Cloning and functional analysis of the juvenile hormone receptor gene CsMet in Coccinella septempunctata.

The potential role of the juvenile hormone receptor gene (methoprene-tolerant, Met) in reproduction of Coccinella septempunctata L. (Coleoptera: Coccinellidae)(Coleoptera: Coccinellidae), was investigated by cloning, analyzing expression profiles by quantitative real-time PCR, and via RNA interference (RNAi). CsMet encoded a 1518-bp open reading frames with a predicted protein product of 505 amino acids; the latter contained 2 Per-Arnt-Sim repeat profile at amino acid residues 30-83 and 102-175. CsMet was expressed in different C. septempunctata larvae developmental stages and was most highly expressed in third instar. CsMet expression in female adults gradually increased from 20 to 30 d, and expression levels at 25 and 30 d were significantly higher than levels at 1-15 d. CsMet expression in 20-d-old male adults was significantly higher than in males aged 1-15 d. CsMet expression levels in fat body tissues of male and female adults were significantly higher than expression in the head, thorax, and reproductive system. At 5 and 10 d after CsMet-dsRNA injection, CsMet expression was significantly lower than the controls by 75.05% and 58.38%, respectively. Ovary development and vitellogenesis in C. septempunctata injected with CsMet-dsRNA were significantly delayed and fewer mature eggs were produced. This study provides valuable information for the large-scale rearing of C. septempunctata.

Diagnostic and prognostic role of LINC01767 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a primary contributor to cancer-related mortality on a global scale. However, the underlying molecular mechanisms are still poorly understood. Long noncoding RNAs are emerging markers for HCC diagnosis, prognosis, and therapeutic target. No study of LINC01767 in HCC was published. AIM: To conduct a multi-omics analysis to explore the roles of LINC01767 in HCC for the first time. METHODS: DESeq2 Package was used to analyze different gene expressions. Receiver operating characteristic curves assessed the diagnostic performance. Kaplan-Meier univariate and Cox multivariate analyses were used to perform survival analysis. The least absolute shrinkage and selection operator (LASSO)-Cox was used to identify the prediction model. Subsequent to the validation of LINC01767 expression in HCC fresh frozen tissues through quantitative real time polymerase chain reaction, next generation sequencing was performed following LINC01767 over expression (GSE243371), and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes/Gene Set Enrichment Analysis/ingenuity pathway analysis was carried out. In vitro experiment in Huh7 cell was carried out. RESULTS: LINC01767 was down-regulated in HCC with a log fold change = 1.575 and was positively correlated with the cancer stemness. LINC01767 was a good diagnostic marker with area under the curve (AUC) [0.801, 95% confidence interval (CI): 0.751-0.852, P = 0.0106] and an independent predictor for overall survival (OS) with hazard ratio = 1.899 (95%CI: 1.01-3.58, P = 0.048). LINC01767 nomogram model showed a satisfied performance. The top-ranked regulatory network analysis of LINC01767 showed the regulation of genes participating various pathways. LASSO regression identified the 9-genes model showing a more satisfied performance than 5-genes model to predict the OS with AUC > 0.75. LINC01767 was down-expressed obviously in tumor than para-tumor tissues in our cohort as well as in cancer cell line; the over expression of LINC01767 inhibit cell proliferation and clone formation of Huh7 in vitro. CONCLUSION: LINC01767 was an important tumor suppressor gene in HCC with good diagnostic and prognostic performance.

Pathogenomics analysis of high-risk clone ST147 multidrug-resistant Klebsiella pneumoniae isolated from a patient in Egypt.

