and serum, kallikrein 7  belong to the non-classical subfamily of cadherins. These are highly expressed in brain and mediate cell to cell interactions . Several pathway . PCDH7 eer cells . Colony er cells . The expgulation . Furtherinvasion . The rolThe pathogenesis of CC is intricate and interconnected to the key determinants of molecular interactions and biological effects. HPV infection along with other risk factors is known to initiate the CC by recreating aberrant local microenvironment. Further, genetic and epigenetic modifications along with altered gene expression may favor the progression and metastasis of the cancer cells. The role of microbiota is significant in the modulation of microenvironment to facilitate the infection by HPV and also to promote metastasis of the tumor. Hence, understanding and elucidating the functional properties of the diverse species in the cervix will aid in deciphering the role of cervical microbiota  in the progression of CIN to CC and metastasis. Sentinel lymph nodes are the first nodes to receive lymphatic drainage from the primary lesion and promote LNM due to immune suppression by the tumor microenvironment. Further, lymphatic system can facilitate metastasis to distant organs, such as bone, lung, liver, and brain. Hence, identification of LNM and modulation of its microenvironment is required to prevent metastasis to distant organs and to provide effective treatment. Number of molecular pathways and their downstream functions are altered because of viral proteins, HPV integration, somatic mutation, copy number variation, epigenetic alteration, differential expression of the genes, miRNAs, lncRNAs and circRNAs, and interestingly, each of these factors is interconnected such that they favor incremental evolution, development and progression of CC. Implication of multi-omics approach to determine the genomic landscape can aid the stratification of CC patients and formulating effective treatment strategies.


Sorting of myeloid cell subsets demonstrated that CD14+CD206–, CD14+CD206+, and CD14–CD206+ cell populations were infected, in the liver and lungs, in SIVmac251-infected RMs. Of importance, early ART drastically reduced viral seeding consistent with the absence of ISG detection but also of genes related to inflammation and tissue damage. Viral DNA was only detected in CD206+HLA-DR+CD11b+ cells in ART-treated RMs. The observation of pulmonary and hepatic viral rebound after ART interruption reinforces the importance of early ART implementation to limit viral seeding and inflammatory reactions.Identifying immune cells and anatomical tissues that contribute to the establishment of viral reservoirs is of central importance in HIV-1 cure research. Herein, we used rhesus macaques (RMs) infected with SIVmac251 to analyze viral seeding in the liver and lungs of either untreated or early antiretroviral therapy–treated (ART-treated) RMs. Consistent with viral replication and sensing, transcriptomic analyses showed higher levels of inflammation, pyroptosis, and chemokine genes as well as of interferon-stimulating gene (ISG) transcripts, in the absence of ART. Our results highlighted the infiltration of monocyte-derived macrophages (HLA-DR The observation that viral rebound occurs after antiretroviral therapy (ART) interruption (ATI) despite early treatment indicated early viral seeding and the absence of the full eradication of HIV and SIV –4. PersiMonocytes play a critical role in tissue homeostasis and host defense . Blood mIn addition to monocyte-derived macrophages , tissue-+ and LYVE+ macrophages also express CD183 (CXCL10 or IP-10 receptor), suggesting recruitment consistent with higher levels of chemokine and innate immune transcripts. In particular, genes related to inflammasome/pyroptosis and tissue damage are increased . During infection, plasma viral loads reached 102 to 108 copies/mL  were lower in the liver, compared with the levels of viral RNA in the lungs   and the lungs   . We then 0.0004) , a stron 0.9214) . We then 0.9214) . Associa 0.9214)  or undet 0.9214) , viral D 0.9214) , D and EThus, our results are consistent with previous reports, showing viral DNA and RNA in the lungs –39 and lNext, we performed transcriptomic analyses from the liver and lung cells of untreated SIVmac251-infected RMs including highly viremic RMs  and low viremic RMs  . Transcrz score scaled  . PCA, ale scaled , indicate scaled .hi) and low viremic (SIVlo) RMs, we then performed additional analyses separating these 2 groups. Volcano plots showed the number of transcripts that were significantly up- and downregulated  and cell activation pathway  (lo), upregulated genes were associated with immune defense response pathway in the liver  and mostly associated with chemical homeostasis pathway in the lungs . The lists of genes are indicated in Having observed differentially expressed genes in both tissues from SIV × 10–5) , B and CISG15, IFI6/27/44/44L (interferon-α–inducible proteins), IFIT1/2/3/5 (interferon-induced proteins with tetratricopeptide repeats), IRF7 (interferon regulatory factor 7), OAS1/2/3/L (2′-5′-oligoadenylate synthetase), and MX1/2 (MX dynamin like GTPase), and several ISG-related transcripts was observed in SIVhi-infected RMs, compared with SIVlo-infected RMs and healthy uninfected RMs , we demonstrated a positive correlation between the levels of mRNA transcripts and VL , significant enrichment of interferon-stimulating gene (ISG) mRNA, including cted RMs . The P vs and VL , with OATRIM5) and Viperin (RSAD2), were also increased in both tissues of SIVhi-infected RMs compared with healthy uninfected RMs and SIVlo-infected RMs , DDX60, and DHX58 (LGP2) in the liver and lungs of SIVhi-infected RMs compared with healthy uninfected and SIVlo-infected RMs  and AIM2  were modulated in SIV-infected RMs (P = 3.770 × 10–22) were observed in the lungs. Among them, we found that several members of the collagen family and metalloproteinases were downregulated. In contrast, several serine protease inhibitors, including members of the serpin family  and of the inter-alpha-trypsin inhibitor (ITIH) family  were mostly upregulated in SIVhi-infected RMs (FGG and FGL1), ITIH1/4, MMP9, and SERPINs , and SIGLEC1 were upregulated in pulmonary tissue (MARCO (a class A scavenger receptor), MSR1 (CD204), FCGR1A (CD64), and CD163 , CSF1R, and CD163, whereas FCGR1A (CD64) was downregulated in the lungs , CRP, and neutrophil transcripts  both in hepatic and in pulmonary tissues . In SIVlo-infected RMs, few genes were significantly modulated. We observed higher levels of CXCL8, CCL19 (MIP-3β), and CCL23 (MIP-3) in the liver only , complement , and coagulation    that wasnd KNG1) .hi-infected RMs revealed genes related to lipid/steroid metabolic pathways , supporting cholesterol and steroid synthesis (HSD3B2) (ME1 (NADP-dependent enzyme that generates NADPH for fatty acid) was downregulated (PLA2G2A and PLA2G4C), known for their contribution in the biosynthesis of inflammatory mediators, were increased. The P values and log2 fold-changes, associated with up- and downregulated genes, are presented in Because injury and inflammation may be associated with altered cellular metabolic pathways, such as lipotoxic injury fueling inflammation by attracting innate cells , we anal(HSD3B2) , whereasegulated . In addiUGT1A1/UGT2B7), foam cell formation (PON1), and PPARα pathway   . Members HMGCS2) .APOA1, APOA5, APOB, APOC2, APOD, APOF, PON1, and FABP1), retinol , steroid , pyruvate , and tryptophan pathways (PAH), as well as several methyl-acyl-glucuronosyltransferases   . Thus, ahi-infected RMs . Initially, the expressions of CD14 and CD16 were analyzed. The percentages of CD14+CD16+ cells were 65.2% ± 24% in the liver of SIV-infected RMs (+CD16– (61.0% ± 11%)   . As showl cells)  (Suppleml cells) . Becausected RMs  expressee spleen . Howevere spleen . Therefohe liver , whereashe liver . Those e6+ cells . Similar6+ cells . The spe6+ cells . As a cocells , we also detected viral DNA in blood monocytes (4 copies/mL) (4 copies/mL)  . On the pies/mL)  in whichpies/mL)  and disppies/mL) . Due to he lungs . In the +HLA-DR+ expressing or not CD206. Indeed, due to the low number of cells recovered from ART SIV-infected RMs, it was not possible to analyze more subsets. In parallel, CD4+ T cells were sorted from the same individuals. Our results indicated that viral DNA was detected in CD206+ myeloid cells and not in CD206– cell subset  and CX3CR1 by CD11bu et al.  have repu et al. , 69, 70.u et al. , we havee injury . In the rophages –75 in whrophages –78.