Lentilactobacillus parabuchneri is of particular concern in fermented food bioprocessing due to causing unwanted gas formation, cracks, and off-flavor in fermented dairy foods. This species is also a known culprit of histamine poisonings because of decarboxylating histidine to histamine in ripening cheese. Twenty-eight genomes in NCBI GenBank were evaluated via comparative analysis to determine genomic diversity within this species and identify potential avenues for reducing health associated risks and economic losses in the food industry caused by these organisms.L. parabuchneri strains analyzed. After assigning CDS to their corresponding function, ~ 79% of all strains were predicted to carry putative intact prophages, and ~ 43% of the strains harbored at least one plasmid; however, all the strains were predicted to encode genomic island, insertion sequence, and CRISPR-Cas system. A type I-E CRISPR-Cas subgroup was identified in all the strains, with the exception of DSM15352, which carried a type II-A CRISPR-Cas system. Twenty strains were predicted to encode histidine decarboxylase gene cluster that belongs to not only dairy but also saliva, KEM complex, and unknown source. No bacteriocin-encoding gene(s) or antibiotic resistome was found in any of the L. parabuchneri strains screened.Core genome-based phylogenetic analysis revealed four distinct major clades. Eight dairy isolates, two strains from an unknown source, and a saliva isolate formed the first clade. Three out of five strains clustered on clade 2 belonged to dairy, and the remaining two strains were isolated from the makgeolli and Korean effective microorganisms (KEM) complex. The third and fourth clade members were isolated from Tete de Moine and dairy-associated niches, respectively. Whole genome analysis on twenty-eight genomes showed ~ 40% of all CDS were conserved across entire strains proposing a considerable diversity among L. parabuchneri by comparing twenty-eight genomes available to date. For example, the hdc gene cluster was generally reported in cheese isolates; however, our findings in the current work indicated that it could also be encoded in those strains isolated from saliva, KEM complex, and unknown source. We think prophages are critical mobile elements of L. parabuchneri genomes that could pave the way for developing novel tools to reduce the occurrence of this unwanted species in the food industry.The findings of the present work provide in-depth knowledge of the genomics of The online version contains supplementary material available at 10.1186/s12864-022-09053-y. Lentilactobacillus (L) species parabuchneri was described by [L. parabuchneri is Gram (+), non-motile, catalase and Voges-Proskauer negative, facultative anaerobe, rod-shaped (~ 0.9 × 3 μm), and appears as single, pairs or short rod chains under the microscope . L. parabuchneri grows at 15 °C but not at 45 °C [2 from L-glucose, presumably, lactic acid is biosynthesized from L-arabinose, D-raffinose, ribose, sucrose, D-fructose, D-glucose, gluconate, galactose, melibiose, melezitose, and maltose. However, some L. parabuchneri strains can utilize lactose (the primary carbon source available in milk) and convert it into acid [L. parabuchneri occurs through the pentose phosphate pathway (PPP) because of the organism’s obligatory heterofermentative lifestyle [L. parabuchneri produces ornithine and 1,2-propanediol from L-arginine and lactic acid, respectively. L. parabuchneri is also known to decarboxylase histidine into histamine, a strain-dependent trait of this species [L. parabuchneri can convert lactic acid to carbon dioxide, acetate, or 1,2-propanediol anaerobically, a valuable metabolic trait combating pH decrease in the cytoplasm due to large amounts of lactate accumulation [L. parabuchneri to degrade lactate to acetate makes this species instrumental in certain fermentation processes, particularly silage stabilization [2, and ATP from L-arginine is another unique metabolic attribute of L. parabuchneri, protecting against acid stress conditions by maintaining a favorable pH in the cytoplasm. Again, the formation of ammonia and CO2 leads to pH elevation and undesirable gas occurrence in cheese resulting in unwanted metabolites that ultimately cause food spoilage [L. parabuchneri ferments C6 sugars through the pentose phosphate pathway, which releases a mole of CO2 upon utilizing one mole of hexose sugars available in ripening cheese that perhaps contributes to unwanted gassiness [The ribed by  and assoribed by –5. L. paat 45 °C ; howeverifestyle . L. paraaped ~ 0. × 3 μm, lization –12. Evenlization . For exalization . The prospoilage . Neverthassiness –16.Lactic acid bacteria (LAB) are heavily accustomed to specific environmental microniches and carry smaller genomes as opposed to other bacteria as a result of genome reduction, which leads to the preservation of a small number of critical genes necessary for niche-specific survivability , 17. DesBacteriophages replicate using the bacterial host’s cellular machinery. While certain bacteriophages are lytic , others could undergo a lysogenic life cycle. In lysogeny, the genome of the bacteriophage integrates into the host chromosome in the form of a prophage, which is later replicated as the host duplicates. The prophage can be induced in response to certain environmental conditions resulting in the initiation of a lytic replication . Later, L. parabuchneri is diverse in isolation source and a problematic species causing food spoilage and histamine poisonings, there are few studies overall for this species. The shortage of studies on L. parabuchneri has resulted in rather limited knowledge regarding genomic diversity at the strain level. To fully leverage the genomic potential of L. parabuchneri and understand the framework of strategies to control this species unwanted growth in food systems, we first should evaluate genetic species diversity. Therefore, the present work aimed to fill this gap in the literature by comparing all the genomes available to date in NCBI GenBank and proposing genome-guided solutions for strains of interest.Even though L. parabuchneri genomes from NCBI GenBank (https://www.ncbi.nlm.nih.gov/genome/browse/#!/prokaryotes/41166/) were used in this study. Two strains  showing incomplete assemblies, as revealed from BUSCO analysis   was utilized for performing a comparative whole genome analysis of twenty-seven KEM Fig. . In geneKEM Fig. -S2.Fig. L. parabuchneri strains [L. parabuchneri species.Genomic conservation analysis across twenty-eight isolates regarding pan- and core genomes revealed that 40.2% of the entire genes were conserved within 95% BLASTP identity Fig.  A. Of th strains . For furThe UpSet plot represented the shared orthogroup numbers of each strain and shared orthogroup numbers between the strains with a bar chart Fig. . A totalL. parabuchneri strains. Among the 259 mobilomes in pangenome, 179 CDS belonged to transposons or closely associated derivatives.The pan- and core genomes were annotated using PSI-BLAST followed by COG database  and assiL. parabuchneri genomes were evaluated for the presence of mobilomes such as IS elements, prophages, plasmids, and CRISPR-Cas system. At least two insertion sequences (IS) were identified in all genomes analyzed  . FAM21731 and NSMJ were predicted to encode two and three plasmids, respectively , histidine decarboxylase maturation protein (hdcB), histidine-tRNA ligase (hisS), and histidine/histamine antiporter (hdcC). It was found that twenty strains were predicted to carry a complete histidine decarboxylase gene cluster , ornitihine transcarbamoylase (arcB), carbamate kinase (arcC), and arginine-ornithine antiporters (arcD)) [L. parabuchneri strains )  and shows Table S.ladH) and lactaldehyde reductase (ldr) [L. parabuchneri genomes analyzed in the present study were predicted to carry both ladH and ldr.Some LAB species have the capability to convert lactate into 1,2-propanediol. The genes required for such a conversion from lactate are lactaldehyde dehydrogenase (se (ldr) . All L. L. parabuchneri strains representing milk, cheese, KEM complex, makgeolli, and saliva microbiomes. The genome sizes range between 2.51 Mb to 2.80 Mb, which falls in the range of lactic acid bacteria . The GC content of L. parabuchneri is consistent with low GC LAB. Whole genome analysis identified either single or multiple plasmid sequences in twelve L. parabuchneri strains that are inconsistent with no plasmids found in the reference strain of KEM. It was hypothesized that lactic acid bacteria are heavily adapted to their specific ecological niche, which is further supported by the existence of plasmids that could rapidly be gained and transferred at times of swift environmental changes [In the present work, we performed a genome-wide evaluation on the twenty-eight  changes .We also showed similarities or discrepancies in the strain phylogenetic locations across core and whole genome-based sequence alignments. For example, four strains isolated from Tete de Moine cheese  were closely related in phosphoglucomutase, core genome, and whole genome-based phylogenetic trees  [L. parabuchneri strains were repeatedly isolated from cheese, showing an increased histamine content [L. parabuchneri strains were primarily isolated from cheese [L. parabuchneri acquired the hdc gene cluster through a horizontal gene transfer [L. parabuchneri strains that were not only isolated from cheese but also originated from saliva, KEM, and unknown sources. This implies that hdc-positive L. parabuchneri strains are more prevalent than initially thought and exceed the environments beyond the cheese microbiome. It was demonstrated that the hdc gene cluster could be utilized for energy production and pH regulation [L. parabuchneri strains in the cheese microenvironment. Although this might be seen as a beneficial trait during the acidification and ripening of cheese making, the end product, histamine, causes intolerance reactions in consumers [Histamine formation is of particular health concern due to several symptoms caused by ingestion of histamine  catalyzend hdcC) , 47. L. m cheese . It was transfer . Here wegulation . Moreovegulation . We woulonsumers .L. parabuchneri genomes analyzed in the present study carried putative arcA, arcB, arcC, and arcD, which catalyze the ADI pathway. In carbohydrate-deficient but amino acid-rich conditions such as ripening cheese, the ADI pathway is an important avenue to produce ATP for bacterial proliferation [L. parabuchneri ADI metabolism is a helpful trait during cheese ripening where pH is reduced due to acidification, and bacteria that are not capable of adapting to this low pH would be outgrown or neutralized [L. parabuchneri can increase its biomass in high salt in moisture and acidic conditions of cheese ripening. The ammonia produced in the ADI pathway also enhances the proteolysis in the cheese ripening [2 released contributes to the increase in the quantity and size of holes in semi-hard cheese [L. parabuchneri in long-ripened cheese varieties could be associated with the unwanted gas formation and splits that can cause downgrading of cheese and cutting losses resulting in severe economic losses to cheese manufacturers [LAB utilizes the arginine deiminase pathway for converting arginine into ornithine by citrulline while producing ATP and ammonia. Production of ammonia elevates the pH so that bacteria are protected against conditions of an acid-stress . We showferation . It was tralized . Thereforipening , 14. Mord cheese . Howeveracturers , 51–54.L. parabuchneri genomes present metabolic pathways for the conversion of lactate to 1,2-propanediol perhaps to cope with acid stress in the microenvironment and to produce ATP. All L. parabuchneri genomes studied in the present