BACKGROUND: The emergence of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) represents a serious clinical health concern. Antibiotic resistance and virulence interactions play a significant role in the pathogenesis of K. pneumoniae infections. Therefore, tracking the clinical resistome and virulome through monitoring antibiotic resistance genes (ARG) and virulence factors in the bacterial genome using computational analysis tools is critical for predicting the next epidemic. METHODS: In the current study, one hundred extended spectrum ß-lactamase (ESBL)-producing clinical isolates were collected from Mansoura University Hospital, Egypt, in a six-month period from January to June 2022. One isolate was selected due to the high resistance phenotype, and the genetic features of MDR-KP recovered from hospitalized patient were investigated. Otherwise, the susceptibility to 25 antimicrobials was determined using the DL Antimicrobial Susceptibility Testing (AST) system. Whole genome sequencing (WGS) using Illumina NovaSeq 6000 was employed to provide genomic insights into K. pneumoniae WSF99 clinical isolate. RESULTS: The isolate K. pneumoniae WSF99 was phenotypically resistant to the antibiotics under investigation via antibiotic susceptibility testing. WGS analysis revealed that WSF99 total genome length was 5.7 Mb with an estimated 5,718 protein-coding genes and a G + C content of 56.98 mol%. Additionally, the allelic profile of the WSF99 isolate was allocated to the high-risk clone ST147. Furthermore, diverse antibiotic resistance genes were determined in the genome that explain the high-level resistance phenotypes. Several ß-lactamase genes, including blaCTX-M-15, blaTEM-1, blaTEM-12, blaSHV-11, blaSHV-67, and blaOXA-9, were detected in the WSF99 isolate. Moreover, a single carbapenemase gene, blaNDM-5, was predicted in the genome, positioned within a mobile cassette. In addition, other resistance genes were predicted in the genome including, aac(6')-Ib, aph(3')-VI, sul1, sul2, fosA, aadA, arr-2, qnrS1, tetA and tetC. Four plasmid replicons CoIRNAI, IncFIB(K), IncFIB(pQil), and IncR were predicted in the genome. The draft genome analysis revealed the occurrence of genetic mobile elements positioned around the ARGs, suggesting the ease of dissemination via horizontal gene transfer. CONCLUSIONS: This study reports a comprehensive pathogenomic analysis of MDR-KP isolated from a hospitalized patient. These findings could be relevant for future studies investigating the diversity of antimicrobial resistance and virulence in Egypt.

Automated classification of group B Streptococcus into different clonal complexes using MALDI-TOF mass spectrometry.

Objectives: To evaluate the performance of Matrix-Assisted Laser Desorption/Ionization Time-of Flight Mass Spectra (MALDI-TOF MS) for automated classification of GBS (Group B Streptococcus) into five major CCs (clonal complexes) during routine GBS identification. Methods: MALDI-TOF MS of 167 GBS strains belonging to five major CCs (CC10, CC12, CC17, CC19, CC23) were grouped into a reference set (n = 67) and a validation set (n = 100) for the creation and evaluation with GBS CCs subtyping main spectrum (MSP) and MSP-M using MALDI BioTyper and ClinProTools. GBS CCs subtyping MSPs-M was generated by resetting the discriminative peaks of GBS CCs subtyping MSP according to the informative peaks from the optimal classification model of five major CCs and the contribution of each peak to the model created by ClinProTools. Results: The PPV for the GBS CCs subtyping MSP-M was greater than the subtyping MSP for CC10 (99.21% vs. 93.65%), but similar for CC12 (79.55% vs. 81.06%), CC17 (93.55% vs. 94.09%), and CC19 (92.59% vs. 95.37%), and lower for CC23 (66.67% vs. 83.33%). Conclusion: MALDI-TOF MS could be a promising tool for the automated categorization of GBS into 5 CCs by both CCs subtyping MSP and MSP-M, GBS CCs subtyping MSP-M is preferred for the accurate prediction of CCs with highly discriminative peaks.

Construction of an Infectious DNA Clone of Grapevine Geminivirus A Isolate GN and Its Biological Activity in Plants Analyzed Using an Efficient and Simple Inoculation Method.

The pathogenicity of grapevine geminivirus A (GGVA), a recently identified DNA virus, to grapevine plants remains largely unclear. Here, we report a new GGVA isolate (named GGVAQN) obtained from grapevine 'Queen Nina' plants with severe disease symptoms. The infectious clone of GGVAQN (pXT-GGVAQN) was constructed to investigate its pathogenicity. Nicotiana benthamiana plants inoculated with GGVAQN by agroinfiltration displayed upward leaf curling and chlorotic mottling symptoms. A simple, quick, and efficient method for delivering DNA clones of GGVAQN into grapevine plants was developed, by which Agrobacterium tumefaciens cells carrying pXT-GGVAQN were introduced into the roots of in vitro-grown 'Red Globe' grape plantlets with a syringe. By this method, all 'Red Globe' grape plants were systemically infected with GGVAQN, and the plants exhibited chlorotic mottling symptoms on their upper leaves and downward curling, interveinal yellowing, and leaf-margin necrosis symptoms on their lower leaves. Our results provide insights into the pathogenicity of GGVA and a simple and efficient inoculation method to deliver infectious viral clones to woody perennial plants.

