Endocrine disruption is an important factor in the development of endometrial cancer. Expression of miR-149-3p is observed in some cancer types, while its role in uterine corpus endometrial carcinoma (UCEC) is unclear.  The clinical and genomic data and prognostic information on UCEC were obtained for patients from the TCGA database. The Kruskal–Wallis test, Wilcoxon signed-rank test, and logistic regression were used to analyze the relationship between clinical characteristics and miR-149-3p expression. Kaplan–Meier survival curve analysis was used to study the influence of miR-149-3p expression and miR-149-3p target genes on the prognosis of UCEC patients. The TargetScan, PicTar, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to determine the involvement of miR-149-3p target genes in function. Immune infiltration analysis was used to analyze the functional involvement of miR-149-3p. QRT-PCR was used to validate the expression of miR-149-3p in UCEC cell lines. P < 0.001), histological type (P < 0.001), histological grade (P < 0.001), tumor invasion (P=0.014), and radiation therapy (P=0.011). High miR-149-3p expression predicted poorer overall survival (OS) , progression-free interval (PFI) , and disease-specific survival (DSS) . Low expressions of miR-149-3p target genes, including ADCYAP1R1, CGNL1, CHST3, CYGB, DNAH9, ESR1, HHIP, HIC1, HOXD11, IGF1, INMT, LSP1, MTMR10, NFIC, PLCE1, RARA, SNTN, SPRYD3, and ZBTB7A, were associated with poor OS in UCEC. MiR-149-3p may be involved in the occurrence and development of UCEC via pathways including PI3K-Akt signaling pathway, Ras signaling pathway, AGE-RAGE signaling pathway in diabetic complications, focal adhesion, and MAPK signaling pathway. miR-149-3p may inhibit the function of CD8 T cells, cytotoxic cells, eosinophils, iDC, mast cells, neutrophils, NK CD56bright cells, NK CD56dim cells, pDC, T cells, T helper cells, TFH, Th17 cells, and Treg. miR-149-3p was significantly upregulated in UCEC cell lines compared with endometriotic stromal cells.  High expression of miR-149-3p in UCEC was significantly associated with age ( High expression of miR-149-3p was significantly associated with poor survival in UCEC patients. It may be a promising biomarker of prognosis and response to immunotherapy for UCEC patients. Uterine corpus endometrial carcinoma (UCEC) is one of the three main gynaecological malignancies, the incidence of which increases over time. Endometrial cancer is an epithelial tumor of the endometrium and a malignant tumor of the female reproductive system with a high incidence, posing a serious threat to the health of women worldwide , 2. It cMicroRNAs (miRNAs) are a unique class of endogenous and small noncoding RNAs that are approximately 18 to 25 nucleotides in length. They alter gene expression at the posttranscriptional level primarily through complete or incomplete base pairing with the 3′ untranslated region (3′UTR) of their target mRNAs. Translational repression and mRNA degradation are the 2 main pathways through which miRNAs direct gene regulation . There iIn oral squamous cell carcinoma, reduced levels of miRNA-149-3p lead to malignant progression and predict poor prognosis . IncreasThe present study examined the expression of miR-149-3p in UCECs using an online database and analyzed the relationship between expression levels and clinical characteristics. A survival curve was drawn to analyze the relationship between miR-149-3p expression level and overall survival (OS). Important contributions of miR-149-3p target genes to function were identified by TargetScan, PicTari, Gene Ontology (GO), and Kyoto Gene and Genome Encyclopedia (KEGG) analyses. The functionally significant involvement of miR-149-3p was analyzed by immune infiltration analysis. QRT-PCR was used to validate the expression of miR-149-3p in UCEC cell lines. The results of this study could provide new prognostic biomarkers for UCEC.https://portal.gdc.cancer.gov/) UCEC project. The miRNAseq data in RPM (reads per million mapped reads) format were log2-transformed.The analysis was carried out according to references , 17. R . R package was the ggplot2 package.Nomogram plot analysis was carried out according to literature , 20. R phttps://bioinfo.life.hust.edu.cn/miR_path/download.html). The UCEC prognosis-related genes were analyzed according to reference [miR-149-3p targets were obtained from Database TargetScan, miRanda, TarBase, miRTarBase, miR2Disease, miRecords, and miRWalk –25. UCECeference . R  [P value less than 0.05) were considered significant categories.The Database for Annotation, Visualization, and Integrated Discovery (DAVID) can provide a comprehensive set of functional annotation tools to facilitate understanding of the biological significance behind a large number of genes. GO and KEGG analyses were performed on the targets of miR-149-3p using the DAVID database (rf.gov/) –28. GO aThe analysis was performed according to reference . R  have been isolated from endometriotic tissue. Human UCEC cells Ishikawa and KLE were obtained from our laboratory. KLE cells were grown in F12. Ishikawa cells were grown in RPMI-1640. 10% fetal bovine serum was added to the medium to maintain the cell state. Add 1% antibiotics to the soil to prevent contamination. Cells were grown in a 37°C incubator containing 5% COeference . The priP values less than 0.05 were considered statistically significant.Statistical analysis was carried out according to reference . All staR0 (90.9%), 22 R1 (5.4%), and 15 R2 (3.7%). The histologic grade included 98 G1 (18.3%), 121 G2 (22.6%), and 316 G3 (59.1%). Tumor invasion (%) included 260 patients (<50) (55.3%) and 210 patients (≥50) (44.7%). The menopause status included 34 pre (6.8%), 17 peri (3.4%), and 449 post (89.8%). The hormone therapy included 297 no (86.6%) and 46 yes (13.4%). The diabetes included 323 no (72.6%) and 122 yes (27.4%). The radiation therapy included 278 no (53.4%) and 243 yes (46.6%). The surgical approach included 207 minimally invasive (39.6%) and 316 open (60.4%). The age range was 57 to 71 years, with a median of 64 years.A total of 546 patients were analyzed in the present study . The clin = 546) was significantly higher than that in normal tissues  (P=0.007)  . The areP=0.007) , suggestn = 273) and low (n = 273) expression groups. As shown in P < 0.001), histological type (P < 0.001), histological grade (P < 0.001), tumor invasion (P=0.014), and radiation therapy (P=0.011). As shown in P=0.048), age , histological type , histologic grade , tumor invasion , and radiation therapy .The characteristics of UCEC patients are shown in P < 0.001), PFI , and DSS  of UCEC patients (P < 0.001), clinical stage , primary therapy outcome , age , histological type , residual tumor , histologic grade , tumor invasion , and radiation therapy . As shown in P < 0.001), primary therapy outcome , radiation therapy , and hsa-miR-149-3p  were independently correlated with OS in multivariate analysis. The above data indicated that miR-149-3p is a prognostic factor, and increased miR-149-3p is associated with poor OS. A nomogram was constructed to predict the 1-, 3-, and 5-year survival probability of UCEC patients by combing the expression of miR-149-3p with clinical variables, as shown in The association between miR-149-3p expression and OS of patients with UCEC was evaluated by Kaplan–Meier analysis, which indicated that the expression of miR-149-3p was correlated with poor OS , cytotoxic cells (P < 0.001), eosinophils (P < 0.001), iDC (P < 0.001), mast cells (P=0.002), neutrophils (P < 0.001), NK CD56bright cells (P < 0.001), NK CD56dim cells (P < 0.001), pDC (P < 0.001), T cells (P < 0.001), T helper cells (P=0.004), TFH (P=0.024), Th17 cells (P=0.006), and Treg (P < 0.001).As shown in Figures P < 0.001) (P < 0.001)  . The exp< 0.001) . These rThe development of type I endometrial cancer is associated with continuous estrogen stimulation of the endometrium without progestin antagonism. The endometrium lacks progesterone antagonism, and continuous stimulation by estrogen will result in a prolonged state of hyperproliferation, which will further develop into endometrial cancer. MiRNAs regulate the cell cycle and cell differentiation and migration, which may also act as tumor suppressor genes or oncogenes during tumorigenesis and tumor development . MiRNAs P < 0.001), histological type (P < 0.001), histological grade (P < 0.001), tumor invasion (P=0.014), and radiation therapy (P=0.011). UCEC expressed more miR-149-3p than normal tissue, especially in patients with age (>60), histological type (mixed and serous), histologic grade (G2 and G3), tumor invasion (≥50%), or radiation therapy (yes). These phenomena suggested that miR-149-3p may be involved in tumor development and promote proliferation. And miR-149-3p was highly expressed in UCEC, and patients with high miR-149-3p expression had poorer OS , PFI , and DSS  of UCEC patients. Low expressions of miR-149-3p target genes, including ADCYAP1R1, CGNL1, CHST3, CYGB, DNAH9, ESR1, HHIP, HIC1, HOXD11, IGF1, INMT, LSP1, MTMR10, NFIC, PLCE1, RARA, SNTN, SPRYD3, and ZBTB7A, were associated with poor OS in UCEC. Lymphovascular space invasion (LVSI) has an independent influence on the poor prognosis of UCEC patients [In the present study, miR-149-3p was significantly correlated with age , histological type (P < 0.001), histological grade (P < 0.001), tumor invasion (P=0.014), and radiation therapy (P=0.011). miR-149-3p may be involved in the occurrence and development of UCEC via pathways, including PI3K-Akt signaling pathway, Ras signaling pathway, AGE-RAGE signaling pathway in diabetic complications, focal adhesion, and MAPK signaling pathway. Expression of miR-149-3p was correlated with immune infiltration in UCEC. This study partially elucidates the role of miR-149-3p in UCEC and provides a promising biomarker for prognosis and immunotherapy response in UCEC patients.miR-149-3p was highly expressed in UCECs and correlated with poorer OS compared with normal tissues. The high miR-149-3p expression in UCEC was significantly associated with age (


Substantial changes in access and delivery of primary HIV care occurred during the COVID-19 pandemic. To assess how care access changed during the COVID-19 pandemic, we estimated ED use among PWH in care 2017-2021 in the southeastern US.For each calendar year, among PWH in care in the UNC CFAR HIV Clinical Cohort (defined as having a clinic visit in the current or prior year), we estimated the percent of patients with ≥ 1 ED visit in a given year, overall and by age, gender, race/ethnicity, HIV viral load (VL), and CD4 count. We estimated risk ratios (RRs) comparing patient characteristics and years 2020-2021 vs. 2017-2019, using Poisson regression with generalized estimating equations to account for repeated measures.B-F, all P< 0.05).Among 2129 PWH in care 2017-2021 (N≈1700-1800 in each year), 57% identified as Black, 31% White, 8% Hispanic, 26% women, with median age of 47 years (IQR 35-55). During the study period, there were 3645 ED visits over 8813 person-years, a rate of 41.4 ED visits-per 100 person-years(95% CI 36.8-46.5) per 100 person-years. The 845 PWH with at least one ED visit during the study period contributed a median of 2 visits each (IQR 1-5). The unadjusted probability of having ≥1 ED visit in a given year was higher among women vs. men , Black vs. White PWH , with VL ≥ 40 copies/mL , and with CD4 < 200  or 200-349  vs. ≥ 500 cells/μL; age was not associated with ED use. Compared with 2017-2019, the annual probability of having ≥ 1 ED visit was lower in 2020-2021, with RRs of 0.83 (95% CI 0.76-0.90) in unadjusted analyses and 0.80 (95% CI 0.71-0.90) after adjusting for demographics, VL, and CD4. There was also a significant unadjusted decrease for 2020-2021 vs. 2017-2019 among women, men, PWH who were Black, White, < 40 or 50-59 years old, and with CD4 >500 (Fig. Among PWH in HIV care, ED use was higher among women, Black PWH, and PWH with poorly controlled HIV. ED use decreased 2020-2021 in most groups, indicating that PWH during the COVID-19 pandemic may be delaying seeking care for acute conditions, or accessing care in other ways. Work is ongoing to characterize reasons for ED visits across calendar years and examine the impact of reduced ED utilization among PWH.Joseph J. Eron, MD, Adagio Therapeutics: data safety monitoring committee|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research Support|Glaxo Smith Kline: Advisor/Consultant|Merck: Advisor/Consultant|ViiV Healthcare: Advisor/Consultant|ViiV Healthcare: Grant/Research Support.