+CD14–206+, CD11b+CD14+206+, and CD11b+CD14+206–, are infected. However, it is noteworthy that we detected viral DNA only in RMs in which viremia was the highest (>104 copies/mL) and a frequency higher than 102 SIV DNA copies per million of blood monocytes. Of importance, we noticed that viral DNA was only detected in CD206+ cells after early ART. Because of the limited quantity of materials, we cannot exclude that viral DNA detected in macrophages reflects the phagocytosis of dying CD4+ T cells, as previously reported in the liver  that subsequently acquire resident macrophage phenotypes. Of interest, we demonstrated that this macrophage population expresses CD183 and CX3CR1. Finally, our findings also provide evidence that early ART administration efficiently inhibits inflammation, as well as viral seeding, in hepatic and pulmonary tissues. Therefore, it is of crucial importance to limit this vicious cycle of viral infection of myeloid cells in the lungs and liver early, reducing the comorbidities associated with liver and lung tissue damage.Overall, our data highlight the presence of CD1650). At day 4 postinfection, 10 RMs were treated with tenofovir  and emtricitabine  subcutaneously and raltegravir  or dolutegravir  and ritonavir  by oral route. Five individuals of the early ART-treated RMs interrupted the treatment 8 weeks after ART administration. RMs were sacrificed at different time points postinfection during natural infection , under ART , and after ATI (n = 5), as shown in Twenty-five RMs seronegative for SIV, simian T leukemia virus type 1, SRV-1 (type D retrovirus) and herpes B virus were infected intravenously with SIVmac251  for 15 minutes, cells were washed and resuspended in cold paraformaldehyde 1%. Then, 60,000 events corresponding to mononuclear cells were recorded in FACSCelesta (BD Biosciences). Analyses were performed using FlowJo software (Tree Star).After a mechanical process, fresh cells from the liver, lungs, and spleen were resuspended in RPMI medium (Thermo Fisher Scientific) and stained with monoclonal antibodies  in the dRNA from liver and lung cell samples in TRIzol (Thermo Fisher Scientific) were extracted using QIAGEN RNeasy Micro Kit with DNase I treatment. Quantity and quality of total RNA were assessed by ND-1000 spectrophotometer (NanoDrop Technologies) and Bioanalyzer 2100 (Agilent RNA 6000 Pico Kit). The NEBNext Ultra II directional RNA library prep kit for Illumina (New England Biolabs) was used to prepare libraries, according to the manufacturer’s instructions. Briefly, 400 ng of total RNA was purified using the NEBNext poly(A) and used as a template for cDNA synthesis by reverse transcriptase with random primers. The specificity of the strand was obtained by replacing the dTTP with the dUTP. This cDNA was converted to double-stranded DNA that was end-repaired. Ligation of adaptors was followed by a purification step with AxyPrep Mag PCR Clean-up kit (Axygen), by an excision of the strands containing the dUTPs, and finally, by a PCR enrichment step of 9 cycles to incorporate specific indexed adapters for the multiplexing. The quality of final amplified libraries was examined with a DNA screentape D1000 on a TapeStation 2200, and the quantification was done on the QuBit 3.0 fluorometer (Thermo Fisher Scientific). mRNA-Seq libraries with unique index were pooled together in equimolar ratio and sequenced for paired-end 100 bp sequencing on an NovaSeq 6000 flowcell S1 at the Next-Generation Sequencing Platform (CHU de Québec Research Center). The average insert size for the libraries was 290 bp. The mean coverage/sample was 24 million paired-end reads.Macaca mulatta transcriptome (Mmul_10 downloaded from Ensembl release 103). The PCA was completed with the FactoMineR v2.4 R package. The PCA and volcano graphical representations were produced with the ggplot2 v3.3.3 package. Differential expression analysis was performed using the DESeq2 v1.30.1 package. This package overcomes the lack of power, due to the high uncertainty of the variance estimation, by pooling information across genes  were analyzed using the ToppGene (ToppFun) tool  tool . Gene clun) tool , GeneCodun) tool , and Topun) tool . Genes run) tool .+ T cells were sorted using a BD Influx cell sorter upon staining with specific antibodies: anti-CD3, -CD4, -CD20, –HLA-DR, -CD11b, -CD14, and -CD206 mAbs  versus enrichment, leading to a high level of purity of over 95% . Samples were run in duplicate, and results are expressed as SIV RNA copies/mL . The PCR mixture was composed of 4× TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems), 750 nM of primers, and 200 nM of probe. Primers and probe sequences are listed in opies/mL .5 cells. RNA was extracted with TRIzol procedure and resuspended in 50 μL of RNase-free water. DNA was eliminated using a TURBO DNA-free Kit (Invitrogen). We amplified 5 μL of RNA by qRT-PCR with a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Eukaryotic 18S rRNA Endogenous Control mix (Applied Biosystems) was used to estimate cell numbers in each sample. Primers and probe sequences are listed in 6 cells.Viral RNA was assessed from 105 to 106 cells) was purified using the Genomic DNA Tissue Kit (Macherey-Nagel). After elution (50 μL), 10 μL was amplified by nested PCR with SIVmac251-specific primers surrounding the nef coding region , following the manufacturer’s instructions. Quantitative PCR was performed in 10 μL using 2× QuantiTect SYBR Green PCR kit (QIAGEN) and 250 nM specific primers in a QuantStudio 6 Flex Real-Time PCR System. The sequence of each primer is provided in P values less than 0.05 indicate a significant difference. Spearman’s analysis was used for correlations.Statistics were analyzed using GraphPad Prism 8 software. The nonparametric Mann-Whitney test and the 1-tailed Wilcoxon’s test were used for comparison, as indicated in the figures. http://www.ccac.ca). The protocol was approved by the Laval University Animal Protection Committee (Project number 106004). Animals were fed a standard monkey chow diet, supplemented daily with fruit and vegetables and water ad libitum. Social enrichment was delivered and overseen by veterinary staff, and overall animal health was monitored daily. Animals were evaluated clinically and were humanely euthanized, using an overdose of barbiturates, according to the guidelines of the Veterinary Medical Association.RMs were housed at Laval University, in accordance with the rules and regulations of the Canadian Council on Animal Care (All data are available in the JAC, MAJ, PA, and JE conceived and designed the experiments. JAC, HR, GR, CS, OZA, and JE performed the experiments. JAC, HR, GR, SB, OZA, CS, CJB, and JE analyzed the data. JAC, MAJ, AD, PA, and JE wrote the paper.