The Full-Genome Analysis and Generation of an Infectious cDNA Clone of a Genotype 6 Hepatitis E Virus Variant Obtained from a Japanese Wild Boar: In Vitro Cultivation in Human Cell Lines.

Hepatitis E virus (HEV) can cause self-limiting acute and chronic hepatitis infections, particularly in immunocompromised individuals. In developing countries, HEV is mainly transmitted via drinking contaminated water, whereas zoonotic transmission dominates the route of infection in developed countries, including Japan. Pigs are an important reservoir for HEV infection. Wild boars, which share the same genus and species as domestic pigs, are also an HEV reservoir. During our nationwide study of HEV infection in wild boar populations in Japan, a genotype 6 (HEV-6) strain, wbJHG_23, was isolated in Hyogo Prefecture in 2023. The genomic length was 7244 nucleotides, excluding the poly(A) tract. The wbJHG_23 strain exhibited the highest nucleotide identity throughout its genome with two previously reported HEV-6 strains (80.3-80.9%). Conversely, it displayed lower similarity (73.3-78.1%) with the HEV-1-5, HEV-7, and HEV-8 strains, indicating that, although closely related, the wbJHG_23 strain differs significantly from the reported HEV-6 strains and might represent a novel subtype. The wbJHG_23 strain successfully infected the human-derived cancer cell lines, PLC/PRF/5 and A549 1-1H8 cells, suggesting that HEV-6 has the potential for zoonotic infection. An infectious cDNA clone was constructed using a reverse genetics system, and a cell culture system supporting the efficient propagation of the HEV-6 strain was established, providing important tools for further studies on this genotype. Using this cell culture system, we evaluated the sensitivity of the wbJHG_23 strain to ribavirin treatment. Its good response to this treatment suggested that it could be used to treat human infections caused by HEV-6.

Highs and Lows in Calicivirus Reverse Genetics.

In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an "infectious clone". This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones.

Characterization of a Molecular Clone of Deformed Wing Virus B.

Honey bees (Apis mellifera) play a crucial role in agriculture through their pollination activities. However, they have faced significant health challenges over the past decades that can limit colony performance and even lead to collapse. A primary culprit is the parasitic mite Varroa destructor, known for transmitting harmful bee viruses. Among these viruses is deformed wing virus (DWV), which impacts bee pupae during their development, resulting in either pupal demise or in the emergence of crippled adult bees. In this study, we focused on DWV master variant B. DWV-B prevalence has risen sharply in recent decades and appears to be outcompeting variant A of DWV. We generated a molecular clone of a typical DWV-B strain to compare it with our established DWV-A clone, examining RNA replication, protein expression, and virulence. Initially, we analyzed the genome using RACE-PCR and RT-PCR techniques. Subsequently, we conducted full-genome RT-PCR and inserted the complete viral cDNA into a bacterial plasmid backbone. Phylogenetic comparisons with available full-length sequences were performed, followed by functional analyses using a live bee pupae model. Upon the transfection of in vitro-transcribed RNA, bee pupae exhibited symptoms of DWV infection, with detectable viral protein expression and stable RNA replication observed in subsequent virus passages. The DWV-B clone displayed a lower virulence compared to the DWV-A clone after the transfection of synthetic RNA, as evidenced by a reduced pupal mortality rate of only 20% compared to 80% in the case of DWV-A and a lack of malformations in 50% of the emerging bees. Comparable results were observed in experiments with low infection doses of the passaged virus clones. In these tests, 90% of bees infected with DWV-B showed no clinical symptoms, while 100% of pupae infected with DWV-A died. However, at high infection doses, both DWV-A and DWV-B caused mortality rates exceeding 90%. Taken together, we have generated an authentic virus clone of DWV-B and characterized it in animal experiments.

Insights into the Pathogenesis and Development of Recombinant Japanese Encephalitis Virus Genotype 3 as a Vaccine.