work were predicted to carry genes required for lactate to 1,2-propanediol conversion. First, lactate is transformed to L-lactaldehyde by lactaldehyde dehydrogenase enzyme then L-lactaldehyde is reduced to 1,2-propanediol through lactaldehyde reductase. This degradation of two moles of lactate generates one mole of ATP [L. parabuchneri developed to protect against surrounding acidic conditions during cheese make and ripening conditions. Conversion of lactate by L. parabuchneri was reported to be a minor factor in the CO2 production [e of ATP . It was e of ATP . This cooduction .L. parabuchneri contamination and outgrowth are significant industrial concerns and health risks [L. parabuchneri should be explored in vivo to reduce the incidence of unwanted product quality and health-associated symptoms caused by this species. In particular, the deliberate induction strategies against putative prophages for controlling contamination and proliferation of this organism should require further attention. Bioprotection agents such as bacteriocins or bioprotective adjunct cultures could also be explored to limit this species’ population and cheese defects caused by L. parabuchneri.Cheese defects of burning taste, crack formation, and histamine outbreaks due to th risks , 55. TheL. parabuchneri, a lactobacilli species primarily associated with unwanted gas formation, off-flavor, and elevated histamine formation in ripening cheese, (ii) identify novel tools and strategies based on genomic analysis to combat with this organism causing both economic loss and health concerns worldwide. The phylogenetic analysis based on the core genome sequence alignment revealed four distinct clades. Saliva, KEM, and makgeolli form the same clade, with dairy-originated strains showing evidence of diversity. No pronounced differences seen in carbohydrate-active enzymes according to the origins of each strain imply a free-living lifestyle. The frequency of type I-E CRISPR-Cas system in all strains but one is consistent with the high occurrence of type I-E across all types. The abundance of IS elements, genomic islands, and intact prophages found in the majority of strains revealed the plasticity of the genome. In particular, prophages could be further studied in vivo to determine their activity which could evolve into prophage mediated lysis strategy, thus potentially helping to reduce the contamination and growth of this unwanted microbe in fermented dairy foods.The goal of the present work was to (i) improve core knowledge on L. parabuchneri genomes were downloaded from NCBI GenBank, followed by a BUSCO analysis to inspect the completeness of the genome assemblies using the lactobacillales_odb10 lineage dataset [Whole genome sequences of thirty  dataset , 57. The dataset –60 and P dataset . The twohttps://www.ncbi.nlm.nih.gov/COG/. Core and Pan COGs were visualized using R [RAST annotation outputs were analyzed to predict putative cas genes . The cor using R  and ggpl using R .L. parabuchneri strains were annotated to clusters of orthologous groups (COG) categories using eggNOG-mapper [A global phylogenetic analysis was conducted based on the phosphoglucomutase gene , core geG-mapper .https://bcb.unl.edu/dbCAN2/index.php) [L. parabuchneri strains were classified based on the number of CAZYmes they harbored. KEGG Automatic Annotation Server (KAAS) was utilized for functional annotation of L. parabuchneri strains with the assignment method of the bi-directional best hit (BBH) method [CAZy database (v10) in dbCAN server (dex.php)  and HMMEdex.php)  were utidex.php) . Then, L) method . Results) method , 72.L. parabuchneri was performed with PLSDB (version 2021_06_23_v2) using default settings [Identification, alignment, and visualization of CRISPR elements, such as repeats and spacers, were conducted with the CRISPRviz tool . Classifsettings , 77. Phasettings . Insertisettings . To idensettings . Screenisettings .Additional file1.


Background: Immune targeted therapy has become an attractive therapeutic approach for patients with dilated cardiomyopathy (DCM) recently. Genetic predisposition and gender play a critical role in immune-related responses of DCM. This study aimed to perform a bioinformatics analysis of molecular differences between male and female samples and identify immune-related ceRNA network in DCM.Methods: The gene expression microarray and clinical features dataset of GSE19303 was downloaded from the GEO. The raw data were preprocessed, followed by identification of differentially expressed genes (DEGs) between male and female DCM samples. Crucial functions and pathway enrichment analysis of DEGs were investigated through GO analysis and KEGG pathway analysis, respectively. A lncRNA–miRNA–mRNA network was constructed and a central module was extracted from the ceRNA network.Results: Compared with the female group, the male group benefits more from IA/IgG immunotherapy. Male patients of DCM had a significant positive correlation with the abundance of inflammatory cells . Sex difference DEGs had a widespread impact on the signaling transduction, transcriptional regulation, and metabolism in DCM. Subsequently, we constructed an immune-related ceRNA network based on sex differences in DCM, including five lncRNAs, six miRNAs, and 29 mRNAs. Furthermore, we extracted a central module from the ceRNA network, including two lncRNAs (XIST and LINC00632), three miRNAs , and six mRNAs . Among these DEGs, CBL, CXCL12, and IL6ST expression was considered to be associated with inflammatory cell infiltration in DCM.Conclusions: The identified ceRNA network and their enriched pathways may provide genetic insights into the phenotypic diversity of female and male patients with DCM and may provide a basis for development of sex-related individualization of immunotherapy. Globally, dilated cardiomyopathy (DCM) is one of the most common forms of cardiomyopathy, and it represents a leading cause of cardiac transplantation in children and adults . ContempMale sex is a key risk factor for progression to heart failure following a large number of cardiovascular conditions, including DCM . StudiesThe most common causes of DCM are infections and autoimmunity. Enteroviruses, adenoviruses, and herpesviruses are commonly found in patients with DCM . Virus iWith the rapid development of sequencing technologies, an increasing number of competing endogenous RNAs (ceRNAs), such as microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA), have been found to be involved in DCM progression . The genHistorically, pharmacological therapy (ACE inhibitors and β-blockers) and cardiac resynchronization therapy (CRT) are standard treatments for heart failure in DCM, but they all have limitations. Recently, immunotherapies have become an attractive therapeutic strategy in DCM. Moreover, sex differences and gene expression influence the efficacy of immunotherapies . Therefohttp://ncbi.nlm.nih.gov/geo/). The dataset contained a total of 81 endomyocardial biopsy samples, 40 baseline biopsies from patients with DCM, 33 of 40 patients had received immunotherapy (immunoadsorption with subsequent immunoglobulin substitution (IA/IgG) treatment), 33 follow-up biopsies of DCM patients collected 6 months after the treatment, and eight biopsies from individuals without DCM . After log2 transformation and quantile normalization of the expression data, we annotated the converted probe ID for each gene to a gene symbol utilizing hgu133plus2. db, org. Hs.eg.db, and the annotate package in Bioconductor (http://www.bioconductor.org/). If a gene’s symbol corresponded with the multiple probe IDs, the expression level of that gene was represented by the mean of the probes.We retrieved the expression matrix from the GEO database and preprocessed it by using the robust multiarray analysis (RMA) method  is a computational tool for immuno-tumor biology research . Here, bp-values were calculated using the Benjamini–Hochberg method. The significant DEGs were selected with a threshold of p-value < 0.05 and fold change>1.5. We obtained the volcano plot utilizing the pheatmap package in R.The Linear Models for Microarray Analysis (Limma) package in R software was applied to identify the differentially expressed genes (DEGs) in the male DCM samples compared with the female DCM ones, based on Student’s t-test. Adjusted p-value < 0.05 and fold change>1.5 were selected to be the cutoff criteria of the significant DEGs. We obtained another volcano and heatmap plot. In order to plot the differentially expressed lncRNAs (DE-lncRNAs), we used the pheatmap package in R to construct a heatmap.Moreover, we excluded the genes in the Y chromosomes and then compared the male and female DCM samples again using the Limma package. http://www.geneontology.org/) database was used to determine the biological processes (BPs) that the DEGs may be involved in. In addition, according to the modified Fisher’s exact test, the Kyoto Encyclopedia of Genes and Genomes  database was used for pathway enrichment analysis of the identified DEGs. The selection criteria for the significant GO terms and pathways were p < 0.05, and the number of enriched genes were  > 2. The plots were performed by the ggplot2 package in R.In order to explore the potential functions and pathways that may be altered by the DEGs, we applied the clusterProfiler package in R to perform the functional and pathway enrichment analyses of the identified DEGs. The Gene Ontology   or FDR <0.25 (as needed) were considered statistically significant.GSEA software (version 3.0) and c2. all.v7.4. symbols.gmt subcollection were obtained from the GSEA website (dex.jsp) . We divihttps://diana.e-ce.uth.gr/lncbasev3) (https://starbase.sysu.edu.cn/index.php) (http://www.immport.org/) (http://jvenn.toulouse.inra.fr/app/example.html)   and ENCOdex.php) , respectrt.org/) . The Venle.html) .https://cytoscape.org) was used to develop the ceRNA network. For gene set functional enrichment analysis, we used the GO and KEGG annotations of genes in the R package org. Hs.eg.db (version 3.1.0) as the background to map the genes to the background set using the R package clusterProfiler (version 3.14.3) to perform enrichment analysis to obtain gene set enrichment results. p-value < 0.05 was considered statistically significant.Coexpression patterns in 29 immune-related DEGs were analyzed using Pearson’s correlation coefficient, and the results were visualized using the heatmap packages in R software. Cytoscape software   to carry out the heart (left ventricle)-specific PPI, TF-miRNA interactions, and protein–chemical interaction analysis on the ceRNA network coexpression module (A protein–protein interaction (PPI) network, comprising 50 ceRNA network coexpression proteins, was constructed by GeneMANIA (ia.org/) . These nn module . In thesp-value less than 0.05 is considered statistically significant. The plots were performed by R, Cytoscape software , or SangerBox tools .All the statistical analyses were performed using R (version 3.6.4) or SPSS (version 19.0), and a p = 0.032) and Epstein–Barr virus (EBV) infection . However, gender was not significantly correlated with other clinical features such as LVEF, LVIDD, BMI, inflammation index, total virus infection, PVB19 infection, HHV6 infection, HSV1 infection, and IgG treatment.The RNA array data for a total of 40 DCM patients were acquired from the GES19303 dataset. The detailed baseline clinical features are listed in p = 0.002) and LVIDD . For male patients, immunotherapy was more significantly correlated with LVEF  and LVIDD   . However417E-04) . These r+ cells, and NK cells  were negative regulation of cellular process, negative regulation of programmed cell death, cardiovascular system development, ncRNA metabolic process, and RNA modification . The sigTo confirm