Gait speed is a predictor of overall health and mortality in older adults. Metabolomics may provide insights into biological mechanisms underlying gait speed. Herein, we examined the association between 193 lipid metabolites with gait speed in 1,717 adults (52.1% women) aged 82.0 ± 14.5. Lipidomic analysis was performed using liquid chromatography-mass spectrometry. Gait speed was measured over 4-meters and slowness was defined as <0.8m/s. Logistic regression, adjusted for age, sex, field center, height, fasting-duration and familial-relatedness, were used to examine the association between log-transformed metabolites with slowness. A false discovery rate (FDR) of p<0.05 was employed to account for multiple comparisons. Gait speed was 0.83 ± 0.32 and 53.4% had slowness. Three lipid metabolites were significantly associated with lower odds of slowness: an acylcarnitine, sphingomyelin and a ceramide non-hydroxy fatty acid-sphingosine. Our results potentially link lipids involved with mitochondrial beta-oxidation and nerve signal transduction to gait speed in older adults.


Klebsiella sp. CTHL.F3a was isolated from kimchi (Korean fermented cabbage/vegetables). Its complete genome sequence , comprising a chromosome and a single plasmid, was established through hybrid assembly.The cellulolytic strain  Klebsiella oxytoca species complex (KoSC) may be encountered as human commensals and opportunistic pathogens  in Bushnell-Haas medium (BHM) (Six samples of fresh kimchi (Korean fermented cabbage/vegetables) from Hong Kong markets were screened for cellulolytic bacteria using carboxymethylcellulose (CMC) agar, as described previously . Twelve um (BHM)  containiN50, 12,200 bp). Default parameters were used for all software unless otherwise specified.Paired-end short-read sequencing libraries, prepared using the NexteraXT DNA library preparation kit, were sequenced via the Illumina MiSeq platform with v3 chemistry (2 × 300 bp). Adapter sequences were removed, and 1,416,362 raw reads were quality filtered and trimmed using Trimmomatic v0.32  to give dnaA/repA on the forward strand), with genome coverage of 81×, which were submitted to NCBI PGAP v5.0 (The Illumina and Nanopore datasets were combined using Unicycler v0.4.3  to yieldGAP v5.0  and to PGAP v5.0  for annohttps://www.ezbiocloud.net/tools/ani) of 99.60% (Klebsiella sp. BDA134-6 (GenBank accession number CP064784) (bcs operons (Klebsiella pneumoniae recovered from the gut of a Chinese bamboo rat (Rhizomys sinensis) (The CTHL.F3a chromosome has an average nucleotide identity (by OrthoANIu online at f 99.60%  with ricP064784) . It cont operons , punctua operons . Phyre2  operons  finds thinensis) .https://bioinfo-mml.sjtu.edu.cn/KpVR/index.php) (Plasmid pCTHL.F3a was classified as IncFIB(K)_30 (clade II) by the KpVR Web-based tool (dex.php) . It contdex.php)  but no aKlebsiella sp. CTHL.F3a are available through NCBI under BioProject PRJNA758781, with GenBank accession numbers CP082360 (chromosome) and CP082361 (plasmid) and SRA accessions SRX12151552 (MiSeq) and SRX12151553 (MinION). Klebsiella sp.Complete genome sequences and raw sequence data for


Klebsiella pneumoniae (CR-cKp) and carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) isolates in southwestern Iran. From 2019 to 2021, 136 (88.9%) cKp and 17 (11.1%) hvKp isolates were identified using biochemical tests and polymerase chain reaction (PCR). Antibiotic resistance, beta-lactamases, and clonal relatedness of carbapenem-resistant isolates were investigated using disk diffusion, PCR, and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR), respectively. The different markers of hvKp isolates were as follows: string test , magA , rmpA , rmpA2 , iucA , and peg344 . Also, 55.1% (n = 75/136) of cKp and 47.1% (n = 8/17) of hvKp isolates were CR-cKp and CR-hvKp, respectively. All CR-hvKp  isolates were MDR. Colistin, tetracycline, and tigecycline were the most effective antibiotics. The occurrence of beta-lactamase genes in 75 CR-cKp and 8 CR-hvKp isolates was as follows: blaNDM , blaIMP , blaVIM , blaGES , blaOXA–48–like , blaCTX–M , blaSHV , blaTEM , blaFOX , blaDHA , blaCMY , blaLAT , and blaACT . ERIC-PCR showed a high diversity among isolates. In this study, the occurrence of MDR CR-hvKp isolates harboring blaNDM and blaGES was detected for the first time in southwestern Iran. To prevent the spread of CR-hvKp and reduce selection pressure, long-term surveillance and more effective treatment strategies should be implemented.This study investigated the molecular epidemiology of carbapenem-resistant classic  Klebsiella pneumoniae is one of the most important Gram-negative bacteria (GNB) causing a variety of community-acquired and nosocomial infections. It is estimated that about one-third of all Gram-negative infections are caused by this bacterium (K. pneumoniae (hvKp) characterized by hypermucoviscosity was first reported from a liver abscess with extrahepatic complications, including endophthalmitis, in Taiwan in 1986. HvKp has been implicated in community-associated infections in healthy people with the most common cases of pyogenic liver abscesses in Asia (acterium . Hypervi in Asia .Klebsiella pneumoniae (cKp) strains from hvKp. These include the string test, the regulator of mucoid phenotype A and A2 (rmpA and rmpA2), an aerobactin siderophore (iucA), the peg-344 gene, and the mucoviscosity-associated gene A (magA) (Some phenotypic and genotypic distinctive properties and determining factors differentiate classic A (magA) . The strA (magA) . TherefoK. pneumoniae. A recent systematic review and meta-analysis from Iran found a prevalence rate of 0.004–58% for carbapenem-resistant K. pneumoniae (CR-Kp) in different regions of the country  GNB, including  country . Accordi country . HoweverK. pneumoniae include the production of various beta-lactamases capable of hydrolyzing carbapenems as well as the reduced membrane permeability that occurs via loss or downregulation expression of outer membrane porins (OMPs)  . CR-Kp is (OMPs) .Klebsiella pneumoniae carbapenemase (KPC), oxacillinases (OXA)-type enzymes such as OXA-48-like carbapenemases, Guiana extended-spectrum beta-lactamase (GES), imipenem-hydrolyzing beta-lactamase (IMI) and metallo-beta-lactamases (MBLs) including Verona integrin-encoded MBL (VIM), New Delhi MBL (NDM), and imipenemase (IMP) play a major role in the development of carbapenem resistance in Enterobacteriaceae (K. pneumoniae (Pseudomonas extended resistance), and VEB (Vietnamese extended-spectrum beta-lactamases) enzymes  affiliated to the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. Ahvaz is the capital of Khuzestan province which located at 31°20’ north latitude and 48°40’ east longitude, and ranks the second-largest city (with an area of about 215 km2) after Tehran, the capital of Iran , non-repetitive clinical isolates of  of Iran . The afoK. pneumoniae isolates was performed using a series of standard biochemical tests including Gram stain, triple sugar iron (TSI) agar, sulfur-indole-motility (SIM) agar, lysine iron agar (LIA), ornithine decarboxylase, citrate utilization, and urea hydrolysis  and incubated aerobically for 18–24 h at 37°C. Primary identification of drolysis . All medK. pneumoniae isolates were confirmed by polymerase chain reaction (PCR) using specific primers for the 16S–23S internal transcribed spacer (16S–23S ITS) gene (130 bp), as previously described  to determine the size of PCR products compared to a 100 bp DNA ladder. The agarose gel was scanned using a UV light transilluminator . Confirmed isolates were stocked in tryptic soy broth (TSB)  containing 20% glycerol and stored at –80°C for ongoing analysis.The presumptive escribed . To extrescribed . All PCRescribed  for 30 smagA, rmpA, rmpA2, iucA, and peg344 were investigated by PCR using previously described primers , ampicillin/sulbactam (10/10 μg), aztreonam (30 μg), cefazolin (30 μg), ceftriaxone (30 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), cefepime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), ertapenem (10 μg), gentamycin (10 μg), fosfomycin (200 μg), imipenem (10 μg), meropenem (10 μg), piperacillin/tazobactam (100/10 μg), tetracycline (30 μg), tobramycin (10 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg)  were tested on Mueller–Hinton agar (MHA)  using the Kirby-Bauer disc diffusion method according to Clinical Laboratory Standard Institute (CLSI) procedures . Also, tectively . Any isoectively . Multiplectively . EscheriK. pneumoniae isolates that had breakpoints of ≤27 mm for cefotaxime (30 μg), ≤22 mm for ceftazidime (30 μg), and ≤25 mm for ceftriaxone (30 μg), were selected for primary ESBL screening  were considered as primary AmpC producers. These isolates were further confirmed by a combined disk method using a cefoxitin disk (30 μg) alone and in combination with phenylboronic acid (PBA) (400 μg) . The cefoxitin disk and cefoxitin/PBA disk were dispensed onto an MHA plate that had already been inoculated with the test isolate  by the standard disk diffusion method. Subsequently, the inoculated plates were incubated for 24 h at 37°C. The isolates were confirmed as AmpC producers when the zone diameter of cefoxitin/PBA increased by ≥5 mm compared to cefoxitin alone .E. coli ATCC® 25922™. After 18–24 h incubation at 35 ± 2°C, mCIM and eCIM results were interpreted as follows: carbapenemase positive (only mCIM positive): zone diameter of 6–15 mm, carbapenemase negative (mCIM negative): zone size ≥ 19 mm, MBL positive (both mCIM/eCIM positive): increase in eCIM zone size by ≥5 mm compared to zone size of mCIM  together with EDTA-modified carbapenem inactivation method (eCIM) . For mCI of mCIM . It shoublaCTX–M, blaSHV, blaTEM, blaPER, and blaVEB), AmpC , carbapenemase , and MBLs  as previously described  was performed with the previously described primers in a final volume of 25 μl containing 1 μl of each primer, 3 μl of template DNA, 12.5 of master mix , and 7.5 μl of nuclease-free water using the Biorad S1000 thermocycler (USA) . FollowiP-value ≤ 0.05) of variables were evaluated with Chi-square and Fisher’s exact tests  version 22.0 . Significant associations  males and 71 females (46.4%). All isolates showed the 130 bp band of the 16S–23S ITS gene in PCR and were confirmed as K. pneumoniae. The mean ± SD age of patients was 43.1 ± 16.3 (10–81 years) for males and 36.9 ± 12.7 (11–82 years) for females. Using phenotypic and genotypic criteria, 136 (88.9%) and 17 (11.1%) isolates were identified as cKp and hvKp, respectively. Of all hvKp isolates, 6 (35.3%), 17 (100.0%), and 6 (35.3%) were positive for the string test, gene markers, and both, respectively. The occurrence of hvKp gene markers was as follows: magA , rmpA , rmpA2 , iucA , and peg344 . The co-occurrence of two or more markers was detected in 5 (29.4%) isolates as follows: magA/rmpA2 (n = 1), rmpA2/iucA/peg344 (n = 3), and magA/rmpA/rmpA2/iucA/peg344 (n = 1).Using standard bacteriology tests, a total of 153 presumptive n = 83/153), including 90.4% (n = 75/83) cKp and 9.6% (n = 8/83) hvKp isolates. In other words, 55.1% (n = 75/136) of cKp and 47.1% (n = 8/17) of hvKp isolates were CR-cKp and CR-hvKp, respectively. Imipenem was the most effective carbapenem against K. pneumoniae isolates, with a susceptibility rate of 48.4% (n = 74/153), followed by ertapenem , and meropenem . Of 75 CR-cKp isolates, 60 (80.0%), 8 (10.7%), and 7 (9.3%) isolates