Japanese encephalitis virus (JEV), a flavivirus transmitted by mosquitoes, has caused epidemics and severe neurological diseases in Asian countries. In this study, we developed a cDNA infectious clone, pBAC JYJEV3, of the JEV genotype 3 strain (EF571853.1) using a bacterial artificial chromosome (BAC) vector. The constructed infectious clone was transfected into Vero cells, where it exhibited infectivity and induced cytopathic effects akin to those of the parent virus. Confocal microscopy confirmed the expression of the JEV envelope protein. Comparative analysis of growth kinetics revealed similar replication dynamics between the parental and recombinant viruses, with peak titers observed 72 h post-infection (hpi). Furthermore, plaque assays demonstrated comparable plaque sizes and morphologies between the viruses. Cryo-electron microscopy confirmed the production of recombinant virus particles with a morphology identical to that of the parent virus. Immunization studies in mice using inactivated parental and recombinant viruses revealed robust IgG responses, with neutralizing antibody production increasing over time. These results showcase the successful generation and characterization of a recombinant JEV3 virus and provide a platform for further investigations into JEV pathogenesis and vaccine development.

Fine Root Density Dynamics and Carbon Stock of Eucalyptus spp.: Interplay of Age, Genotype, and Edaphoclimatic Conditions.

Roots play a fundamental role in forest ecosystems, but obtaining samples from deep layers remains a challenging process due to the methodological and financial efforts required. In our quest to understand the dynamics of Eucalyptus roots, we raise three fundamental questions. First, we inquire about the average extent of the roots of two contrasting Eucalyptus genotypes. Next, we explore the factors that directly influence the growth and depth of these roots, addressing elements such as soil type, climate, and water availability. Lastly, we investigate how the variation in Eucalyptus species may impact root growth patterns, biomass, and carbon stock. In this study, we observed that the maximum root depth increased by an average of 20% when genotypes were grown on sites with higher water availability (wet site). E. urophylla stands had a higher biomass and carbon stock (5.7 Mg C ha-1) of fine roots when cultivated on dry sites (annual rainfall~727 mm) than the wet sites (annual rainfall~1590 mm). In E. grandis × E. camaldulensis stands, no significant differences were observed in the stock of fine root biomass (3.2 Mg C ha-1) between the studied environments. Our results demonstrated that genotypes with greater drought tolerance (E. grandis × E. camaldulensis) tend to maintain higher stocks of fine root biomass (3.2-6.3 Mg ha-1) compared to those classified as plastic (E. urophylla), regardless of the edaphoclimatic conditions of the cultivation site. Finally, our research helps understand how Eucalyptus trees adapt to their environment, aiding sustainable forest management and climate change mitigation. We also provide a practical tool to estimate underground biomass, assisting forest managers and policymakers in ensuring long-term forest sustainability.

Microfluidic capillary electrophoresis - mass spectrometry for rapid charge-variant and glycoform assessment of monoclonal antibody biosimilar candidates.

Early-stage cell line screening is a vital step in developing biosimilars of therapeutic monoclonal antibodies (mAbs). While the quality of the manufactured antibodies is commonly assessed by charge-based separation methods employing UV absorbance detection, these methods lack the ability to identify resolved mAb variants. We evaluated the performance of microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) as a rapid tool for profiling mAb biosimilar candidates from clonal cell lines. A representative originator sample was used to develop the MCE-MS method. The addition of dimethylsulfoxide (DMSO) to the background electrolyte yielded up to 60-fold enhancement of the protein MS signal. The resulting electropherograms consistently provided resolution of mAb charge variants within 10 min. Deconvoluted mass spectra facilitated the identification of basic variants such as C-terminal lysine and proline amidation, while the acidic variants could be assigned to deamidated forms. The MCE-MS method also allowed the identification of 18 different glycoforms in biosimilar samples. To mimic early-stage cell line selection, samples from five clonal cell lines that all expressed the same biosimilar candidate mAb were compared to their originator mAb. Based on the similarity observed in charge variants and glycoform profiles acquired by MCE-MS, the most promising candidate could be selected. The MCE-MS method demonstrated good overall reproducibility, as confirmed by a transferability study involving two separate laboratories. This study highlights the efficacy of the MCE-MS method for rapid proteoform screening of clonal cell line samples, underscoring its potential significance as an analytical tool in biosimilar process development.

Genomic insights into NDM-1-producing Pseudomonas aeruginosa: Current status in a Bulgarian tertiary hospital and on the Balkans.