the abovementioned results, we performed GSEA on the sex-related DEGs to identify Gene Ontology and signaling pathways that were differentially activated in DCM. The top 20 significant positive and negative enrichment pathways are shown in Among the 68 DE-lncRNAs and five DE-miRNAs, five DE-lncRNAs ) were predicted to interact with four DE-miRNAs  by the DIANA database. The four DE-miRNAs were predicted to interact with a total of 7,747 genes by the ENCORI database . To furtGO and KEGG enrichment analyses were conducted to investigate the functions of the ceRNA network. GO_BP analysis showed that ceRNA network was significantly enriched in programmed cell death, immune system process, and heart development . GO_CC aThe protein–protein interaction (PPI) network was assembled based on the ceRNA network in the DCM cohort by GeneMANIA. The analysis showed that the 50 most significantly coexpressed genes play roles in peptidyl-tyrosine phosphorylation, leukocyte chemotaxis, and the vascular process in the circulatory system . Next, tin vivo circulating and tissue levels of AA were associated with lower risk of major cardiovascular events   Figure . Excludir events . In addir events . Based op = 0.03), neutrophils (p = 0.008), and fibroblasts (p = 0.02). Moreover, CBL expression had a significant negative correlation with CD8+Tem (p = 0.0096) and conventional DC  (p = 0.03), activated DCs , MSCs (p = 0.02), lymphatic endothelial cells , and mv endothelial cells (p = 0.0032). Furthermore, CXCL12 expression had a significant negative correlation with B-cells (p = 0.01), Treg (p = 0.03), M2 macrophages (p = 0.04), hematopoietic stem cells , and common lymphoid progenitor  (p = 0.0014) and myocytes (p = 0.03). Moreover, IL6ST expression had a significant negative correlation with iDC (p = 0.02), ly endothelial cells (p = 0.0044), and mv endothelial cells (p = 0.01)  . CXCL12  0.0067) . IL6ST e = 0.01) . HoweverThe etiology of DCM can be broadly categorized into genetic, acquired, or mixed . SusceptViral infections of the heart were considered to be possible triggers or contributors for the development of the disease in a large number of the DCM patients . The mosIn addition to sex differences, genetic predisposition plays a crucial role in the DCM process. The ceRNA network has been proven to be involved in various heart diseases, including DCM . In thisImmune-targeted therapy has become an attractive therapeutic strategy in DCM recently. Damaged cardiac tissue and infections strongly induce the innate immune response, activating Toll-like receptors (TLRs) and the inflammasome, resulting in the release of the proinflammatory cytokines . Immune + Tem cells, and NK cells). Sex difference DEGs had a widespread impact on the signaling transduction, transcriptional regulation, and metabolism in DCM. Subsequently, we constructed an immune-related ceRNA network based on sex differences in DCM, including five lncRNAs, six miRNAs, and 29 mRNAs. This ceRNA network can regulate a variety of immune-related signaling pathways in DCM. Among this ceRNA network, CBL, CXCL12, and IL6ST were considered to be important DEGs associated with immune cell infiltration. Together, our findings suggest that the sex difference ceRNA network plays a crucial role in immune response regulation in DCM, yet the underlying mechanism still needed further validation.In summary, in this comprehensive study, we found sex differences in the outcome of immunotherapy in DCM patients. In addition, male DCM patients had a significant positive correlation with the abundance of inflammatory cells (B cells, memory B cells, CD8


Justicia secunda Vahl. is a traditional medicinal plant in tropical regions, including West Africa. The present study examined the chemical profiles and biological properties of J. secunda extracts obtained with different solvents . Chemical components were characterized by liquid chromatography-mass spectrometry (LC-MS), and over 50 compounds were identified, including flavonoids, phenolic acids, and alkaloids. Antioxidant, enzyme inhibitory, cytotoxic, and antiviral properties were selected as biological properties. Total phenolic and flavonoid contents in methanol /g and 13.07 mg rutin equivalent (RE)/g) and water (infused) (36.34 mg GAE/g and 8.52 mg RE/g) were higher than in other extracts. Consistent with the levels of total bioactive components, the methanol and water extracts exhibited stronger antioxidant abilities. However, the dichloromethane and ethyl acetate extracts were more active on α-amylase and α-glucosidase than other extracts. Aqueous extracts exerted selective anticancer properties toward human pharyngeal cancer cell lines, whereas the methanolic extract decreased the human herpesvirus type-1 (HHV-1) infectious titer by 2.16 log and the viral load by 1.21 log. Overall, J. secunda could be considered a multifunctional bioactive raw material in the preparation of potent applications to manage diseases related to oxidative stress, including cancer, diabetes, and Alzheimer’s. In the past ten years, the world population has been increasing daily, and the way of life of humans has undergone an intense change. Due to this fact, the prevalence of some diseases, including cancer , diabeteJusticia secunda Vahl. (eng. St. John’s bush) is a plant belonging to the Acanthaceae family. In Barbados, it is known as “Bloodroot” and in Venezuela as “Sanguinaria”, which refers to the red color of water observed when the plant is boiled. Traditional medicine of Barbadian locals describes the oral use of decoctions and infusions from the leaves of this plant to treat wound infections  β-glucopyranoside and luteolin 7-O-[β-apiofuranosyl-(1→2)]-β-xylopyranoside were previously identified in J. secunda leaves by Koffi et al. - at m/z 355.0612 (predicted) but with different retention times, which were tentatively identified as lignan derivatives, probably 10--5H-benzo[c]furochromen-5-one isomers. They were present only in the infusion. Koffi et al. [m/z 355 ([M-H]−), which they identified as justiflorinol with molecular formula C19H16O7. Nevertheless, the molecular formula calculated for the precursor ion [M-H]− at m/z 355.0612 (C22H12O5) found in our extract varied and was identified as a different compound. One lignan derivative  was found in the methanolic extract.According to Corrêa and Alcântara , lignansi et al.  also fouJ. aequilabris [− at m/z 431.1923 (predicted) was identified as roseoside. Two different cyclohexanone derivatives were tentatively identified as dihydroroseoside and 9-hydroxy-7-megastigmen-3-one glucoside. Furthermore, decalin glucoside derivatives tentatively identified as ophiogonoside A isomers were present in all extracts except the aqueous one.Moreover, cyclohexanone derivatives, mainly glucosides, were found in all obtained extracts except the aqueous one. According to data obtained for uilabris , a compoJusticia [J. secunda. These compounds can be classified as pyrrolidone alkaloids. Secundarellone B/C and one isomer of secundarellone A were found in dichloromethane extract, and the second one of the isomers of secundarellone A was identified in all obtained extracts.Alkaloids are one of the compound classes found in the species belonging to the genus Justicia ,21. NMR Justicia  revealedAntioxidant has been one of the most used terms in pharmaceutical, nutraceutical, and cosmetic applications over the past decade. Antioxidant compounds could be considered powerful protective agents against the attacks of free radicals, which are the main trigger for developing severe health problems such as cancer, diabetes, or cardiovascular diseases. In this sense, discovering new sources of antioxidants is crucial to treating the mentioned diseases. In the chemical industry, some compounds, including butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate (PG), have been chemically produced, but their use raises public concerns such as hepatotoxicity or cancerogenic influence on the human organism . TherefoJ. secunda extracts using various chemical assays, such as free radical quenching, reducing power, and metal chelation. The results are presented in 2+ to Cu+ and Fe3+ to Fe2+, respectively. 2 was 0.96 for DPPH and TPC; was 0.97 for ABTS and TPC) and reducing power assays  correlated strongly with the total bioactive content at this point  of J. secunda leaves was reported in a previous study by Onoja et al. [2: 0.43). Several researchers who reported a weak correlation between total phenolic and phophomolybdenum assay concurred with our findings [J. secunda. The methanolic and infused extracts of J. secunda exhibited greater antioxidant activity than other extracts, and this finding could be used to design functional applications utilizing J. secunda in future research.Given the preceding information, we examined the antioxidant capacities of is point B. Consisi et al.  examinedr et al. , the hota et al. . Phosphofindings ,35. TranEnzymes play crucial roles in developing effective therapeutic strategies to combat global health issues. In this sense, the majority of drugs have been designed to inhibit specific enzymes. This phenomenon is known as the enzyme inhibition theory . For exaJ. secunda extracts against AChE, BChE, tyrosinase, amylase, and glucosidase. The results are presented in p > 0.05). Regarding the BChE inhibitory abilities, only two extracts (methanolic and ethyl acetate) were active on the enzyme, and the methanolic extract was more active than the ethyl acetate one. In both AChE and BChE assays, macerated and infused aqueous extracts were not active. The observed anti-cholinesterase ability of the methanolic extract might be explained by the presence of rutin, which was only detected in the methanolic extract. Similar to our approach, the anti-cholinesterase ability of rutin and its mechanism have been reported in several studies [Justicia genus. In contrast to our results, Rawa et al. [J. gendarussa leaves was not active on both AChE and BChE. Previous research by Gupta and Gupta [J. gendarussa showed weak AChE inhibitory properties.In light of the above information, we examined the enzyme-inhibitory properties of  studies ,43. In a studies ,45, caffa et al.  reportednd Gupta  found thJ. carnea was reported to be moderate [J. carnea leaves and found that the ethanolic extract was the most active. In contrast to our results, the methanolic extract of J. adhatoda leaves showed low amylase inhibitory activity with an inhibitory value of less than 50% [Similar to the cholinesterase inhibitory properties, the aqueous extracts exhibited lower inhibitory effects on amylase and glucosidase compared to other extracts. The ethyl acetate extract was the most active on the anti-diabetic enzymes, but its activity was not statistically different from dichloromethane extract. The observed inhibitory effects on amylase and glucosidase did not correlate with the total bioactive compounds. As can be seen from moderate , which imoderate  studied than 50% .2 < 0.7). Based on the identified compounds from n-butanol extract of J. vahlii leaves was reported as 193.21 mg KAE/g, which was higher than our results. The anti-tyrosinase inhibitory effect of the ethanolic extract (50%) of J. adhatoda leaves was found to be less than 50% in a previous study by Ito et al. [J. secunda. The results could shed light on the potential applications of J. secunda in nutraceutical, cosmetic, and pharmaceutical practices.Melanogenesis is an essential process that shields humans from the sun’s ultraviolet rays. Tyrosinase is a crucial enzyme in melanogenesis reactions, and inhibiting it could alleviate hyperpigmentation problems . As can o et al. . The curJ. secunda aqueous extract and infusion showed the lowest cytotoxicity toward normal VERO cells, and the exact CC50 