were resistant to ertapenem/imipenem/meropenem, ertapenem/meropenem, and imipenem/meropenem, respectively. However, all CR-hvKp isolates were simultaneously resistant to meropenem/imipenem/ertapenem. The carbapenem-resistant isolates had MICs ranging from 0.03 to 64 μg/mL, MIC50 = 8 μg/mL, MIC90 = 32 μg/mL for ertapenem; MICs ranging from 0.03 to 64 μg/mL, MIC50 = 16 μg/mL, MIC90 = 32 μg/mL for imipenem; and MICs ranging from 8 to 64 μg/mL, MIC50 = 16 μg/mL, MIC90 = 32 μg/mL for meropenem. The detailed antibiotic resistance rates of cKp, hvKp, CR-cKp, and CR-hvKp isolates were summarized in n = 150/153), tetracycline , and tigecycline  and the highest resistance to ampicillin/sulbactam . Resistance rates to other antibiotics ranged from 29.4% (n = 45/153) for aztreonam to 64.7% (n = 99/153) for cefazolin. CR-cKp and CR-hvKp isolates showed resistance rates of more than 50.0% against gentamicin, tobramycin, amikacin, and piperacillin/tazobactam. Also, all hvKp isolates (n = 17) including CR-hvKp (n = 8) strains were susceptible to colistin, tigecycline, and tetracycline. There were no significant differences  in the antibiotic resistance patterns of the cKp with hvKp isolates and the CR-cKp with CR-hvKp isolates except for tigecycline  of all isolates were resistant against third-generation cephalosporins. Of 136 cKp, 1 (0.7%), 1 (0.7%), 1 (0.7%), 3 (2.2%), and 81 (59.6%) isolates were resistant to cefotaxime, cefotaxime/ceftazidime, ceftazidime/ceftriaxone, cefotaxime/ceftriaxone, and cefotaxime/ceftazidime/ceftriaxone, respectively. Of 17 hvKp, 11 (64.7%) isolates were simultaneously resistant to cefotaxime/ceftazidime/ceftriaxone. Of 75 CR-cKp isolates, 69 (92.0%) strains showed resistance against third-generation cephalosporin as follows: cefotaxime , ceftazidime/ceftriaxone , cefotaxime/ceftazidime , cefotaxime/ceftriaxone , and cefotaxime/ceftazidime/ceftriaxone . Also, all CR-hvKp were simultaneously resistant to third-generation cephalosporins. In total, 66.7% (n = 102/153) and 7.8% (n = 12/153) of isolates were MDR and XDR, respectively. None of the isolates were PDR. Of 75 CR-cKp, 84.0% (n = 63) and 16.0% (n = 12) were MDR and XDR, respectively. While, all CR-hvKp  isolates were MDR (K. pneumoniae isolates had 57 (A1–A57) different antibiotypes (antibiotic resistance patterns) (n = 10), A4 , and A5  were the most frequent patterns. Also, MARI ranged from 0.1 to 1.0 and the majority of isolates  had MARI of ≥0.5.Using the carbapenem antibiotic disk diffusion test and the broth microdilution method, the overall resistance to carbapenems was 54.2% (ecycline . The carwere MDR . The 83 atterns) . A2 (12.n = 71/136), male patients , patients aged 26–41 years , urine samples , and men ward . Meanwhile, the highest occurrence of hvKp isolates was found in Golestan Hospital , male patients , patients aged 42–57 years , urine samples , and men ward  . Similar results were obtained for CR-cKp and CR-hvKp isolates (n = 41/75) than in males . Also, the CR-hvKp isolates were more prevalent in intensive care unit (ICU)  than other wards. However, the distribution of CR-cKp and CR-hvKp was not significantly different according to the various items  . Althougisolates . Neverthus items .n = 69/75) and 100.0% (n = 8/8) of CR-cKp and CR-hvKp were presumptive ESBL producers, respectively (n = 20/75) and 12.5% (n = 1/8) of CR-cKp and CR-hvKp were ESBL producers, respectively. All CDT positive isolates were also positive for at least one ESBL gene by PCR. The distribution of ESBL genes among CR-cKp and CR-hvKp was as follows: for CR-cKp: blaCTX–M , blaSHV , blaTEM ; and for CR-hvKp: blaCTX–M , blaSHV , and blaTEM   . None ofn = 59/75) of CR-cKp and 100.0% (n = 8/8) of CR-hvKp were presumptive AmpC producers, the confirmatory cefoxitin (30 μg)/phenylboronic acid method was positive in only 13.3% (n = 10/75) and 0.0% (n = 0/8) of CR-cKp and CR-hvKp isolates, respectively (n = 21/75) of CR-cKp and none of CR-hvKp isolates were AmpC producers. The PCR method detected a greater number of AmpC positive CR-cKp isolates compared with the phenotypic confirmatory test. The distribution of AmpC genes among CR-cKp was as follows: blaLAT , blaACT , blaFOX , blaDHA , and blaCMY . The blaACC was not detected in any isolate  resistance criteria, 78.7% (ectively . However isolate . The disn = 26/75) and 25.0% (n = 2/8) of CR-cKp and CR-hvKp were carbapenemase producers, respectively , blaGES . Moreover, the occurrence of carbapenemase genes in CR-hvKp isolates was as follows: blaOXA–48–like , blaGES  , 34.7% (ectively . PCR shon = 2/8) . The blan = 18/75) and 25.0% (n = 2/8) of CR-cKp and CR-hvKp were MBL positive, respectively (n = 33/75) of CR-cKp and 25.0% (n = 2/8) of CR-hvKp isolates were MBL producers. The distribution of MBL genes among CR-cKp was as follows: blaNDM , blaVIM , and blaIMP . Moreover, 25.0% (n = 2/8) of CR-hvKp isolates carried blaNDM gene , 24.0%  of CR-cKp and 25.0% (n = 2/8) of CR-hvKp isolates. Also, the blaCTX–M/blaSHV was the second most prevalent pattern, detected in 6.7% (n = 5/75) of CR-cKp and 12.5% (n = 1/8) of CR-hvKp isolates. The frequency of co-occurrence of various beta-lactamases was as follows: MBL/carbapenemase/AmpC , MBL/carbapenemase/ESBL , carbapenemase/ESBL/AmpC , MBL/carbapenemase and MBL/AmpC , ESBL/carbapenemase, ESBL/MBL, and ESBL/AmpC , and carbapenemase/AmpC .The different genotypes of beta-lactam resistance genes were shown in n = 62/75) and 13 singletons  with 57 different ERIC-types (E1–E57), indicating high genetic diversity among the isolates (blaNDM positive). The remaining isolates had different beta-lactamase genotypes. The eight CR-hvKp isolates were also divided into two clusters (A and B) and two singletons with four ERIC -types (E1–E4)  consisting of 2–7 isolates  . ElectroK. pneumoniae isolates, including 136 (88.9%) cKp and 17 (11.1%) hvKp isolates, were identified in different clinical samples. Previous reports from Iran by n = 14/52), n = 22/146), and n = 3/122) showed different prevalence rates of hvKp than the current study. However, n = 12/111) from Turkey reported almost the same prevalence of hvKp as in the current study. These discrepancies can be explained by the different nature and size of the samples studied and the detection method of the hvKp isolates. To date, there are no standard methods to differentiate hvKp from cKp strains, and several studies classified hvKp based on positivity of the string test and some virulence genes including rmpA, rmpA2, iucA, peg-344, and magA (n = 6/17) of hvKp had a positive string test, and all  were also positive for at least one of the studied genes. However, 64.7% (n = 11/17) of hvKp isolates carrying gene markers had a negative string test. This phenomenon has also been reported in previous studies from Turkey and Iran , rmpA , rmpA2 , iucA , and peg344 . Simultaneous occurrence of two or more genes was also found in five (29.4%) isolates. In a previous study from Turkey, string test, iucA, and magA were positive in three (25.0%), eight  of the hvKp isolates, respectively. However, the rmpA and peg344 genes were not detected  of cKp and 47.1% (n = 8/17) of hvKp isolates were CR-cKp and CR-hvKp, respectively and showed various resistance rates against ertapenem, imipenem, and meropenem ranging from 47.1 to 54.4%  revealed that a relatively high proportion (more than 50.0%) of cKp and hvKp isolates were resistant to aminoglycosides, beta-lactam combination agents (piperacillin-tazobactam and ampicillin/sulbactam), quinolones, folate pathway inhibitors, and different classes of cephalosporins. These findings were consistent with recent reports of increasing emergence of highly resistant XDR and PDR cKp and hvKp strains from different countries, including Iran, Turkey, Spain, India, Saudi Arabia, and Lebanon . Generalto 54.4% . Howevern = 3/75) of CR-cKp isolates were resistant to colistin. These observations coincided well with previous reports from Iran  and 16.0% (n = 12/75) of CR-cKp were MDR and XDR, respectively. While all CR-hvKp  isolates were MDR (n = 70/83) had a MARI of ≥0.5 and no PDR isolate was detected. In contrast to the current study, in previous reports by In this study, the majority of CR-cKp and CR-hvKp isolates had high resistance rates (more than 60.0%) against 18 of 23 tested antibiotics, so that 84.0%  and CR-hvKp  isolates were identified from urine samples, followed by blood samples for CR-cKp  and tracheal tubes  for CR-hvKp. Also, the CR-hvKp isolates were more prevalent in the ICU ward  than in other wards. However, the distribution of CR-cKp and CR-hvKp did not differ significantly by sample type, hospital wards, and other demographic variables (P > 0.05)  in this study. The reason for this observation was unclear.In this research, most CR-cKp  . These f > 0.05) . Howevern = 20/75) and 12.5% (n = 1/8) of CR-cKp and CR-hvKp were ESBL producers, respectively. The PCR assay confirmed the results of the CDT phenotypic test and all CDT positive isolates harbored at least one ESBL gene. Also, the co-occurrence of ESBL genes was detected in both CR-cKp and CR-hvKp isolates, which was in line with previous reports from different countries , blaSHV , blaTEM ; and for CR-hvKp: blaCTX–M , blaSHV , and blaTEM  , blaSHV , and blaTEM . However, similar to the findings of the current study, the blaPER and blaVEB were not detected , blaTEM , and blaCTX–M . These frequencies were higher than in the current study. Moreover, in contrast to this research, the prevalence of blaSHV was significantly higher (P = 0.048) in hvKp isolates than in cKp strains (n = 9) harbored blaSHV and blaCTX–M, whereas blaTEM was not detected. In this study, the blaCTX–M was the most frequent ESBL among carbapenem-resistant K. pneumoniae isolates that was in line with the previous report from Iraq  . None ofdetected . Also, T strains . In anotrom Iraq . In the al ESBLs . Antibioal ESBLs .n = 21/75) of CR-cKp isolates as follows: blaLAT , blaACT , blaFOX , blaDHA , blaCMY , and blaACC . There is evidence for the role of AmpC beta-lactamase in resistance to carbapenems and increasing their MIC (blaCMY–2 (60.7%) and blaDHA–1 (34.4%) were found in K. pneumoniae isolates  of CR-cKp and 25.0% (n = 2/8) of CR-hvKp, were carbapenemase producers using mCIM test. All mCIM positive isolates carried at least one carbapenemase gene. The distribution of carbapenemase genes among CR-cKp and CR-hvKp was as follows: blaOXA–48–like  and blaGES . Also, the blaKPC and blaIMI were not detected in any isolate. This study was the first to detect the blaGES carbapenemase in CR-hvKp isolates from Iran. In previous studies from Iran, blaGES was not detected in hvKp isolates (blaGES gene (27.8%) was the third most prevalent carbapenemase among carbapenem-resistant K. pneumoniae isolates  was reported to be the most prevalent gene in carbapenem-resistant K. pneumoniae isolates.In this study, 34.7%  of CR-cKp and 25.0% (n = 2/8) of CR-hvKp isolates were MBL positive using the mCIM/eCIM positivity criteria. PCR showed that all mCIM/eCIM positive isolates carried at least one MBL gene. However, PCR detected a greater number of MBL positive isolates than the phenotypic confirmatory test. This may be due to the co-occurrence of carbapenemases and MBLs, resulting in a false-negative eCIM test (n = 33/75) of CR-cKp and 25.0% (n = 2/8) of CR-hvKp isolates were MBL producers. The blaNDM gene was the most frequently detected MBL gene