Fredericella sultana is a well known primary host of the myxozoan parasite Tetracapsuloides bryosalmonae. There have been no attempts to identify the cellular responses induced in F. sultana during the T. bryosalmonae development. We therefore performed transcriptome analysis with the aim of identifying candidate genes and biological pathways of F. sultana involved in the response to T. bryosalmonae. A total of 1166 differentially up- and downregulated genes were identified in the infected F. sultana. Gene ontology of biological processes of upregulated genes pointed to the involvement of the innate immune response, establishment of protein localization, and ribosome biogenesis, while the downregulated genes were involved in mitotic spindle assembly, viral entry into the host cell, and response to nitric oxide. Eukaryotic Initiation Factor 2 signaling was identified as a top canonical pathway and MYCN as a top upstream regulator in the differentially expressed genes. Our study provides the first transcriptional profiling data on the F. sultana zooid’s response to T. bryosalmonae. Pathways and upstream regulators help us to understand the complex interplay in the infected F. sultana. The results will facilitate the elucidation of innate immune mechanisms of bryozoan and will lay a foundation for further analyses on bryozoan-responsive candidate genes, which will be an important resource for the comparative analysis of gene expression in bryozoans.Bryozoans are sessile, filter-feeding, and colony-building invertebrate organisms.  Fredericella sultana is a freshwater bryozoan and is found worldwide. It is typically colonial, consisting of up to several hundred connected individual zooids. Each zooid has its own independent tentacular lophophore used for suspension feeding [F. sultana reproduces both asexually and sexually. The colonies propagate quickly during spring and form tubular and branching colonies that attach to submerged surfaces like pieces of wood and tree roots by summer. Over the winter period, they produce dormant seed-like stages called statoblasts [F. sultana provides insight into the unique cellular, metabolic, and catalytic processes of this bryozoan species [ feeding . F. sulttoblasts . Recent  species .F. sultana is the most common bryozoan host of Tetracapsuloides bryosalmonae (Myxozoa), the causative agent of proliferative kidney disease in salmonids. The cycle of the parasite alternates between covert and overt infection in bryozoans. The spores of T. bryosalmonae develop first as a covert infection (single-cell stages) within the bryozoan body wall. At the time of overt infection, multicellular sacs produce from single cell stages and multiply in the body cavity of the bryozoans [ryozoans ,5. Coverryozoans . Infecteryozoans . The sporyozoans ,9.F. sultana has become one of the most studied bryozoan species. A number of studies have investigated the ecology of the bryozoan in relation to its infection status [T. bryosalmonae infection reduces utilization of food levels, growth rates, and reproduction in F. sultana [F. sultana and T. bryosalmonae are associated with multiple transmission pathways and host condition-dependent developmental cycling [F. sultana transcriptome that identifies the unique characteristics of this organism [T. bryosalmonae. Understanding the cellular triggers that occur when the bryozoan is infected with the parasite will allow us to elucidate molecular immunity and related components in the defense system of bryozoan. This will be a useful starting point to enhance our understanding of the mechanisms of immunity of bryozoan to T. bryosalmonae infection at the molecular level, by combining a variety of bioinformatic analyses for studies of host-parasite interactions, gene discovery, and novel antigen identification.Due to being the primary host of an economically important fish parasite, n status . T. bryo sultana . Interac cycling . Now thaorganism , we can T. bryosalmonae infection. Towards this end, we aimed to analyze transcriptomic changes in the zooids of F. sultana during the T. bryosalmonae development using high-throughput transcriptome sequencing. To study the molecular mechanism of F. sultana, we performed enrichment analysis and functional annotation of the differentially expressed genes (DEGs) involved in a plethora of biological roles in different immune system processes and pathways.Understanding the dynamic interactions at the bryozoan-host interface can be achieved in part by characterizing bryozoan gene expression relative to F. sultana during parasite development, cDNA libraries were constructed for RNA-seq from infected and uninfected zooids. After data analysis and quality filtering of the sequencing data, we obtained a total of 274 million raw reads (paired) of the zooids transcriptome. We obtained a total of 250 million high-quality reads (paired) ranging from 37 to 44 million trimmed reads per sample after removal of low-quality reads and contaminating adapter sequences  plot , we assup-value ≤ 0.01 and fold change ≥2 or ≤−2) in the infected zooids of F. sultana  software. The top 20 enriched canonical pathways identified by IPA are shown in F. sultana during parasite development using IPA software. Upstream regulator analysis identified 82 direct targets among the 1166 DEGs (Our objective here was to identify upstream regulators of DEGs that are associated and activated in 166 DEGs . The topr (8) = 0.994, p < 0.00001) between the log2 fold change values of RNA-seq and qRT-PCR confirms the consistency and reproducibility of the RNA-seq analysis , C-type lectin 37Db-like (−12.99 fold), perlucin-like protein (−42.01 fold), and galectin 2 (−2.54 fold) were significantly downregulated in the infected F. sultana. Specific lectin-carbohydrate binding molecules at the parasite membrane and host–parasite interaction have been described earlier for myxozoans such as T. bryosalmonae and Myxobolus cerebralis [2 fold) was also observed in oligochaete Branchiura sowerbyi in response to myxozoan Myxobolus cultus infection [T. bryosalmonae and the rhamnose-binding lectin STL1 from the kidney of brown trout was identified by antibody-based protein purification followed by ESI-MS [T. bryosalmonae attachment and the subsequent activation of the immune response system of F. sultana to eliminate the parasite infection and maintain homeostasis. However, the roles of identified downregulated lectins need to be investigated using molecular characterization of lectins to provide further insight into the bryozoan’s immune response and downstream pathway activation.Lectins are carbohydrate-binding proteins that have a role in recognition at the cellular and molecular level. Lectins interact with pathogen-associated molecular patterns (PAMPs) to activate innate immunity . In the rebralis ,17. Uprenfection . The inty ESI-MS . The ideB. sowerbyi [T. bryosalmonae infection. To maintain the endogenous redox homeostasis, bryozoans must cope with the oxidative challenges caused by the host immune system against T. bryosalmonae.We found that the expression of 20 ubiquitin related genes such as ubiquitin-like protein, polyubiquitin, SCF ubiquitin ligase, ubiquitin-conjugating enzyme E2, ubiquitin-40S ribosomal protein S27a, and ubiquitin-60S ribosomal protein L40 were significantly upregulated in the infected zooids. Ubiquitin-mediated proteolysis activates the innate immune system and motivates a variety of immune-related regulators from transcription to apoptosis [sowerbyi . This suT. bryosalmonae adhere at the inside of bryozoan peritoneal wall and they develop and float in the coelomic fluid of the body cavity of bryozoan [T. bryosalmonae. Lysosome, peroxisome, and oxidative phosphorylation pathways genes like cathepsin B, lysosomal alpha-mannosidase, peroxiredoxin-5, superoxide dismutases (Fe and Cu-Zn), cytochrome c oxidase subunit V, and NADH dehydrogenase (ubiquinone) iron-sulfur proteins  were upregulated in the infected zooids. Similarly, the upregulation of TLR13-like, cathepsin B, peroxiredoxin, and cytochrome c oxidase subunit 5B was observed in the kidney of brown trout in response to T. bryosalmonae [B. sowerbyi [T. bryosalmonae. However, some immune-related genes such as lysosomal protective protein-like (−4.37 fold), lysosome-associated membrane glycoprotein 1-like (−3.96 fold), macrophage mannose receptor 1-like (−58.36 fold), CD63 antigen (−2.04 fold), CD82 antigen-like (−4.10 fold), CD225/dispanin family protein (−2.18 fold), T-cell immunomodulatory protein (−2.19 fold), and TNF receptor-associated factor 2 isoform X1 (−2.93 fold) were downregulated in the infected zooids. Further study would be required to provide insight into the innate immune responses that function in bryozoan’s immune network and the relevant effector responses.Oxidative stress is an important process in the recognition and elimination of pathogens. The spores of bryozoan . Based osalmonae ,23. The sowerbyi . CombineT. bryosalmonae. In support of this, we found upregulation of Toll signaling pathway genes such as TLR3 (2.59 fold), TLR4 (2.53 fold), and TLR13 (2.55 fold) in the infected zooids. Additionally, upregulation of TLRs such as TLR1 (4.1 fold), TLR13-like Chr1 (16.3 fold), and TLR19 (5.5 fold) has been reported in the kidney of brown trout during active T. bryosalmonae sporogenesis [Numerous proteases that belong to the peptidase, hydrolase, and metalloprotease families were upregulated in the infected zooids. Cathepsins, a superfamily of hydrolytic enzymes enclosed within lysosomes, contribute to several physiological and immune response activities . Here, togenesis . We presB. sowerbyi, Enteromyxum scophthalmi, and T. bryosalmonae [T. bryosalmonae using RNA-seq analysis (authors own unpublished data). Consequently, the inhibition of parasite proteases is likely an important innate immune response employed by bryozoans in resisting T. bryosalmonae