Phytophthora infestans poses a serious threat to potato production, storage, and processing. Understanding plant immunity triggered by fungal elicitors is important for the effective control of plant diseases. However, the role of the potato stress response to Fusarium toxin deoxynivalenol (DON)-induced stress is still not fully understood. In this study, the metabolites of DON-treated potato tubers were studied for four time intervals using UPLC-MS/MS. We identified 676 metabolites, and differential accumulation metabolite analysis showed that alkaloids, phenolic acids, and flavonoids were the major differential metabolites that directly determined defense response. Transcriptome data showed that differentially expressed genes (DEGs) were significantly enriched in phenylpropane and flavonoid metabolic pathways. Weighted gene co-expression network analysis (WGCNA) identified many hub genes, some of which modulate plant immune responses. This study is important for understanding the metabolic changes, transcriptional regulation, and physiological responses of active and signaling substances during DON induction, and it will help to design defense strategies against Phytophthora infestans in potato. Solanum tuberosum L.) have been cultivated in more than 160 countries and regions, making it the world’s most important non-cereal food crop [Fusarium infection account for 17.2% of total potato production, causing huge economic losses [Pseudomonas spp. [P. infestans also produces virulence factors  such as T-2 toxin, DON, ENN-A, ZEB, and FB, which can promote infestation while causing disease [To date, more than 10,000 varieties of potato  and systemic acquired resistance (SAR) in tobacco [Valsa mali exciton, VmE02, induces tobacco cell death, ROS accumulation, callus deposition, and activation of salicylic acid (SA) and jasmonic acid (JA) induced immune responses [Pathogens and elicitors can activate plant-induced defenses and produce heritable resistance ,15. In tbidopsis ; Pseudomtotoxins ; cryptog tobacco ; and theesponses .Fusarium, contributes to the colonization of P. infestans, and the study of DON resistance can provide new insights into plant disease resistance [TaMetRS) gene, which encodes a novel MetRS with defense and detoxification effects [TaUGT6 enhances resistance to Fusarium head blight (FHB) and accumulation of DON in wheat [TaCYP72A enhances wheat resistance to DON [Fusarium crown rot (FCR) has been reported to be suppressed by limiting the action of DON during plant glycosylation [DON, a virulence factor of sistance . Studies effects . TaUGT6 in wheat . Additioe to DON . DON actsylation ,27.PAL) genes were found to be important genes for cell wall-mediated immunity and were involved in broad-spectrum disease resistance [Stylosanthes to Colletotrichum infection [When plants activate their defenses, secondary metabolites synthesized through primary metabolites or other biosynthetic pathways regulate adaptive plant growth, mainly including terpenoid, phenylpropanoid, alkaloid, and polyketide metabolic pathways . The phesistance . Activatsistance . Differesistance , phenolisistance ; lignifisistance  and prodsistance . For exanfection . Accordinfection .P. infestans infected potatoes revealed SA-JA-ABA(abscisic acid)-mediated signaling pathways [Salvia miltiorrhiza stems and leaves through transcriptional metabolic analysis.In recent years, combined transcriptional and metabolic analyses have become an important tool for discovering new signaling pathways and responses to environmental stress induction, and they are an effective method for revealing plant stress responses, plant-pathogen interactions, mechanisms of metabolite synthesis, and identification of resistance genes . For exapathways ; Li et apathways  elucidatpathways  elucidatP. infestans [Our previous study found that treatment of potato tubers with low concentrations of DON also affected resistance to late blight caused by nfestans . HoweverTo better understand the dynamics of relevant metabolites during DON stress, total metabolites were extracted from each sample, and metabolite detection was performed using UPLC-MS/MS. The mass spectrometry data of each group of samples  were processed using Analyst 1.6.3 software, and a total of 676 metabolites were detected based on the local metabolic database. The integration of the peaks and the correction of the chromatographic peaks were performed on the downstream mass spectrometry results of each sample using MultiaQuant software (v3.0.3) to obtain the relative content of each substance. The 15 samples were clearly divided into 5 groups by heat map and correlation analysis A, and thTo show the changes in the metabolite content of potato tubers under different DON stress durations, we used orthogonal partial least squares discriminant analysis (OPLS-DA) to screen for differential metabolites, and metabolites with fold change ≥2 or ≤0.5, along with VIP ≥ 1, were considered as significantly differential metabolites. Compared with CK0h, 75, 117, 180, and 197 differential metabolites were identified in DON0h, DON4h, DON12h, and DON48h, respectively, and more upregulated metabolites were specific at 48 h . Among tN-benzylformamide (C15561), calystegine A3 (C10850), 2-(formylamino) benzoic acid (C05653), l-tyrosine (C00082), 3-indoleacrylic acid (C21283), l-tryptophan (C00078), methoxyindoleacetic acid (C05660), N-acetyl-l-tryptophan, N-caffeoylputrescine (C03002), p-coumaroylmalic acid, 5-O-caffeoylshikimic acid (C10434), syringaldehyde-4-O-glucoside, laempferol-3-O-(2-O-xylosyl-6-O-rhamnosyl)glucoside, and quercetin-3-O-rutinoside. To further understand the in vivo interactions of the differential metabolites in plants, we annotated and enriched the functions of the differential metabolites based on the KEGG database and found that all the differential metabolites were mainly annotated in metabolic pathways and biosynthesis of secondary metabolites. The enrichment results showed that nine pathways—metabolic pathways, biosynthesis of secondary metabolites, ABC transporters, biosynthesis of amino acids, 2-oxocarboxylic acid metabolism, aminoacyl-tRNA biosynthesis, phenylpropanoid biosynthesis, arginine and proline metabolism, and cysteine and methionine metabolism—were enriched at various time periods under DON stress  in the different samples were classified into 12 categories, of which the main ones were flavonoids (18.3%), phenolic acids (16.6%), and alkaloids (16.2%). Most of the DAMs showed large differences at different times, with 55 of the 82 flavonoids showing variability and significant down-regulation at 0 h and 12 h; 50 of the 99 phenolic acid metabolites showed variability and significant accumulation at 12 h and 48 h; and 49 of the 83 alkaloids showed variability and significant accumulation at 48h A. NotablN stress C.Taken together, our results indicate that potatoes under DON stress mainly exhibit phenolic acid and alkaloid metabolite accumulation patterns, with significant differences in defense responses for approximately 50% of the metabolites that are directly or indirectly attributed to the phenylpropane metabolic pathway.To study the gene expression profile of potato tubers under DON stress at different times, we performed RNA-seq on the same samples as those used in metabolomics. Fifteen samples from five groups of treatments were sequenced with libraries, and each library of Clean Data was greater than 6 GB, Q30 base percentage was greater than 90%, average length was 150 bp, and GC content of each sample was greater than 43% see , and a UTo assess the reproducibility of the RNA-seq data, we calculated Pearson correlation coefficients between the three biological replicates for each sample. The results showed that the Pearson correlation coefficients between the three biological replicates ranged from 0.88 to 0.99, with large differences between groups, and PCA graph also showed good sample consistency , indicatWe identified DEGs between the control (CK0h) and other treatments using |log2 (fold change)| ≥ 1 and Padj < 0.05 as screening criteria. Compared with the control, 1053, 20,943, 20,347, and 21,172 were identified at 0 h, 4 h, 12 h, and 48 h under DON stress, respectively A. We fouAnalysis of DEGs at different treatment times showed that each treatment time had its specific DEGs, and the highest number of specific DEGs was found at DON4h, followed by DON48h treatment, whereas 97 and 30 consistently upregulated and downregulated, respectively, and expressed DEGs were found in the four sets of comparisons, indicating that these genes may play a crucial role in response to DON stress in potato C,D. ExprGene Ontology (GO) and KEGG enrichment analyses of DEGs were performed. All protein sequences were annotated based on the EGGNOG website, and the Orgdb package was constructed to functionally annotate and enrich each group of DEGs according to GO classification. In the GO analysis of the CK0h-DON0h group, 420, 328, and 391 DEGs were classified as biological processes, molecular functions, and cellular components, respectively. In the GO analysis of the CK0h-DON12h group, 7854, 6783, and 7846 DEGs were classified into biological processes, molecular functions, and cellular components, respectively. In the GO analysis of the CK0h-DON48h group, 8013, 6928, and 7991 DEGs were classified as biological processes, molecular functions, and cellular components, respectively. Compared with the CK0h control, DEGs under DON stress from 0 to 48 h were significantly enriched in the secondary metabolite biosynthetic process (GO:0044550), phenylpropanoid biosynthetic process (GO:0009699), and phenylpropanoid metabolic process (GO:0009698) .DEGs were annotated in the KEGG database to understand their biological functions and gene interactions. In the CK0h-DON0h group, 423 of the 1053 DEGs were enriched in 27 pathways, including phenylpropanoid biosynthesis, flavonoid biosynthesis, and phenylalanine metabolism . In the Genes with very low expression (average TPM < 1) were removed from this analysis to avoid the inclusion of spurious edges in the networks. We screened 52,387 highly expressed DEGs from 63,883 DEGs using co-expression network analysis. The power values were first filtered to ensure that the gene distribution conformed to a scale-free network. Hierarchical clustering of all the samples showed no outlier samples. When the power value  was 20, several genes with similar expression profiles belonging to the same subnetwork were clustered into the same co-expression modules . Finallyp < 0.01): darkseagreen1 (6 positive correlations), darkseagreen3 (3 positive correlations), and darkgoldenrod3 (1 positive correlation), and seven modules showed a clear trend of positive expression. Among all modules, the darkseagreen1 module was significantly correlated with seven differential metabolites, suggesting