values could not be evaluated since they were above the tested concentration range (50 between 61.18 and 79.57 μg/mL (p > 0.5). J. secunda dichloromethane extract showed substantial anticancer activity (p < 0.01) toward FaDu and Detroit with SI of 2.83 and 1.75, respectively. Notably, also methanolic extract exerted anticancer selectivity, but the observed CC50 values point to low toxicity. The highest anticancer potential and selectivity toward both pharyngeal carcinomas were found for J. secunda aqueous extract and infusion. The influence of infusion in the 500, 250, 125, 62 and 31 μg/mL concentrations on the morphology of FaDu cells monolayer is presented in on range . Based o57 μg/mL . DespiteJ. secunda leaves ethanolic extract caused increased lipid profile values, creatinine and blood urea levels in Wistar rats, which may indicate potential negative cardiac and renal influence. Moreover, the calculated LD50  of the extract was determined to be 3800 mg/kg body weight, which also raises potential safety concerns [J. secunda infusions or decoctions, which may exert different safety profiles. Nevertheless, our in vitro experiments also indicate lower toxicity of aqueous extract and infusion to normal cells than the extracts obtained using organic solvents, especially dichloromethane and ethyl acetate. It should be emphasized that this report is the first one depicting the cytotoxicity and anticancer potential of extracts obtained from the aerial parts of J. secunda.Onochie et al.  reportedconcerns . HoweverJ. secunda methanolic extract (500 μg/mL) with HHV-1 infected VERO cells decreased the intensity of virus-induced cytopathic effect (CPE). However, there were still noticeable signs of CPE present. The rest of the tested extracts did not influence the formation of CPE. The formation of CPE was observed every 2 h during a 72 h incubation period using Olympus Provi CM20. Live-cell imaging allowed for real-time monitoring of morphological changes induced by the HHV-1 infection in the VERO cells’ monolayer. A comparison between the development of CPE in virus-infected cells treated with J. secunda methanolic extract (500 μg/mL) or J. secunda ethyl acetate extract (16 μg/mL), and the CPE in the virus control (VC) is presented in J. secunda ethyl acetate extract, only early signs of CPE, including cell rounding and several areas of focal degeneration, were visible, and J. secunda methanolic extract protected cells from virus-induced damage. After the 48 h incubation period, the VC showed profound CPE, with ballooning degeneration, vacuolization, and polykaryon (syncytia) formation. Moreover, the HHV-1-infected cells treated with J. secunda ethyl acetate extract showed significant CPE, and occasional cells treated with J. secunda methanolic extract showed early signs of CPE . Continued incubation , a reference antiviral drug, inhibited the development of the CPE.Incubation of J. secunda methanolic extract (500 μg/mL) managed to decrease the HHV-1 infectious titer by 2.16 ± 0.25 log  inhibited the replication of HHV-1, decreasing the viral load by 1.21 log compared to the virus control. Virus-infected cells treated with J. secunda dichloromethane extract showed a titer of HHV-1 comparable to the virus control. In contrast, ethyl acetate, aqueous, and infusion extracts reduced the viral load by 0.46, 0.6, and 0.73 log, respectively. The viral DNA was not detected in samples obtained from HHV-1-infected cells treated (60 μg/mL) with acyclovir. The melt curve analysis showed a single melting peak at 83.5–84 °C , and lignans and their glycosides  were found to be responsible for the observed effect [Justicia spp. also demonstrated antiviral activity against HIV-1 [J. procumbens, justatropmers A–H, showed potent anti-HIV-1 activity [J. gendarussa on HIV-infected MOLT-4 cells using syncytia formation assay and HIV p24 antigen assay [J. adhatoda, was shown in silico to inhibit the main protease (Mpro) of the SARS-CoV-2 [J. secunda extracts, which may be related to the observed inhibition of HHV-1-induced CPE formation and reduction of infectious titer and viral load by the methanolic extract, since lignans were also reported to inhibit herpesviruses [J. secunda extracts might have been insufficient for higher inhibition of HHV-1 replication. That is why it could be beneficial to perform a more detailed analysis of J. secunda, including the isolation of bioactive fractions and pure compounds responsible for the observed bioactivity.Subsequent virus titration showed that 0.25 log . Other technique A. The HHechnique C. It was.5–84 °C B, confir treated  μg/mL wist HIV-1 ,62,63. Aen assay . InteresRS-CoV-2 . We havesviruses . HoweverJ. secunda obtained with different solvents. Both chemical profiles and biological abilities were influenced by the extraction solvent. In general, the best antioxidant activity was observed for the methanolic and aqueous extracts. However, different results were obtained considering each enzyme inhibitory assay. LC-MS analysis revealed the presence of biologically active compounds, including apigenin glucosides (infusion and methanolic extract), rutin (methanolic extract), luteolin and its glucosides , and alkaloids, namely secundarellones B/C in the dichloromethane extracts and isomers of secundarellone A in all tested extracts. Lignan derivatives were found in the infusion and methanolic extract. The highest toxicity toward all cell types was observed for the ethyl acetate extract. Whereas the highest anticancer potential and selectivity toward both pharyngeal carcinomas, FaDu and Detroit 562, were found for aqueous extract and infusion. Our results show that J. secunda has significant biological properties, making it a promising candidate as a coadjutant agent for developing therapeutics, dietary supplements, and cosmetics. However, further studies are strongly suggested to isolate active compounds responsible for biological activitiesIn the current study, we focused on the chemical profile and biological activities of extracts of


H. pylori infection and gastric cancer (GC) has been widely studied, and H. pylori is considered as the main factor. Utilizing bioinformatics analysis, this study examined gene signatures related to progressing H. pylori-associated GC.  The relationship between H. pylori-associated GC and normal tissues. The TCGA-STAD database was chosen to verify the expression of key genes in GC and normal tissues.  The dataset GSE13195 was chosen to search for abnormally expressed genes in T3, T2, and T4), while the orangered4, salmon2, pink, and navajowhite2 modules were correlated with lymph node metastasis . Based on the results of DEGs and hub genes, a total of 7 key genes  were screened out. These gene mRNA levels were able to distinguish between normal and H. pylori-associated GC tissue using receiver operating characteristic curves. After transcriptional level verification and survival analysis, ADAM28 and C1QC were excluded. An immune infiltration study revealed that key genes were involved in regulating the infiltration levels of cells associated with innate immune response, antigen presentation process, humoral immune response, or Tcell-mediated immune response. In addition, drugs targeting FCER1G and TYROBP have been approved and are under investigation.  In GSE13195, a total of 332 differential expression genes (DEGs) were screened. The results of weighted gene co-expression network analysis showed that the light cyan, plum2, black, and magenta4 modules were associated with stages (H. pylori-associated GC tumorigenesis. Patients with higher levels of C3 expression had a poorer prognosis than those with lower levels. In addition, these key genes may serve as biomarkers and therapeutic targets for H. pylori-associated GC diagnosis, targeted therapy, and immunotherapy in the future. Our study identified five key genes involved in  Incidence of gastric cancer (GC) is the sixth highest of all cancer types, with approximately 1,089,103 cases worldwide. GC is also the third leading cause of cancer death, with approximately 769,000 deaths each year . CurrentHelicobacter pylori (H. pylori) infection, gender, poor dietary habits, and smoking [H. pylori infection, which often leads to gastritis, followed by gastric atrophy and gastrointestinal metaplasia, is most closely related to GC [H. pylori and its eradication therapy can reduce the risk of GC [H. pylori-producedcytotoxicity-associated gene A (CagA) and vacuolar cytotoxicity A (VacA) proteins on gastric mucosal cells can trigger a series of complex biological effects, including release of proinflammatory cytokines, recruitment of immune cells, and stimulation of the survival of gastric epithelial cells [H. pylori inhibits phagocytic activity and T cell function during infection, while catalyzing the formation of urea to ensure its survival in harsh low pH conditions. Furthermore, H. pylori metabolism byproducts damage epithelial cells of the host and contribute to the carcinogenesis of H. pylori infection [H. pylori, it remains unclear whether H. pylori is only involved in the initiation of gastric tumor processes, or whether it affects the mechanisms of tumor progression.Various risk factors affect the incidence of GC, including  smoking . Of thesed to GC . Currentsk of GC . Mechanial cells , 11. H. nfection . DespiteH. pylori will make it more complicated.In recent years, immunotherapy, as a novel treatment method, mainly induces antitumor effects by modulating the immune system and has made revolutionary progress in the treatment of gastric cancer . The tumH. pylori-associated GC tissues and to find possible biomarkers and targeted drugs.In this article, based on the GSE13195 dataset and the TCGA-STAD dataset, we used a series of bioinformatics research methods to explore the dysregulated genes and mechanisms in https://www.ncbi.nlm.nih.gov/geo/) for our study [H. pylori-associated GC and normal tissues from 25 patients. The dataset also included patients' pathological information, tumor stages , and lymph node metastasis . Subsequently, Sangerbox Tools (https://www.sangerbox.com/) were used for normalized raw data as well as multiarray analysis (“lima” package) [P value <0.05 and |logFC| > 1.We selected the dataset GSE13195 from the Gene Expression Omnibus  . Finallyhttps://david.ncifcrf.gov/home.jsp) [Genes differentially expressed were functionally enriched using DAVID v6.8  . These iH. pylori-associated GC tissues [To more accurately determine the functions of differential genes, we performed GSEA using Sangerbox Tools on the basis of normal tissues and  tissues . The refH. pylori-associated GC tissues were constructed using Sangerbox Tools [https://string-db.org/).Gene co-expression networks in ox Tools . First, ox Tools . The proH. pylori-associated GC samples, 157 H. pylori-unassociated GC samples, and 153 other samples) were used for validation  in UALCAN online website  [Key genes were selected from abnormally expressed genes and hub genes. Receiver operating characteristic (ROC) curves were drawn to calculate specificity and sensitivity. In order to verify the accuracy and reliability of the screened key genes, the gene expression data of GC patients in the TCGA-STAD dataset  .H. pylori-associated GC tissues and normal tissues was performed [According to the calculation method of the immune microenvironment score of CIBERSORT, the immune microenvironment analysis of erformed . We calchttps://go.drugbank.com/) was used to find compounds that might act on key genes [The DrugBank online analysis website , including 198 that were upregulated and 134 that were downregulated  in order to make the network scale-free. As shown in Figures R2 reached 0.85 for the first time. After the adjacency matrix was constructed, we transformed it into a topological