infection.Protease inhibitors are an enormously various and large group of proteins that inhibit the proteolytic activity of proteases in multiple physiological processes and are involved in the innate immune response . We founsalmonae ,22,28. Tsalmonae ,30. Protsalmonae ,32,33,34M. cultus infection has been documented [We found upregulation of other genes such as actins , actin-1 (6.13 fold), actin binding protein (5.05 fold), actin depolymerizing protein (5.28 fold), and actin-related protein 2/3 complex subunit 1A (7.65 fold)) and heat shock proteins (HSPD1 (21.71 fold), HSPA2 (65.94 fold), HSP5 (3.33 fold), HSP60 (6.67 fold), HSP70 (61.84 fold), HSP70-3 175.18 fold), HSP70A (12.23 fold), HSP71 (11.43 fold), HSP90 (47.08 fold), and HSP90B1 (5.70 fold)) in infected bryozoans. The upregulation of HSPs in oligochaete against  fold, HSPlumatella repens [T. bryosalmonae infection, suggesting that chitinase may be closely linked with inflammation and infection progression. However, the regulation and role of CHIT1 in bryozoans to parasite infection is not known.We also identified some other important downregulated genes in the infected zooids such as peptidoglycan-binding protein (−125.94 fold) and chitotriosidase-1 −37.05 fold). Peptidoglycan-binding protein is required as a cell surface receptor for peptidoglycan elicitor signaling leading to innate immunity of the host . Downreg.05 fold.a repens . Hence, F. sultana was cultivated in our controlled laboratory conditions after hatching of specific pathogen-free (SPF) statoblasts. F. sultana colonies were maintained in an aquarium at 18 ± 1 °C and were fed with algae species mixtures  according to Kumar et al. [T. bryosalmonae-infected brown trout. F. sultana colonies were maintained under optimal laboratory conditions according to Kumar et al. [F. sultana colonies were observed using a dissecting microscope to check for the presence of T. bryosalmonae. Colonies cohabitated with T. bryosalmonae-infected brown trout showed the presence of parasite stages. However, no parasite stages were seen in SPF uninfected control F. sultana colonies. Additionally, these colonies were tested using PCR to confirm pathogen-free status [The colony of r et al. . We cohar et al. . F. sulte status .F. sultana colonies with infected brown trout was conducted in accordance with the relevant guidelines and regulations §26 of the Austrian Law for Animal Experiments, Tierversuchsgesetz 2012. The institutional ethics committee of the University of Veterinary Medicine, Vienna, Austria and the national authority approved this experiment under the permission numbers BMWFW GZ: 68.205/0181-WF/V/3b/2017 (23 October 2017) and BMWFW GZ: 2020-0.237.729 (17 April 2020).The cohabitation of F. sultana colony with the help of needles and forceps. Uninfected zooids  and uninfected control zooids (n = 3) from RNAlater-preserved samples using the RNeasy Mini Kit , utilizing the optional DNase digestion step. The quantity and integrity of the RNA were determined using a NanoDrop 200C spectrophotometer  and 4200 TapeStation using the RNA ScreenTape assay . RNA samples with an RNA integrity number above 8.0 were used for library preparation.Total RNA was extracted from infected zooids  according to the manufacturer’s protocol. Illumina sequencing was performed on one lane of an Illumina NextSeq550 instrument implementing paired-end 150-bp reads.F. sultana transcriptome assembly (NCBI TSA accession: GHLZ01000000) [Data analysis and quality filtering of the sequencing data was performed with CLC Genomics Workbench 12 software . Adapter sequences, low quality bases (Phred score ≤30) and reads shorter than 50 nt were removed. The filtered reads were mapped against the 1000000) . In the p-values ≤ 0.01 were considered differentially expressed. For downstream analysis, orthologous human gene symbols obtained from the UniProt database (https://www.uniprot.org) were added to the F. sultana reference transcripts and genes that are represented by multiple contigs on the reference transcriptome were merged. For merged transcripts, the statistical results of the contig with the highest mean coverage (TPM) across all samples were used for further analysis assuming that more mapping reads represent more reliable expression data. A heatmap of the Z scaled resulting normalized TPM values of RNA-seq was generated using the heatmap.2 function of the gplots R package and hierarchical clustering was based on Euclidean distance and complete linkage.Differential expression between infected and uninfected control zooid samples was analyzed using the ”exact test” for two-group comparisons . All feaF. sultana, the analysis was based on human GO data. p-values ≤ 0.05 were considered significant. Additionally, Ingenuity Pathway Analysis  was used to detect enriched canonical pathways in the list of DEGs. A p-value (Fisher’s exact test) ≤0.05 was considered significant. Activation z-scores calculated by IPA were used to predict the activation or inhibition of the networks. A z-score ≥2 denotes activation and a z-score ≤−2 denotes inhibition. PathVisio 3.3.0 [The list of DEGs was analyzed for significantly enriched Gene Ontology (GO) terms using the ClueGO v2.2.4 plugin of the biomolecular network software Cytoscape v3.1.1 . Due to io 3.3.0  and Grapn = 6) and uninfected control (n = 6) samples using an iScript cDNA Synthesis Kit (Bio-Rad), according to the manufacturer’s protocol. Ten gene-specific primers . cDNA was synthesized from total RNA as described above for infected ( primers  were descriptome . qPCR hacriptome . The staon 3.5.3 .F. sultana to the parasitic malacosporean T. bryosalmonae. We found that T. bryosalmonae development caused dramatic alterations in gene regulation of F. sultana, leading to a sequence of cellular events that involve activating signaling modulation responses in F. sultana. Further analysis revealed that several well-known functional categories of genes and signaling pathways, which are associated with innate immune response, had been significantly enriched, including the functional categories of antigen presentation and processing, phagosome maturation, antioxidative responses, proteases, and proteases inhibitors. The transcriptome profile of F. sultana has yielded several novel putative innate immune system genes that may play critical roles in the response to T. bryosalmonae. This study improves our understanding of the molecular mechanisms underlying the interaction between T. bryosalmonae and their bryozoan host. The results of this study show a foundation for further analyses on bryozoan-responsive candidate genes, which would help us to understand bryozoan adaptation under T. bryosalmonae stress and eventually advantage future bryozoan molecular biology.We provide a global overview of the transcriptional responses of the bryozoan


Trichobilharzia szidati. The circular genome is 14293 bp in length and contains 12 protein-coding genes, 12S and 16S rRNAs genes, 22 tRNAs and one non-coding region (202 bp) (accession number MF136777). Phylogenetic relationships based on 12 protein-coding gene sequences (PCG) of mitogenomes of a number of trematode and cestode species have shown that T. szidati is the closest genetic relative to nasal bird schistosome T. regenti. The complete mitogenome sequence of T. szidati may serve as a resource for comparative mitogenomics and trematode evolution studies.We report the first complete mitochondrial genome of visceral bird schistosome  Trichobilharzia spp. including T. szidati live in the blood system of visceral organs, only mature T. regenti occurs in the nasal mucosa of its definitive host  occurs when larval stages of bird schistosomes that normally develop in birds penetrate human skin. The majority of bird schistosomes T. szidati and revise its phylogenetic position. It will be useful for the studies in population genetics, comparative mitogenomics, phylogenetics and evolutionary studies of trematodes.We report for the first time the complete sequence of mitogenome of visceral species T. szidati were collected from host snail Lymnaea stagnalis from Lake Naroch  in Belarus. The specimen (N15Lst1) is stored in the Laboratory of Genome Organization, Institute of Gene Biology of the Russian Academy of Sciences, Moscow, Russia. Total genomic DNA was extracted by NEB #E6000 Kit  following the instructions of the manufacturer. DNA sequencing was conducted using Illumina Hiseq 2000 . Cercariae of T. regenti (DQ859919). Phylogenetic position of T. szidati was estimated on the base of protein-coding genes (PCG) of the mitogenomes of platyhelminth species from class Trematoda: Schistosomatidae: T. regent Schistosoma haematobium, S. mansoni, S. japonicum, S. mekongi; Paragonimidae: Paragonimus westermani; Fasciolidae: Fasciola gigantica and F. hepatica and two species from class Cestoda: Diphyllobothrium latum and Taenia solium (as an out group). We constructed a phylogenetic tree using PhyML  and S. japonicum , but smaller than mitogenomes of two African flukes S. mansoni , S. haematobium  and nasal schistosome T. regenti .The complete mtDNA sequence of T. szidati mitochondrial genome is identical to T. regenti and Asiatic mammalian schistosomes described earlier , nad5 (11590 bp vs. nd 1599 bp) and cytb (1107 bp vs. 1110 bp). The overall sequence difference between concatenated PCG of T. regenti and T. szidati is 17.6%.The gene order of Trichobilharzia species, the genes of two mitochondrial rRNA subunits, rrnL and rrnS, are separated by trnC. The mitogenome of T. szidati includes a non-coding region (202 bp) with two similar short sequences (25 bp). In contrast, the long non-coding region in T. regenti includes three tandem repeats of 184 bp each.In both T. szidati is closely related to T. regenti (The phylogenetic study provides a strongly supported framework for the monophyly of the Schistosomatidae subgroup and supports that the  regenti .