in CR-cKp (41.3%) and CR-hvKp isolates (25.0%), followed by blaVIM (8.0%) and blaIMP (4.0%) for CR-cKp. These results were in good parallel with available data that identified the blaNDM (30.1%) as the most predominant MBL type among K. pneumoniae isolates from Iran, followed by blaVIM (10.6%) and blaIMP (4.5%)  of hvKp isolates (blaNDM and blaOXA–48 genes were frequently associated with hvKp isolates (n = 6/75) was the most co-occurrence pattern among CR-cKp isolates. In contrast, CR-hvKp isolates had only the ESBL co-existence pattern (blaCTX–M/blaSHV). Contrary to these results, the co-occurrence of ESBLs and carbapenemases in hvKp isolates has been reported in previous studies from Iran  . Howeverisolates . In mostisolates . This phisolates . Anotherrom Iran .K. pneumoniae isolates, several expensive and time-consuming molecular methods such as pulsed-field gel electrophoresis (PFGE), multiple locus sequence typing (MLST), and ribotyping are available  based analysis for phylogenetic relatedness, pan-genome analysis and insights on the circulating plasmids and Inc groups especially among the colistin-resistant isolates.blaCTX–M, blaSHV, blaNDM, and blaGES, in southwestern Iran. As a result of their hypervirulence coupled with multidrug resistance, these isolates pose a particular threat to healthcare systems. Hence, long-term surveillance and more effective treatment strategies should be implemented to prevent the spread of CR-hvKp and reduce selection pressure. Also, it is recommended to use a fast and cheap molecular method such as ERIC-PCR for primary evaluation of clonal relatedness of MDR isolates.This study was the first report of emergence of MDR CR-hvKp isolates harboring different beta-lactamases, including The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was approved by the Ethics Committee of the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (ethics code: IR.AJUMS.REC.1398.489) according to the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations of the Ethics Committee of the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. Clinical samples were not collected as part of this study. The clinical samples were collected as routine clinical care and to check the presence of any infection for referred and admitted patients. As a result, written informed consent was waived by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.MSak performed the experiments, analyzed majority of the data, and wrote the whole manuscript. MSav, MH, and MA prepared all the necessary materials and performed some of the experiments. MSav, MH, and SSS contributed to the discussion of experimental results. MA designed and supervised the experiments at different stages. All authors contributed to the article and approved the submitted version.


Similarly, Arabidopsis thaliana SAUR63 was able to increase growth of various organs, antagonize PP2C.D5 phosphatase, and increase H+-ATPase activity. Using a gain-of-function approach to bypass genetic redundancy, we dissected structural requirements for SAUR63 growth-promoting activity. The divergent N-terminal domain of SAUR63 has a predicted basic amphipathic α-helix and was able to drive partial PM association. Deletion of the N-terminal domain decreased PM association of a SAUR63 fusion protein, as well as decreasing protein level and eliminating growth-promoting activity. Conversely, forced PM association restored ability to promote H+-ATPase activity and cell expansion, indicating that SAUR63 is active when PM-associated. Lipid binding assays and perturbations of PM lipid composition indicate that the N-terminal domain can interact with PM anionic lipids. Mutations in the conserved SAUR domain also reduced PM association in root cells. Thus, both the N-terminal domain and the SAUR domain may cooperatively mediate the SAUR63 PM association required to promote growth.In plants, regulated cell expansion determines organ size and shape. Several members of the family of redundantly acting Small Auxin Up RNA (SAUR) proteins can stimulate plasma membrane (PM) H Plant organs reach their final shape and size after substantial cell expansion. Proton pumps at the plasma membrane promote cell expansion by acidifying the cell wall to loosen it, and by increasing electrochemical potential across the plasma membrane for solute uptake that maintains intracellular turgor. Plasma-membrane-associated proteins tightly regulate proton pump activity, in order for organs to grow to an appropriate extent. We have studied requirements for activity of one such regulatory protein in the model plant Arabidopsis called SAUR63. This protein is made rapidly in response to plant growth hormones, and it increases proton pump activity to promote organ growth. These activities depend on its binding to anionic lipids in the plasma membrane, and forced plasma membrane association of SAUR63 can increase growth. Many proteins in the same family are found within Arabidopsis and in all land plants, and likely differ in their affinity for the plasma membrane or in other properties. Further studies of other family members may show how such proteins regulate growth under diverse physiological contexts. Small Auxin Up RNA (SAUR) genes. SAUR genes are numerous in all land plants, and have evolved through extensive independent gene amplifications in multiple lineages ) of seedlings of indicated genotypes grown for 3d in darkness on plates with 0.5x MS medium. C,D) Root length (C) and root tortuosity index (D) of seedlings of indicated genotypes grown for 4d in long days on plates with 1x MS 1% Sucrose medium. E,F) Root length (E) and root tortuosity index (F) of seedlings of indicated genotypes grown for 4d in short days on plates with 0.5x MS medium. Graphs show means ± s.d. Letters in graphs indicate statistical classes based on Tukey’s Honestly Significant Difference test. n from left to right: Panels A,B: 51, 24, 26, 28, 23, 25, 25; Panels C,D: 34, 17, 23, 14, 19, 15, 16; Panels E,F: 43, 21, 18, 17, 16, 15, 17. The same genotypes were measured in panels A-F, with genotype designations shown only in panels E and F. G,H,I) Hypocotyl epidermal cells of seedlings of indicated genotypes grown in short days for 2 days, visualized with the ML1:RFP shoot epidermis plasma membrane marker. Shown are z-stack confocal image projections of the near side of the hypocotyl. Scale bar, 0.1 mm. (TIF)Click here for additional data file.S4 FigA) Cotyledon area of seedlings of indicated genotypes grown on vertically oriented plates for 7d in long days in the absence (open bars) or presence (filled bars) of 1% sucrose. B) Root length of seedlings of indicated genotypes grown on vertically oriented plates for 4d in long days in the absence (open bars) or presence (filled bars) of 1% sucrose. C,F) Hypocotyl lengths (E) and hypocotyl tortuosity index  of seedlings grown on vertically oriented plates for 3d in darkness without sucrose. D) Cotyledon areas of P35S:SAUR63 and P35S:CBL11-12:SAUR6326-142 seedlings grown for 6d on vertically oriented MS 1% Suc plates. E) Hypocotyl lengths of P35S:SAUR63 and P35S:CBL11-12:SAUR6326-142 seedlings grown for 4d on vertically oriented 0.5x MS plates. Panels D and E show data for the three homozygous single-locus P35S:SAUR63 lines that differed most from wild type among seven lines analyzed. Graphs show means ± s.d. Letters in graphs indicate statistical classes based on Tukey’s Honestly Significant Difference test. n, from left to right: panel A: 26, 20, 25, 15, 22, 23, 24, 20, 22, 23, 19, 19, 21, 19, 21, 20; panel B: 19, 16, 18, 14, 20, 20, 21, 16, 21, 17, 19, 16, 18, 15, 16, 17; panel D: 77, 18, 17, 17, 19, 20; panel E: 119, 25, 24, 27, 21, 24; panels C and F: 35, 36, 45, 43, 47, 38, 41, 47.(TIF)Click here for additional data file.S5 FigA) Appearance of genotypes used for crosses presented in A and in B) Hypocotyl lengths of seedlings of indicated genotypes grown for 4d in short days in the absence of sucrose. Plants measured were F1 progeny of crosses of transgenic lines with each other or with wild-type Columbia, and were hemizygous for the indicated transgene(s). C) Appearance of PEST:SAUR63:CerFP:HA and wild-type seedlings grown with estradiol and in the absence or presence of 15 mM LiCl. Seedlings were grown for 3d under control conditions, and then transferred to plates with estradiol and with or without 15 mM LiCl, and grown for an additional 3d before imaging. Dots mark positions of root tips at the time of transfer to estradiol plates. Scale bar, 5 mm. D) Root growth of indicated genotypes in the absence (open bars) or presence (closed bars) of 15 mM LiCl. Seedlings were grown without LiCl for 5d, transferred to plates containing 0 or 15 mM LiCl, and root growth over the next three days was measured. E) HPTS fluorescence ratios around root cells of indicated genotypes. Data are pooled from measurements taken on three different days, each normalized to the average of wild-type values on those days. Graphs show means ± s.d. Letters in graphs indicate statistical classes based on Tukey’s Honestly Significant Difference test. n, from left to right: panel B: 28, 24, 24, 30, 26, 27, 26, 25, 26, 25, 24, 20, 24, 26, 25; panel D: 21, 27, 16, 23, 19, 25, 20, 30, 18, 28, 21, 30, 23, 31, 18, 29; panel E: 26, 13, 14, 16.(TIF)Click here for additional data file.S6 FigA-D)P35S:SAUR63:YFP:HA. E-H)P35S:SAUR6326-142:YFP:HA. I-L)PUBQ10:WAVE 138Y expressing a PM-localized YFP fusion protein. M-P)PUBQ10:WAVE 1Y expressing a cytoplasmically-localized YFP fusion protein. Q-T)PUBQ10:WAVE 9Y expressing a YFP fusion protein localized to the tonoplast. Shown are fluorescence confocal microscopy images of YFP , FM4-64 membrane staining , and both channels together  with vertical yellow lines indicating locations of quantitation of fluorescence intensity signals, scaled to the maximum signal along the line . Image color channel brightnesses were adjusted for visibility. Scale bar, 20 μm.(TIF)Click here for additional data file.S7 FigA-M) Confocal images showing YFP fluorescence of transgenic lines expressing the indicated fusion proteins behind the P35S promoter. Scale bar, 20 μm.(TIF)Click here for additional data file.S8 FigA-L) Confocal images showing YFP fluorescence of indicated SAUR63:YFP:HA variants expressed in transiently transformed N. benthamiana leaves. Scale bar, 20 μm.(TIF)Click here for additional data file.S9 FigA-E) Protein levels in multiple P35S:SAUR63:YFP:HA, P35S:SAUR6326-142:YFP:HA, and P35S:CBL11-12:SAUR6326-142:YFP:HA pooled T2 seedlings from different T1 transformants. The blot in panel E shows protein extracts from selected genotypes in panels A-D for side-by-side comparison in the same experiment. F,G) SAUR63:YFP:HA, SAUR6326-142:YFP:HA, SAUR631-25:YFP:HA, CBL11-12:SAUR6326-142:YFP:HA, and SAUR63m2:YFP:HA fusion proteins, detected by western blots in total (T), soluble (S) and microsomal (M) protein fractions. Lower panels show controls for loading or fractionation. α-HA detects SAUR63 fusion protein; α-AHA2 detects a membrane protein; α-UGPase and α-APX detect soluble proteins. Arrows indicate position of full-sized SAUR63:YFP:HA fusion proteins. FL, Full-length SAUR63:YFP:HA fusion protein. wt, wild-type Columbia lacking any transgene. In genotype designations, S63 is short for SAUR63. Letters and numbers after genotype designations indicate independent transgenic lines. H) Amount of SAUR63:YFP:HA and SAUR6326-142:YFP:HA proteins in whole seedling extracts at indicated times after start of cycloheximide treatment to block new protein synthesis. Fusion proteins were detected by western blots using anti-HA antibody. The Rubisco large subunit band from Ponceau S staining of the same gels is shown as a loading control. A repeat of this experiment gave a very similar result. In the lower gel, the larger band is the presumed intact SAUR6326-142:YFP:HA protein, and the lower band is a presumed smaller breakdown product. In genotype labels, S63 denotes SAUR63, letters and numbers after genotype names indicate distinct transgenic lines, FL denotes a strong full-length P35S:SAUR63:YFP:HA line used as a common standard line in most experiments, and wt indicates wild-type Columbia lacking any transgene.