overlap matrix. Genes were then sorted into different modules, performing a dynamic tree-cutting method. Different genes would be categorized into the same module if their expressions were significantly correlated. Finally, we got 66 modules; the module feature vector and clustering dendrogram are shown in Figures H. pylori-associated GC, the correlation between tumor characteristics and each module was examined. As shown in T3, T2, and T4) with P values below 0.05; modules orangered4, salmon2, pink, and navajowhite2 were associated with lymph node metastasis  were most correlated with P values below 0.05. We calculated MM and defined genes with MM ≥ 0.8 as central genes among the genes in selected modules and obtained a total of 318 hub genes. The protein interaction networks of these 318 hub genes in their respective categories are shown in To find modules highly correlated with the progression of C3   . This suH. pylori-associated GC samples, 157 H. pylori-unassociated GC samples, and 153 other samples) were generated for further validation of the key genes. As shown in C3, SOSTDC1, and TYROBP) were significantly different between tumor tissues and normal tissues, while ADAM28 and C1QC showed no significant differences. In addition, FCER1G, MRPL14, and C3 were abnormally high in H. pylori-associated and H. pylori-unassociated GC tissues compared to normal tissues; SOSTDC1 was abnormally low in H. pylori-associated and H. pylori-unassociated GC tissues. Interestingly, TYROBPHP was abnormally high in H. pylori-associated GC tissues compared to normal tissues but not in H. pylori-unassociated GC tissues. Furthermore, the expression of TYROBP was significantly increased in H. pylori-associated GC tissues relative to H. pylori-unassociated GC tissues. The expression levels of key genes were correlated with the prognosis of GC patients through survival analysis. According to the median expression value, GC patients were divided into a high expression group and low expression group. We found that patients with GC who expressed high levels of C3 had poorer overall survival, while the results of survival analysis of other genes were not statistically significant  of an immunoglobulin fragment involved in various immune responses such as phagocytosis and cytokine release [R of immune cells, which in turn trigger destructive inflammation, immune cell activation, phagocytosis, oxidative burst, and cytokine release [M2 macrophages by the increased secretion of IL-4. In addition, M2 macrophages can acquire their tumor suppressor function in part by suppressing cytotoxic T cells. This may explain the relevance of FCER1G to macrophage and T cell function [M2 macrophages and negatively correlated with CD8 T cells.FCER1G is located on chromosome 1q23.3 and encodes the gamma subunit of the crystalline (Fc) region (Fc  release , 27. Cel release , 29. FCE release . In renafunction . These fC-terminal region of MRPL14, which consists of a five-stranded beta barrel and two small alpha helices [MRPL14 is a highly conserved protein. One protein-binding site and two RNA-binding sites are located in the  helices . MRPL14  helices . The con helices . HoweverN-terminus that contains a C-terminal cysteine knot domain [H. pylori-associated GC tissues, which needs to be further explored in the follow-up studies.SOSTDC1 is a secreted protein with a glycosylated t domain . SOSTDC1t domain , 36. Rest domain . In addit domain . This seM2 to enhance TGF-β secretion in vitro [TYROBP, also known as DAP12, can noncovalently bind to activating receptors on the surface of various immune cells and mediate signal transduction and cell activation , 40. Thein vitro . Our resT-cell immunosuppression and chronic inflammation, thereby promoting tumor-promoting effects, ultimately promoting tumor immune escape, growth, and distant metastasis [C3 and downstream signaling molecules are involved in multiple biological processes of tumor cells, including tumor cell anchoring, proliferation, tumor-associated angiogenesis, matrix remodeling, migration, and invasion [M2 macrophages, DCs, Tregs, and T cell exhaustion were significantly associated with C3 expression. An immunotherapeutic approach based on C3 could provide a potential biological target for GC [Complement is an important part of the innate immune system. Previously, it was thought to be a network of proteins that released inflammatory mediators in response to microbial invasion . A growitastasis , 45. C3 invasion –48. In Gt for GC .H. pylori-associated GC, we were unable to perform multifaceted validation due to the small sample size of GSE13195 and the lack of studies of the same type. In addition, we did not perform experimental tests on key genes. It is critical to conduct larger sample studies as well as multicenter clinical trials to gain a deeper understanding of how genes are involved in H. pylori-associated gastric cancer.Although we identified and confirmed 5 key genes that were highly correlated with the progression of C3, associated with the occurrence of GC in H. pylori infection. Among them, H. pylori-associated GC patients with higher C3 expression had worse prognosis than those with lower expression. In addition, in the future, H. pylori-associated GC may be diagnosed and treated precisely by biomarkers and therapeutic targets related to these key genes.In conclusion, we identified five key genes, FCER1G, MRPL14, SOSTDC1, TYROBP, and


The hemi-metabolous aquatic order Plecoptera (stoneflies) constitutes an indispensable part of terrestrial and aquatic food webs due to their specific life cycle and habitat requirements. Stoneflies are considered one of the most sensitive groups to environmental changes in freshwater ecosystems and anthropogenic changes have caused range contraction of many species. Given the critical threat to stoneflies, the study of their distribution, morphological variability and genetic diversity should be one of the priorities in conservation biology. However, some aspects about stoneflies, especially a fully resolved phylogeny and their patterns of distribution are not well known. A study that includes comprehensive field research and combines morphological and molecular identification of stoneflies has not been conducted in Croatia so far. Thus, the major aim of this study was to regenerate a comprehensive and taxonomically well-curated DNA barcode database for Croatian stoneflies, to highlight the morphological variability obtained for several species and to elucidate results in light of recent taxonomy.A morphological examination of adult specimens was made using basic characteristics for distinguishing species: terminalia in males and females, head and pronotum patterns, penial morphology, and egg structures. DNA barcoding was applied to many specimens to help circumscribe known species, identify cryptic or yet undescribed species, and to construct a preliminary phylogeny for Croatian stoneflies.Capnopsis schilleri balcanica Zwick, 1984. A total of 84% morphological species could be unambiguously identified using COI sequences. Species delineation methods confirmed the existence of five deeply divergent genetic lineages, with monophyletic origin, which also differ morphologically from their congeners and represent distinct entities. BIN (Barcode Index Number) assignment and species delineation methods clustered COI sequences into different numbers of operational taxonomic units (OTUs). ASAP delimited 76 putative species and achieved a maximum match score with morphology (97%). ABGD resulted in 62 and mPTP in 61 OTUs, indicating a more conservative approach. Most BINs were congruent with traditionally recognized species. Deep intraspecific genetic divergences in some clades highlighted the need for taxonomic revision in several species-complexes and species-groups. Research has yielded the first molecular characterization of nine species, with most having restricted distributions and confirmed the existence of several species which had been declared extinct regionally.Sequences (658 bp in length) of 74 morphospecies from all families present in Croatia were recovered from 87% of the analysed specimens (355 of 410), with one partial sequence of 605 bp in length for  Typicalllution)  in the mllution) . Due to llution) , stoneflllution) .Perlodes cf. intricatus,  Isoperla cf. lugens , Leuctra cf. pusilla Krno, 1985, Leuctra sp., Nemoura sp., Protonemura sp., Isoperla sp. and Perlodes sp. Also, during these studies, several remarkable species were documented: Marthamea vitripennis Burmeister, 1839, which was re-discovered again after one century  gene to aid in species identification and circumscription, is a useful tool for associating immature life stages and to identify cryptic species. Such an approach is extremely important for biodiversity assessment  developing a DNA reference barcode library for the Croatian stonefly fauna with macrophotographs of 26 species, (ii) getting first insight into inter- and intraspecific genetic diversity, (iii) assessing morphological variability of stoneflies in Croatia, (iv) highlighting localities with high biodiversity, especially in isolated habitats of the Dinaric Karst area to assist conservation planning and strategies for protecting the genetic diversity of stoneflies, and (v) filling the gaps in the Barcode of Life Data System database (BOLD). Furthermore, the study will contribute to knowledge about species distribution, species complexes and systematic and phylogenetic relationships.Conducted in the framework of the project Specimen collection was conducted with approval from the Ministry of Economy and Sustainable Development of the Republic of Croatia (UP/I-612-07/21-48/73). A total of 337 stonefly specimens  from 95 dx.doi.org/10.5883/DS-CROPL) and GenBank under the accession numbers as listed in Comparative study on morphology was made using specimens kept in the Collection of stoneflies in the Slovenian Museum of Natural History, Ljubljana, Slovenia (PMSL). Newly collected specimens are deposited in the Croatian Natural History Museum, Zagreb, Croatia (CNHM), under the Collection of Plecoptera Sivec & Hlebec (CPSH). Voucher information for individuals used in phylogenetic analysis are publicly accessible in BOLD  in MEGA-X ver. 10.2.6  were selected as outgroups. Additionally, phylogenetic relationships between haplotypes were inferred using the Neighbour joining (NJ) method in MEGA-X ver. 10.2.6 (via the online CIPRES Science Gateway ver. 3.3 (COI sequences) was ascertained using the “Barcode Gap Analysis” tool, provided in BOLD, using the Kimura-2-Parameter (K2P) distance metric  and Isoperla inermis), (b) specimens that differed from the described morphospecies  and Taeniopteryx n.sp. CRO-1) and (c) species, which were taxonomically interesting  and B. illyricus Kovács & Zwick, 2008). Analyses were performed with sequences of these species and their closely related congeners from BOLD (http://www.boldsystems.org) and GenBank (https://www.ncbi.nlm.nih.gov/) databases (accessed 20 August 2021). Accession numbers of all sequences used in additional analysis are listed in Datasets 1–6, COI sequences into Operational Taxonomic Units (OTUs) based on sequence similarity.In total, 355 obtained sequences were checked and inspected manually for base pair ambiguities, indels and stop codons in Geneious Prime 2021.2  to confirm overall sequences quality. Sequences were aligned using MAFFT ver. 7 . The finn length . Sequencver. 1.5 . Evoluti. 10.2.6  COI gene . 