Staphylococcus aureus in Chinese neonatal intensive care units (NICUs). We describe the molecular epidemiology of S. aureus isolated from neonates on admission to Beijing Children's Hospital.Little is known about the molecular epidemiology of S. aureus isolates were characterized by staphylococcal chromosomal cassette (SCCmec) type, staphylococcal protein A (spa) type, multilocus sequence type (MLST), sasX gene, antimicrobial susceptibility and cytotoxicity. Logistic regression assessed risk factors for colonization.From May 2015-March 2016, nasal swabs were obtained on admission from 536 neonates. Cultures were also obtained from body sites with suspected infections. S. aureus and 20 (3.7%) were diagnosed with culture-positive S. aureus infection. Of the colonized infants, 70% (64/92) harbored methicillin-susceptible S. aureus (MSSA), 30% (28/92) harbored methicillin-resistant S. aureus (MRSA) while 70% (14/20) of infected infants were culture-positive for MRSA, 30% (6/20) were culture-positive for MSSA. Risk factors for colonization included female sex, age 7–28 days, higher birthweight (3270 IQR [2020–3655] grams) and vaginal delivery (p<0.05). The most common MRSA and MSSA clones were community-associated ST59-SCCmecIVa-t437 (60%) and ST188-t189 (15%), respectively. The sasX gene was not detected. Some MSSA isolates (16%) were penicillin-susceptible and some MRSA isolates (18%) were oxacillin-susceptible. MRSA and MSSA had similar cytotoxicity, but colonizing strains were less cytotoxic than strains associated with infections.Overall, 92 (17%) infants were colonized with S. aureus colonization was common in infants admitted to our NICU and two community-associated clones predominated. Several non-modifiable risk factors for S. aureus colonization were identified. These results suggest that screening infants for S. aureus upon admission and targeting decolonization of high-risk infants and/or those colonized with high-risk clones could be useful to prevent transmission. Staphylococcus aureus infections represent a significant clinical burden for infants worldwide and were recently found to be the second most common cause of late-onset sepsis in very-low birth weight (VLBW) infants admitted to neonatal intensive care units (NICUs) in the United States and United Kingdom [S. aureus colonization [S. aureus (MRSA) colonization was associated with a 24.2 times increased MRSA infection risk [S. aureus (MSSA) occur frequently in NICUs [S. aureus in the NICU population can promote an increased understanding of pathogenesis and ultimately guide preventive strategies. Kingdom , 2. Pretnization , a potenin NICUs . StudyinS. aureus colonization and infection have been described for NICU populations across the globe and have increased our knowledge of the global burden [The molecular characteristics of and risk factors for l burden , 6. Howel burden –9, wherel burden –9. Many S. aureus colonization. We further aimed to describe the molecular epidemiology of both MSSA and MRSA, including the most dominant clones, and their in vitro cytotoxicity. Ultimately, we will use these data to inform future surveillance and S. aureus prevention efforts.In this study, we aimed to determine the proportion of neonates colonized and/or infected with MSSA and MRSA on admission to the NICU of BCH, as well as assess risk factors for From May 2015 to March 2016, we performed a prospective surveillance study on admission for MSSA and MRSA among neonates ≤28 days of age hospitalized in the level 3, 50-bed NICU of BCH. This hospital does not have an obstetrics unit; therefore, neonates  are admitted to the NICU from home or other obstetric units. The most common admitting diagnoses are infectious diseases (~60%), prematurity (~20%), and various congenital comorbidities including cardiac, gastrointestinal, and neurologic disorders (~15%). The Ethics Committee of BCH, affiliated with Capital Medical University, approved this study; parents and/or legal guardians of infants provided written informed consent, and none refused participation.Selected demographic  and clinical characteristics  were abstracted from the electronic medical records of enrolled neonates. Medical records were transferred from their delivery hospital. Age was trichotomized as <7 days, 7–14, and15-28 days of life for analysis, which was consistent with the way neonates have been previously defined , 11. AntS. aureus colonization in the study cohort, both anterior nares were swabbed within 24 hours of admission, following a standard operational procedure. BBL™ Culture Swab™ Collection and Transport System  was used. Specifically, only one swab was used for both nares. The swab was pre-moistened with sterile saline and was inserted in the nasal vestibule, introducing only the cotton part of the swab. The operator rotated the swab while circulating in the nasal vestibule for approximately 5 seconds. This procedure had to be repeated in both nares.To detect S. aureus was isolated from either a normally sterile site  or cultures obtained for clinical purposes . The Clinical Microbiology Laboratory at BCH processed both surveillance and clinical specimens. S. aureus was identified based on colony morphology and the coagulase test . PCR was used to detect the mecA gene [mecA gene positive were considered MRSA. All S. aureus isolates were stored at -20° C.Infants showing suggestive clinical symptoms were considered infected if ecA gene . IsolateecA gene , and whimec (SCCmec) typing [spa) typing [spa types were assigned using the Ridom Staph Database  [https://pubmlst.org/saureus/) [sasX [For molecular studies, isolates were cultured on blood agar overnight at 37°C. DNA was extracted and used to perform staphylococcal cassette chromosome ) typing , multi-l) typing . The spaGermany) . Sequencaureus/) . Additio/) [sasX  and for S. aureus culture filtrate preparations, which we obtained from early logarithmic-phase growth (6 hours of incubation). These were used to assess cytotoxicity, as described previously [S. aureus isolates were grown in 96-well, round bottom plates in tryptic soy broth for 16–18 hours with shaking at 37°C. Cultures were then diluted 1:75 with fresh Roswell Park Memorial Institute with casamino acids, and 150 μL of the diluted culture was regrown in 96-well, round-bottomed plates for 6 hours at 37°C. Plates were centrifuged and culture supernatants were collected and stored at −80°C for future use. We assayed cytotoxic activity of the culture supernatants using the human myeloid cell line HL-60, differentiated into neutrophil-like cells (PMN-HL60). These have been shown to mimic the sensitivity of human neutrophils to S. aureus. Twenty microliters of the S. aureus supernatant were incubated with approximately 1.0×105 PMN-HL60 cells in a final volume of 100 μL (20% v/v supernatants) for 2 hours at 37°C, followed by 2-hour incubation with the CellTiter reagent, monitoring metabolic activity. This dye stains living cells brown, and the intensity of binding was measured in a spectrophotometer at 480nM. Values were compared to positive and negative controls ). We assayed each sample in triplicate and independently repeated the assay for each isolate at least twice. If >10% variation was observed between triplicate samples, we repeated the assay.We prepared eviously . In brieS. aureus ATCC 29213 was used as the quality control.In addition to cefoxitin testing carried out by disc diffusion as outlined above, antimicrobial susceptibility to other antibiotics was determined by the agar dilution method, in accordance with the CLSI . MRSA anThe characteristics of colonized and non-colonized neonates were compared using Chi-squared and Fisher’s exact tests, as appropriate. Colonized infants with concurrent infections were excluded from this analysis. Continuous variables, such as birthweight, were assessed using Mann Whitney U test.S. aureus colonization were assessed by comparing the characteristics of colonized vs. non-colonized neonates. For this analysis, birthweight, in grams, was categorized into quartiles . Factors with p<0.10 in bivariate analysis were then assessed in a multivariate logistic regression model to determine risk factors for overall S. aureus colonization. To determine independent risk factors for MRSA or MSSA colonization, a multivariate multinomial logistic regression model was used. All statistical tests were two-sided and performed in SAS 9.4 ; a p-value<0.05 was considered significant.Categorical risk factors for Cytotoxicity was expressed as the percentage of cells killed, and the median was compared among MRSA versus MSSA isolates, isolates associated with infections versus colonization, and among the most common STs. Cytotoxicity analyses were performed in GraphPad Prism 7.04  using the Mann Whitney U test.S. aureus nasal colonization on admission. Overall, 17% (n = 92) of the 536 neonates had nasal colonization with S. aureus, 12% (n = 64) were colonized with MSSA and 5.2% (n = 28) were colonized with MRSA respectively. Four neonates were both colonized and infected. As previous work has shown differences in risk factors between S. aureus colonization and infection [p = 0.001).From May 2015 to March 2016, 536 hospitalized neonates were admitted to the BCH NICU, most of whom  were admitted from home after presenting as outpatients, another 16 neonates were transferred from other obstetric units within 24 hours of birth. All were swabbed for nfection  and to aS. aureus. Of the 20 S. aureus infections, 70% (n = 14) were infected with MRSA and 30% (n = 6) were infected with MSSA. Of the 20 infants who were culture-positive for S. aureus, one infant had two isolates from the eye and conjunctival secretions, respectively, and another infant had three isolates from the eye, conjunctival secretions, and sputum. Conjunctivitis  and omphalitis  were the most commonly diagnosed S. aureus infections, followed by pneumonia , impetigo , cellulitis , and septicemia , with 3 infants having more than one site of infection. Four infected neonates were also colonized (2 with MSSA and 2 with MRSA).In this cohort, 255 infants were admitted with suspected infections; of these 20 (7.8%) were culture-positive for S. aureus colonization are shown in S. aureus colonization, while antibiotic use in the week prior to admission was protective. Newborn provenance  was not associated with colonization. An additional adjusted logistic regression model assessing interaction between birthweight quartiles and neonate age was assessed, but the interaction term was not significant (p>0.05).Risk factors for p = 0.02), vaginal delivery (p<0.0001), and age days (p<0.0001) remained significant risk factors for MSSA colonization, while female sex (p = 0.02) and age (p = 0.001) remained significant risk factors for MRSA colonization. Antibiotic use remained protective for both MSSA and MRSA , while birthweight was not associated with either MSSA (p = 0.18) or MRSA (p = 0.1) colonization.In multinomial logistic regression, female sex  was the most common MSSA clone.MLST revealed 16 different sequence types (STs) among the 74 MSSA isolates , the mosspa types were identified, SCCmecIVa was the most common SCCmec type detected , followed by SCCmecV , SCCmecIVg , and SCCmecIII ; one isolate could not be typed. ST59-SCCmecIVa-t437  was the most common MRSA clone , 3 (17%) belonged to ST398. No MSSA or MRSA isolate harbored sasX. The molecular characteristics of the MRSA and MSSA colonizing and infectious isolates are summarized in Of 19 mec, spa types). For the 4th pair, we noted a single nucleotide point mutation in the tpi gene of MLST (ST217: 7-6-1-5-8-8-6 and ST22: 7-6-1-5-8-5-6).In this study, there were 4 neonates with both colonization and infection isolates. Genotyping revealed that for 3 pairs, both isolates were identical  were also susceptible to levofloxacin, 26% (19/74) to erythromycin and 16% (12/74) to penicillin. All 45 MRSA isolates were susceptible to rifampin, TMP-SMX, mupirocin, levofloxacin, vancomycin, linezolid and tigecycline; 98% (44/45) were also susceptible to gentamicin and fusidic acid (44/45).mecIVa-t437. Of the 3 other OS-MRSA, one was ST22 and two were ST1. One of the OS-MRSA isolates was recovered from a neonate with infection; the others were from colonization.Eight MRSA isolates (18%) were oxacillin-susceptible (OS-MRSA), five (63%) of these belonged to ST59-SCCp = 0.85) as shown