The present study aimed to explore the genomic characteristics of eight New Delhi metallo-ß-lactamase-1 (NDM-1)-producing carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Bulgarian tertiary hospital (2021-2023) in comparison to blaNDM-1-positive strains originating from the Balkans. Antimicrobial susceptibility testing, phenotypic assays for carbapenemase activity, PCR screening, whole-genome sequencing (WGS), and phylogenomic analysis were performed. Seven of the CRPA isolates investigated (Minimum inhibitory concentration values of imipenem and meropenem >32 mg L-1) were also resistant to piperacillin-tazobactam, ceftazidime, ceftazidime-avibactam, cefepime, ceftolozane-tazobactam, amikacin, tobramycin, ciprofloxacin, and levofloxacin, but were susceptible to colistin (0.5-2 mg L-1) and cefiderocol (0.25-1 mg L-1). The P. aeruginosa Pae57 isolate (designated Pae57) remained susceptible to aminoglycosides as well. WGS uncovered the co-existence of blaNDM-1 and blaGES-1. The isolates belonged to the ST654 high-risk clone, except for Pae57 (ST611). Alignment against reference sequences revealed the presence of a Tn21 transposon harboring bleMBL-blaNDM-1-ISAba125. It was similar to that found in the P. aeruginosa ST654 NDM1_1 strain (GCA_020404785.1) from Serbia. Phylogenomic analysis of our isolates indicated that seven of them (ST654) differed from each other in no more than 44 single-nucleotide polymorphisms (SNPs). Pae57 (ST611) was strikingly different (>21,700 SNPs) compared to all Balkan strains. In conclusion, to our knowledge this is the first report of blaNDM-1-positive P. aeruginosa ST611 isolation, which indicates the transmission dynamics of this determinant between high-risk and potentially high-risk P. aeruginosa clones. Obtained results unveil the dissemination of clonally related NDM-1-producing P. aeruginosa strains in the monitored hospital for approximately a 2-year period.

Comparison of two biological systems used for phototoxicity testing: Cellular and tissue.

The OECD has approved two similar methods for testing the phototoxic potency of chemicals. The first method, OECD 432, is based on the cytotoxicity properties of materials to the mouse 3T3 (clone A31) cell line (fibroblasts) after exposure to light. The second method, OECD 498, is based on the same properties but using reconstructed human epidermis - EpiDerm (stratified keratinocytes). The aim of this study was to compare these two methods using statistical tests (specificity, sensitivity, negative predictive value, positive predictive value and accuracy) and non-statistical characteristics (e.g. price and experimental duration, amount of material, level of complications, cell type, irradiation dose). Both tests were performed according to the relevant guidelines using the same 11 control substances. Higher performance values were observed for OECD 432 in both phototoxic and non-phototoxic classifications. The accuracy of OECD 432 was 90.9%, while that of OECD 498 was 72.7%. OECD 432 was also shorter and less expensive. On the other hand, OECD 498 was less complicated, and used human cells with stratum corneum, which better reflects real skin. This method can also be used with oily substances that are poorly soluble in water. However, both methods are important for testing the phototoxic properties of materials, and can be used alone or in a tiered strategy.

Cytotoxicity effect and transcriptome analysis of PCV3-infected cells revealed potential viral pathogenic mechanisms.

Porcine circovirus type 3 (PCV3) has become an important pathogen in the global swine industry and poses a threat to pig health, but its pathogenic mechanism remains unknown. In this study, we constructed an innovative, linear infectious clone of PCV3 for rescuing the virus, and explored the transcriptome of infected cells to gain insights into its pathogenic mechanisms. Subsequently, an in vivo experiment was conducted to evaluate the pathogenicity of the rescued virus in pig. PCV3 nucleic acid was distributed across various organs, indicating systemic circulation via the bloodstream and viremia. Immunohistochemical staining also revealed a significant presence of PCV3 antigens in the spleen, lungs, and lymph nodes, indicating that PCV3 had tropism for these organs. Transcriptome analysis of infected ST cells revealed differential expression of genes associated with apoptosis, immune responses, and cellular metabolism. Notably, upregulation of genes related to the hypoxia-inducible factor-1 pathway, glycolysis, and the AGE/RAGE pathway suggests activation of inflammatory responses, ultimately leading to onset of disease. These findings have expanded our understanding of PCV3 pathogenesis, and the interplay between PCV3 and host factors.