that genes in this module are highly correlated with the resistance mechanism of potato plants under DON stress. KEGG enrichment of three highly correlated modules revealed that the phenylpropanoid biosynthesis, oxidative phosphorylation, cysteine and methionine metabolism, necroptosis, and phenylalanine metabolism pathways were significantly enriched  convert toxins to their glucosides (DON3G and NIV3G) via xenobiotic detoxification . DON tresistance . In 5 ngsistance .JAR1 and JAZ, were upregulated, while COI1 and MYC2 were significantly downregulated, and key genes associated with SA transduction, such as NPR1, TGA, and PR-1, were significantly upregulated -l-homocysteine, S-adenosyl-l-methionine, and l-alanyl-l-phenylalanine, which may play a key role in DON-induced plant defense. Consistent with this finding, genes related to these amino acids and their derivatives were also induced.Many changes occur in the primary metabolisms of plants, such as amino acids, nucleotides, and their derivatives, in response to stress, and the composition and levels of amino acids affect the resistance of plants ,58. ManyStreptomyces scabiei. In addition, it is possible that because the family of tyrosine-derived metabolites is flavonoids, DON induction caused significant downregulation of l-tyrosine, resulting in a reduction of 84% of all flavonoids in DAMs, suggesting that flavonoid levels may be negatively correlated with DON-induced plant defense. Combined with the transcriptome enrichment results, the phenylpropane synthesis pathway is a rich source of plant metabolites and a starting point for the production of many other important compounds such as alkaloids, coumarins, and lignans [Plant secondary metabolites such as alkaloids, phenolic compounds, and flavonoids are typical plant antitoxins that play an important role in protection against diseases . However lignans . Therefo lignans ,64 that ATTPS6 and COMT [COMT in the darkseagreen3 module is a key enzyme in phenylpropanoid biosynthesis, catalyzing a variety of phenylpropanoids and flavonoids, including caffeic acid, caffeol, and 5-hydroxyferulic acid, and catalyzes the production of melatonin from N-acetyl-5-hydroxytryptamine [ASMT encoded N-acetylserotonin methyltransferase in the darkseagreen1 module is the final enzyme in the biosynthetic pathway that produces melatonin. ASMT is induced in rice by both ABA and methyl jasmonic acid treatment [MYB48 encoded transcription factor plays an important role in regulating stress response in response to JA signaling [MYB48 plays a central role in improving tomato tolerance [MYB48 to stress treatment in rice [CYP71B36 and CYP76C2 belong to the cytochrome P450 family, of which CYP71B has been shown to be involved in terpene biosynthesis (nerol and geraniol). For example, CYP71BJ1 in rose, which forms part of the pathway leading to 19-O-acetyl horhammericine, will help elucidate how this branch point is controlled [CYP76C is a sesquiterpene plant antitoxin hydroxylase that hydroxylates rishitin in potato to form rishitin-M1 [ABCG22 encodes an ABC transporter protein and the ABC transporter protein TaABCC3.1 has been reported to contribute to DON tolerance [P. infestans in potatoes.WGCNA divided all DEGs into 14 modules, and three modules were highly correlated with 15 shared DAMs. KEGG enrichment analysis showed that genes of related modules were mainly enriched in phenanthrene biosynthesis, which is consistent with the above results. The annotation of hub genes was mainly for functional protein-encoding genes such as and COMT . Most huyptamine . The ASMreatment ,68. Studreatment . MYB48 eignaling , and a polerance  and a po in rice ,73. The  in rice . CYP71B3ntrolled . CYP76C olerance . qRT-PCRIn this study, Atlantic variety potato tubers were used as experimental material . To study the dynamic response of the whole potato genome and metabolome to DON stress, potato tubers were treated with 5 ng/mL Vomitoxin  and sampled at 0 h, 4 h, 12 h, and 48 h , and 0 h under sterile water treatment as control (recorded as CK0h), and all samples were frozen in liquid nitrogen and stored at −80 °C. Three biological replicates were performed for all five groups of samples.The preserved samples were freeze-dried under vacuum using a lyophilizer  and the freeze-dried samples were crushed to powder form using a grinder  at 30 Hz for 90 s. 100 mg of sample powder was weighed and dissolved in 1.2 mL of 70% methanol extract at 4 °C, and vortexed every 30 min for 30 s for a total of 6 times, and the samples were placed in a refrigerator at 4 °C overnight. After centrifugation at 12,000 rpm for 10 min, the supernatant was aspirated, and the sample was filtered through a microporous membrane (0.22 μm pore size) and stored in the injection vial for subsequent UPLC-MS/MS analysis.For each sample, three biological replicates were analyzed independently. Chromatography mass spectrometry acquisition was performed using ultra performance liquid chromatography  and tandem mass spectrometry .LIT and triple quadrupole (QQQ) scans were performed by a triple quadrupole linear ion trap mass spectrometer (Q TRAP), AB4500 Q TRAP UPLC/MS/MS system. The chromatographic column was an Agilent SB-C18 ; mobile phase was ultra-pure water in phase A (with 0.1% formic acid): acetonitrile in phase B (with 0.1% formic acid); and elution gradient: 5% in phase B at 0.00 min, linearly increasing to 95% in phase B at 9.00 min and maintaining at 95% for 1 min (10.00–11.10 min). The B-phase ratio decreased to 5% and equilibrated at 5% until 14 min, with a flow rate 0.35 mL/min, a column temperature of 40 °C, and injection volume 4 μL. For the mass spectrometry analysis , the ESIMetabolites were characterized and quantified based on Metware Biotechnology Ltd.’s own database MWDB and public metabolite databases  . The MS Principal component analysis for unsupervised pattern recognition was performed on the filtered data using the prcomp function  in R software (v4.2.1) ; orthogohttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 27 October 2022) and Trimmomatic (v0.36) to obtain clean reads [Total RNA was extracted from each sample, and RNA-seq libraries were constructed using Illumina Stranded Total RNA Prep  based on the Illumina Novoseq 6000 system, and the fragment size and concentration of the libraries were detected by Agilent 2100 Bioanalyzer. QC of the RNA-seq raw data was performed using FastQC  . Lowly eABCG22, CYP71B36, SR1IP1, C3H20, PG2, CKX1, CYP76C2, VSP3, APD2, ADAP, COMT, RBF1, PTPMT1, WD40, L15e, and the primers are shown in ® One-Step gDNA Removal and cDNA Synthesis SuperMix . qRT- PCR reactions were performed using the ChamQ Universal SYBR qPCR Master Mix  kit. data from three independent replicates were calculated by the 2−ΔΔCt method [Fifteen genes were randomly selected from hub genes for qRT-PCR analysis using the LineGene 9620 Real-Time PCR system from BIOER, with β-action as an internal reference gene. These genes include t method .


The genes involved in cisplatin resistance across KIRC were retrieved from the National Center for Biotechnology Information (NCBI) database using search terms as “Kidney renal clear cell carcinoma” and “Cisplatin resistance”. The genes retrieved were analyzed for hub gene identification using the STRING database and Cytoscape tool. Expression and promoter methylation profiling of the hub genes was done using UALCAN, GEPIA, OncoDB, and HPA databases. Mutational, survival, functional enrichment, immune cell infiltration, and drug prediction analyses of the hub genes were performed using the cBioPortal, GEPIA, GSEA, TIMER, and DrugBank databases. Lastly, expression and methylation levels of the hub genes were validated on two cisplatin-resistant RCC cell lines (786-O and A-498) and a normal renal tubular epithelial cell line (HK-2) using two high throughput techniques, including targeted bisulfite sequencing (bisulfite-seq) and RT-qPCR. A total of 124 genes were identified as being associated with cisplatin resistance in KIRC. Out of these genes, MCL1, IGF1R, CCND1, and PTEN were identified as hub genes and were found to have significant (p < 0.05) variations in their mRNA and protein expressions and effects on the overall survival (OS) of the KIRC patients. Moreover, an aberrant promoter methylation pattern was found to be associated with the dysregulation of the hub genes. In addition to this, hub genes were also linked with different cisplatin resistance-causing pathways. Thus, hub genes can be targeted with Alvocidib, Estradiol, Tretinoin, Capsaicin, Dronabinol, Metribolone, Calcitriol, Acetaminophen, Acitretin, Cyclosporine, Azacitidine, Genistein, and Resveratrol drugs. As the pathogenesis of KIRC is complex, targeting hub genes and associated pathways involved in cisplatin resistance could bring a milestone change in the drug discovery and management of drug resistance, which might uplift overall survival among KIRC patients.The low survival rate of Kidney renal clear cell carcinoma (KIRC) patients is largely attributed to cisplatin resistance. Rather than focusing solely on individual proteins, exploring protein-protein interactions could offer greater insight into drug resistance. To this end, a series of  Kidney renal clear cell carcinoma (KIRC) is a type of kidney cancer that originates from the cells lining the small tubules in the kidney that filter waste from the blood . KIRC isThe prognosis for KIRC varies based on factors such as Tumour, Node, Metastasis (TNM) staging, age, and gender, with the patient’s five-year survival rate heavily influenced by the tumor’s pathological grade ,6. KIRC 2 IV on days 1, 22, and 43, or at a weekly dose of 30-40 mg/m2 IV for 6 to 7 weeks [The management of KIRC involves a variety of approaches, including surgery, radiotherapy, chemotherapy, and immunotherapy –13. Chem 7 weeks . Despite 7 weeks .Earlier, several studies reported the development of cisplatin resistance in KIRC patients. In a study by Taniguchi et al. they found that cisplatin resistance was associated with the overexpression of the MDR1 gene in KIRC cells . SimilarMoreover, cancer patients who are resistant to cisplatin treatment typically have a bleak prognosis . Proteinin silico and in vitro methodologies, that are associated with cisplatin resistance in KIRC. In silico and in vitro combined approach for identifying cisplatin resistance-associated hub genes is the integration of computational and experimental methods. The in silico approach involves the utilization of bioinformatics tools and algorithms to analyze large-scale genomic, transcriptomic, and proteomic data sets. On the other hand, the in vitro approach involves the application of experimental methods to validate the identified hub genes, including their expression and methylation levels. The integration approach of in silico and in vitro methodologies can help