in S. aureus isolates (median: 85%) was less than that of the 23 infectious isolates (median: 88%), p = 0.0008)  compared to ST188-t159 (median: 85%) and ST59-t437 . We then compared the distribution of data using the Mann Whitney U test between groups. The cytotoxicity of MRSA  and of MSSA  were similar ( 0.0008) . Of the mecIVa-t437 . The cytotoxicity of the 21 PVL positive isolates (median: 84%) and 98 PVL negative isolates (median: 86%) were similar (p = 0.98).The overall prevalence of PVL was 18/45 (40%) of MRSA and 3/74 (4.1%) of MSSA isolates, Most of PVL positive MRSA isolates belonged to ST59-SCCS. aureus at the time of admission to a tertiary care Chinese NICU in Beijing. On admission, 17% of neonates ≤28 days of age had nasal colonization with MSSA or MRSA. This rate was higher than previously reported for neonates within 6 days of birth in Japan (10%) [S. aureus case was considered community acquired if it was isolated from an outpatient or an inpatient within 48 h of hospitalization14, so all the S. aureus were more likely to have been transferred from the community. Furthermore, 7.8% of neonates admitted with suspected infections were culture-positive for S. aureus, 70% of which were due to MRSA. This contrasts with previous reports in the United States which MSSA represented a greater proportion of S. aureus infections than MRSA [S. aureus into the NICU, and thus served as potential reservoirs of pathogens for other infants. This suggests that routine surveillance followed by targeted intervention strategies could be useful in reducing S. aureus infections. However, future studies should assess the effectiveness of surveillance and decolonization in identifying high-risk infants and/ or targeting highly cytotoxic S. aureus clones in our NICU.To our knowledge, this is the first study to assess the burden and molecular epidemiology of an (10%) , neonatean (10%) , and neoan (10%) . In the an (10%) –22. Our han MRSA . Our finS. aureus colonization in the NICU population, including prematurity and intubation, few have examined the effect of age on colonization risk [S. aureus colonization with the current study design. We also found that higher birthweight quartiles were associated with an increased risk of S. aureus colonization, which contrasts with previous literature [S. aureus occurs, but is more relevant for MSSA than MRSA, presumably because fewer women are colonized with MRSA.While others have assessed factors associated with ion risk , 6. We fion risk . As the terature ; we beliterature , while aterature . Howeverterature . Similarterature . These fIn contrast, neonates who had received antibiotics within seven days of admission were at decreased risk for both MSSA and MRSA colonization. Notably, 188 (37%) neonates had received antibiotics within seven days of admission, as many infants had suspected infections managed as outpatients. Oral antibiotics have not been shown to eradicate MRSA colonization in hospitalized adults . Howeverspa and STs were diverse, as noted in previous studies [MSSA  studies . However studies . ST188 v studies . Additio studies . ImportamecIVa was the most common MRSA clone, consistent with previous studies of epidemic MRSA clones across Asia [S. aureus [S. aureus were ST398. As previous studies have primarily focused on adult patients, the current study may help elucidate the role of ST398 in S. aureus infections in the neonatal population.ST59-SCCoss Asia  and of coss Asia . In thisoss Asia , but hasoss Asia –36. In C. aureus . Here, wsasX, which has been implicated in epidemic spread of MRSA across China and has been demonstrated to play a key role in colonization and pathogenesis, including promotion of immune evasion [sasX was not detected in the current study, potentially because no neonates harbored ST239, the dominant global healthcare-associated MRSA clone which also predominantly carries sasX [We also explored the presence of the virulence gene  evasion . Howeveries sasX .mecIV-t437 was the most common OS-MRSA clone (63%), which is consistent with a recent study from China which showed that the most frequent OS-MRSA clones were ST338-t437-SCCmecV (32%) and ST59-t437-SCCmecIV/V (21%) [mecVT was the most prevalent OS-MRSA clone in a study from Taiwan [S. aureus infections. Surveillance for such emergent strains should thus be a public health priority.Overall, 16% of MSSA strains were penicillin-susceptible. A recent study in Massachusetts found a 3-fold (13% to 32%) increase in penicillin susceptibility among MSSA bloodstream isolates over a decade, speculated to be the result of less selective pressure by β-lactam agents . Future /V (21%) . Likewism Taiwan . Howeverm Taiwan , potentiS. aureus was higher in infectious than in colonizing isolates. Universal decolonization targeting MRSA has been attempted in different healthcare settings to reduce the incidence of infections. These studies have had mixed outcomes. Potential downsides of this approach include the emergence of resistance. Moreover, in many settings MSSA account for higher infection rates than MRSA. We therefore need additional tools to better risk stratify S. aureus clones that might warrant targeted decolonization, using bacterial determinants other than MRSA status. Given recent investigations into the potential role of virulence ascertained using comparable in vitro assays [S. aureus isolates to better understand if these are potentially useful markers to consider in future decolonization programs. We recognize that our study is not designed to fully answer this question; however, we believe that these data provide important insights into the distribution of cytotoxicity of S. aureus isolates in this high-risk population. Our observations differs from a recent report by Maisem et al. [S. aureus toxicity and disease severity when comparing colonizing isolates and those isolated from SSTIs or bacteremia in adult patients. They suggested that bacterial fitness in human serum could explain the unexpected association of low-toxicity isolates with severe, invasive disease. Similarly, Rose et al. [S. aureus, as ST398-t571 exhibited significantly lower cytotoxicity than the two most common clones, as well as all other clones combined. The most common ST398 spa-type in our study was t571, associated with human colonization and infection in the US and China [S. aureus background. The association between cytotoxicity, clinical presentations, and outcomes should be studied to further elucidate S. aureus pathogenesis. Such studies could also have important clinical implications and support targeted rather than universal decolonization of neonates colonized with strains with specific molecular and virulence properties.Furthermore, we found that cytotoxicity of o assays  we beliem et al. , who foue et al.  reportede et al. . We alsond China , 46–48, S. aureus isolates were collected from neonates upon admission, we could not explore hospital transmission of MSSA or MRSA, nor ascertain whether colonized neonates subsequently developed infections. Surveillance swabs were directly plated onto blood agar plates and did not contain an enrichment step, potentially leading to an underestimation of MRSA carriage. Furthermore, given the study design, we could not assess the potential relationship between cytotoxicity and subsequent infections or clinical outcomes. Because surveillance cultures were only obtained from the nares of neonates and not from other body sites, it is possible that we underestimated the true proportion of colonized neonates. We may have also underestimated infections caused by S. aureus, as pre-admission antibiotics could have resulted in negative clinical cultures.Several limitations to our study need to be considered. This was a single NICU cohort study with unique admission patterns, which limits the generalizability of our findings. Since all S. aureus in neonates was high in the NICU of BCH. Female sex, age 7–28 days, higher birthweight and vaginal delivery were risk factors for colonization. While MSSA more frequently colonized neonates, MRSA more frequently infected neonates. Future studies are needed to determine potential sources for MRSA acquisition in these neonates. Most S. aureus strains were community-associated, reflective of NICU admission patterns. Isolates associated with clinical infection exhibited higher cytotoxicity than colonizing isolates. Our findings suggest that active surveillance of neonates for S. aureus should be considered as part of strategies to detect importation and prevent transmission of both MRSA and MSSA within the NICU.In conclusion, the nasal colonization rate of S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Bergeyella cardium infection is becoming increasingly prevalent in patients with infective endocarditis, suggesting its significance in disease pathogenesis. However, few studies have fully characterized this species.B. cardium HPQL strain isolated from a patient with infective endocarditis. Results from the cellular morphology and biochemical analysis provide basic knowledge on the new pathogen. The whole genome sequencing of B. cardium HPQL consists of a circular chromosome with a total length of 2,036,890 bp. No plasmid was detected. Comparative genomics were carried out then. Antibiotics resistance related genes, pathogenesis related genes, predicted insertion sequences, genome islands and predicted CRISPRs sequences were demonstrated. To our knowledge, this is the first study to provide a complete genome sequence for Bergeyella spp.Herein, we report the morphological and physiological characteristics, as well as whole genome sequencing of a newly identified B. cardium. Our results provide insights into effective clinical diagnosis and treatment of this pathogen.This study provides fundamental phenotypic and genomic information for the newly identified fastidious infective endocarditis causative bacteria,  Streptococcus, Staphylococcus, and Enterococcus are the primary causative organisms of infective endocarditis [Bergeyella spp. [Infective endocarditis is a serious infectious disease with high associated morbidity and mortality. Identification of the causative agents is, therefore, crucial for improving the clinical outcome . Clinicacarditis . Howeverlla spp. –5.Bergeyella spp. are non-fermenting gram-negative bacilli, belonging to the family Flavobacteriaceae [B. zoohelcum, known to cause cellulitis, leg abscess, tenosynovitis, septicemia, pneumonia, and meningitis, is one of the best described zoonotic pathogens afflicting humans [B. zoohelcum is usually isolated from the normal oral microflora of animals such as cats and dogs [B. zoohelcum [B. zoohelcum bacteremia after eating food prepared with coagulated goat blood [B. zoohelcum infection was also reported in a tsunami victim [Bergeyella spp. are rarely reported. A previously uncultivated Bergeyella sp. (clone AF14) with strong homology to a previously reported uncultivated oral Bergeyella strain was suspected to be an opportunistic pathogen during preterm birth [Bergeyella strains was reported from patients with infective endocarditis. Both strains shared 94.9% homology with B. zoohelcum, suggesting that they are a new species belonging to of the genus Bergeyella. The two strains were designated as Bergeyella cardium13-07T and Bergeyella cardium13–16 [Bergeyella strain from an infective endocarditis patient that had 98.2% shared identity with B. zoohelcum, which was slightly lower than the ≥99.0% homology required for two organisms to be considered the same species [Bergeyella sp. was isolated from a patient with infective endocarditis. The organism was determined to be genetically most closely related to B. cardium [B. cardium prosthetic valve endocarditis was also reported quite recently [eriaceae . B. zoohg humans , 7. B. zand dogs . Hence, oohelcum . In addiat blood , 10. A ci victim . Alternarm birth . Furtherclone AF1 with str cardium . Moreoverecently .B. cardium infection in patients with infective endocarditis suggests its importance in disease pathogenesis. However, studies examining the microbial characteristics and genetic features of this species are very rare. In this study, we therefore, sought to describe the isolation, identification and characterization of a new B. cardium sp. from blood cultures of a patient with infective endocarditis. We also performed whole genome sequencing