10.2.6    . BIN cou., 2.2%) , but sin., 2.2%) . Use of https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html), applying the K2P model and using default parameters, except for the relative gap width, which was set to X = 1.0. ASAP was also carried out on the web server (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html) using p-distances with default settings. The mPTP method, run on the web server (http://mptp.h-its.org/), was implemented using default settings and inputting the ML tree from IQ-TREE2 (Automatic Barcode Gap Discovery (ABGD) was carried out on the web server  and Nemoura sciurus Aubert, 1949) were identified by comparing with available sequences in the BOLD and GenBank databases.Field sampling and morphological identification produced 74 species of stoneflies in seven families and nineteen genera. Four species  individuals. All COI sequences alone. The median number of barcodes per species was four, and seventeen predominantly rare species were known only from single specimens.Molecular results were largely congruent with morphological assessment such that 62 of the 74 morphological species (84%) could be unambiguously identified using p-distance was 20.8% (ranging from 18.2–23.4%) while the average congeneric distance was 15.6% (ranging from 12.1–19.2%). The mean intraspecific p-distance was 1.3% (ranging from 0–6.8%)  and the distance to the furthest conspecific is above zero  . The maxove zero  and 1C.p-distance distribution is partially overlapping with distance to the nearest neighbour distribution /Leuctra albida; Perla pallida Guérin-Méneville, 1838/Perla marginata  and Isoperla illyrica/Isoperla tripartita. For the following neighbour species pairs, p-distance to the nearest neighbour was below 2%: Leuctra albida/Leuctra mortoni, Nemoura cf. rivorum Ravizza & Ravizza Dematteis, 1995/Nemoura flexuosa Aubert, 1949, Perla illiesi Braasch & Joost, 1973/Perla burmeisteriana, Perla sp./Perla marginata, Perla pallida/Perla marginata and Isoperla illyrica/Isoperla tripartita.The mean intraspecific ribution , but forribution . Maximum species   recoverObtained sequences were allocated to 85 BINs (of which 29 were unique to BOLD), and delimited OTUs were mostly consistent with the clustering pattern observed in the ML tree, which was also concordant with morphological identification. Twenty-six BINs were represented by a single individual (singletons).COI divergence resulting in multiple BINs within a species: Protonemura praecox  (BOLD:AEH4111 and BOLD:AEH7722), Perlodes microcephalus  (BOLD:AAL2343 and BOLD:AEH5507), Nemoura marginata Pictet, 1836 , Isoperla illyrica (BOLD:AEH3875 and BOLD:AEH7030), Leuctra fusca (BOLD:AAE6442 and BOLD:ACY3863), Leuctra albida (BOLD:AAM4011 and BOLD:AEH5504), Leuctra hippopus Kempny, 1899 (BOLD:ACL7184 and BOLD:AEH4770), Isoperla tripartita , Isoperla grammatica  (BOLD:AEH6396 and BOLD: AEG4373) and Isoperla inermis . Intraspecific p-distances were as follows for the following species: P. praecox (0–3.2%), P. microcephalus (0–4.2%), N. marginata (0–4.8%), Isoperla illyrica (0.002–6.8%), Leuctra fusca (0–5.8%), Isoperla tripartita (0–6.6%), Isoperla grammatica (0–5.0%) and Isoperla inermis (0–4%).One BIN often belongs to a single species delineated by traditional taxonomy , and eveIsoperla, Perla and Nemoura, and species Isoperla illyrica/Isoperla tripartita, Nemoura flexuosa/Nemoura cf. rivorum and Perla pallida/Perla sp./Perla marginata.A shared BIN assignment was obtained within three genera: Overall, use of different species delineation algorithms resulted in a different number of putative species. ASAP, with the best ASAP-score (6.00) which was achieved at a distance threshold of 2.5%, delimited 76 putative species and achieved a maximum match score with morphology (97%). The partition with the second-best ASAP-score  delimits 70 species and the third partition  delimits 64 species. The ABGD method delineated 62 putative species, while mPTP delineated 61 putative species. ABGD and mPTP represent conservative estimates of DNA species. Results of all species delineation methods are shown in Isoperla cf. lugens. The MJ network among 12 unique haplotypes which were separated by a different number of mutational steps is shown in Median joining (MJ) network depicted relatedness and distribution within the newly obtained divergent lineage  named as The present study represents the first comprehensive research combining morphological and molecular identification of stonefly species in Croatia and establishes DNA barcoding as an effective tool for reliable species identification. Such an approach enhances taxonomic resolution and assists in the quality of faunal research and can be used in discovering cryptic diversity and species complexes . A taxonCOI gene fragment has been used to elucidate the systematics and phylogeography of Plecoptera  (COI) barcode region cannot be used as the only/main data source for describing or delimiting species (COI barcode clusters (“BINs”) as a basis for species descriptions, given the assumption that a BIN equals a species, is often not consistent with the results of other species delineation methods (High numbers of intraspecific BINs (obtained for ten species) can indicate discrete geographical populations of a species or overlooked cryptic species , e.g., Ds, 1827) , so the  species  and stre methods  due to t methods .p-distances can be represented against a universal cut-off value , indicating the need for further field research across the entire range of the species. For another species, from the same genus, P. microcephalus, we obtained high intraspecific p-distance, and it is accompanied by increasing morphological variability from type specimens  from central Europe, resulted in a highly supported monophyletic clade I. inermis-I. difformis, which could ultimately result in the synonymy of these species   as suggested by In a comprehensive study of the genus T. hubaulti confirmed the variability of the femoral thorn on the hind legs, present in some individuals but varying in size, even though this character was originally described as absent , for which it has already been pointed out that additional research is needed to clarify their distinction  collected in the Plitvice Lakes National Park, Leuctra sp. collected at Cetina River  collected at Žumberak Hills and L. cf. prima  collected at Papuk Mountain and near Plitvice Lakes National Park. Due to the unavailability of specimens of L. carphatica Kis, 1966, only found in the Carphatian Mountains, Slovenia and Austria , which is not surprising, given that these areas have suitable conditions and mostly include fast streams with high oxygen saturation. Current research shows a significant decrease in the number of species from the northern part of the Dinaric Karst (Gorski kotar and Lika) to the Cetina River and the headwater of the Una River, which is consistent with the biological features of stoneflies. Great species richness is also confirmed by The diversity of the stonefly fauna in Croatia is a probable consequence of the different climatic conditions in a variety of regions , a substantial altitudinal gradient, and an immense number of protected habitats. Within this study, we identified hotspots of elevated stonefly species. The highest levels of species richness were primarily located in the northwest Dinarides : the borBrachyptera tristis, Perlodes dispar, Leuctra bronislawi Sowa, 1970, Isoperla bosnica , Isoperla illyrica, Isoperla albanica , Perla carantana Sivec & Graf, 2002, Perla illiesi and Agnetina elegantula .This study provides the first molecular characterization of nine species: Protonemura auberti , P. hrabei, P. intricata , P. nitida  and P. praecox should be considered in future revision of the genus, a need that has already been emphasized  collected during the spring at the Una River appeared as a separate clade among other specimens of P. auberti  .DNA barcoding of the species evations , so bracCapnia bifrons Zhiltzova, 2001 species-group, following Zwicknia bifrons  from only a single locality, a surprising find given that this was 17 years since the first finding , N. dubitans Morton, 1894, N. marginata, N. minima Aubert, 1946, N. sciurus, N. flexuosa, N. cf. rivorum, N. mortoni  and N. uncinata Despax, 1934. The Nemoura flexuosa-marginata complex is one of the most enigmatic assemblages of species within European stoneflies and requires revision  and six Italian and French Alps endemic species: N. hesperiae Consiglio, 1960, N. lucana Nicolai & Fochetti, 1991, N. oropensis Ravizza & Ravizza Dematteis, 1980, N. pesarinii Ravizza & Ravizza Dematteis, 1979 , N. palliventris Aubert, 1953 and N. rivorum Ravizza & Ravizza-Dematteis, 1995   have morphological characteristics similar to N. rivorum (determined as N. cf. rivorum). However, based on the similarity of the sequences, these specimens clustered with N. flexuosa , which further emphasizes the importance of species-group revision, and revision of the sequences in BOLD and GenBank databases, respectively.The ch Alps) . DescripNemoura marginata Pictet, 1836 . This autumnal species, which is relatively rare and a relict species with disjunct distribution in the Balkan and the Carpathians, was recently found in the Czech Republic , NW Macedonia and Montenegro  . Within the present study, this species was recorded at two localities in Lika and DNA barcoded for the first time  , often eet, 1833  and specMountain . Perla ip-distances between newly obtained sequences of B. imhoffi and B. illyricus retrieved from GenBank (10.6–12.4) do not support the synonymy of B. illyricus and B. imhoffi, as stated before , Isogenus nubecula Newman, 1833, Taeniopteryx nebulosa  and Perla abdominalis Burmeister, 1839  and Isogenus nubecula  has long been considered extinct because it does not occur in Central Europe , have been recorded at several localities in the foothills of Papuk Mountain. Besdolus imhoffi was re-discovered in Croatia in 2005    after a al Park) . This fiibutions  and are ibutions . Marthamf Europe  due to df Europe , but alsf Europe , and durf Europe  is Agnete.g., Isoperla, Taeniopteryx and Perla) an integrative revisionary examination based on more comprehensive geographic sampling and application of a multi-gene approach, especially on type specimens, is necessary for resolving taxonomic relationships. Identifying areas with high biodiversity, including both morphological variability and genetic diversity, will allow further protection of stoneflies and their habitats.The current study generated a validated national reference DNA barcode library for stoneflies in Croatia, which can support the implementation of cost-efficient DNA-based identifications and assessments to ecological status. DNA barcoding proved to be an effective tool for the identification and delimitation of some closely related species. Furthermore, this study provides several findings of species thought to be extirpated from Croatia and neighbouring regions, as well as the first molecular characterization of species with restricted distributions. For some genera  categories. Terminal codes present BOLD IDs, as in Click here for additional data file.10.7717/peerj.13213/supp-7Supplemental Information 7The information comprises BOLD IDs, GenBank accession numbers, BINs, Collection inventory numbers , species clade numbers indicating clades correspond to those in Click here for additional data file.10.7717/peerj.13213/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.13213/supp-9Supplemental Information 9p-distances are outlined by the black line.Values were obtained through complete deletion option. Intraspecific Click here for additional data file.10.7717/peerj.13213/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.13213/supp-11Supplemental Information 11Additional phylogenetic analyses were performed in several cases: for the species with high level of intraspecific morphological variability and species which differ mainly in taxonomic features from the species described so far.Click here for additional data file.