(TIF)Click here for additional data file.S10 FigA) Western blot showing presence of fusion proteins in extracts used in lipid blot experiments in 26-142:YFP:HA fusion proteins (upper arrow) and SAUR631-25:YFP:HA N-terminal domain fusion protein (lower arrow). B) Longer exposures of two lipid blots from C) Mock experiment in which extracts were incubated for 70 minutes in protein extraction buffer at the indicated temperatures, and then run on a gel for western blots. For both SAUR63:YFP:HA and SAUR6326-142:YFP:HA, similar amounts of protein are present after incubation at -20 C or after incubation at 22 C, as during the lipid blot binding experiment.(TIF)Click here for additional data file.S11 FigA)PEST:SAUR63NAAIRS:CerFP:HA lines grown on plates with estradiol  or without estradiol (wild-type Columbia and PEST:SAUR63:CerFP:HA only). B) Western blots of total protein in estradiol-induced PEST:SAUR63NAAIRS:CerFP:HA lines using α-HA antibody.(TIF)Click here for additional data file.S12 FigA-D)P35S:SAUR63:YFP:HA. E-H)P35S:SAUR6326-142:YFP:HA. I-L)P35S:SAUR63m9:YFP:HA. M-P)P35S:SAUR63m13:YFP:HA. Q-T)P35S:SAUR63m15:YFP:HA. Shown are fluorescence confocal microscopy images of YFP , FM4-64 membrane staining , and both channels together  with vertical yellow lines indicating locations of quantitation of fluorescence intensity signals, scaled to the maximum signal along the line . Image color channel brightnesses were adjusted for visibility. Panels A-D are the same as in (TIF)Click here for additional data file.


Plasmodium vivax apical membrane antigen-1 (pvama-1) is an important vaccine candidate against Malaria. The genetic composition assessment of pvama-1 from wide-range geography is vital to plan the antigen based vaccine designing against Malaria.P. vivax positive malaria patients from different districts of Khyber Pakhtunkhwa (KP) province of Pakistan. The highly polymorphic and immunogenic domain-I (DI) region of pvama-1 was PCR amplified and DNA sequenced. The QC based sequences raw data filtration was done using DNASTAR package. The downstream population genetic analyses were performed using MEGA4, DnaSP, Arlequin v3.5 and Network.5 resources.The blood samples were collected from 84 pvama-1 (DI) in KP samples with majorly prevalent H-14 and H-5 haplotypes. Pairwise comparative population genetics analyses identified limited to moderate genetic distinctions among the samples collected from different districts of KP, Pakistan. In context of worldwide available data, the KP samples depicted major genetic differentiation against the Korean samples with Fst = 0.40915 , while least distinction was observed against Indian and Iranian samples. The statistically significant negative values of Fu and Li’s D* and F* tests indicate the evidence of population expansion and directional positive selection signature. The slow LD decay across the nucleotide distance in KP isolates indicates low nucleotide diversity. In context of reference pvama-1 sequence, the KP samples were identified to have 09 novel non-synonymous single nucleotide polymorphisms (nsSNPs), including several trimorphic and tetramorphic substitutions. Few of these nsSNPs are mapped within the B-cell predicted epitopic motifs of the pvama-1, and possibly modulate the immune response mechanism.The analyses unveiled total 57 haplotypes of pvama-1 DI among the P. vivax isolates acquired from widespread regions of KP province of Pakistan. The information may implicate in future vaccine designing strategies based on antigenic features of pvama-1.Low genetic differentiation was observed across the The online version contains supplementary material available at 10.1186/s12879-022-07798-1. Plasmodium. The P. vivax and P. falciparum are predominant species responsible for malaria [P. vivax is most widely distributed human malaria parasite, endemic in tropical and subtropical countries of Asia, South Pacific, Central and South America, Middle East, and North Africa [Malaria is an acute febrile infectious disease caused by vector-borne apicomplexan parasites of the genus  malaria . The P. h Africa . Accordih Africa .P. vivax [Plasmodium species such as apical membrane antigen-1 (AMA-1), Circumsporozoite proteins (CSP), Merozoite surface proteins (MSP) and Duffy binding protein (DBP) are reported as potent malarial vaccine candidates’ targets [Treatment and control of malaria have become a serious challenge due to drug resistance and lack of effective vaccines. The wide-range distribution, antigenic variation, relapsing and co-infection led to a collective interest towards the development of effective vaccine against P. vivax . The impP. vivax . Several targets .Plasmodium species as promising malaria vaccine candidate antigens [Plasmodium parasites [P. falciparum and P. vivax [The genetic composition assessment of vaccine candidates’ loci is indispensable in modern-age to plan an effective vaccination strategy. There are ample of studies suggesting the AMA-1 of antigens . The AMAarasites , 9. The arasites –12. The arasites . The ectP. vivax –17. FurtP. vivax . This suPlasmodium vivax ama-1 (pvama-1) have been conducted in malaria endemic countries [pvama-1 genetic features from Pakistan. Particularly, no study till date is reported from remote malaria endemic regions of Khyber Pakhtunkhwa (KP) province of Pakistan. The current study was therefore designed to evaluate the genetic composition of pvama-1 among P. vivax isolates collected from widespread KP regions of Pakistan . Blood samples were obtained from 100 consented patients tested positive for pvama-1 was amplified by polymerase chain reaction (PCR) using the specific primers and amplification conditions as reported previously [Escherichia coli DH5α competent cells, and positive clones were selected by colony PCR. The nucleotide sequence of cloned insert was analyzed by automatic DNA sequencing with M13 forward and reverse primers . The raw data was filtered for quality assessment using DNASTAR Lasergene package.A DNA fragment flanking the DI region of eviously , 29. Thepvama-1 sequence i.e. Sal-I (AF063138) and Genbank-deposited pvama-1 sequences from China Myanmar Boarder (KX495505–KX495577), Iran (KF422636.1–KF422681.1), Korea (KM230319.1–KM230384.1), Myanmar (FJ157248.1–FJ157285.1), Papua New Guinea (PNG) (KC702402.1–KC702501.1), Sri Lanka (EF218679.1–EF218701.1), Venezuela (EU346015.1–EU346087.1), Thailand (FJ784891.1–FJ784990.1), and India (EU282774.1–EF025196.1). The comparative sequences analyses were performed using MEGA4 software suite [The DNA sequences data generated in the current study was analyzed in comparison with reference pvama-1 with a threshold score of ≥ 0.5. The higher BepiPred score predicts higher binding affinity of epitopes with immune receptors. The non-synonymous SNPs (nsSNPs) mapping within the top predicted epitopes of pvama-1 was checked. The intrinsically unstructured regions (IURs) and RBC binding sites within the pvama-1 have previously been characterized [pvama-1. Additionally, the positive selection sites in B-cell epitopes were identified via the maximum likelihood method of Codeml [The BepiPred-2.0  server wcterized , 32; andf Codeml  implemenf Codeml .2 index via DnaSP [D* and F* indices were calculated via a sliding window method using DnaSP. The population genetics statistical analyses, including pairwise fixation index (Fst), analysis of molecular variance (AMOVA), haplotype frequencies, and nucleotide diversity based on Nei’s net distance (DA) were computed using Arlequin v3.5 [The DnaSP v6.12 software package  was usedia DnaSP . The Tajuin v3.5 . The hapuin v3.5 .pvama-1, flanking the DI domain were amplified from genomic DNA of 84 P. vivax positive samples. The sequences data spanning the 322–737 nucleotide positions of the reference pvama-1 sequence i.e. Sal-I (AF063138). The analyses identified a large numbers of single nucleotide polymorphisms (SNPs) in KP samples. Among these, the 68 were nsSNPs, i.e. causing amino acid substitutions, including 53 dimorphic, 10 trimorphic, 3 tetramorphic, and 2 pentamorphic nsSNPs. The two pentamorphic amino acid changes observed were R112K/T/E/S and S228D/N/R/K. The ten trimorphic amino acid substitutions include the N132D/G, A141E/G, E145A/G, K190E/Q, T191K/P, A199T/V, S209G/C, P210S/L, P223L/S, and V233L/P. While the three tetramorphic amino acid changes are K120R/S/G, E189N/K/G, and E227V/K/G. These amino acid substitutions were observed at varied frequencies in the KP samples. Among the 68 nsSNPS, 59 have previously been reported in literature for P. vivax isolates from different geographical origin. However, the rest of 9 nsSNPs were found specific to KP samples set of this study. These nsSNPs were observed at low frequencies, i.e. 1.1 to 1.19%. Few nsSNPs, including K120R, N132D, L140I, A141E, K190E, E227V, and S228D were commonly observed in KP samples, as well as in some other continental pvama-1 sequences with high frequency of 3.8–100%  of 0.978 ± 0.008 . The difference between dN/dS ratio for pvama-1 DI region was also found negative (− 0.05413 ± 0.02) in case of KP samples set.The 416 bp sequences of pvama-1 DI  were compared to the global pvama-1 sequences deposited in Genbank. The values of K and π observed for KP sequences were more or less similar to previously reported sequences from Iran and India, however different from the rest of global sequences , followed by Swabi and Kohat  samples. The lowest Fst was estimated between Swat and Bannu samples , followed by Swat and Hungo samples   and mean pairwise differences (πxy) was observed between Bannu and Swabi samples, i.e. congruent to the Fst result . The Korean samples showed significant genetic distinction in pairwise comparison to rest of the global samples as well. Meanwhile, least genetic differentiation was observed among KP, Iranian, and Indian samples . Likewise, higher variance component was noticed within the population group i.e. 3.67534 as compare to among populations  pattern of pvama-1 DI sequences of P. vivax isolates from KP, Pakistan. The LD index (R2) (Y-axis) plotted against nucleotide distance (X-axis) using a two tailed Fisher’s exact test.Additional file 4: Fig S3. Pearson correlation plot of KP and global pvama-1 samples based on pairwise Fst values. The plot shows clustering and correlation between the groups in hierarchical order. The dark brackets and large sizes depict the minimum genetic distinction and high correlation.Additional file 5: Fig S4. The principle component analysis (PCA) of pvama-1 DI sequences. Different colors depict different populations groups. This include KP Pakistan (as a single group) and worldwide samples.