HATCHet2: clone- and haplotype-specific copy number inference from bulk tumor sequencing data.

Bulk DNA sequencing of multiple samples from the same tumor is becoming common, yet most methods to infer copy-number aberrations (CNAs) from this data analyze individual samples independently. We introduce HATCHet2, an algorithm to identify haplotype- and clone-specific CNAs simultaneously from multiple bulk samples. HATCHet2 extends the earlier HATCHet method by improving identification of focal CNAs and introducing a novel statistic, the minor haplotype B-allele frequency (mhBAF), that enables identification of mirrored-subclonal CNAs. We demonstrate HATCHet2's improved accuracy using simulations and a single-cell sequencing dataset. HATCHet2 analysis of 10 prostate cancer patients reveals previously unreported mirrored-subclonal CNAs affecting cancer genes.

LinG3D: visualizing the spatio-temporal dynamics of clonal evolution.

BACKGROUND: Cancers are spatially heterogenous, thus their clonal evolution, especially following anti-cancer treatments, depends on where the mutated cells are located within the tumor tissue. For example, cells exposed to different concentrations of drugs, such as cells located near the vessels in contrast to those residing far from the vasculature, can undergo a different evolutionary path. However, classical representations of cell lineage trees do not account for this spatial component of emerging cancer clones. Here, we propose routines to trace spatial and temporal clonal evolution in computer simulations of the tumor evolution models. RESULTS: The LinG3D (Lineage Graphs in 3D) is an open-source collection of routines (in MATLAB, Python, and R) that enables spatio-temporal visualization of clonal evolution in a two-dimensional tumor slice from computer simulations of the tumor evolution models. These routines draw traces of tumor clones in both time and space, and may include a projection of a selected microenvironmental factor, such as the drug or oxygen distribution within the tumor, if such a microenvironmental factor is used in the tumor evolution model. The utility of LinG3D has been demonstrated through examples of simulated tumors with different number of clones and, additionally, in experimental colony growth assay. CONCLUSIONS: This routine package extends the classical lineage trees, that show cellular clone relationships in time, by adding the space component to show the locations of cellular clones within the 2D tumor tissue patch from computer simulations of tumor evolution models.

Immunohistochemical study of Programmed Cell Death Ligand 1 (PDL1) expression by combined positive score using 22C3 clone in head and neck squamous cell carcinomas, its correlation with clinicopathological features and outcome.

Context: Programmed cell death ligand 1 (PD L1) is a transmembrane protein that is highly expressed in neoplastic cells. Therapy with immune checkpoint inhibitors target PD-1/PD-L1 blockade-inducing tumour regression. Immunohistochemistry (IHC) for PD-L1 expression enables patient selection for immunotherapy and can be considered as a potential predictive biomarker for immunotherapy in head and neck squamous cell carcinoma (HNSCC). Aims: To determine the PDL1 expression in HNSCC, to correlate with clinicopathological features and outcome. Settings and Design: We retrospectively analysed 59 cases of HNSCC at our Tertiary Hospital between January 2017 and November 2018 and followed up until death/Nov 2022 for Overall survival. Methods and Material: IHC analysis of PD-L1 using Combined Positive Score (CPS) with antibody clone 22C3 in 59 cases of HNSCC was performed. PD-L1 expression was correlated with clinicopathological features and outcomes. Statistical Analysis Used: Pearson Chi-square test was used to analyse the correlation between PD-L1 expression and clinicopathological parameters using SPSS20.0. Survival curves were calculated by Kaplan-Meier method, and differences were analysed by log-rank test. Results: A total of 25 cases (42.4%) had positive PDL expression (CPS ≥1). 16/25 cases (27.1%) belonged to CPS (≥1, <10). An almost-perfect interobserver agreement was noted by two pathologists for PD-L1 IHC expression. No statistically significant correlation was noted between PD-L1 score and clinicopathologic features. Conclusions: Detection of PD-L1 status gives further insight into frequency of PD-L1 expression in Indian HNSCC patients to possibly improve clinical treatment strategies, ensuring that our patients get the maximum therapeutic benefit of immunotherapy.

Pinus thunbergii Parl. Somatic Plants' Resistance to Bursaphelenchus xylophilus Depends on Pathogen-Induced Differential Transcriptomic Responses.