improve our understanding of cisplatin resistance development in KIRC, leading to the development of more effective therapies.Hence, this study was initiated for identifying biomarker hub genes through https://www.ncbi.nlm.nih.gov/) database [Homo sapiens have appeared. Out of the appeared items, a total of 124 cisplatin resistance genes were used in the present work  [STRING (db.org/)  is a datdb.org/) . Overalldb.org/)  is an opdb.org/) . Users cdb.org/)  of the Cdb.org/) . Cytohubhttps://ualcan.path.uab.edu/analysis-prot.html) [UALCAN (ot.html)  is an onot.html) . UALCAN ot.html) . UALCAN http://gepia.cancer-pku.cn/) [https://oncodb.org/index.html) [GEPIA (pku.cn/)  and Oncoex.html)  are widehttps://www.proteinatlas.org/) database is an extensive and accessible repository of information on the human proteome [The Human Protein Atlas  is a user-friendly, open-access platform that enables researchers and clinicians to perform complex genomic analysis on cancer data [cBioPortal (cer data . It offecer data . The plaThe Gene Ontology (GO) analysis provides functional annotation of the gene(s) of interest. While KEGG (Kyoto Encyclopedia of Genes and Genomes) provides an interpretation of the user-defined genes in biological pathways . The GO http://timer.cistrome.org/) is a comprehensive tool that allows for the estimation of immune cells in a variety of tumor types [The TIMER database (or types . The dator types . This inhttps://rna.sysu.edu.cn/encori/) was developed for analyzing the miRNA-mRNA, RNA-RNA, and lncRNA-miRNA networks across various types of cancer [The ENCORI platform (f cancer . In thisf cancer .https://go.drugbank.com/) to uncover a variety of drugs associated with the identified hub genes because we believe that the identified hub genes could be promising therapeutic targets. This database compiles information on chemotherapeutic drugs that target hub genes from a variety of reliable sources [We used the DrugBank database ( sources .Cell lines: Human RCC cell lines (786-O and A-498) and a normal renal tubular epithelial cell line (HK-2) were purchased from the American Type Culture Collection  and cultivated in accordance with the manufacturer’s instructions.Construction of cisplatin resistance cell strains: Cisplatin resistance of cells was induced using a previously established pulse therapy technique  combinedTotal RNA and DNA extraction: Total RNA extraction from both KIRC and normal cell lines was done by isopycnic centrifugation as described previously . The extTargeted bisulfite-seq and RT-qPCR analyses: DNA samples were sent to Beijing Genomics Institute (BGI) company for targeted bisulfite-seq analysis. Following targeted bisulfite-seq analyses, methylation values were normalized as beta values. The obtained beta values against hub genes in RCC and a normal control cell line were compared to identify differences in the methylation levels.RT-qPCR validation analysis: The specific protocols are as follows: First, the PrimeScript™ RT reagent kit  was used for reverse transcription of the extracted RNA from cisplatin-resistant RCC cell lines (786-O and A-498) and a normal control cell line (HK-2) into complementary DNA. Then, the RT-qPCR was carried out on an ABI ViiA 7 Real Time PCR System  with a SuperReal SYBR Green Premix Plus  as a fluorescent dye. GAPDH was chosen as the internal reference in the present study. All the experiments were done in triplicate independently. All the primers for each hub gene are shown as following:GAPDHF 5-ACCCACTCCTCCACCTTTGAC-3′,GAPDHR 5′-CTGTTGCTGTAGCCAAATTCG-3′ .MCL1F 5′-GGACATCAAAAACGAAGACG-3′,MCL1R 5′- GCAGCTTTCTTGGTTTATGG-3′ .IGF1RF 5′-GGCACAATTACTGCTCCAAAGAC-3′,IGF1RR 5′-CAAGGCCCTTTCTCCCCAC-3′ .CCND1F 5′-CGATGCTGGAGGTCTGCGA-3′,CCND1R 5′-AGAGGCCACGAACATGCAAG-3′ .PTENF 5′-TGGCATACACCAAATATAAGAGC-3′,PTENR 5′-TCCCTTATCAGATACATGACTTTCA-3′ .−ΔΔCt method was employed to evaluate the relative expression of each hub gene through RT-qPCR [t-test was adopted in the current study. All the analyses were carried out in R version 3.6.3 software.For enrichment analysis, we used Fisher’s Exact test for computing statistical difference . Correla RT-qPCR . Log-ran RT-qPCR . For comHomo sapiens. The obtained 124 genes were evaluated for constructing the PPI network through the STRING database. As highlighted in After a brief literature search, a total of 124 genes were found associated with cisplatin resistance across KIRC in p < 0.05).The TCGA dataset was used in conjunction with the UALCAN tool to analyze the expressions of MCL1, IGF1R, CCND1, and PTEN. The results showed a significant up-regulation of MCL1 and CCND1, while IGF1R and PTEN showed a significant down-regulation at both mRNA and protein levels in KIRC samples compared to controls –2C. ThisFurthermore, we also investigated the potential implications of dysregulation of these genes in KIRC patients with different clinicopathological parameters. Information on cancer stage, race, gender, and age was retrieved from UALCAN, and these parameters were analyzed in relation to mRNA expression of the hub genes in the KIRC cohort. Results revealed a significant up-regulation of MCL1 and CCND1, and a significant down-regulation of IGF1R and PTEN in KIRC patients with varying cancer stages, races, genders, and age groups compared to normal controls .p < 0.05) while IGF1R and PTEN mRNA levels were significantly lower (p < 0.05) in KIRC samples compared to healthy samples (p < 0.05) correlation between dysregulated MCL1, IGF1R, CCND1, PTEN, and poor OS for KIRC patients , IGF1R (0%), and CCND1 (2.5%), indicating their lesser involvement in hub gene expression regulation . PTEN hap-value < 0.05. GO and KEGG pathways in KIRC. In the CC, “Insulin receptor complex, Alphav-beta integrin-IGF-1-IGF1R complex, Bcl-2 family protein complex, and Arp2/3 protein complex”, etc., were significantly associated with the analyzed genes  was observed between CD8+ T and CD4+ T cell infiltration and the expression levels of MCL1, IGF1R, CCND1, and PTEN genes . A significant positive correlation  as a potential inducer of KIRC, as it targets all hub genes simultaneously. These findings suggest that the identified axis of has-mir-27a-3p and MCL1, IGF1R, CCND1, and PTEN genes may have a role in drug resistance development across KIRC .For KIRC patients suffering from cisplatin drug resistance, the use of alternative drugs for medical treatment is the first option. Therefore, a selection of appropriate alternate candidate drugs is required. In the current study, via the DrugBank database, we explored some potential drugs, that can reverse the gene expressions of MCL1, IGF1R, CCND1, and PTEN hub genes. Alvocidib, Estradiol, Tretinoin, Capsaicin, Dronabinol, Metribolone, Calcitriol, Acetaminophen, Acitretin, Cyclosporine, Azacitidine, Genistein, and Resveratrol drugs  could beIn the current study, by performing targeted bisulfite-seq analyses of 2 cisplatin-resistant RCC cell lines, including 786-O and A-498, and the normal renal tubular epithelial cell line HK-2, methylation levels of the MCL1, IGF1R, CCND1, and PTEN were validated. Methylation levels of these genes were validated using beta values. As shown in To further validate the aforementioned bioinformatics analysis, the mRNA expression levels of these hub genes  were obtained by RT-qPCR experiment in cisplatin resistant RCC cell lines (786-O and A-498) and a normal control cell line (HK-2). As suggested in in silico and in vitro methodologies.Cisplatin resistance refers to the ability of cancer cells to become resistant to the effects of cisplatin, to treat a variety of cancers, including KIRC . CisplatTo do so, initially, 124 cisplatin resistance-linked genes were identified from the published literature. Then, PPI of the identified genes was constructed and screened out via Cytoscape software to identify hub genes. Based on the degree method, the MCL1, IGF1R, CCND1, and PTEN genes were identified as the most prominent drug resistance-associated genes in KIRC.The MCL1 gene encodes for a protein called myeloid cell leukemia 1, which plays a crucial role in regulating programmed cell death, or apoptosis ,58. In nThe IGF1R (Insulin-like Growth Factor 1 Receptor) gene encodes for a transmembrane receptor protein that binds to insulin-like growth factors (IGFs) and plays a key role in cell growth and survival ,66. In nThe CCND1 gene, also known as cyclin D1, plays a crucial role in the cell cycle by regulating the G1/S transition ,73. HoweThe PTEN gene is a tumor suppressor gene that plays a critical role in regulating cell growth and proliferation . PTEN isWe next analyzed and validated the mRNA and protein expression levels of MCL1, IGF1R, CCND1, and PTEN using UALCAN, GEPIA, OncoDB, and HPA databases on TCGA datasets. Our research took into account different factors such as cancer stages, races, genders, ages, and pathological data. Analysis of the UALCAN, GEPIA, and OncoDB databases revealed a significant up-regulation of MCL1 and CCND1 mRNA expressions, and a significant down-regulation of IGF1R and PTEN mRNA expressions in KIRC samples compared to controls. We also found that the protein expression of MCL1, IGF1R, CCND1, and PTEN in KIRC tissue samples was consistent with their corresponding mRNA expression levels using the HPA database and immunohistochemical staining. Therefore, we infer that the high expressions of MCL1 and CCND1 and low expressions of IGF1R and PTEN may play critical roles in developing cisplatin drug resistance across KIRC. The analysis of promoter methylation has revealed that an abnormal pattern of methylation significantly contributes to the dysregulation of MCL1, IGF1R, CCND1, and PTEN expression in KIRC patients compared to normal controls. MCL1 methylation has been associated with poor overall survival in KIRC patients , while IThe TIMER analysis revealed a significant correlation between the expression of MCL1, IGF1R, CCND1, and PTEN and the presence of CD8+ T cells, CD4+ T cells, and macrophages. The CD8+ T and CD4+ T cells are the most abundant types of T lymphocytes in tumor microenvironments and capable of eliminating tumor cells, their differentiation into dysfunctional states may render them ineffective at responding to immunotherapy ,86. ThisThis study revealed that MCL1, IGF1R, CCND1, and PTEN, are the central genes implicated in cisplatin resistance in KIRC. These genes demonstrated significant expression variability and had an impact on the OS of KIRC patients. These genes were also found modulating multiple cisplatin-associated pathways. Targeting MCL1, IGF1R, CCND1, and PTEN genes with different other drugs explored via the present study, such as palbociclib, methotrexate, bortezomib, fluorouracil, sorafenib, dasatinib, carboplatin, paclitaxel, gemcitabine, imatinib, doxorubicin, and vorinostat, may have the potential to overcome cisplatin drug resistance in KIRC.