Subjects and Methods. The coexpression of PD-L1/PD-1 with CXCR3/CD36 on circulating lymphocytes was analyzed by flow cytometry in 78 lymphoma patients before and after therapy and in 50 healthy controls. The concentration levels of IL-19 in serum were assessed by an ELISA. Results. PD-L1 and PD-1 were expressed on circulating CXCR3+ and CD36+ lymphocytes in lymphoma and were significantly higher in patients with extranodal involvement than in lymphoma patients without extranodal involvement (P < 0.001). Elevated IL-19 levels were observed in lymphoma patients and increased significantly in extranodal involvement (P < 0.001). High percentages of PD-L1+CXCR3+ and PD-1+CXCR3+ lymphocytes were associated with high LDH levels, hepatomegaly, lymphedema, advanced tumor stage, and recurrence. Furthermore, patients with splenomegaly and generalized lymphadenopathy had high percentages of PD-L1+CXCR3+ lymphocytes. In addition, levels of PD-L1/PD-1 coexpression with CXCR3 and IL-19 were significantly associated with bone marrow, lung, and lymph vessel involvement. Further analysis revealed that high percentages of PD-L1+CD36+ and PD-1+CD36+ lymphocytes were associated with lung and bone marrow involvement. Patients with high levels of PD-L1/PD-1 coexpression with CXCR3 and IL-19 had inferior event-free survival (EFS) compared with that in lymphoma patients with low levels. EFS was decreased in patients with high percentages of PD-L1+CD36+ and PD-1+CD36+ lymphocytes. When using the receiver operating characteristic (ROC) curve, the superiority of IL-19 (area under the curve (AUC): 0.993) and PD-L1+CXCR3+% (AUC: 0.961) to PD-1+CXCR3+% (AUC: 0.805), PD-L1+CD36+% (AUC: 0.694), and PD-1+CD36+% (AUC 0.769) was evident in the diagnosis of extranodal involvement, identifying lymphoma patients with extranodal involvement from patients without extranodal involvement. Conclusions. Coexpression of PD-L1/PD-1 with CXCR3/CD36 in circulating lymphocytes and serum IL-19 levels contributes to poor prognosis and might be potential markers for extranodal involvement in lymphoma.Many studies have demonstrated that PD-L1/PD-1 signaling is an immune evasion mechanism in tumors. PD-L1/PD-1 coexpression with CXCR3/CD36 in peripheral lymphocytes in lymphoma still needs to be clarified. The current study investigated PD-L1/PD-1 coexpression with CXCR3/CD36 in circulating lymphocytes, serum IL-19 levels, and their correlation with clinical outcome and extranodal involvement in lymphoma.  Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) are the two forms of lymphoma , 2. Bothy , 8. The y .T cells express the regulatory inhibitory protein programmed death-1 (PD-1), a transmembrane element . MacrophCXCR3, a G protein-coupled cell surface receptor (GPCR), is present on CD4+ and CD8+ lymphocytes surface and other cells, like epithelial cells . Still, Cluster differentiation 36 (CD36) is a scavenger receptor on the surface of monocytes, adipocytes, dendritic cells, macrophages, and lymphocytes –33. CD36Interleukin-19 (IL-19) is a member of the IL-10 family . EssentiTumors have developed various strategies, disrupting “immune checkpoints” to get through the host's immune system . The stuA total of 78 lymphoma patients and 50 healthy volunteers participated in the study. Healthy controls included thirty-six males and fourteen females. Healthy volunteers' range in age was from 26 to 70 years old. Seventy-eight lymphoma subjects ranged in age from 11 to 81 years, including 39 men and 39 women. Patients received treatment as soon as the primary diagnosis is confirmed. Patients with lymphoma were followed, and the patients were separated into two groups based on how well they responded to treatment: group I with no extranodal involvement and group II with extranodal involvement. Lymphoma patients' clinical outcomes were evaluated according to Cheson response criteria  . AccordiA complete clinical examination was performed on all subjects to monitor lymphadenopathy and hepatosplenomegaly, and detailed history questionnaires were completed. To determine the performance, type, and stage of lymphoma, all patients underwent bone marrow aspiration and lymph node biopsy. The Ann Arbor classification has been considered for clinical staging . The modFollow-up for lymphoma patients was performed at the hematology clinic and by telephone. The posttherapy lymphoma status was evaluated in lymphoma patients by means of clinical examination and PET/CT scans. Patients will be evaluated every three months for the identification of lymphoma progression. The follow-up was only for 78 patients with lymphoma out of 92 patients. The follow-up could not be performed for these patients as some patients were referred to other centers, some have died, and some had rejected blood samples rewithdrawal. Event-free survival (EFS) measures the time between the end of therapy and the commencement of an event . Seven follicular cell lymphoma (FCL) and twenty-eight diffuse large B cell lymphoma (DLBCL) patients' subjects had CHOP therapy  . In contAll subjects provided peripheral samples, which were taken under very sterile conditions. Eight milliliters of blood were collected. For a complete blood count (CBC), 2 mL of blood was put into an EDTA tube. For flow cytometric analysis, 2 mL of blood was inserted into an EDTA tube. A plain tube was filled with 4 mL of blood, then centrifuged for 5 minutes at 3000 rpm. Serum was isolated and used to analyze liver function tests , and aspartate aminotransferase (AST)), lactate dehydrogenase (LDH), kidney function tests (serum creatinine and blood urea), and random blood sugar. The leftover serum was kept at -70°C until the serum IL-19 concentrations were measured.A computerized hematology analyzer was used to determine CBC . Renal function analysis (blood urea nitrogen and serum creatinine), liver function analysis , and random blood sugar were performed using an autoanalyzer . An automated ACE chemistry analyzer assessed LDH .PD-1/PD-L1 coexpression with CXCR3/CD36 was determined in collected blood samples. To identify the various immune cells, the following human monoclonal antibodies were used, as directed by the manufacturer: PD-1 , CXCR3 monoclonal antibody , PD-L1 , and CD36 monoclonal antibody . Unstained cells were utilized as a negative control for every patient. Negative isotypic controls were performed using other tubes. As isotype controls, monoclonal PE-conjugated IgG2a  and FITC IgG1 were used .The percentages of PD-L1+CXCR3+, PD-1+CXCR3, PD-L1+CD36+, and PD-1+CD36+ lymphocytes were calculated using flow cytometry BD FACSCanto II . In brief, a hundred microliters of anticoagulated-EDTA whole blood were stained with five uL of monoclonal antibodies and incubated at room temperature in the dark for twenty minutes. The cells were lysed using the lysing buffer and set aside for about ten minutes at room temperature in the dark. Cells were then washed two times with PBS and resuspended in 300 uL of PBS solution. A minimum of 10,000 events were analyzed. Lymphocyte gating was carried out through the FSC/SSC plots (front scatter vs. side scatter technique) –60, and The concentrations of IL-19 in serum were assessed using an ELISA kit  following the manufacturer's instructions. In the 96 wells of the microtiter strips, a specific monoclonal for IL-19 was coated (sensitivity for IL-19: 1.3 pg/mL). A microtiter plate reader was used to determine optical densities at 450 nm.The data were analyzed by applying the SPSS application  version 25. Normally, quantitative data was analyzed by minimum and maximum range and mean and standard deviation (SD). The median and interquartile range (IQR) was utilized for quantitative nonparametric data, while percentage and number were employed for categorical data. Mann–Whitney analysis was carried out to analyze quantitative nonparametric data between two groups. Kruskal-Wallis test was carried out to analyze nonparametric data between more than two groups, proceeded by pairwise comparisons between each two groups applying Bonferroni correction. Fisher's exact test, or the Chi-square test, was carried out to compare the qualitative data between groups. Association between continuous and qualitative ordinal variables was assessed by Spearman's correlation, while Pearson's correlation was performed for the association between 2 continuous variables.P values less than 0.05 were considered significant.The Kaplan-Meier analysis was carried out to assess EFS, comparing the survival curves using the log-rank test. The variables' cutoff point, area under the curve (AUC), specificity, and sensitivity were calculated using the receiver operator characteristic (ROC) curve. The study included 50 healthy individuals and 78 lymphoma patients. Normal controls included thirty-six males and fourteen females. Healthy volunteers ranged in age from 24 to 81 years old. Seventy-eight lymphoma subjects ranged in age from 11 to 81 years, including 39 males and 39 females. The criteria for all subjects are listed in (Supplementary Table N = 34) and patients with extranodal involvement (N = 34). Lymphoma patients with and without extranodal involvement revealed statistical significance regarding sex (P = 0.039), stage (P < 0.001), recurrence (P < 0.001), and death (P = 0.018). Among lymphoma subjects with extranodal involvement, 73.5% had a recurrence, and 20.6% died. 26.5% of patients with extranodal involvement presented with stage III diseases, while 58.8% had stage IV diseases. Only 2.9% of patients with extranodal involvement had stage I, and 11.8% had stage II   .P < 0.001). Interestingly, PD-L1+CD36+% and PD-1+CD36+% in newly diagnosed lymphoma subjects were significantly higher than in healthy volunteers   .n = 34) or without extranodal involvement (n = 44) (P < 0.001) (P < 0.001)  . Postthe< 0.001) . When co< 0.001) .P < 0.001, respectively)  . Moreover, lymphoma patients had higher posttherapy IL-19 levels than the normal controls   .P < 0.001) (P < 0.001) , which was significantly higher than that of subjects without extranodal invasion with a median of 46.5 pg/mL (range: 33-137.5) (< 0.001) . Interes< 0.001) .P < 0.05). Furthermore, compared to the PR, recurrence, and refractory groups, the CR group had a significant reduction in posttherapy IL-19, CXCR3+%, PD-L1+CXCR3+%, and PD-1+CXCR3+% (P < 0.05). Additionally, PD-L1+CD36+% and PD-1+CD36+% were significantly lower in subjects with CR compared to the other groups. Contrarily, comparing the CR group to the other groups, there was a substantial rise in posttherapy CD36+%   .P < 0.001). However, no significant difference was observed when subjects with CR were compared to the treatment-refractory patients (P = 0.092 and P = 0.055). In addition, the CR patients' PD-L1+CD36+% and PD-1+CD36+ % were significantly lower than those in the PR group (P = 0.037 and P = 0.002). However, no significant difference was detected between the CR group and the recurrence group or the refractory group (P > 0.05) (The CR group showed a significant reduction in pretherapy PD-1+CXCR3+% and posttherapy PD-L1+CXCR3+% compared to the PR group and recurrence group ( > 0.05) .P < 0.001). Additionally, the recurrence group's posttherapy IL-19 levels, PD-L1+CXCR3+, and PD-1+CXCR3+% were significantly higher than those of PR patients  (P = 0.022 and P = 0.002). However, no significant differences were observed when the treatment-refractory group compared to PR or recurrence groups (P > 0.05)  . Further > 0.05) .r = 0.344, P = 0.002; r = 0.375, P = 0.001; r = 0.315, P = 0.005, respectively). However, negative