Ophiocordyceps sinensis parasitizes the ghost moth larva (Thitarodes spp.) and produces a fruit-body, which is a well-known Traditional Chinese Medicine (TCM). The infection process is time-consuming and inefficient, restricting the large-scale cultivation of this fungus. This study aimed to reveal the biology of immune modulation during the interaction between O. sinensis and its host  using high-throughput RNA sequencing. In H. xiaojinensis, 345 immune-related gene candidates were identified and were further classified according to the functions of pathogen recognition, signal transduction and immune response. According to the results of the analysis, the putative pathways of immune modulation in H. xiaojinensis in response to O. sinensis infection were sketched. Meanwhile, gene families probably involved in O. sinensis pathogenicity were identified, mainly including serine carboxypeptidase, peroxidase, metalloprotease peptidase, aminopeptidases, cytochrome P450, and oxidoreductase. Our findings provided an insight into the biology of fungi–insect host interaction, paving the way for large-scale artificial cultivation of the valued fungus as well as biological pest control.Ophiocordyceps sinensis (Berk.) is an entomopathogenic fungus that can infect the larva of the ghost moth, Hepialus xiaojinensis, causing mummification after more than one year. This prolonged infection provides a valuable model for studying the immunological interplay between an insect host and a pathogenic fungus. A comparative transcriptome analysis of pre-infection (L) and one-year post-infection (IL) larvae was performed to investigate the immune response in the host. Here, a total of 59,668 unigenes were obtained using Illumina Sequencing in IL and L. Among the 345 identified immune-related genes, 83 out of 86 immune-related differentially expressed genes (DEGs) had a much higher expression in IL than in L. Furthermore, the immune-related DEGs were classified as pathogen recognition receptors (PRRs), signal modulators or transductors, and immune effector molecules. Serpins and protease inhibitors were found to be upregulated in the late phase of infection, suppressing the host’s immune response. Based on the above analysis, the expression levels of most immune-related genes would return to the baseline with the immune response being repressed in the late phase of infection, leading to the fungal immunological tolerance after prolonged infection. Meanwhile, the transcriptomes of IL and the mummified larva (ML) were compared to explore O. sinensis invasion. A total of 1408 novel genes were identified, with 162 of them annotated with putative functions. The gene families likely implicated in O. sinensis pathogenicity have been identified, primarily including serine carboxypeptidase, peroxidase, metalloprotease peptidase, aminopeptidases, cytochrome P450, and oxidoreductase. Furthermore, quantitative real-time PCR (qPCR) was used to assess the expression levels of some critical genes that were involved in immune response and fungal pathogenicity. The results showed that their expression levels were consistent with the transcriptomes. Taken together, our findings offered a comprehensive and precise transcriptome study to understand the immune defense in H. xiaojinensis and O. sinensis invasion, which would accelerate the large-scale artificial cultivation of this medicinal fungus. Ophiocordyceps sinensis (Berk.), belonging to the family Ascomycete, is a highly valued traditional Chinese medicine (TCM) fungus that has been used in several Asian countries for over 2000 years [O. sinensis hasn’t been accomplished due to major obstacles such as low rates of fungal infection, primordium induction, and fruit-body development [00 years ,2. This 00 years . In rece00 years ,5. NeverO. sinensis is an entomopathogenic fungus with a complicated life cycle: it specifically colonizes ghost moth caterpillars (Thitarodes spp.), mummifies the larva, and grows a stalked fruiting body from the sclerotium head [O. sinensis infection at the early phase but the host will tolerate the fungus’s proliferation in the late phase, leading to a chronic infection that can last for more than one year [O. sinensis has a significant lineage-specific expansion of gene families enriched in fungal pathogenicity, such as peroxidase activity, serine hydrolase, deuterolysin metalloprotease (M35) peptidase, cytochrome P450 (Cyt P450), and glucose-methanol-choline (GMC) oxidoreductase [O. sinensis pathogenesis remain elusive. The long-lasting mutualistic occurrence between this fungus and its host larva provides a model system for scientific research and artificial cultivation. ium head ,2. Unlikone year . The proone year . In termeductase ,8,9. Theeductase . However were successfully reared and infected by O. sinensis under our laboratory conditions [O. sinensis accelerating the large-scale artificial cultivation of this highly valued medicinal fungus, as well as the biological control of insects.In this study, the insect larvae nditions . The RNAH. xiaojinensis larvae were reared and infected by O. sinensis in our lab using the following method. The pupae of the host insects were collected from 3500–4000 m mountains in Xiaojin County, Sichuan province, China, and housed with the roots of Potentilla anserina as food in plastic containers at 9–13 °C and 40–50% relative humidity (RH). Fifty 4–5th instar larvae were collected for O. sinensis infection. O. sinensis were harvested in 3000 to 4000 m mountains in Aba Prefecture, Sichuan province, China in May 2020 and stored at 15–18 °C and 75% RH. Aseptic bags were wrapped around the fruiting bodies to collect discharged ascospores. Ascospores were washed from bags with ddH2O and diluted to about 1 × 106 spores/μL in potato dextrose agar (PDA) liquid medium  and sprayed uniformly onto the 4–5th instar larvae 2 or 3 times per day, then kept at 13 °C and 40–50% RH. After one year, about 10 μL hemolymph of each infected larva was sampled for the presence of O. sinensis blastospores to confirm that the larva was successfully infected.The mature fruit-bodies of Three biological triplicates of fresh IL samples were kept in RNAlater  for RNA extraction prior to use, as shown in The mRNA was enriched from total RNA using poly(T)+ oligo attached magnetic beads, eluted with Tris–HCl buffer and fragmented in fragmentation buffer using an RNA fragmentation kit . First-strand cDNA was synthesized with fragmented mRNA as template and random hexamers as primers, followed by second-strand synthesis with the addition of PCR buffer, dNTPs, RNase H, and DNA polymerase I. Purification of cDNA was processed with AMPure XP beads. Double-strand cDNA was subjected to end repair. Adenosine was added to the end and ligated to adapters. AMPure XP beads were applied here to select fragments ranging in size from 300 to 400 bp. cDNA library was obtained by certain rounds of PCR on the cDNA fragments. O. sinensis, each RNA-seq was assembled separately. For the assembly of RNA-seqs of O. sinensis, Hishat2 [http://ccb.jhu.edu/software/tophat/index.shtml (accessed on 10 May 2021)) was used to align sequencing reads to the O. sinensis reference genome under the accession No. PRJNA608258 in NCBI database. StringTie [O. sinensis reference genome were removed and then the left reads were used in the subsequent de novo transcriptome assembly with Trinity  using default settings [Sequencing of the cDNA library was performed by Illumina NovaSeq based on Sequencing by Synthesis technology (SBS) at Biomarker Technology Co., Ltd. . Clean data with high quality was obtained by filtering raw data, removing adapter sequences and reads with low quality (<Q30), with default parameters. In terms of IL comprising both the host and  Hishat2  aligner tringTie  was applsettings ,14. Finahttp://www.ncbi.nih.gov/blast/db/ (accessed on 10 May 2021)), Swiss-Prot (http://www.expasy.org (accessed on 10 May 2021)), Gene Ontology database ), Cluster of Orthologous Groups ), KOG (http://www.ncbi.nlm.nih.gov/KOG/ (accessed on 10 May 2021)), eggnog ), Kyoto Encyclopedia of Genes and Genomes ). KEGG Orthology of unigenes was obtained by KOBAS2.0. Coding sequences were predicted by Transdecoder  and then were annotated by blasting against Pfam database  using HMhttp://deweylab.github.io/RSEM/ (accessed on 10 May 2021)) and the fragments per kilobase per million mapped reads (FPKM). Differentially expressed genes (DEGs) were calculated using DEseq2 in R. The expression difference with Fold Change(FC) ≥ 2 and FDR < 0.01 was considered to be significant. To globally characterize the expression profiles of these RNA-Seq samples, paired-end reads were aligned back to the assembled transcripts using Bowtie 2.0 as the aligner. RSEM, the utility package of the Trinity software, was performed to estimate the abundance of transcripts ), PROSITE (http://prosite.expasy.org/ (accessed on 15 May 2021)), and SMART (http://smart.embl.de/ (accessed on 15 May 2021)). Signal peptide and transmembrane domains were predicted using the SignalP 4.1 server ) and TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/ (accessed on 15 May 2021)). Protein structure was predicted by AlphaFold2. Multiple sequence alignments were performed using CLUSTALX 2.0 . The probable immune-related genes in −∆∆ct values were considered to be the relative expression levels or fold change between samples. The results are presented as the mean ± SD. Two-tailed Student’s t-tests and graphs were performed using GraphPad Prism software  and used for statistical analysis.Quantitative real-time PCR (qPCR) was performed on a CFX96 Touch qPCR system  according to manufacturer’s instructions. Quantitative measurements were carried out in three biological triplicates for each sample. Elongation factor 1 α subunit (gene-G6O67_008216) and 18sRNA were used as an internal standard to normalize the cDNA for the assessment of expression level of transcripts in IL vs. L and IL vs. ML, respectively. All primers used for the selected genes are listed in https://www.ncbi.nlm.nih.gov/ (accessed on 1 May 2021)) under the accession No. SRR1735514, SRR5282572, SRR5282573. The raw data of ML was derived from the NCBI database under the accessions No. SRX9405963, SRX9405964, SRX9405965. The sequencing reads of IL was submitted to GSA database (https://ngdc.cncb.ac.cn/gsub/ (accessed on 1 July 2022)) under the accession No. subCRA012084 with the BioProject No. PRJCA011311.The raw RNA-seqs of L were derived from the NCBI database | is more than 2 and FDR is less than 0.001. This threshold resulted in a total of 7194 genes  as significant DEGs in IL vs. L , indicatO. sinensis infection.Based on the COG database, 4799 DEGs were enriched in GO categorization, with 4426 upregulated and 373 downregulated in IL compared to L . The DEGO. sinensis infection (β-ketoacyl synthase (BMK_Unigene_035362), thiolase (BMK_Unigene_022220), and ubiquitin carboxyl-terminal hydrolase (BMK_Unigene_052423). Pathway-based analyses were performed to further understand the biological functions of DEGs. 3154 DEGs were enriched in KEGG categories. The KEGG pathway enrichment analysis revealed that the upregualted DEGs in IL versus L were highly associated with several pathways, such as carbon metabolism (ko01200), fatty acid metabolism (ko01212), biosynthesis of acid amino (ko01230), ABC