High Homology Research Articles

Genome-wide identification, transcript profiling and functional analyses of PCP gene family in Wucai (Brassica campestris).

Pollen coat proteins (PCPs) are cysteine-rich small-molecule proteins, which exhibit high levels of polymorphism and are expressed in gametocytes. Previous investigations have revealed that PCP genes are involved in pollen wall synthesis, pollen-stigma recognition, and pollen development and germination. However, gene expression and function of PCP family in pollen development is not well understood in Wucai (Brassica campestris L.). In this study, genome-wide identification and expression analysis of the BcPCP gene family members were conducted, including their physical and chemical properties, chromosome localization, phylogenetic relationships, gene structure, and tertiary structure. A total of 20 BcPCP genes were identified and classified into three subfamilies showing high homology to Arabidopsis thaliana. Expression pattern analysis indicated that the BcPCP gene family exhibits higher expression levels in reproductive organs, suggesting their potential involvement in the reproductive development. Notably, BraA02g002400.3C, potentially associated with male sterility, was identified through multiple transcriptomic and proteomic datasets. Subsequently, sequence analysis revealed its homology with the Arabidopsis GRP20 gene, and thus it was named BcGRP20. Functional analysis of this gene showed that overexpression of BcGRP20 gene in the Arabidopsis grp20 mutant could restore anther fertility. Overall, our findings indicate that BcGRP20 plays a critical role in pollen development and may be the causative gene for male sterility in Wucai. This study provides candidate genes for further functional identification of BcPCP genes in Wucai, which are crucial for anther development.

Genome-Wide Identification of Heat Shock Transcription Factor Family and Key Members Response Analysis to Heat Stress in Loquat

Eriobotrya japonica (loquat) is an evergreen fruit tree of the apple tribe in Rosaceae with high edible and medicinal value. The yield and quality of loquat fruits are significantly influenced by environmental stress, particularly heat stress during fruit ripening. In this study, thirty EjHSFs were identified in the loquat genome. Twenty-nine EjHSFs were unevenly distributed across sixteen chromosomes, except Chr-6. A synteny analysis revealed that twenty-six EjHSF genes had undergone duplication events. Twenty-nine EjHSF genes were found to be in sync with HSF in apples while also diverging with other Rosaceae species. A phylogenetic analysis revealed that EjHSFs could be divided into three categories, including eighteen EjHSF-A, ten EjHSF-B, and two EjHSF-C. Twenty-nine members of the EjHSF family showed high homology to those of Malus domestica and Gillenia trifoliate. A promoter analysis retrieved thirty-three CAREs that were functionally relevant and connected to the expression of HSFs. Among these, the promoters of twenty-three EjHSF genes possessed at least one STRE element that could be activated by heat shock. Eleven of these EjHSFs were highly expressed in loquat tissues, namely EjHSF-B4a, EjHSF-A4a, EjHSF-A4d, and EjHSF-C1a in roots; EjHSF-B4b in roots and inflorescence; EjHSF-C1b in stems and roots; EjHSF-A2a in three tissues; EjHSF-A8b in four tissues; and EjHSF-A4c, EjHSF-B1a, and EjHSF-B2b in six tissues. Moreover, fifteen EjHSFs were differentially expressed (eleven upregulated and four downregulated) in fruits under heat stress treatment in the color-changing period. Among them, EjHSF-A2a and EjHSF-A2b upregulated transcriptional abundance over 300 times after heat treatment; EjHSF-B2b consistently displayed an increasing trend over time; and EjHSF-B1a was significantly downregulated. Hence, these results suggest that EjHSFs take part in loquat tissue development and may be involved in the fruit’s heat stress response. These findings enhance the understanding of EjHSFs’ role within loquats and the heat stress response of its fruit and provide target genes for heat stress improvement.

Charting Cortical-Layer Specific Area Boundaries Using Gibbs Ringing Attenuated T1w/T2w-FLAIR Myelin MRI.

Cortical areas have traditionally been defined by their distinctive layer cyto- and/or myelo- architecture using postmortem histology. Recent studies have delineated many areas by measuring overall cortical myelin content and its spatial gradients using the T1w/T2w ratio MRI in living primates, including humans. While T1w/T2w studies of areal transitions might benefit from using the layer profile of this myelin-related contrast, a significant confound is Gibbs ringing artefact, which produces signal fluctuations resembling cortical layers. Here, we address these issues with a novel approach using cortical layer thickness-adjusted T1w/T2w-FLAIR imaging, which effectively cancels out Gibbs ringing artefacts while enhancing intra-cortical myelin contrast. Whole-brain MRI measures were mapped onto twelve equivolumetric layers, and layer-specific sharp myeloarchitectonic transitions were identified using spatial gradients resulting in a putative 182 area/subarea partition of the macaque cerebral cortex. The myelin maps exhibit unexpectedly high homology with humans suggesting cortical myelin shares the same developmental program across the species. Comparison with histological Gallyas myelin stains explains over 80% of the variance in the laminar T1w/T2w-FLAIR profiles, substantiating the validity of the method. Altogether, our approach provides a novel, noninvasive means for precision mapping layer myeloarchitecture in the primate cerebral cortex, advancing the pioneering work of classical neuroanatomists.