associations between CXCR3+%, PD-L1/PD-1+CXCR3%, and albumin levels were identified . Additionally, there was a negative association between PD-L1+CXCR3+% and hemoglobin   .r = 0.464, P < 0.001; r = 0.398, P < 0.001, r = 0.335, P = 0.003). Data revealed that CXCR3+%, PD-L1+CXCR3+%, and PD-1+CXCR3+% positively correlated with lymphoma stages   and general lymphadenopathy . Furthermore, CXCR3+% and PD-L1+CXCR3+% positively correlated with BCR-ABL . Additionally, CXCR3+%, PD-L1+CXCR3+%, and PD-1+CXCR3+% had a significant association with lymphoma recurrence  (P > 0.05)  . Moreove= 0.006) . CD36+%, > 0.05) .r = 0.349 and P = 0.002), hepatomegaly (r = 0.362 and P = 0.001), and splenomegaly (r = 0.231 and P = 0.042). Furthermore, pretherapy IL-19 levels were significantly associated with lymphedema, general lymphadenopathy, and recurrence   .r = 0.771, P < 0.001; r = 0.793, P < 0.001; r = 0.528, P < 0.001). CXCR3+%, PD-L1+CXCR3+%, and PD-1+CXCR3+% had a positive association with bone marrow involvement . Moreover, CXCR3+%, PD-L1+CXCR3+%, and PD-1+CXCR3+% were positively associated with lymph vessel involvement . Additionally, a significant association between CXCR3+%, PD-L1+CXCR3+%, PD-1+CXCR3+%, and lung involvement was identified   .r = 0.333, P = 0.003; r = 0.469, P < 0.001), while CD36+% had a negative association with extranodal involvement (r = −0.792 and P < 0.001). Of interest, PD-L1+CD36+% and PD-1+CD36+% were positively correlated with bone marrow infiltration . PD-1+CD36+% was positively correlated with lung involvement . CD36% was negatively correlated with bone marrow and lymph vessel involvement . Furthermore, percentages of CD36+ lymphocytes were correlated with spleen infiltration   .r = 0.848; P < 0.001). IL-19 levels are positively associated with bone marrow and lymph vessel involvement . Similarly, a positive association between posttherapy IL-19 levels and lung involvement was identified   .The effect of the markers on EFS was assessed using Kaplan-Meier statistics . SurvivaP < 0.001)   (P < 0.001) (P < 0.001)  . High pr< 0.001) . A prolo Figures .The effect of the immune markers on overall survival and recurrence-free survival was determined using Kaplan-Meier statistics (data not shown). Patients with low pre- and posttherapy CXCR3+%, PD-L1+CXC3+%, and PD-1+CXCR3+% do better than those with a high percentage. Furthermore, low PD-L1+CD36+% and PD-L1+CD36+% predicted a more prolonged survival and recurrence-free time.P < 0.001, respectively) . The pretherapy IL-19 cut-off was >209 pg/mL, with specificity and sensitivity 75% and 91.18%  at a cutoff >49, >40, and >3, respectively . Posttherapy CD36+% had the best sensitivity of 100% and the best specificity of 95.45% . Posttherapy CD36+%, PDL-1+CD36+%, and PD-1+CD36+% had cutoff levels ≤37, > 11, and>3 , with a cutoff >280 pg/mL for diagnosis of extranodal involvement. IL-19 showed 100% sensitivity and 97.73% specificity . The specificity and sensitivity were 97.73% and 100% at a cut-off >280 pg/mL.PD-L1/PD-1 coexpression with CXCR3/CD36 in identifying patients with extranodal involvement was assessed using ROC curves. The AUCs of the pre- and posttherapy PD-L1+CXCR3+% were 0.981 and 0.961, respectively, with high specificity and sensitivity. The cut-off values were >33 and >40. CXCR3+% and PD-L1+CXCR3+% yielded the best sensitivity and specificity. Moreover, the ROC curve was assessed for posttherapy PD-L1/PD-1+CD36+ percentages. The AUCs were 0.694 and 0.769, with reduced sensitivity and specificity. Thus, according to the findings, PD-L1/PD-1+CD36+% is insufficient for identifying extranodal involvement in lymphoma patients. Furthermore, the pretherapy IL-19 ROC curves demonstrated a pattern of extranodal involvement (This study had some limitations: (1) the small size of the subjects in this study; (2) short follow-up time, longer follow-up, and multicenter collaborations are needed to confirm PD-L1/PD-1's role; (3) PD-L1/PD-1 coexpression with CXCR3/CD36 should be investigated in tumor tissue; (4) cell function activities such as differentiation, proliferation, apoptosis, and cytokine release were not carried out; future research will investigate these issues; (5) PD-L1/PD-1 coexpression with CXCR3/CD36 was not investigated in different lymphocyte subsets; further studies are required.In conclusion, PD-L1/PD-1 coexpression with CXCR3/CD36 and serum IL-19 may be involved in lymphoma extranodal involvement and have prognostic and predictive values in lymphoma. The findings could also shed light on the role of circulating CXCR3 and CD36-positive lymphocyte cells in lymphoma. Future clinical trials and research are required to create new treatments based on PD-L1/PD-1-induced lymphoma immune evasion mechanisms and host immune response regulation. The combination of PD-L1/PD-1 blockades, anti-CXCR3/CD36, and IL-19 monoclonal antibody therapy might start a new era for immunotherapy. PD-L1+CXCR3+ lymphocytes and serum IL-19 might play a more important role in poor clinical behavior in lymphoma.


The second paragraph of Acknowledgments should read as follows: “We gratefully acknowledge funding from the following sources: Czech Science Foundation grants 20-23513S to H.H., 18-17529S to A.Z., and 20-04150Y to O.G.; Czech Ministry of Education grant OPVVV16_019/0000759; and Czech BioImaging grant LM2015062.”Volume 6, no. 3, e00327-21, 2021,


Brassica rapa, we identified 28 putative HSP70 gene family members using state-of-the-art bioinformatics-based tools and methods. Based on chromosomal mapping, HSP70 genes were the most differentially distributed on chromosome A03 and the least distributed on chromosome A05. Ka/Ks analysis revealed that B. rapa evolution was subjected to intense purifying selection of the HSP70 gene family. RNA-sequencing data and expression profiling showed that heat and cold stress induced HSP70 genes. The qRT-PCR results verified that the HSP70 genes in Chinese cabbage (Brassica rapa ssp. pekinensis) are stress-inducible under both cold and heat stress. The upregulated expression pattern of these genes indicated the potential of HSP70 to mitigate environmental stress. These findings further explain the molecular mechanism underlying the responses of HSP70 to heat and cold stress.Heat shock proteins protect plants from abiotic stress, such as salt, drought, heat, and cold stress. HSP70 is one of the major members of the heat shock protein family. To explore the mechanism of HSP70 in  The heat shock transcription factor family strengthens plants under biotic and abiotic stress conditions; as a result, plants proceed with normal growth and development . TemperaArabidopsis, HSP70 deficient plants showed the stunted plant growth and abnormal leaf phenotype [Arabidopsis plant development under abiotic stress conditions [Brassica rapa family member. This vegetable-based family includes cabbage, Chinese cabbage, mustard, and turnip, which are sources of oil, amino acids, and proteins for humans and animals. These vegetables are highly susceptible to temperature stress [The HSP70 family, which encodes HSP70 proteins, has been identified and studied in many plants, such as Arabidopsis (17 genes), rice (26 genes), spinach 12 genes), and soybean (61 genes) . In Arabhenotype , double henotype . Further genes, ae stress  and are e stress .Brassica rapa has gained remarkable economic and nutritional value due to its heading trait, which makes it highly susceptible to stress. Under temperature stress, the leaves become pale and weakened due to chloroplast damage, and the plant becomes unable to properly regulate photosynthesis [Brassica rapa to date. We identified and explored the members of the HSP70 gene family, constructed their evolutionary tree and synteny correlations, and predicted protein structures with available bioinformatics software. Moreover, we analyzed the expression of these proteins by qRT-PCR to determine their responses to heat shock stress in Chinese cabbage (Brassica rapa subsp. Pekinensis). These findings will open up new gates for understanding the structure, function, and expression of HSP70 genes in B. rapa.ynthesis . Under sA. thaliana HSP70 family genes (AT3G12580.1) was obtained from the TAIR Arabidopsis genome database (TAIR-Home Page arabidopsis.org (accessed on 20 April 2022). This sequence was used as a query to retrieve the HSP70 genes in the Brassica database . Secondly, the HMMER 3.1  and BLASTP with the threshold e-value set to 1e−5 were performed using the Hidden Markov Model (HMM V 3.0) profiles of the HSP70 gene family (PF00012) were downloaded from the Pfam protein database  and used as the inquiry, The default limitation of HMMER 3.1 was set to 0.01. Finally, total of 28 HSP70 were retrieved in the Brassica rapa genome. B.rapaHSP70 proteins were further characterized by determining the molecular weight, number of amino acids, isoelectric point, and grand average of hydropathicity (GRAVY) through the ProtParam tool . The protein sequences were uploaded to the online database Wolf PSORT for the prediction of subcellular localization . Furthermore, the protein conserved domain was identified using the NCBI conserved domain online server  and motif analysis was performed by using MEME Suite . The gene structure was predicted by the Gene Structure Display Server (GSDS) , using the CDS and genomic sequences of 28 selected B.rapaHSP70. Furthermore, prediction of the subcellular location pattern of each B.rapaHSP70 was carried out using the WoLF PSORT server .Arabidopsis, B. oleracea, and B. napus were retrieved to construct the phylogenetic tree using MEGA X (V 6.06) software . The sequences were multiple aligned and employed using the neighbor-joining (NJ) method with 1000 bootstrap replicates [B. rapa with B. oleracea, B. napus, and Arabidopsis were developed using MCScanX to obtain collinearity files that were used to build dual synteny plots in TBtools .The protein sequences of HSP70s from plicates . SyntenyB. rapa [B. rapa and its closely related species through TBtools [Duplicated genes were retrieved using the Blast, MCScanX, and Advance Circos features of TBtools . For theB. rapa ,20. Synt TBtools .B.rapaHSP70 genes were classified, and the 2000 bp upstream of the start codon were downloaded from the BRAD database . The PlantCARE web-based tool  was used for further classification, and the results were presented using TBtools. The graphical construction of the 3-dimensional (3D) structure of B.rapaHSP70 proteins was done using the online web-based software PHYRE2 (PHYRE2 Protein Fold Recognition Server (ic.ac.uk), selecting the fold recognition method [Putative cis-elements of the 28 B.rapaHSP70 were used to determine the interaction of genes with microRNAs using the psRNATarget database , and the interactions were further prophesied by Cytoscape Software. Gene ontology (GO) annotation was performed to explore functional characteristics. The B.rapaHSP70 gene sequences were uploaded to the online eggNOG database . Then, the GO annotation data were handled by using  and graphical demonstration was given by using OmicShare .CDS sequences of B.rapaHSP70 genes in different tissues, and a heat map was constructed using TbTools [http://www.ncbi.nlm.nih.gov/geo/, accessed on 20 April 2022) under accession no. GSE43245. In addition, expression profiles of B.rapaHSP70 under cold and heat stress conditions were obtained by performing qRT-PCR.RNA-seq data were used to determine the expression of  TbTools . The datSeeds of Chinese cabbage were grown in pots containing soil: vermiculite mixture (3:1) in a controlled chamber  