Pinus thunbergii Parl. is an economically and medicinally important plant, as well as a world-renowned horticultural species of the Pinus genus. Pine wilt disease is a dangerous condition that affects P. thunbergii. However, understanding of the genetics underlying resistance to this disease is poor. Our findings reveal that P. thunbergii's resistance mechanism is based on differential transcriptome responses generated by the early presence of the pathogen Bursaphelenchus xylophilus, also known as the pine wood nematode. A transcriptome analysis (RNA-seq) was performed to examine gene expression in shoot tissues from resistant and susceptible P. thunbergii trees. RNA samples were collected from the shoots of inoculated pines throughout the infection phases by the virulent Bursaphelenchus xylophilus AMA3 strain. The photosynthesis and plant-pathogen interaction pathways were significantly enriched in the first and third days after infection. Flavonoid biosynthesis was induced in response to late infestation (7 and 14 days post-infestation). Calmodulin, RBOH, HLC protein, RPS, PR1, and genes implicated in phytohormone crosstalk (e.g., SGT1, MYC2, PP2C, and ERF1) showed significant alterations between resistant and susceptible trees. Furthermore, salicylic acid was found to aid pine wood nematodes tolerate adverse conditions and boost reproduction, which may be significant for pine wood nematode colonization within pines. These findings provide new insights into how host defenses overcame pine wood nematode infection in the early stage, which could potentially contribute to the development of novel strategies for the control of pine wilt disease.

Surveillance of Enterobacteriaceae from Diabetic Foot Infections in a Tunisian Hospital: Detection of E. coli-ST131-blaCTX-M-15 and K. pneumoniae-ST1-blaNDM-1 Strains.

The study determined the prevalence, antimicrobial resistant (AMR) determinants, and genetic characteristics of Escherichia coli and Klebsiella pneumoniae isolates from patients with diabetic foot infection (DFI) in a Tunisian hospital. A total of 26 Escherichia spp. and Klebsiella spp. isolates were recovered and identified by MALDI-TOF-MS. Antimicrobial susceptibility testing, the detection of AMR determinants and Shiga-like toxin genes, phylogenetic grouping, and molecular typing were performed. Twelve E. coli, 10 K. pneumoniae, 3 K. oxytoca, and 1 E. hermanii were isolated. A multidrug-resistant phenotype was detected in 65.4% of the isolates. About 30.8% of isolates were extended-spectrum ß-lactamase (ESBL) producers and mainly carried blaCTX-M-15 and blaCTX-M-14 genes. One blaNDM-1-producing K. pneumoniae-ST1 strain was identified. Class 1 integrons were detected in 11 isolates and 5 gene cassette arrangements were noted: dfrA1+aadA1 (n = 1), dfrA12+aadA2 (n = 3), and dfrA17+aadA5 (n = 1). Other non-ß-lactam resistance genes detected were as follows (number of isolates): aac(3')-II (3), aac(6')-Ib-cr(8), qnrB (2), qnrS (4), cmlA (2), floR (4), sul1 (11), sul2 (11), and sul3 (2). The phylogroup B1 was the most frequent (41.7%) among E. coli, and two ESBL-producing isolates corresponded to the ST131-B2 lineage. The ESBL- and carbapenemase-producing Enterobacteriaceae in DFIs are described for the first time in Tunisia.

Extraction of tannin, colour and aroma compounds in pinot noir wines as affected by clone selection and whole bunch addition.

Our previous study revealed stem inclusion fermentation reduced anthocyanin, and increased tannin and aroma compounds responsible for green notes. This study further investigated the effect of clone selection and whole bunch fermentation on Pinot noir wine composition, with focus on tannin composition. Three treatments were conducted using two clones (AM10/5 and UCD5) in 2021 and 2022: 100% destemmed (DS), 30% whole bunch (WB30), and 60% whole bunch (WB60). WB60 increased stem and skin derived tannins but reduced seed derived tannin proportion in wines. Clone selection had an impact on tannin composition and an even greater impact on tannin concentration, colour, and aroma compounds. AM10/5 produced wines with higher tannin, polymeric pigments and darker colour. AM10/5 wines also had higher concentration of phenylethyl alcohol, but lower concentrations of 3-isobutyl-2-methoxypyrazine and ethyl esters, indicating more floral but less fruity and green notes.