transporters, and peroxisome (ko04146). The results showed that extra energy, compounds, and unique proteins might be produced as a defense against nfection . Fatty aO. sinensis infection. A previous study found that the treatment of macrophages with peroxisome-derived lipid mediators enhanced their capacity to engulf bacteria [Furthermore, 77 DEGs annotated were found to be enriched in the KEGG pathway of peroxisome (ko04146); 84.2% of these DEGs were upregulated in IL compared to L, including Pex2_Pex12 domain-containing protein genes (BMK_Unigene_014313), peroxisomal acyl-coenzyme A oxidase (BMK_Unigene_054313), and cytochrome b2 (BMK_Unigene_044383) . Peroxisbacteria . Here, abacteria . Here, sA total of 345 immune-related genes were annotated, with 86 of them screened for DEGs with log2 FC > 2, FDR < 0.01. The heatmap of immune-related DEGs was shown in Bombyx mori [Drosophila [meso-2,6-diaminopimelic acid (DAP)-type PGNs, enabling them to defend against some gram-negative and -positive bacteria [A PGRP was initially isolated from the hemolymph of the silkworm, byx mori . When PGbyx mori . Drosophosophila . In thisosophila . In thisbacteria . PGRP-LEbacteria . It is abacteria . Our datbacteria . Here, βbacteria . Hence, H. xiaojinensis, including CTL 5, CTL 6, and immulectin-21 and -22, which bind specifically to certain sugars and cause agglutination of particular cell types. In B. mori, CTL 5 might be an important PRR for activating the JAK/STAT signaling pathway and mediating the nodule melanization for fungal infection [B. mori, CTL-S6 may recognize foreign pathogens, activate the prophenoloxidase pathway and innate immune response [H. xiaojinensis. SRs are also an important subclass of PRRs and have a ligand-binding specificity [H. xiaojinensis. A higher expression level of the two transcripts in IL compared to L showed that SRs might also participate in activating immune response in the late phase of infection.C-type lectin (CTL) serves as a key PRR in innate immunity by recognizing a wide spectrum of pathogens . In thisnfection . In B. mresponse . In thiscificity . In thisH. xiaojinensis and even more in other Lepidopteran insects, indicating that CSPs would play a key role for the host immunity [H. xiaojinensis, including prophenoloxidase-activating enzyme 3, CSPs 2, and melanization protease 1. ModSP, a modular serine protease, plays an essential part in the activation of the Toll pathway by gram-positive bacteria and fungi [H. xiaojinensi [H. xiaojinensis [H. xiaojinensis.Clip domain serine proteases (CSPs) in insect hemolymph are rapidly activated by specific proteolysis after pathogen recognition and participate in extracellular signal amplification and countervailing serine proteases . Here, 3immunity . Microbiimmunity ,33. CSPsnd fungi . In thisojinensi . On the ojinensi . They acojinensi . A previjinensis . In thisjinensis . Based oH. xiaojinensis. Besides, lysosome-autophagy is the first-line defense against pathogens. Autophagy defends against pathogenic infection as a part of the cellular defense system by producing autophagosomes that transport these cargoes to lysosomes for degradation [H. xiaojinensis were sketched (Mitogen-activated protein kinases (MAPKs) also regulate immune response by producing immunomodulatory cytokines . Followiradation . In thissketched .B. bassiana [O. sinensis would rapidly grow after successfully colonizing the host.DEGs were considered statistically significant if the |log2 FC (log2 fold-change)| is more than 2 and the FDR is less than 0.001. This threshold resulted in a total of 3243 genes  as significant DEGs in ILvs. L . Based obassiana . FAD-depbassiana . FurtherO. sinensis pathogenicity , pentose and glucuronate interconversion , glycosphingolipid biosynthesis , glycine, serine and threonine metabolism (ko00260), and taurine and hypotaurine metabolism ko00430), etc., were enriched and upregulated in IL compared to ML, indicating that these pathways might play crucial roles in genicity . For exa0, etc., nization . So, lipradicals . After iradicals . Here, hradicals . Here, gO. sinensis genome showed a considerable expansion of gene families that are mainly involved in fungal pathogenicity, including peroxidase activity, serine hydrolase, deuterolysin metalloprotease (M35) peptidase, Cyt P450, and glucose-methanol-choline (GMC) oxidoreductase involved in the ecdysteroid metabolism of molting in insects [The  insects ,48. ThesO. sinensis pathogenicity via ROS pathway. In this study, 37 DEGs were enriched in the Pfam of Cyt P450. Of them, 13 genes were upregulated in IL compared with ML, including Cyt P450 monooxygenase sdnE, Cyt P450/NADPH reductase, and Cyt P450 monooxygenase ABA1. Cyt P450, membrane-bound hemoproteins, play pivotal roles in the detoxification of xenobiotics, cellular metabolism, and homeostasis . CytP450O. sinensis, compared to that in other entomopathogenic fungi [A. fumigatus and some basidiomycetes [O. sinensis pathogenicity.Classically, protease is assumed to be a destructor of host tissues while simultaneously providing nutrients for pathogen replication . Aspartyic fungi . Fungalyomycetes . In thisomycetes . Serine omycetes . In thisH. xiaojinensis were detected to be upregulated in IL compared to L. In IL vs. ML, all the qPCR results of all the seven genes were consistent with that of RNA-seq (O. sinensis pathogenicity.To confirm gene expression levels in RNA-seq, some critical genes were selected for qRT-PCR validation. The gene-specific primers used in qRT-PCR are listed in  RNA-seq B, includH. xiaojinensis and the pathogenicity of O. sinensis were studied during the infection process by using high-throughput RNA-seq. A total of 345 immune-related genes were identified in H. xiaojinensis. Most of these genes had no differential expression. However, serpins and protease inhibitors were found to be upregulated in the late phase of infection. It was indicated that the fungal immune tolerance would develop in the late phase of infection in the host. Furthermore, gene families probably implicated in O. sinensis pathogenicity were also identified, primarily including peroxidase, serine hydrolase, serine carboxypeptidase, an aspartyl protease, M35 peptidase, and Cyt P450. Our findings revealed the biology of the immune modulation in the long-term chronic infection process between H. xiaojinensis and O. sinensis using a comprehensive and precise transcriptome analysis.In this study, the immune response of


Pharmaceutics [aceutics  is instiPharmaceutics Editorial Office based on the number of citations and downloads from the website. The winners from the nominations were determined by an award committee, the Editor-in-Chief, and the Editorial Office. The following three top-voted papers, in no particular order, have won the Pharmaceutics 2022 Best Paper Awards:The papers published in 2020 were preselected by the Development of Remdesivir as a Dry Powder for Inhalation by Thin Film FreezingBy Sahakijpijarn, S.; Moon, C.; Koleng, J.J.; Christensen, D.J.; Williams, R.O., III. .Pharmaceutics 2020, 12, 1002. https://doi.org/10.3390/pharmaceutics12111002.https://www.mdpi.com/1999-4923/12/11/1002 (accessed on 27 July 2022).Available online: Synopsis of the paper by the authors:® demonstrated an increased systemic uptake compared to leucine. Remdesivir was hydrolyzed to the nucleoside analog GS-441524 in the lung, and the levels of GS-441524 were greater in the lung with leucine formulation compared to Captisol®. In conclusion, TFF technology produces high-potency remdesivir dry powder formulations for inhalation that are suitable to treat patients with COVID-19 on an outpatient basis and earlier in the disease course when effective antiviral therapy can reduce the related morbidity and mortality [Remdesivir exhibits in vitro activity against SARS-CoV-2 and was granted approval for emergency use. To maximize its delivery to the lungs, we formulated remdesivir as a dry powder for inhalation using thin film freezing (TFF). TFF produces brittle matrix-nanostructured aggregates that are sheared into respirable low-density microparticles upon aerosolization from a passive dry powder inhaler. In vitro aerodynamic testing demonstrated that the drug loading and excipient type affected the aerosol performance of remdesivir. Remdesivir combined with optimal excipients exhibited a desirable aerosol performance . Remdesivir was amorphous after the TFF process, which benefitted the drug’s dissolution in simulated lung fluid. TFF remdesivir formulations are stable after one month of storage at 25 °C/60% relative humidity. An in vivo pharmacokinetic evaluation showed that TFF remdesivir–leucine was poorly absorbed into systemic circulation while TFF remdesivir-CaptisolManufacturing Considerations for the Development of Lipid Nanoparticles Using MicrofluidicsBy Roces, C.B.; Lou, G.; Jain, N.; Abraham, S.; Thomas, A.; Halbert, G.W.; Perrie, Y. .Pharmaceutics 2020, 12, 1095. https://doi.org/10.3390/pharmaceutics12111095.https://www.mdpi.com/1999-4923/12/11/1095 (accessed on 27 July 2022).Available online: Synopsis of the paper by the authors:In recent years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or for treating a wide range of diseases. Microfluidics offer considerable advantages for their manufacture due to their scalability, reproducibility, and fast preparation. Thus, in this study, we have evaluated the operational and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice , buffer choice (citrate buffer pH 6 or TRIS pH 7.4), and type of nucleic acid payload  have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics [Skin Wound Healing Process and New Emerging Technologies for Skin Wound Care and RegenerationBy Tottoli, E.M.; Dorati, R.; Genta, I.; Chiesa, E.; Pisani, S.; Conti, B. .Pharmaceutics 2020, 12, 735. https://doi.org/10.3390/pharmaceutics12080735.https://www.mdpi.com/1999-4923/12/8/735 (accessed on 27 July 2022).Available online: Synopsis of the paper by the authors:Skin wound healing shows an extraordinary cellular function mechanism, which is unique in nature and involves the interaction of several cells, growth factors, and cytokines. Physiological wound healing restores tissue integrity, but in many cases the process is limited to wound repair. Ongoing studies aim to obtain more effective wound therapies with the intention of reducing inpatient costs, providing long-term relief, and enabling effective scar healing. The main goal of this comprehensive review is to focus on the progress in wound medication and how it has evolved over the years. The main complications related to the healing process and the clinical management of chronic wounds are described in the review. Moreover, the advanced treatment strategies for skin regeneration and experimental techniques for cellular engineering and skin tissue engineering are addressed. Emerging skin regeneration techniques involving scaffolds activated with growth factors, bioactive molecules, and genetically modified cells are exploited to overcome wound-healing technology’s limitations and to implement personalized therapy designs .Pharmaceutics. We warmly congratulate both teams on their accomplishments and wish them continued success.These three papers represent valuable contributions to Pharmaceutics 2022 Best Paper Awards Committee,Pharmaceutics Editorial Board.