CEP290 (21%), CRB1 (21%), RPE65 (14%), RDH12 (13%), AIPL1 (6%), TULP1 (6%), and IQCB1 (5%), and few cases harbored pathogenic variants in LRAT, CABP4, NMNAT1, RPGRIP1, SPATA7, CRX, IFT140, LCA5, and RD3 . The most common clinical diagnosis was LCA  followed by retinitis pigmentosa , but also other IRDs were seen . Among LCA patients, 50% were caused by variants in CEP290 (29%) and RPE65 (21%), whereas variants in other genes were much less frequent . In general, the patients showed a severe phenotype hallmarked by severely reduced visual acuity, concentric narrowing of the visual field, and extinguished electroretinograms. However, there were also exceptional cases with best corrected visual acuity as high as 0.8 (Snellen), well-preserved visual fields, and preserved photoreceptors in spectral domain optical coherence tomography. Phenotypic variability was seen between and within genetic subgroups. The study we are presenting pertains to a considerable LCA group, furnishing valuable comprehension of the genetic and phenotypic spectrum. This knowledge holds significance for impending gene therapeutic trials. In this German cohort, CEP290 and CRB1 are the most frequently mutated genes. However, LCA is genetically highly heterogeneous and exhibits clinical variability, showing overlap with other IRDs. For any therapeutic gene intervention, the disease-causing genotype is the primary criterion for treatment access, but the clinical diagnosis, state of the retina, number of to be treated target cells, and the time point of treatment will be crucial.To report the spectrum of Leber congenital amaurosis (LCA) associated genes in a large German cohort and to delineate their associated phenotype. Local databases were screened for patients with a clinical diagnosis of LCA and for patients with disease-causing variants in known LCA-associated genes independent of their clinical diagnosis. Patients with a mere clinical diagnosis were invited for genetic testing. Genomic DNA was either analyzed in a diagnostic-genetic or research setup using various capture panels for syndromic and non-syndromic IRD  genes. Clinical data was obtained mainly retrospectively. Patients with genetic and phenotypic information were eventually included. Descriptive statistical data analysis was performed. A total of 105 patients  with disease-causing variants in 16 LCA-associated genes were included. The genetic spectrum displayed variants in the following genes:  Leber congenital amaurosis  refers to a heterogeneous group of severe early infantile retinal dystrophies with typically extinguished electroretinograms (ERGs). It was first described by Theodor Leber in 1869 . The worABCA4-, KCNV2-, RPE65-associated retinopathy, bestrophinopathies—IRDs associated with disease-causing variants in BEST1) [With the advances in molecular diagnostic genetic technology, knowledge about the genetic background of LCA has expanded widely in recent years. Disease-causing variants associated with LCA have been identified in more than 25 genes, and different path mechanisms have been implicated, all of which result in a diverse range of retinal dysfunction and affected pathways, including the phototransduction, the visual cycle, and photoreceptor development/integrity ,6,7. Then BEST1) ,16,17,18CEP290 , for instance are known to be frequent in Northern Europe [So far, at least 25 genes have been associated with LCA , and then Europe ,20,21,22n Europe . Due to n Europe ,24 and An Europe ,27,28. ARPE65-mediated IRDs, was approved in December 2017 in the USA [As of today, the structural and functional associations seen in the various LCA/EOSRD genotypes are well described and understood ,3,30. Th the USA  and in N the USA . Therefo the USA .The purpose of the current study is to describe the prevalence of disease-causing variants in LCA-associated genes in a large German cohort at a single clinical site and to give detailed clinical information on subgroups, aiming to pave the way for future gene therapies.AIPL1, CABP4, CEP290, CRB1, CRX, IFT140, IQCB1, LCA5, LRAT, NMNAT1, RD3, RDH12, RPE65, RPGRIP1, SPATA7, and TULP1) (CEP290 (22/105), CRB1 (22/105), and RPE65 (15/105)  from 87 families with confirmed disease-causing variants in LCA-associated genes were included. Altogether, disease-causing variants in 16 LCA-associated genes were found in our cohort (d TULP1) . Among t(15/105) . SegregaIn most cases, disease onset was reported in infancy or early childhood, but in a few cases first symptoms were noticed in middle aged children or in young adults. The distribution of disease onset shown in  also varCEP290  and RPE65 , while variants in other genes were much less frequent . If the initial clinical diagnosis was RP or SECORD/EOSRD, the compilation of responsible genes was different: CRB1  was the most common, followed by RDH12  and CEP290 , while other genes were less frequent , followed by nonsense (n = 23), frameshift (n = 16), splicing variants (n = 7), and single exon deletion (n = 1).2; m: 23 deg2; for OD): In 41% (33/80), visual field (VF) was not preserved and in another five patients, VF was constricted <5 degrees (for OD). Thirty-seven percent (28/105) had no or old VF records, so no field in deg2 could be measured. The electroretinograms (ERG) (feasible in 84/105 cases) was mostly non-reproducible . Patients with non-reproducible responses were most frequently diagnosed with LCA , followed by RP  and CRD . Residual ERG responses were present in 21% (23/84); here, the most frequent diagnosis was RP , followed in descending order by LCA , CSNB , and CRD .In general, disease-causing variants in LCA-associated genes led to a severe phenotype . HoweverCEP290 n = 3, CRB1 n = 3). Other features included macular scars , a peripapillary staphyloma , bull`s eye configuration , central whitish vascular septa (CRB1 n = 3), glistening spots , cobblestones , and nevi .Funduscopy revealed typical signs of IRDs  but with clinical variability. Six patients (6%) had optic nerve head drusen .OCT ) generally showed advanced disease with severe photoreceptor loss. In 60% of cases (32/53), the photoreceptors were destroyed, and the ellipsoid zone (EZ), the transition of the photoreceptor inner/outer segments, was not measurable. In 40% of patients (21/53), photoreceptors were measurable centrally. The average EZ was 1215 µm. A morphologically completely intact photoreceptor layer  was only found in CABP4, IFT140, IQCB1, and RPGRIP1 (AIPL1 (102 ± 34 µm), CRB1 (146 ± 60 µm), LRAT (145 ± 4 µm), RDH12 (138 ± 72 µm), RPE65 (137 ± 47 µm), and TULP1 (131 µm) cases. The outer nuclear layer  was reduced to 92 µm on average. In addition, three patients had cystoid macular edema . Seven patients had epiretinal gliosis .Central foveal thickness (CFT) was reduced to 160 µm on average . Excepti RPGRIP1 . The fovCRB1 (n = 2) and RDH12 (n = 3), pyelonephritis was seen in a patient with AIPL1 disease-associated variants, and renal transplantation due to renal failure was necessary for one patient with IQCB1 variants. Other additional findings included polydactyly, asthma and elevated pancreatic enzymes (n = 1 in CRB1), hearing loss (n = 1 in CRB1), respiratory arrhythmia (n = 1 in RDH12), and scoliosis (n = 1 in TULP1).Concomitant diseases were observed in some patients: thyroid disease occurred in the genotype groups For further detailed description, we selected the four most frequent genetic subgroups accounting for 70% (73/105) of all cases in our cohort. Some of our cases have been published previously ,35,36,37CEP290-associated retinal dystrophy seen at our clinical site have been previously published by our group [CEP290 genotypes, and 17 different CEP290 variants were revealed. By far, the most common variant, detected in 77% of patients (17/22) with CEP290-related pathology, was the deep intronic c.2991+1655A>G variant. Compared to previous studies, we found a surprisingly high number of homozygous c.2991+1655A>G cases , indicating this CEP290 genotype is the most important in Germany. Clinically, all patients showed a severe phenotype. The most common clinical diagnosis was LCA or EOSRD (together with 82%), followed by RP (18%). The clinical diagnosis could not be predicted by the underlying genotype, and phenotypic overlaps existed; however, milder phenotypes were rather observed in compound heterozygous patients or in cases without the deep intronic c.2991+1655A>G variant. Examples of this subgroup are shown in The clinical and genetic findings of ur group . This waCRB1 gene. The most common diagnosis was RP , followed by LCA  and CRD . Within the cohort, a total of 22 different CRB1 variants were present. The most frequent variant was c.2843G>A;p.