until they reached the five-leaf stage. Cold and heat stress conditions were applied to five-leaf seedlings. Seedlings were transferred to 4 °C for cold stress and 45 °C for heat stress under the same day/night and humidity conditions. Three biological replications were used in both stress conditions. Leaf samples were harvested after 0, 1, 3, 6, 12, and 24 h, placed in liquid nitrogen, and stored at −80 °C [B.rapaHSP70 gene expression; actin was used as the internal reference gene in Chinese cabbage. All primers used in this experiment are listed in −ΔΔCt method was used to check the relative expression levels, and the results were graphically represented [Leaf samples were used to extract the RNA, and qRT-PCR was performed with three replicates to check resented .B.rapaHSP70-1–B.rapaHSP70-28. These proteins were further analyzed for the HSP70 domain (PF00012) , equal number and similar motif are present within each group except in group 4 , and the longest was B.rapaHSP70-28 (894 aa). The genes that translate into HSP70 proteins were distributed on 10 chromosomes, A01–A10. Among these, the maximum genes were distributed on chromosome A03 and the minimum on chromosomes A02, A04 and A05 , and all selected genes were named PF00012) . Conserv group 4 A. Detail group 4 . Gene st group 4 B. The am and A05 .B.rapaHSP70, B.nHSP70, B.olHSP70, and AtHSP70 were analyzed. Based on domains and a phylogenetic tree, 77 HSP70s were clustered into six major groups   and Arabidopsis.The evolutionary relationships between , and E) . The resB.rapaHSP70 genes in the B. rapa genome. We detected duplicated gene couples among the 28 B.rapaHSP70 genes identified in B. rapa , B. napus (AC), and B. oleracea (CC) using collinearity analysis. In addition, a comparative synteny analysis of HSP70 gene pairs among B. rapa, B. napus, B. oleracea, and A. thaliana was conducted (B. rapa (such as Chinese cabbage) and B. oleracea (Cabbage) and B. napus (Mustard) was included due the presence of both sub genome (A and C). B.rapaHSP70 displayed the most collinearity with B. napus, followed by A. thaliana and B. oleracea. Results demonstrated that homologues genes from A genome of B. rapa are present in A as well as C genome of B.napus. Similar behavior was also observed in C genome of B. oleracea. These results suggest that, in addition to the whole genome duplication event, an independent duplication event also occurred during the evolution of these species.BraA10g033140.3C/BraA10g033130.3C, BraA09g038510.3C/BraA01g027130.3C, BraA01g038810.3C/BraA03g035360.3C, BraA08g029700.3C/BraA06g012060.3C, BraA03g019440.3C/BraA03g016490.3C, BraA03g051830.3C/BraA08g020460.3C, BraA02g003050.3C/BraA03g003930.3C, BraA08g021750.3C/BraA01g001130.3C, BraA03g047170.3C/BraA01g019900.3C, and BraA06g001280.3C/BraA04g004290.3C were determined A, which onducted B. We selB.rapaHSP70 genes was retrieved and uploaded to the PlantCare database to examine the cis-elements in the promoter region. The graphical representation  helices and beta (β) sheets, because proteins with similar structures often have similar functions  topology, thus preserving conserved signature fold of HSP70 family with central mixed β-sheet sandwiched between α-helices [B.rapaHSP70 protein family.With the advent of graphical visualization of genomic data, 3D protein structures also explain the properties, as well as facilitate the comparative studies, of proteins. All 28 unctions . Protein-helices . On the B.rapaHSP70 genes, we identified the miRNAs associated with the identified genes , cellular component (CC), and biological process (BP), were investigated to further explore the ed terms . Some ofed terms .The GO-MF (Molecular Function) enrichment results detected 19 enriched terms, namely, protein serine/threonine phosphatase activity (GO:0004722 and GO:0004674), manganese ion binding (GO:0030145), pectate lyase activity (GO:0030570), 1-phosphatidylinositol binding (GO:0005545), glycoprotein binding (GO:0005515), DNA-binding transcription factor activity (GO:0003700), calmodulin binding (GO:0005516), cobalt ion binding (GO:0050897), clathrin binding (GO:0030276), ATP binding (GO:0005524 and GO:0005524), oligopeptide transmembrane transporter activity (GO:0015198), oxidoreductase activity (GO:0016491 and GO:0016491), glutathione peroxidase activity (GO:0004602), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase activity (GO:0046537), protein serine/threonine kinase activity (GO:0004674), beta-amylase activity (GO:0016161), and maltose biosynthetic process (GO:0000024).The GO-BP  enrichment results detected 26 enriched terms, namely, floral organ development (GO:0048437), oxidative stress responsive (GO:0006979), signal transduction (GO:0007165), signal transduction (GO:0006857), defense response (GO:0006952), pollen development (GO:0009555), male gamete generation (GO:0048235), signal transduction by cis-phosphorylation (GO:0007165), clathrin coat assembly (GO:0048268), regulation of transcription (GO:0006355), metabolic process (GO:0008152), salt stress responsive (GO:0009651), transmembrane transport (GO:0055085), plasma membrane organization (GO:0007009), glycolytic process (GO:0006096), abscisic acid responsive (GO:0009737), transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169), protein phosphorylation (GO:0006468), starch catabolic process (GO:0005983), cold stress responsive (GO:0009409), cadmium sensitivity/resistance (GO:0046686), regulation of meristem growth (GO:0010075), cadmium ion responsive (GO:0046686), temperature stimulus responsive (GO:0009409), maltose biosynthetic process (GO:0000024), and protein phosphorylation (GO:0006468).The GO-CC (cellular component) enrichment results detected 29 enriched terms, including mitochondrial protein-transporting ATPase activity (GO:0005739 and GO:0005739), plasma membrane (GO:0005886), chloroplast envelope (GO:0009941), endomembrane system (GO:0012505), nucleus (GO:0005634 and GO:0005634), chloroplast (GO:0009507), apoplast (GO:0048046), endomembrane system (GO:0012505), chloroplast stroma (GO:0009570), cytosol (GO:0005829), clathrin coat (GO:0030118), mitochondrial envelope (GO:0005740), vacuole (GO:0005773), and integral component of membrane (GO:0016021) .B.rapaHSP70 genes, we examined 8 tissues and organs of B. rapa at various growth phases based on RNA-seq data of B. rapa  under accession no. GSE43245. For instance, the expression patterns of most B.rapaHSP70 genes in the silique, callus, and flower were higher than those of other tissues  showed no transcript changes in any tissue/organ.To illustrate the transcript levels of the  tissues . Three BB.rapaHSP70 genes varied in various tissues and organs. In silique, the expression level of B.rapaHSP70-4, B.rapaHSP70-9, and B.rapaHSP70-24 showing higher expression, and that of B.rapaHSP70-19 was lowest. In callus, B.rapaHSP70-25 was highly expressed, and B.rapaHSP70-10, B.rapaHSP70-11, B.rapaHSP70-12, B.rapaHSP70-16, B.rapaHSP70-18, B.rapaHSP70-20, B.rapaHSP70-4, and B.rapaHSP70-4 was the least expressed. In callus, B.rapaHSP70-4, B.rapaHSP70-9, B.rapaHSP70-24 and B.rapaHSP70-25 are expressing highly and B.rapaHSP70-10, B.rapaHSP70-11, B.rapaHSP70-12, B.rapaHSP70-18, B.rapaHSP70-22 and B.rapaHSP70-28 are showing no expression change. In stem, B.rapaHSP70-4, B.rapaHSP70-9, B.rapaHSP70-24 and B.rapaHSP70-25 are highly expressing, whereas B.rapaHSP70-10, B.rapaHSP70-11, B.rapaHSP70-12, B.rapaHSP70-16, B.rapaHSP70-20, and B.rapaHSP70-26 showing no expression change. In roots, B.rapaHSP70-4, B.rapaHSP70-9, B.rapaHSP70-24, and B.rapaHSP70-25 are highly expressed, whereas B.rapaHSP70-10, B.rapaHSP70-11, B.rapaHSP70-12, B.rapaHSP70-16, B.rapaHSP70-20, and B.rapaHSP70-26 showing no expression change. In flower, B.rapaHSP70-4, B.rapaHSP70-9, and B.rapaHSP70-24 are highly expressed genes, whereas B.rapaHSP70-10, B.rapaHSP70-11, B.rapaHSP70-12, B.rapaHSP70-16, and B.rapaHSP70-22 showing zero expression change. Lastly, the expression was analyzed in leaf, according to the expression pattern the B.rapaHSP70-4, B.rapaHSP70-9, B.rapaHSP70-17, B.rapaHSP70-24, and B.rapaHSP70-25 showing higher expression whereas B.rapaHSP70-10, B.rapaHSP70-11, B.rapaHSP70-12, B.rapaHSP70-16, B.rapaHSP70-20, and B.rapaHSP70-26 showing no expression change. These findings suggest that these candidate genes may play diverse roles in regulating B. rapa growth processes  were upregulated under high-temperature stress at different time intervals, and most were highly expressed after 24 h of heat stress. The expression levels of 16 B.rapaHSP70s  were high at different time intervals under cold stress  as B. rapa. We identified 28 gene family members of HSP70 in B. rapa, which is higher than that found in Arabidopsis (11 AtHSP70) [The model plant B.rapaHSP70 were confirmed by the correlation between intron numbers and motif arrangements in combination with phylogeny. A phylogenetic tree was created to show the evolutionary relationships between B. rapa, B. napus, B. oleracea, and Arabidopsis. Recently, research has examined the evolutionary associations among these species [Notably, genes within the same phylogenetic subgroup had similar motif compositions and exon/intron structures . Genes w species ,34.B.rapaHSP70 family genes and we find all the duplicated B.rapaHSP70s are segmental duplicated.During the genome evolution process, gene duplications and chromosomal segments are major forces in the evolution of plant genome structure and content . Tandem A. thaliana were identified and subjected to promoter analysis to investigate the presence of temperature-associated (heat shock element (HSE)) and dehydration-associated cis-elements  [Previously, a total of 11 HSP70 genes from nt; DRE) . Based oB. rapa stress tolerance. Furthermore, GO enrichment analysis supports the findings of the HSP70 gene association with stress tolerance  [B.rapaHSP70 genes  in post-transcriptional gene regulation . In the nditions ,38. For nditions . Similarinensis) . These s70 genes .StHSP20 under heat stress. In another study, the results verified the involvement of B.rapaHSP70 proteins in thermotolerance [B. rapa and other plant species. In this study, we laid a foundation for recognizing signaling controlled by HSP70 proteins under biotic and abiotic stress conditions.The expression patterns of genes are associated with their function . Severalolerance . Until nB. rapa, and 28 putative members were identified. In silico analyses were performed, including gene structure, distribution, phylogenetic relationship, and syntenic studies, which helped to explore the evolutionary properties of the HSP70 gene family in B. rapa. In addition, targeted miRNAs, promotor cis-acting regulatory elements, and gene ontology (GO) were executed. The finding of all these analysis elucidating that B.rapaHSP70s are targeted by 34 different families of microRNAs, these genes are highly responsive to light, temperature and phytohormones, and B.rapaHSP70s are involved in the Biological, cellular and molecular functioning of B. rapa, respectively. Three dimensional protein structural knowledge can help improve crop properties, such as improving stress resistance and biomass yield. Furthermore, quantitative real-time PCR results indicated that HSP70 genes are strongly involved in heat and cold stress responses in B. rapa plants. To a large extent, all analyses performed on the HSP70 gene family will lay the foundation for further studies of molecular and physiological functions in Brassica crops.In the current study, we performed a genome-wide analysis of the HSP70 gene family in