Dengue Virus Infection: Immune Response and Therapeutic Targets.

Flavivirus infection, especially dengue virus infection caused by DENV, is known to be a significant health concern globally owing to the high incidence and mortality rate. The expanding and increasing disease burden calls for the need to develop an effective treatment and prevent the event of fatal complications, including dengue hemorrhagic fever/dengue shock syndrome. The DENV-induced immune response has been described as paradoxical because it has a protective role in viral clearance but, at the same time, causes more severe infection through viral-specific immunity. This is further complicated by high homology and cross-reactivity between different serotypes of DENV, causing a more severe disease presentation during secondary infection by a heterologous serotype. This serotype complexity poses a challenge for the development of a universal flavivirus vaccine. This review highlights the significance of high motility group box 1 (HMGB1) and nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) inflammasome activation pathways in initiating an inflammatory response through the downstream activation of nuclear factor κB and proinflammatory cytokine Interleukin (IL)-1B, IL-18 release in DENV infection. It also discusses the role of NLRP3 in activating cellular apoptosis and pyroptosis leading to systemic failure, especially in peripheral tissues. Over the decades, there has been much progress in understanding the immunopathogenesis of DENV infection. Researchers have been studying key pathogenic molecules for potential therapeutic targets including HMGB1 and NLRP3 inflammasome inhibitors, which is explored in this review. Ultimately, although there is not yet an effective antiviral or vaccine for DENV, immunomodulators continue to pave the way to decrease disease severity in infected individuals.

Comparative Characterization of Three Homologous Glutathione Transferases from the Weed Lolium perenne

The comparative analysis of homologous enzymes is a valuable approach for elucidating enzymes’ structure–function relationships. Glutathione transferases (GSTs, EC. 2.5.1.18) are crucial enzymes in maintaining the homeostatic stability of plant cells by performing various metabolic, regulatory, and detoxifying functions. They are promiscuous enzymes that catalyze a broad range of reactions that involve the nucleophilic attack of the activated thiolate of glutathione (GSH) to electrophilic compounds. In the present work, three highly homologous (96–98%) GSTs from ryegrass Lolium perenne (LpGSTs) were identified by in silico homology searches and their full-length cDNAs were isolated, cloned, and expressed in E. coli cells. The recombinant enzymes were purified by affinity chromatography and their substrate specificity and kinetic parameters were determined. LpGSTs belong to the tau class of the GST superfamily, and despite their high sequence homology, their substrate specificity displays remarkable differences. High catalytic activity was determined towards hydroxyperoxides and alkenals, suggesting a detoxification role towards oxidative stress metabolites. The prediction of the structure of the most active LpGST by molecular modeling allowed the identification of a non-conserved residue (Phe215) with key structural and functional roles. Site-saturation mutagenesis at position 215 and the characterization of eight mutant enzymes revealed that this site plays pleiotropic roles, affecting the affinity of the enzyme for the substrates, catalytic constant, and structural stability. The results of the work have improved our understanding of the GST family in L. perenne, a significant threat to agriculture, sustainable food production, and safety worldwide.

FNDC4 alleviates cardiac ischemia/reperfusion injury through facilitating HIF1α-dependent cardiomyocyte survival and angiogenesis in male mice

Fibronectin type III domain-containing (FNDC) proteins play critical roles in cellular homeostasis and cardiac injury, and our recent findings define FNDC5 as a promising cardioprotectant against doxorubicin- and aging-related cardiac injury. FNDC4 displays a high homology with FNDC5; however, its role and mechanism in cardiac ischemia/reperfusion (I/R) injury remain elusive. Here, we show that cardiac and plasma FNDC4 levels are elevated during I/R injury in a hypoxia-inducible factor 1α (HIF1α)-dependent manner. Cardiac-specific FNDC4 overexpression facilitates, while cardiac-specific FNDC4 knockdown inhibits cardiomyocyte survival and angiogenesis in I/R-stressed hearts of male mice through regulating the proteasomal degradation of HIF1α. Interestingly, FNDC4 does not directly stimulate angiogenesis of endothelial cells, but increases the expression and secretion of fibroblast growth factor 1 from cardiomyocytes to enhance angiogenesis in a paracrine manner. Moreover, therapeutic administration of recombinant FNDC4 protein is sufficient to alleviate cardiac I/R injury in male mice, without resulting in significant side effects. In this work, we reveal that FNDC4 alleviates cardiac I/R injury through facilitating HIF1α-dependent cardiomyocyte survival and angiogenesis, and define FNDC4 as a promising predictive and therapeutic target of cardiac I/R injury.