and, through phylogenetic analysis, we were able to predict the possible origin of this newly identified species.The increasing number of cases of B. cardium. To further verify the nucleotide BLAST results, a detailed phylogenetic tree was constructed as shown in Fig. B. cardium strains that had been isolated from two infective endocarditis patients in Korea [B. cardium. We, therefore designated the isolated strain in our study as B. cardium HPQL .Four days after the initial blood culture was obtained from a 63-year-old man with infective endocarditis, the growth of microorganisms was reported through an automated blood culture system. Gram staining revealed the presence of gram-negative bacilli. However, no reliable identification was made by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Thus, to accurately identify the pathogenic species, a 1425 bp sequence from the 16S rRNA gene of the isolated strain was amplified and sequenced via polymerase chain reaction (PCR). The sequence was then submitted to NCBI BLASTN to identify matched bacterial sequences. Results revealed that the sequence with the highest homology (approximately 99%) was isolated from B. cardium HPQL were observed on blood agar after 48 h of incubation at 35 °C , including 9 rRNAs, 42 tRNAs and 1 sRNA. A total of 1,813,065 predicted coding sequences were identified, which occupied 89.01% of the whole genome sequence. Additionally, 70.25% (1332/1896) of the protein-coding genes were assigned putative functions in the COG database, while the remaining genes were annotated as encoding hypothetical proteins. The distribution of genes in COGs functional categories are presented in Table The whole genome of the newly isolated B. cardium HPQL. Twelve genes were identified homology to well-known antimicrobial resistance genes , B. zoohelcum ATCC 43767, B. zoohelcum CCUG 30536, B. zoohelcum NCTC 11660 and B. zoohelcum NCTC 11661 genome also demonstrated the evolutionary divergence of B. cardium HPQL from B. Zoohelcum spp.  until a positive result was obtained. The positive blood cultures were inoculated onto Columbia blood agar, MacConkey agar, and Chocolate agar and incubated  at 35 °C until visible colonies appeared. Colonies were purified using blood agar for further analysis.http://www.ncbi.nlm.nih.gov) and aligned using the ClustalX computer program. The aligned sequences were refined and phylogenetically analyzed using distance/neighbor joining (NJ) and maximum-likelihood (ML) algorithms with the Poisson correction distance model in the MEGA software package [The nucleotide sequences of 16S rRNA genes from different bacterial strains were downloaded from the NCBI database  and PDM Epsilometer test (E test) were employed to determine the antibiotic susceptibility of the isolated strain. For the Vitek 2 system, the cell density of the bacterial colony was adjusted to a density of 0.5 McFarland with 0.45% saline; 145 μL of the bacterial suspension was then added into 3 mL of 0.45% saline solution to further adjust the bacterial cell density. The suspension vials were then applied to the Vitek GN09 card and loaded into the Vitek 2 automated reader-incubator for analysis. For the E test, the 0.5 McFarland bacterial cell suspension were surface plated onto Blood agar plates, using a sterile swab to produce an even inoculum . The plaGenomic sequencing and assembly were carried out at Novogen Bioinformatics Technology Co., Ltd. . Single-molecule real-time (SMRT®) sequencing was performed using a Pacific Biosciences RSII sequencer  according to the manufacturer’s instructuions  using P4-C2 chemistry. The low-quality reads were filtered by the SMRT 2.3.0 and the filtered reads were then assembled to generate one contig without gaps. Hierarchical Genome Assembly Process (HGAP) pipeline was used for the whole genome assemble.https://www-is.biotoul.fr/blast.php) was used to predicted IS sequences while CRISPRdigger (https://omictools.com/crisprdigger-tool) [http://phaster.ca) and IslandPath-DIOMB [http://genoweb1.irisa.fr/duals/RASTA-Bacteria/) was used to identify toxin-antitoxins. The methylation data had been submitted to REBASE database for restriction modification system analysis.The assembled genome sequence was annotated further. Small RNAs (sRNAs) were predicted by BLAST against the Rfam  databaseer-tool)  was usedth-DIOMB  was usedThe genome sequences of the HPQL bacterial strain were submitted to the Virulence Factors of Pathogenic Bacteria (VFDB) , ComprehB. cardium HPQL genome, B. cardium (downloaded from NCBI PRJNA490389) B. zoohelcum ATCC 43767 genome (downloaded from NCBI), the B. zoohelcum CCUG 30536 genome (downloaded from NCBI), B. zoohelcum NCTC 11660 genome (downloaded from NCBI), and B. zoohelcum NCTC 11661 (downloaded from NCBI). Core genes and specific genes were analyzed via CD-HIT rapid clustering of similar proteins software with the threshold set to 50% pairwise identity and a 0.7 cutoff in length difference of amino acids [Comparative genomic analysis was performed between the no acids , 33, 34.no acids  accordinB. cardium HPQL was deposited in GenBank under the accession numbers CP029149.The obtained genome sequence for Additional file 1: Table S1. The predicted CRISPR sequences in the genome of B. cardium HPQL.Additional file 2: Table S2. The IS sequences in the genome of B. cardium HPQL.Additional file 3: The methylation data of the whole genome.Additional file 4: The predicted genes related to antibiotic resistance.Additional file 5: Genes related to putative virulence factors.Additional file 6: Genes related to bacteria-host interactions.Additional file 7: The predicted genome islandsAdditional file 8: The predicted toxin-antitoxins


Sambucus, a group of herb plants, possesses a broad spectrum of medicinal properties such as antioxidant, insulin-like activity, anticancer and antiviral. However, the study about its activity toward aplastic anemia incidence is based on limited data. Thus, the research aim of this study was to evaluate the immunomodulatory activities of Sambucus javanica in chloramphenicol-induced anemia aplastic mouse model. In this present study, BALB/c mice were administrated with chloramphenicol (CMP) to induce aplastic anemia then followed by S. javanica extracts treatment. Additionally, cellular and molecular aspects were evaluated by flow cytometry and Hematoxylin-Eosin staining. Further analysis showed that S. javanica extracts could promote the population number of regulatory T-cells and naive cytotoxic T-cells. Moreover, those extract also reduced the inflammation and necrotic incidence in CMP-induced mouse aplastic anemia model. Together, these results suggest that S. javanica has therapeutically effect to aplastic anemia by altering the immune system as an immunomodulatory agent.Aplastic anemia, life-threatened disease, is a hematologic disorder characterised by bone marrow hypoplasia. Multiple modalities such as bone marrow transplantation and immunosuppression treatment have been proposed to ameliorate this entity, however it remains ineffective.  S. javanica extracts promoted CD4+CD25+CD62L+ regulatory T-cells and CD8+CD62L+ naive T-cells in aplastic anemia mice model.S. javanica extracts repressed inflammation rate of CD4+TNF-α+ cells and CD4+IFN-γ+ in aplastic anemia mice model.S. javanica extracts reduces the incidence of necrosis in renal section of aplastic anemia mice model.The immunomodulatory effect of Aplastic anemia is a clinical syndrome which caused severe damage to hematopoietic precursor cell compartment in bone marrow . This enSambucus is a group of herbs that contains bioactive-rich compounds such as phenolic acid and flavonoid. Due to its compounds, Sambucus was considered as a medicinal plant for a long time ago by folk-society  to induce acquired-aplastic anemia. Moreover, berries and leaves extract of The spleen was isolated from experimental mice, then followed by homogenisation for intracellular and extracellular immunostaining procedure as similar as our previous study  staining with the similar protocol as our previous research  production through nitric oxide synthase (NOS) activation . AdditioS. javanica extracts reduces the incidence of necrosis in experimental mouse (Dioscorea zingiberensis can prevent pancreatic necrosis induced by sodium taurocholate. Interestingly, Parinari curatellifolia toward hepatic necrosis in acetaminophen-induced rats.Moreover, the most remarkable point to emerge from the data is the CMP administration can induce necrosis in the renal. However, the immunomodulatory effect of al mouse . Necrosial mouse . The CMPal mouse . HoweverS. javanica exert their immunomodulatory activities by increasing the population number of regulatory T-cells and naive cytotoxic T-cells. Additionally, those extracts slightly decreased the inflammation and necrotic incidence in CMP-induced mouse aplastic anemia model. Finally, these results suggest that S. javanica has therapeutically effect to aplastic anemia by altering the immune system as an immunomodulatory agent.This study has explained that leaves and berries extract of


Medicago truncatula is a model legume plant; however, investigations of MtCML proteins are limited. Using genome analysis and BLAST database searches, fifty MtCML proteins that possess EF-hand motifs were identified. Phylogenetic analysis showed that CML homologs between M. truncatula, Arabidopsis thaliana and Oryza sativa shared close relationships. Gene structure analysis revealed that these MtCML genes contained one to four conserved EF-hand motifs. All MtCMLs are localized to eight chromosomes and underwent gene duplication. In addition, MtCML genes were differentially expressed in different tissues of M. truncatula. Cis-acting elements in promoter region and expression analysis revealed the potential response of MtCML protein to abiotic stress and hormones. The results provide a basis of further functional research on the MtCML gene family and facilitate their potential use for applications in the genetic improvement on M. truncatula in drought, cold and salt stress environments.Calcium is an important second messenger in mediating adaptation responses of plants to abiotic and biotic stresses. Calmodulin-like (CML) protein is an important calcium-signaling protein that can sense and decode Ca Diversentration . The CBPn plants ,5,6. A tn plants , while as a hand . CMLs arbidopsis , rice [1bidopsis , grapeviurensis) , Chineseca rapa)  and tomaersicum) .2+ fluctuations and signal transduction [Arabidopsis, CML15 and CML16 show changes in electrophoretic mobility in the presence of Ca2+, which confirms their ability to bind Ca2+ [Arabidopsis [CML9, CML24, CML37, CML38, and CML39 transcripts are induced by salt or/and drought stress [Glycine soja [CMLs regulate downstream targets in response to various stimuli-induced Casduction . In Arabind Ca2+ ,18,19. Cbidopsis ,21,22,23bidopsis . The othbidopsis ,27,28,29bidopsis ,31,32,33t stress ,35. CML2t stress ,36. OsCMM. truncatula is an important model leguminous plant [M. truncatula by downregulating MtHKTs expression and leading to Na+ toxicity [M. truncatula including gene structure, chromosomal location, duplication, EF-hand motif organization and expression characteristics. The results provide a comprehensive understanding of the CML gene family in M. truncatula.us plant . MtCML40CML genes was performed using the BLASTP program based on the completed genome sequence of M. truncatula, using Arabidopsis and rice CML genes as the query sequences. Fifty MtCML genes were obtained and their deduced peptides were subjected to domain analysis using Pfam and SMART databases for further confirmation. A total of 50 members were identified, and they were named as MtCML1 to MtCML50 (A genome-wide search for  MtCML50 . The ami MtCML50 . The pre MtCML50 .M. truncatula (50), Arabidopsis (50) and rice (32) were aligned using the maximum-likelihood (ML) method to generate an unrooted phylogenetic tree ; three introns in four MtCMLs , four in MtCML11 and six in MtCML34 and MtCML38  contained four conserved EF-hand motifs, and seven members contained three EF-hand motifs. Two EF-hand motifs were present in nine MtCML members , while one EF-hand motif in MtCML28 B.MtCMLs were mapped onto the chromosomes against the M. truncatula genome database to examine their chromosomal distribution. MtCMLs were located on chromosome 1 to 8. Ten MtCMLs  