(Cys948Tyr), which was found homozygously in five patients, accounting for 22% of CRB1 cases and leading to a severe phenotype. In general, CRB1 patients showed a severe disease, but onset, severity, and rate of progression varied between patients. Characteristic findings included macular atrophy, nummular pigmentation, relative para-arteriolar preservation of the retinal pigment epithelium (RPE), and retinal thickening with loss of lamination on OCT scans—in direct contrast to other forms of LCA/EOSRD where progressive retinal thinning is commonplace  had disease-causing variants in the monplace .RPE65-associated retinal dystrophy . We identified 13 different RPE65 variants, 10 patients were homozygous, five (compound) heterozygous. The most frequent variant was c.1451G>T;p., which was seen homozygously in five patients , all members of a consanguineous family .The third most frequent genetic subgroup contained 15 patients with RPE65-deficiency was associated with reduced or absent autofluorescence on FAF imaging, and on OCT imaging the retinal thickness was variable with a tendency to thin with disease duration (We observed a severe disease pattern with LCA/EOSRD phenotype in 67% of the cases (10/15), with markedly reduced BCVA, nystagmus, and night blindness in most cases . duration .RDH12 gene. A total of 12 different genotypes and 12 RDH12 variants were identified, with c.806_810del;p.(Ala269GlyfsTer2)  being the most frequent. Diagnoses were EOSRD/RP  and LCA , indicating an onset of the disease somewhat later compared to the other genetic subgroups  had variants in the ubgroups . We obseubgroups .To establish the diagnosis of LCA, EOSRD/SECORD, or RP in the light of gene therapy, detailed ophthalmic history, imaging studies, electrophysiological examinations, and, most importantly, genetic analysis is needed. Due to the diversity in genetic background, disease course, and variable onset in infancy or childhood, it is challenging but important for ophthalmologists to obtain a definitive diagnosis in preliminary consultations ,7,30,38.When talking about LCA, the difficulty of creating a group collective is striking. We designed the study to include patients with a clinical diagnosis of LCA and patients who are homozygous or (compound) heterozygous for variants in known LCA-associated genes. On the one hand, there were 105 patients with biallelic variants in LCA-associated genes. On the other hand, only 56 patients had a clinical diagnosis of LCA. The diagnosis made by the treating physicians was revised in each case.One problem lies in the clinical and genetic heterogeneity ,36,37,39Recently, a separate group of milder disease phenotypes, the so-called “early-onset severe retinal dystrophy” (EOSRD) or “severe early childhood onset retinal dystrophy” (SECORD), has been described. LCA and EOSRD/SECORD together are the most severe and earliest forms of all IRDs, but there is a fine overlap between the clinical diagnoses of LCA/EOSRD/SECORD/RP. Making a clinical diagnosis remains difficult. Our results underscore that it is useful to consider these as a continuum of phenotypes .GUCY2D, was mapped in 1995 [Foxman’s classification dates from 1985 and does not consider the genetic background. The first LCA-associated gene,  in 1995 . However in 1995 . Given tOf interest, we reported a high rate of variants of uncertain significance VUS, see . If a vaCEP290, RPE65, CRB1, and RDH12 are the most important LCA-associated genes in Germany. Their prevalence was 21% and 28% (CEP290), 21% and 11% (CRB1), 14%, and 23% (RPE65), and 13% and 8% (RDH12) for the total cohort and within LCA cases, respectively. To set these numbers into perspective is difficult, as large cohort studies on LCA are missing. Most studies either provide mutation spectra for all IRDs tested in a given setup or for a single gene tested in a certain clinical diagnosis. Yet, Kumaran and coworkers [GUCY2D, CEP290, CRB1, RDH12, and RPE65 being the most common in general. A recent study in 27 Polish families (31 patients) [CEP290, 9 of 10 cases carrying the intronic variant c.2991+1655A>G, 22% in CRB1, 11% in GUCY2D and NMNAT1, two cases in RPGRIP1, and single cases with disease-causing variants in LRAT, LCA5, and CRX. In an Italian study on 24 LCA patients [CRB1 (7%), CEP290, IQCB1, and GUCY2D (each 5%), while only single families with variants in NMNAT1, TULP1, AIPL1, SPATA7, and RPGRIP1 were identified. In a Brazilian LCA/EORD cohort of 137 molecularly diagnosed families (152 affected patients) [CEP290 (21%), RPE65 (16%), CRB1 (14%), RPGRIP1 (10%), GUCY2D (8%), and RDH12 (8%). A New Zealand study on childhood-onset IRD [ABCA4 and RS1, but also in LCA-related genes . In a recent Chinese study [CRB1 (27%), RDH12 (24%), and RPGRIP1 (19%). Another Chinese study [AIPL1 (11%), RPGRIP1 (9%), and CEP290, GUCY2D, and RPE65 (each 8%) in the LCA patient group, and RPGR (12%), CRB1, and RPE65 (each 11%), RDH12 (7%), and RP2 (5%) in the EOSRD patient groups. A Japanese study indicates that the most frequently mutated genes were CRB1, NMNAT1, and RPGRIP1 [Our study shows that atients)  identifipatients , the mosatients) , the mosnset IRD  identifise study  with 37 se study  on LCA a RPGRIP1 .RPE65 [RPE65 complementary DNA (cDNA) into RPE target cells in animal models and subsequently in humans via viral vector-mediated gene supplementation therapy has resulted in significant improvements in photosensitivity, visual field, and visual function, as demonstrated in several phase II and III clinical studies [®, Novartis) was officially approved by the Food and Drug Administration (FDA) in the US in 2017 and by the European Medicines Agency (EMA) in Europe in 2018 for the treatment of IRDs caused by a biallelic disease-causing variants in RPE65 [RPE65 gene therapy has paved the way for more than 30 gene replacement trials worldwide. Therapeutic clinical trials are ongoing for several genotypes of different IRDs, and hold promise for a cure for a large number of patients in the future [The prevalence of LCA- and RP-associated genes and disease-causing variants is particularly relevant for future gene therapies. The first ophthalmological disease to become the focus of gene therapy was indeed LCA, caused by disease-associated variants in RPE65 ,48,49,50 studies ,53,54,55in RPE65 ,32. The e future ,57,58. TIn summary, our study fills the gap in the literature on the prevalence of LCA subtypes in Germany and provides for the first time a detailed clinical and genetic characterization of one of the largest cohorts in Europe. The results will help to prepare upcoming gene therapeutic trials and to better counsel affected patients.The study was conducted at the Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen, Germany. It was approved by the local ethics committee, and informed consent was obtained from all patients. Examinations were performed in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).Local databases were queried for (1) patients with a clinical diagnosis of LCA and (2) patients with already established likely biallelic disease-causing variants in LCA-associated genes, despite the clinical diagnosis. Individuals who have been clinically diagnosed with LCA but whose genetic status was unknown were invited to undergo molecular genetic testing.CEP290-related cases were invited for a revisit with respect to a previously published study [Clinical data was obtained mainly retrospectively, but in older cases where clinical information was missing, follow-up visits were initiated. Specifically, ed study . All casAIPL1 , CABP4 (Gene ID 57010: OMIM 608965), CEP290 , CRB1 , CRX , GUCY2D , IFT140 , IQCB1 , KCNJ13 , LCA5 , LRAT , NMNAT1 , RD3 , RDH12 , RPE65 , RPGRIP1 , SPATA7 , and TULP1 .Male and female patients independent of age with the clinical diagnosis of LCA and patients who were homozygous or carried two heterozygous variants in at least one of the following LCA-associated genes were included: Medical history was obtained, and a complete ophthalmological examination was performed—upon availability and feasibility—including psychophysical tests , visual field (VF)), electrophysiology ), and imaging , and spectral-domain optical coherence tomography (SD-OCT)).