were located on chromosome 1, while nine  on chromosome five. There were seven MtCMLs locating on chromosomes four, seven and eight, respectively. In addition, five MtCMLs  were located on chromosome three, three  on chromosomes six and two (MtCML11 and MtCML12) on chromosome two  had four homologous genes in M. truncatula. In addition, a synteny analysis of CML genes among M. truncatula and A. thaliana was performed. Twenty pairs of orthologous CMLs were identified between M. truncatula and A. thaliana  had two homologous genes in A. thaliana, while two genes (MtCML12 and MtCML18) had three orthologous genes in A. thaliana. The percentage of identity between pairs of paralogous MtCML proteins ranged from 21.69% to 100% in M. truncatula . Twenty-nine MtCMLs have corresponding probe sets in the dataset   were highly expressed in floral organs  A, indical organs A. On the qRT-PCR B–G. MtCMin seeds B; MtCML1nd seeds C. MtCML1and pods D. MtCML2and pods E, MtCML2in roots E. MtCML4n leaves F. The exMtCML genes, a 1000-bp sequence upstream start codon in the promoter region of MtCMLs was analyzed using the PlantCARE. The major cis-acting elements related to stress and phytohormone responses are shown in MtCMLs, while salicylic acid (TCA-motif), gibberellin (GARE-motif), circadian, drought (MBS) and cold (LTR) response elements were also found in the promoters  have drought response element, and six MtCMLs  have cold response element in the promoter , three salicylic acid responsiveness elements (TCA-element) and one light responsiveness (Box 4) were observed in the promoter region of MtActin . There are 29 MtCML genes that have corresponding probe sets in the dataset [MtCMLs expression patterns were changed in response to salt and drought stress  [CML genes) [CML genes), Chinese cabbage (79 CML genes) [CML genes) [2+-binding properties and their responses to plant hormones and environmental stresses [Arabidopsis CML proteins typically possess two to six EF-hand motifs [MtCML genes have no intron. This case was consistent with AtCMLs, OsCMLs and BrCMLs [Camodulin-like proteins play a key role in signal transduction during plant growth and development . A totalmembers) , but difL genes) , grapeviL genes)  and tomaL genes) . MtCMLs L genes) ,43. The stresses ,44. The d BrCMLs ,12.Arabidopsis and rice. In consistence, most of BrCMLs from Brassica rapa were also closely related to their corresponding homologs in Arabidopsis and rice [MtCML genes were randomly scattered on different chromosomes. Homologous gene pairs were found in almost all MtCMLs. There are many homologous CML genes between the chromosomes in M. truncatula and Arabidopsis. Whole-genome duplication (WDG) in legumes played a major role in shaping the genome and contributed the raw material for the evolution of nodulation [M. truncatula has undergone high rates of local gene duplication and share an ancient round of gene duplications from other legume species [MtCML genes evolution.MtCMLs were classified into eight groups, which is likely similar to those in other plant species ,12,13. Pand rice . In our dulation ,46; M. t species ,47. TherMtCML genes are presumed to be associated with their potential biologic roles. Four MtCMLs  were highly expressed in floral organs, indicating that they are associated with flowering regulation. This can be supported by that AtCML25, the homolog of MtCML25, is involved in pollen germination and pollen tube elongation via regulating Ca2+ and K+ transmembrane trafficking in pollen grains and pollen tubes in Arabidopsis [MtCML17 and MtCML22 were highly expressed in roots, indicating that they are associated with roots in M. truncatula. However, qRT-PCR showed that MtCML14 and MtCML47 were mostly expressed in leaves and seeds rather than roots  and nearly half of the MtCML genes showed extensive responses to salt and drought stresses, respectively. Similar to these results, studies have shown that AtCML9 was induced under dehydration treatment [AtCML9, AtCML37, AtCML38, AtCML39 and OsMSR2 (a rice CML) were up-regulated substantially following salt stress [MtCML40 is not found in the database, its expression was induced by salt treatment and negatively regulates salt tolerance in M. truncatula [MtCML genes showed response to cold stress. AtCML24 transcript is induced after cold treatment in Arabidopsis [Solanum habrochaites) CML gene, ShCML44, was found to improve the tomato tolerance in cold, drought and salinity stresses [MtCML genes in M. truncatula.The temporal, spatial, and cell type-specific expression of genes are associated with the regulatory elements in the promoter region . Some pre AtCML9 , AtCML20[AtCML20 , AtCML37[AtCML37  and MtCMt stress ,50. Althbidopsis . The tomstresses . These pM. truncatula Mt4.0v1 protein sequences were downloaded from Phytozome 12 (https://phytozome.jgi.doe.gov/). The reported CML gene sequences in A. thaliana were downloaded from the TAIR database (https://www.arabidopsis.org/) and used as a query to perform BLASTP searching. The NCBI database was used to search the potential CML genes in M. truncatula. In addition, the protein sequences of OsCMLs was downloaded from TIGR (http://rice.plantbiology.msu.edu/) and used as queries to search against the M. truncatula proteome. To differentiate CaM and CML genes which were homologs in M. truncatula, we follow the principle of the major character of EF-hand-containing proteins [http://smart.embl-heidelberg.de/) and InterProScan (http://www.ebi.ac.uk/interpro/).The proteins ,43 and shttp://web.expasy.org/protparam/). The exon–intron structure analysis of MtCML genes was conducted using the TBtools (Toolbox for Biologists) program with default parameters. The conserved motifs were analyzed using the MEME tool (http://meme-suite.org/tools/meme), with the minimum width of motifs as 10, the maximum width of motifs as 40 and the other parameters as default values.The MtCML protein sequences were analyzed for physical and chemical characteristics, including the molecular weight (MW), theoretical point (pI) and grand average of hydropathicity (GRAVY), using the ProtParam tool of ExPASy  method 1000 bootstrap replicates [The multiple alignments of MtCML proteins were performed using ClustalX with default parameters. Phylogenetic analysis of CMLs in plicates .MtCML were used to retrieve their chromosomal locations in M. truncatula genome databases Phytozome 12 (https://phytozome.jgi.doe.gov/). TBtools software was used for analyze the chromosomal locations and homologous relationship of MtCML genes [The sequences of ML genes . The MulML genes .MtCML was obtained from the Phytozome database (https://phytozome.jgi.doe.gov), and the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [cis-acting elements in the promoter region of MtCMLs.A 1000 bp of genomic DNA sequence upstream of the transcriptional start site in each e/html/)  was usedM. truncatula in different tissues at various developmental stages and the response to drought and salt were retrieved from M. truncatula Gene Expression Atlas . The transcript data of MtCMLs were analyzed using TBtools software. The normalized expression data were used to generate heatmap using the TBtools software [The genome-wide microarray data of software .M. truncatula  A17 plants were were grown in 15 cm-diameter plastic pots containing a mixture of peat and perlite  in a greenhouse with temperature ranging from 20 to 28 °C under natural light. Four-week-old plants were exposed to low temperature in a growth chamber at 4 °C for 24 h under 12 h light as cold treatment. Total RNA was extracted using RNAprep pure Plant Kit  according to the manufacturer’s instructions. The cDNA was synthesized from 1 μg of total RNA, using the PrimeScript RT reagent Kit with gDNA Eraser . MtCML genes transcript were analyzed using qRT-PCR in Thermal Cycler Dice Real Time System II  following the manufacturer’s instructions. The PCR reaction mix was consisted of diluted cDNA as template, 200 nM forward and reverse primers and 5 μL SYBR Premix Ex Taq . Parallel reactions to amplify actin were used to normalize the amount of template. Relative expression was calculated by 2−ΔΔCt. The expression levels in the control plants without treatment (0 h) were normalized to 1. The MtActin gene was used as the internal control. The qRT-PCR primers listed in http://www.idtdna.com/Primerquest/Home/Index).MtCML genes were identified in M. truncatula, which were divided into eight groups. Analysis of intron–exon structure revealed that CML gene family is evolutionarily conserved. The functional divergence between subgroups was mainly attributed to changes in the EF-hand domains, which may confers functional differentiation of MtCMLs. Synteny analysis showed that WGD and tandem duplication probably participated in driving MtCML genes evolution. Expression profile analysis showed that most of MtCML genes are expressed in leaves, flowers and roots and had a tissue and organ expression preference. Many cis-acting element responsive to plant hormones, such as ABA, MeJA and auxin were enriched in the promoter of MtCMLs, while salicylic acid (TCA-motif), gibberellin (GARE-motif), circadian, drought (MBS), and cold (LTR) response elements were found in the promoter of some MtCMLs. Many MtCMLs were regulated by drought and salt stresses. qRT-PCR analysis showed that MtCML16 and MtCML33 were upregulated, MtCML2, MtCML6, MtCML19 and MtCML47 were downregulated in response to cold treatment. Our results provide new information for further understanding of the structure, evolution and function of MtCML gene family.A total of 50


MyoRobot to compare passive visco-elasticity and active contractile biomechanics in single fibers from fast- and slow-twitch muscles from adult to senile mice, hetero- or homozygous for the R349P desmin mutation with wild type littermates. We demonstrate that R349P desmin presence predominantly increased axial stiffness in both muscle types with a pre-aged phenotype over wild type fibers. Axial viscosity and CaMuscle biomechanics relies on active motor protein assembly and passive strain transmission through cytoskeletal structures. The desmin filament network aligns myofibrils at the z-discs, provides nuclear–sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness. As the effect of R349P desmin on axial biomechanics in fully differentiated single muscle fibers is unknown, we used our  Skeletal muscle is the largest organ system of the body and under constant mechanical axial and lateral stress, either due to passive strain or through active contraction. While lateral forces are distributed between single fibers across anchorage points in the extracellular matrix (ECM) to the intracellular cytoskeleton via the dystrophin–glycoprotein complex  and focar strain ,4, the cr strain ,6,7. In r strain ). Serialr strain .In skeletal muscle, IFs form a huge stress-transmitting and stress-signaling network, in which desmin is important to maintain myofibrillar alignment, nuclear positioning and shape, stress production, and sensing ,10. Due Musculus soleus (SOL) fiber bundles from young het and hom R349P desmin mice, showed a marked increase in passive stiffness compared to wt bundles [Musculus extensor digitorum longus (EDL) and also included a wide age range from young (17–23 weeks) to aged (60–80 weeks) animals [MyoRobot biomechatronics system which features sensitive force transducer (FT) technology, high precision voice coil (VC) actuation, and automated chemical solution exchange to capture disease-related influences on active and passive forces [Our previous work already demonstrated that the expression of R349P mutated desmin compromises the 3D arrangement and the order of the myofibrillar lattice in young mice. These findings suggested a pre-aged phenotype of muscle structural aging in the R349P environment . Moreove bundles ,19. Cons animals . Again, e forces . Here, wMyoRobot-recorded force transients of a caffeine-triggered Caorce see B developorce see B. Unlikegenotype C. The rarmer see D. While rmer see C, caffeirmer see B. Withinrmer see D.To elaborate on the Caroup see B. Het fiThe Hill coefficients in EDL single fibers showed no significant differences regarding genotypes. Age, however, had a significant influence on het fibers between aged and senile animals. In SOL single fibers, differences were present among genotypes, with lower coefficient values for fibers expressing the R349P mutation (except het adult). Again, age had a strong influence on wt fibers, leading to a significant increase in the Hill coefficient in aged and senile fibers over adult fibers, indicating a higher Cabers see C. As theTo assess axial viscosity in single muscle fibers, we carried out ultra-fast stretch jumps as shown in bers see B. Analysbers see C. Howeveages see D, arguinThe observed increased passive stiffness in R349P mutant desmin single fibers suggests a negative influence on muscle contraction kinetics, e.g., unloaded speed of shortening. To address this question, we performed so-called slack-tests. engths” . NotablDesminopathies comprise a heterogeneous group of inherited and sporadic myopathies which, in most cases, share a common morphological picture comprising sarcoplasmic and subsarcolemmal desmin-positive protein aggregates and signs of myofibrillar degeneration ,16,23. Imdx mice reported no difference when comparing single skinned SOL and EDL fibers from young (3–6 weeks) and adult (17–23 weeks) animals [Similar to previous studies in fiber bundles ,21, we oterature , but in terature . In thesterature . Last, a animals . The rec animals .MyoRobot system was not yet equipped with an optics system to measure the fiber diameter to normalize forces to cross-sectional area . Yet, the absolute single-fiber force levels presented here are well in the range of those reported by Stelzer et al. (2003) [Unfortunately, when this study was initiated, our . (2003)  in SOL f. (2017) , the meaterature .Regarding the Ca bundles . For SOLber type ,33. Unlie.g., in . Therefobers see B is in gvastus lateralis [In compliance with our previous assessment in small fiber bundles ,19,21, materalis . Thus, on < 3 for aged, n < 8 for senile) and large data scattering in these age cohorts . Previously, it was reported that the loss of desmin results in fiber stiffening, which was found unrelated to alterations in titin [Analysis of compliance in SOL single fibers also shows an augmented passive axial stiffness in het and hom R349P desmin knock-in single fibers. However, in contrast to results from EDL single fibers, it is the wt becoming more compliant with age. Although mutant SOL single fibers are significantly stiffer than their wt counterparts, which was also reported in literature , their cin titin . In the in titin  revealedin titin . The modin titin . In suchMyoRobot approach was able to extend our previous knowledge on R349P axial muscle stiffness to single fibers and also to include a larger age range, which unravelled aging effects in normal muscle as well. For instance, compliance values in wt EDL single-fiber preparations remained mostly stationary (up to the aged group) and only declined in old animals (senile group), while SOL fibers became more compliant (less stiff) with age. This is in agreement with a comparative study on murine tibialis anterior single muscle fibers and small fiber bundles, where single fibers from old mice showed a tendency towards reduced elasticity moduli  [tibialis anterior fiber bundles [vastus lateralis bundles [A direct comparison of bundles  and sin values) . Moreove bundles , which wA similarly increased modulus , our si wt mice  also pro wt mice . It is o wt mice .MyoRobot [MyoRobot system was not yet equipped with an optics system to assess sarcomere length distributions.With our implementation of a VC actuator within the MyoRobot , it was MyoRobot . This deMyoRobot . HoweverMyoRobot . Althougvastus lateralis skinned single fibers, shortening velocities were reduced in type IIA fibers but not type I fibers, while the opposite was found for women [In literature, age-related studies regarding unloaded shortening in single fibers are scarce. One of the few studies on rat EDL single fibers found an unchanged maximum shortening velocity in adult (9 mo) versus senescent (30 mo) animals, whereas SOL fibers from old rats  were sloor women . For muror women . Thus, wor women . Whetherhttp://www.informatics.jax.org/allele/ MGI:5708562) [Heterozygous (het) and homozygous (hom) littermates of the R349P desmin knock-in mouse model B6J.129Sv-Des5708562) ,45 were 5708562)  towards MyoRobot rack to a concentration of 0.1% (w/v).All muscle dissection was performed in Krebs solution containing (mM) 120 NaCl, 4.7 KCl, 1.2 KH uptake . To ini© -coated petri dish. Upon completing the dissection, Krebs solution was exchanged for HKS, allowing for 15 min equilibration, before single fibers were manually dissected with fine forceps.Mice were anesthetized via isoflurane inhalation and sacrificed by cervical dislocation. The hind limbs were cut off and transferred to Krebs solution. SOL and EDL muscles were dissected under a stereo microscope , while being pinned under slight stretch into a SylgardMyoRobot, a novel automated biomechatronics system combining high-precision VC actuation with force sensor technology [MyoRobot multi-well rack in a custom-made Perspex chamber while submerged in HKS solution, placed below the pins of the FT and VC, and fixed to both pins via a tweezer mechanism. For details on the biomechatronics system and sensor and actuation implementation, please refer to the work in [Caffeine-induced, Ca After fiber permeabilization, the fiber was shortly dipped into HR to wash off remaining saponin and to buffer internal Ca 5 s see .Ca The fiber was sequentially exposed to solutions of increasing Ca20 s see .Unloaded speed of shortening (slack test): The muscle fiber was held at resting length L kHz see .Passive stiffness—RLT curves: To assess passive axial stiffness, the muscle fiber was kept in LR solution to avoid active contraction. The fiber was continuously stretched at a slow speed  while plotted and statistically evaluated with SigmaPlot . All data traces were smoothed with a moving average filter. For pCa curves, the plateau force close to the end of each pCa step was determined by the software and plotted against the corresponding pCa value. The scatter plot of normalized force  was fitted to a four-parameter Hill equation (p < 0.05 were considered significant, <0.01 and <0.001 considered strongly and highly significant, respectively. Significance levels involving age effects were depicted as #, while genotype differences were depicted as §: wt vs. het, %: wt vs. hom, and @: het vs. hom, respectively.ints see . For speribed in . PassiveMyoRobot system allows a highly versatile and modular design of automated execution of various additional muscle test protocols, e.g., eccentric contractions, that shall be of great value to the community to facilitate future myopathy and mechanistic studies related to skeletal muscle and aging.Our results confirm an increased passive steady-state stiffness in R349P desminopathy skeletal muscle. Our experiments on single muscle fibers, void of ECM, suggest that compromised biomechanics properties in the R349P desminopathy do not only originate from increased fibrosis, but also from mutant desmin inflicted damage to the cytoskeleton. Particularly in fast-twitch fibers, this results in a pre-aged, stiffened phenotype. Apart from a yet unexplained acceleration of speed of shortening, especially pronounced in aged het fibers, Ca


Helicobacter pylori in gastric cancer as well as Fusobacterium nucleatum, Escherichia coli, Peptostreptococcus anaerobius, and Bacteroides fragilis in CRC. The transcriptional factor NF-κB plays a crucial role in the host response to microbial infection through orchestrating innate and adaptive immune functions. Moreover, NF-κB activity is linked to GI cancer initiation and development through its induction of chronic inflammation, cellular transformation and proliferation. Here, we provide an overview and discussion of modulation of the NF-κB signaling pathway by microbiota, especially infectious bacteria, in GI tumorigenesis, with a major focus on gastric cancer and CRC.Gastrointestinal (GI) cancers, especially gastric cancer and colorectal cancer (CRC), represent a major global health burden. A large population of microorganisms residing in the GI tract regulate physiological processes, such as the immune response, metabolic balance, and homeostasis. Accumulating evidence has revealed the alteration of microbial communities in GI tumorigenesis. Experimental studies in cell lines and animal models showed the functional roles and molecular mechanisms of several bacteria in GI cancers, including  Helicobacter pylori (H. pylori) significantly increases the risk of gastric cancer. The presence of Fusobacterium nucleatum (F. nucleatum), a gram-negative obligate anaerobic bacterium, can contribute to intestinal tumorigenesis , NF-κB is highly activated at the site of infection for its antimicrobial activity and maintenance of tissue homeostasis  and vacuolating cytotoxin A (VacA), the most intensively investigated virulence factors, play significant roles in gastric adenocarcinoma induced by H. pylori infection. The bacterium utilizes a type IV secretion system (T4SS) to inject CagA into host gastric epithelial cells. As a result, CagA is responsible for the dysregulation of cellular proliferation and apoptosis through disturbing the PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways  suggest their important role in GC progression , commonly colonize animals. Mongolian gerbils infected with H. felis developed premalignant gastric lesions  developed more severe gastric lesions and elevated levels of proinflammatory genes than H. pylori-monocolonized INS-GAS mice , Escherichia coli (E. coli), Peptostreptococcus anaerobius (P. anaerobius), and Bacteroides fragilis (B. fragilis) . F. nucleatum is highly increased in CRC patients compared to healthy individuals, and its abundance is closely related to a worse survival rate  mice significantly increased colonic polys compared with those in control groups  and has been found to play an important role in CRC tumorigenesis and development. Sears et al. revealed the increased abundance of the BFT gene in mucosal biopsies from patients with CRC compared to normal individuals. Furthermore, increased bft positivity was found in early to progressive CRC patients  induce intracellular signaling pathways in the innate immune response. TLRs and NOD1 are common PRRs involved in activation of the NF-κB signaling pathway  and mediates the hyperproliferation of gastric cells  extract suppressed the NF-κB pathway by decreasing the expression of NF-κB p65 and IκB, which in turn induced apoptosis and inhibited the growth of gastric cancer cells , i.e., ssp. animalis, ssp. fusiforme, ssp. nucleatum, ssp. polymorphum, and ssp. vincentii, which show the pathogenic differences. Among the five subspecies, ssp. fusiforme and ssp. vincentii are more frequently associated with health while ssp. nucleatum associated with diseases  is a major type of IBD and a known risk factor for CRC . F. nucla IL-17F . In addidiseases . F. nucldiseases , 113. Ade status .E. coli is one of the most common bacterial species that colonizes the human GI tract. Numerous studies have identified E. coli as a risk factor for the development of Crohn's disease, ulcerative colitis, and CRC. Enteropathogenic and enterohemorrhagic E. coli use a type III secretion system (T3SS) to transport dozens of effector proteins into host cells; these effector proteins in turn manipulate the host inflammatory response through activation of the NF-κB pathway , a P. anaerobius surface protein, interacts with the corresponding cell surface receptor integrin α2/β1. Subsequently, experimental data from CRC cells and APCMin/+ mice indicated that P. anaerobius challenge activated the PI3K/AKT and NF-κB signaling pathways via phosphorylation of the AKT and p65 NF-κB subunits. As a result, P. anaerobius modulates the tumor immune microenvironment, including the expansion of myeloid-derived suppressor cells, tumor-associated macrophages and granulocytic tumor-associated neutrophils. Cell proliferation and the proinflammatory response were significantly increased by P. anaerobius infection, further accelerating colorectal tumorigenesis  that collectively contributed to colonic carcinogenesis are ongoing. Moreover, the activated NF-κB signaling pathway is considered an important line of defense against microbial pathogens. Abnormal and sustained activation of NF-κB signaling contributes to malignant transformation from inflammation to