-1= Birth; -2= Infancy (beginning 28 days to 12 months of age); -3= Toddler (beginning 13 months -2 years of age); -4= Early childhood (beginning 2 years to 5 years of age); -5= Middle childhood (beginning 6 years to 11 years); -6= Early adolescent (beginning 12 years to 18 years).Disease onset was evaluated and categorized according to the National Institute of Child Health and Human Development (NICDH) Pediatric Terminology  as folloDisease onset and clinical symptoms determined the final clinical diagnosis. For easier data analysis, we decided to split the cohort into two subgroups, LCA and RP, respectively. LCA as a diagnosis was given for patients with disease onset in the first 2 years of life, RP was set as a diagnosis for patients with disease onset after the age of 2 years. Further IRD diagnoses were also determined on the basis of clinical findings, if reasonable.-Counting fingers (CF) = 0.014;-Hand motion (HM) = 0.005;-Light perception (LP), no light perception (NLP) = 0.BCVA was measured using a Snellen chart. In patients with severely reduced visual acuity, the ability to count fingers or to perceive hand movements or light, respectively, was tested. Data was converted into Snellen as follows ,61:-Coun2 for the right and left eye, respectively. Fullfield and—if possible—multifocal ERG was performed in patients in order to assure clinical diagnosis. All electrophysiological recordings were performed according to the International Society for Clinical Electrophysiology of Vision Standards (ISCEV) with the E3 Espion system  [For visual field testing, an Octopus 900 perimeter  was used. Semiautomated kinetic perimetry was performed using the Goldmann stimuli V4e and III4e, if possible. The size of the fields with stimulus III4e was measured in degMA, USA) ,63. For Genetic testing was performed in most cases with comprehensive next generation sequencing gene panels, either in a diagnostic genetic or research context. Methodological details have been described previously ,33. In iDescriptive statistical analysis was done using JMP , providing median (m), mean (M), and standard deviation (SD) and spread, respectively. Data for the right and left eye were analyzed separately. The normality of data was assessed by evaluation of the histogram plots, correlation parameters were calculated using Pearson or Spearman analysis, where appropriate.


FaMADS1. The transient silence of FaMADS1 in strawberries promoted fruit ripening and induced the content of anthocyanin and soluble pectin but reduced firmness and protopectin through a tobacco rattle virus-induced gene silencing technique. In parallel with the accelerated ripening, the genes were significantly induced in the transiently modified fruit, including anthocyanin-related PAL6, C4H, 4CL, DFR, and UFGT, softening-related PL and XTH, and aroma-related QR and AAT2. In addition, the interaction between FaMADS1 and ABA-related transcription factors was researched. Yeast one-hybrid analysis indicated that the FaMADS1 promoter could interact with FaABI5-5, FaTRAB1, and FaABI5. Furthermore, dual-luciferase assay suggested that FaTRAB1 could actively bind with the FaMADS1 promoter, resulting in the decreased expression of FaMADS1. In brief, these results suggest that the ABA-dependent ripening of strawberry fruit was probably inhibited through inhibiting FaMADS1 expression by the active binding of transcript FaTRAB1 with the FaMADS1 promoter.Abscisic acid (ABA) plays a crucial role in regulating the ripening of non-climacteric strawberry fruit. In the present study, ABA was confirmed to promote strawberry ripening and induce the down-regulation of  Phytohormones strongly dominate transcription factors and genetic regulators to regulate fruit ripening, and the degree of fruit maturity determines fruit quality . As one WRKY40 in response to a high level of ABA signal, thereby releasing the expression of the ABI5 gene , acids (octanoic acid and 3-hydroxydodecanoic acid), terpenes , and benzene . Meanwhile, we observed increased levels of some alcohol, aldehyde, and olefin in FaMADS1-RNAi strawberry fruit . A putative domain (ABRE) for responding to ABA was found (FaMADS1-pro1 and FaMADS1-pro2) of FaMADS1 promoter were cloned to study the interaction between FaMADS1 and ABA-related transcription factors . FaABI5-5, FaTRAB1, and FaABI5 interacted with FaMADS1-pro1, whereas ABI5-2 and ABI5-3 did not interact with FaMADS1-pro1 , softening (PL), and easier biosynthesis (AAT). PAL was discovered to trigger the production of anthocyanin [In this study, ABA significantly promoted the accumulation of anthocyanin and soluble solid in strawberry fruit, but reduced fruit firmness and total titratable acid; thus, ABA regulated almost the entire fruit ripening process . This sihocyanin . CHS washocyanin , and DFRhocyanin . PL was hocyanin . During hocyanin  discoverhocyanin . Alcoholhocyanin . These rFaMADS1 was suppressed with an accelerated ripening process following ABA treatment [FaMADS1 might play a negative role in fruit ripening via the downstream ABA signal transduction pathway connected to related metabolic processes.Studies have suggested that some MADS members contributed to the fruit ripening of tomatoes , grape , strawbereatment . These rFaMADS1 in the ripening of strawberry fruit was verified through a gene silencing technique. Compared with the control (empty vector), FaMADS1-RNAi fruit exhibited accelerated ripening with high levels of anthocyanin and soluble pectin, low firmness, and protopectin content. In addition, an increase in the content levels of ester, ketone, acid, terpenes, and benzene was observed in the FaMADS1-RNAi fruit. The down-regulation of FaMADS1 resulted in increased expression levels of anthocyanin-related genes , softening-related genes (PL and XTH), and aroma-related genes (QR and AAT2). In tomato, the silence experiment also caused changes in fruit ripening. Mekontso et al. [ARF10A, ARF10B, and ZRF17, were up-regulated. Meanwhile, FaMADS1-RNAi significantly promoted the activities of PAL, 4CL, C4H, DFR, PL, and AAT enzymes. These results collectively suggested that the FaMADS1 gene had a strong negative function in strawberry ripening.Previous studies have reported that tobacco rattle virus (TRV)-mediated VIGS was a rapid and feasible technique for analyzing fruit ripening of tomato  and strao et al.  found thABI encoded protein PP2C in the ABA signal pathway. FaABI1 and FaABI2 negatively regulated strawberry fruit ripening, whereas FaABI4 and FaABI5 positively regulated fruit ripening [FaMADS1-pro1. Furthermore, the dual-luciferase assay confirmed that the promoter of FaMADS1 could be significantly inhibited by FaTRAB1, whereas FaABI5-5 and FaABI5 had little influence on the promoter of FaMADS1. These results suggested that the ABA signal was transmitted to the FaMADS1 promoter with the interaction of FaTRAB1. FaMADS1 regulated the transcriptional level of the downstream anthocyanin-related genes, softening-related genes, and aroma-related genes, eventually initiating fruit ripening.The gene ripening . Previouripening ,64,65,66FaMADS1 expression negatively depended on ABA to regulate strawberry fruit ripening. The yeast one-hybrid analysis and dual-luciferase assay suggested that the FaMADS1 promoter was inhibited by the interaction with FaTRAB1, resulting in the blockage of FaMADS1 translation. ABA application and FaMADS1-RNAi data reflected a negative regulation of FaMADS1 to the downstream anthocyanin-related genes , softening-related genes (PL and XTH), and aroma-related genes (QR and AAT2), as well as the enzyme activities (In conclusion, tivities . This ob