cancer. Therefore, selectively targeting molecules of the NF-κB signaling pathway to block the association between pathogens and NF-κB shows therapeutic potential and benefit in cancer treatment. For example, specific NF-κB inhibitors targeting the IKK complex have shown promise as anticancer therapeutics  that investigate the interactive effect of some specific molecular features and environmental factors on disease prognosis and clinical outcome has been widely applied to cancer research , 126. ThNLi, CP, and NLu discussed the contents. NLi, CP, and YO wrote and edited this manuscript. NLi supervised and oversaw the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pleurotusostreatus and Trametesversicolor. Both fungi caused high mass losses in beech wood, while spruce and oak wood were more resistant to decay. The moisture content values of the decayed wood correlated with the mass losses for all three wood species and incubation periods. Combined microscopic and chemical studies indicated that the two fungi differed in their decay behavior. While T. versicolor produced a decay pattern  typical of white-rot fungi in all wood species, P. ostreatus caused cell wall erosion in spruce and beech and soft-rot type I (cavity formation) decay in oak wood. These observations suggest that P. ostreatus may have the capacity to produce a wider range of enzymes/radicals triggered by the chemical composition of wood cell walls and/or local compositional variability within the cell wall.Fungal wood decay strategies are influenced by several factors, such as wood species, moisture content, and temperature. This study aims to evaluate wood degradation characteristics of spruce, beech, and oak after exposure to the white-rot fungi  The three classes of fungi, which attack woody materials, are Basidiomycetes, Ascomycetes, and Deuteromycetes . White, Phellinus pini and Grifola frondosa cause selective delignification, whereas Fomes fomentarius and Trametes versicolor cause simultaneous degradation [Pleurotus ostreatus [Phanerochaete chrysosporium, can degrade wood using dual modes of degradation [Inonotus hispidus [P. ostreatus [Oudemansiella mucida that can produce soft-rot patterns in pine wood species under laboratory conditions [Brown-rot fungi produce only one morphological pattern, where the cell wall is transformed into a porous structure . White-rradation . Howeverstreatus ,11 and Pradation . Furtherhispidus  and P. ostreatus  can prodnditions . Trackinnditions ,17,18, anditions ,21,22,23nditions .T. versicolor and P. ostreatus, at different moisture contents and temperatures and showed that the moisture content was the major factor in influencing the degradation behavior of T. versicolor on Quercus castaneifolia, while the temperature was more important for P. ostreatus. The studies also revealed that T. versicolor caused a simultaneous decay pattern, while P. ostreatus caused both selective and simultaneous decay. However, in Q. castaneifolia wood blocks decayed by P. ostreatus at 85% relative humidity (RH) and 30 °C, there were signs of a soft-rot pattern.There are several environmental and physiological factors that influence fungal degradation, such as moisture content and temperature ,26, oxygT. versicolor and P. ostreatus are based on a comprehensive study, employing relevant chemical and microscopic techniques, and extend the knowledge on the influence of wood species on the behavior of white-rot fungi.With the hypothesis that wood types influence fungal decay behavior, we compared the decay characteristics of two white-rot fungi on three wood substrates. The results presented here on wood decay by P. ostreatus (Jacq.: Fr.) Kummer (isolate 107) and T. versicolor (L.: Fr.) Pilat (isolate 122) were obtained from Bari’s lab  Kummer were collected from a felled oak tree (Quercus castaneifolia C.A.M.). Morphological identification was performed according to Stancheva et al. [Two white-rot fungi, an, Bari ). Fruitia et al. , and mola et al.  and Baria et al. . In briePicea abies (L.) H. Karst.), Oriental beech , and Chestnut-leaved oak (Quercus castaneifolia C.A. Mey.), wood samples were cut 30 × 10 × 5 mm according to Bravery [i is the dry mass before decay (g); Md is the dry mass after decay (g); Mw is the wet mass after decay (g).To determine the rate of decay in Norway spruce . To assess the spectra, only the area between wavelengths 500 to 2000 cm−1 was examined as this region includes the most informative values for lignocellulose materials.Three replicates of decayed wood blocks from each incubation time-period with approximately the same mass losses were selected and ground using a hammer mill, so the wood powder passed through a 60-mesh screen. The power was dried at 103 ± 2 °C for 24 h. Approximately 1 g wood powder was used to acquire Fourier transform infrared (FT-IR) transmission spectra using a Shimadzu 8400 s spectrometer equipped with a deuterated, L-alanine doped triglycine sulfate (DLaTGS) detector. The samples were scanned in two replicates, using a Platinum Diamond Attenuated Total Reflectance (ATR) with a wavelength between 4000 and 400 cmP. ostreatus and T. versicolor for 60 days, small pieces (~1 × 1 × 3 mm) from decayed blocks of spruce, beech, and oak species were fixed in 2.5% v/v glutaraldehyde +2.0% v/v paraformaldehyde dissolved in 0.05 M sodium cacodylate buffer (pH 7.2) for 4 h at room temperature. All chemicals were purchased from Sigma-Aldrich . After washing three times in the buffer (20 min each), samples were dehydrated in a graded ethanol series (20–100%) and embedded in London Resin (LR) White . For LM, semi-thin resin sections (~1 µm) cut on a Reichert Jung ultra-microtome  were mounted on glass slides and stained with 1% w/v toluidine blue . Sections were observed using a DMBL Leica light microscope  equipped with an Infinity X-32 digital camera . For TEM, ultrathin sections (~90 nm) cut on the ultra-microtome were stained with 1% w/v KmnO4 in 0.1% sodium citrate . Sections were examined using a Philips CM12 TEM . TEM negatives were scanned using a film scanner .After incubation with P. ostreatus and T. versicolor are shown in T. versicolor causing slightly higher mass losses than P. ostreatus. Spruce and oak wood was slightly more resistant than beech wood to both fungi. The minimum mass loss obtained for both T. versicolor and P. ostreatus is 20%, which is necessary for beech wood, according to EN-113 [T. versicolor produces simultaneous white-rot, while Pleurotus species are known to cause selective delignification of solid wood and composite materials [P. ostreatus is often selected to determine wood composite durability [P. ostreatus under different environmental conditions and demonstrated the adaptive expression of those genes regulating different transcriptomic and enzymatic pathways. Recent studies have further shown that P. ostreatus is able to switch from selective to non-selective modes of degradation under both natural conditions and controlled [Average mass loss (ML) and moisture content (MC) for o EN-113  and ENV1o EN-113 . On the o EN-113 ,19, T. vaterials ,38. P. orability . Fernándrability  studied ntrolled ,40.Serpula lacrymans have special genes that may be active under certain conditions of temperature, pH, and moisture content to facilitate the enzymatic degradation of wood. In this context, Bari et al. [P. ostreatus produced different degradation patterns in oak wood under natural conditions and observed that this fungus causes Type I soft-rot (cavity formation) in oak wood. Other studies have also suggested that certain Basidiomycetes can produce soft-rot patterns in woody materials [According to Schmidt , some fui et al.  showed taterials ,15.P. ostreatus and T. versicolor deployed different strategies for degrading wood and utilizing nutrients. The relationship between ML and MC is shown in Our results indicate that P. ostreatus and T. versicolor 60 days. The FT-IR spectra of decayed and undecayed (control) wood samples are shown in FT-IR analysis was undertaken on spruce, beech, and oak wood samples, after exposure to −1 and 650 cm−1, and changes in absorption of some important bands in this region are shown in −1 and 1505 cm−1 are characteristic for lignin in hardwoods, as observed in this study for beech and oak samples. The 1504 cm−1 band shifted towards a higher wavelength at 1509 cm−1 in spruce  ≥ guiacyl (G), was lower than beech and oak samples at the same band located at 1594 cm−1 , the highest decrease was observed after 60 days of exposure of beech to P. ostreatus . In addition to the band at 1264 cm−1, which can be assigned to guaiacyl ring breaking, a C-O stretch in lignin was only observed for spruce. Alteration rates were very similar for both fungi at 1509 cm−1 assigned to guaiacyl ring breaking  had a higher decay resistance to both fungi   . This suT. versicolor. The ratio of the peak heights at wavelength 1504/1733 was 0.94 and 1.42, respectively, for control and wood exposed to T. versicolor over 60 days. This verifies that xylan degradation in beech was 51.06% higher after 60 days compared to the control  and oak wood exposed to the fungus for 15, 30, 45, and 60 days, respectively . The hyphae present in beech were much thinner than those present in oak  and latewood (LW)  A. Both wood (LW) C were deood (LW) B,C. By c than EW D. Thinni than EW F. Some E than EW E. The deP. ostreatus and T. versicolor are shown in T. versicolor was more advanced than for P. ostreatus (T. versicolor showed degradation of the middle lamella (ML) regions  [The patterns of cell wall degradation are more clearly resolved in the TEM images for streatus  and T. vrsicolor . Cavity . robur) .P. ostreatus, degradation of fibers and tracheids by T. versicolor occurred by the erosion of the secondary wall from the lumen outwards  was more resistant to decay by both fungi than the hardwoods, with mass losses between 6–10% for the 60-day incubation period. Both P. ostreatus can produce Type I soft-rot in oak trees under natural situations [Armillaria cepistipes [Inonotus hispidus [Fistulina hepatica [Coniophora puteana [The term “facultative soft-rot” proposed by Schwarze et al.  for a sotuations . Some Hypistipes , Inonotuhispidus ,50, Fisthepatica , and Con puteana ,52, are P. ostreatus and T. versicolor produced simultaneous white-rot in beech, while in oak, P. ostreatus caused Type I soft-rot degradation and T. versicolor consistently caused cell wall erosion typical of white-rot attack. Differences between the two fungi in the degradation of oak wood suggest cell wall composition and/or local variations in composition within the cell wall as the likely basis. Stages of advanced decay consist of a mass of vessel elements with only remnants of other cells adhering to the vessel walls. Degradation by various white-rot Basidiomycetes causes loss of fibers, fiber tracheids, and parenchyma cells but not vessels. The resistance of vessels to degradation appears to be due to their high 1ignin: Carbohydrate ratio, lignin monomer composition , and cell wall morphology [Fistulina hepatica; [Coniophora puteana). However, it was reported [The exact reason for the differences in the two fungal species deployed in our study is unclear, since the exposure conditions were the same. Spruce wood was more resistant than both hardwood species, yet both rphology . It has rphology ; e.g., Fepatica; ; e.g., Creported  that difT. versicolor consistently produced a morphological decay pattern reminiscent of simultaneous type white-rot decay in all wood types, the pattern produced by P. ostreatus varied from simultaneous type decay in spruce and both simultaneous and selective types of decay in beech to Type I soft-rot in oak. The switch between decay patterns by P. ostreatus suggests that some white-rot fungi have the genetic capacity to produce a wider range of enzymes and radicals, triggered by the chemical composition of cell walls and perhaps local variations in the molecular composition within the cell wall. Understanding the behavior of wood-rotting fungi with respect to the switch in decay pattern, as shown for P. ostreatus, will require knowledge of gene and protein expression in response to wood variability and other factor(s) that may influence fungal metabolism.From the results, it can be concluded that the wood species had an important impact on the decay behavior of the studied white-rot fungi. While