Caenorhabditis elegans as a Model for Evaluating the Toxicology of Inorganic Nanoparticles.

Inorganic nanoparticles are nanomaterials with a central core composed of inorganic specimens, especially metals, which give them interesting applications but can impact the environment and human health. Their short- and long-term effects are not completely known and to investigate that, alternative models have been successfully used. Among these, the nematode Caenorhabditis elegans has been increasingly applied in nanotoxicology in recent years because of its many features and advantages for toxicological screening. This non-parasitic nematode may inhabit any environment where organic matter is available; therefore, it is interesting for ecotoxicological assessments. Moreover, this worm has a high genetic homology to humans, making the findings translatable. A notable number of published studies unraveled the level of toxicity of different nanoparticles, including the mechanisms by which their toxicity occurs. This narrative review collects and describes the most relevant toxicological data for inorganic nanoparticles obtained using C. elegans and also supports its application in safety assessments for regulatory purposes.

Myh9b is a critical non-muscle myosin II encoding gene that interacts with myh9a and myh10 during zebrafish development in both compensatory and redundant pathways.

Non-muscle myosin (NMII) motor proteins have diverse developmental functions due to their roles in cell shape changes, cell migration, and cell adhesion. Zebrafish are an ideal vertebrate model system to study the NMII encoding myh genes and proteins due to high sequence homology, established gene editing tools, and rapid ex utero development. In humans, mutations in the NMII encoding MYH genes can lead to abnormal developmental processes and disease. This study utilized zebrafish myh9a, myh9b, and myh10 null mutants to examine potential genetic interactions and roles for each gene in development. It was determined that the myh9b gene is the most critical NMII encoding gene, as myh9b mutants develop pericardial edema and have a partially penetrant lethal phenotype, which was not observed in the other myh mutants. This study also established that genetic interactions occur between the zebrafish myh9a, myh9b, and myh10 genes where myh9b is required for the expression of both myh9a and myh10, and myh10 is required for the expression of myh9b. Additionally, protein analyses suggested that enhanced NMII protein stability in some mutant backgrounds may play a role in compensation. Finally, double mutant studies revealed different and more severe phenotypes at earlier timepoints than single mutants, suggesting roles for tissue specific genetic redundancy, and in some genotypes, haploinsufficiency. These mutants are the first in vivo models allowing for the study of complete loss of the NMIIA and NMIIB proteins, establishing them as valuable tools to elucidate the role of NMII encoding myh genes in development and disease.

The Multifunctional Anaphase Promoting Complex 7 (APC7) Gene Is Associated With Increased Plant Growth and Improved Resistance to DNA and RNA Viruses.

The anaphase promoting complex 7 (AtAPC7) is an APC/C subunit expressed in different organs of Arabidopsis thaliana and conserved among eukaryotes. A variant of the complete APC7 protein, containing its C-terminal region (named APC-CT), shows a high homology with a tobacco viral replication inhibitor (IVR-like) protein that reduces plant susceptibility to RNA viruses. Here, the role of the AtAPC7 gene was investigated by characterizing Arabidopsis plants overexpressing the full-length AtAPC7 (APC7OE) and the C-terminal portion (APC7-CTOE), by phenotypical, physiological and molecular approaches. APC7OE plants showed improved growth of vegetative organs, earlier flowering and increased photosynthetic efficiency, CO2 assimilation and productivity, compared with Col-0 control plants. Conversely, APC7-CTOE plants showed reduced susceptibility to both RNA and DNA viruses, along with an improvement in plant growth, although not surpassing APC7OE plants. Altogether, the data provide evidence for the role of the AtAPC7 in regulating cell division, expansion and differentiation, accompanied by an increase in photosynthetic capacity, resulting in enhanced plant biomass and seed yield. AtAPC7-CT might reduce growth-defence trade-offs, enabling plants to simultaneously defend themselves while promoting better growth. Our findings highlight the multifunctional role of AtAPC7, unveiling the potential of its orthologous genes as valuable biotechnological tools in important crops.

Molecular insights into antimicrobial resistant Staphylococcus aureus strains: A potential zoonosis of goat origin

Antimicrobial-resistant (AMR) Staphylococcus aureus (S. aureus) strains have attained global attention due to their life-threatening zoonotic nature. Being a member of ESKAPE group, S. aureus has an ability to escape the biocidal action of antimicrobial drugs. The current study investigated the prevalence and molecular characterization of methicillin-resistant S. aureus (MRSA), β-lactam-resistant S. aureus (BRSA), aminoglycoside-resistant S. aureus (ARSA), tetracycline-resistant S. aureus (TRSA), and fluoroquinolones-resistant S. aureus (FRSA) associated with goat subclinical mastitis (SCM). Furthermore, the antimicrobial resistance and susceptibility profile of various antibiotics and non-antibiotics (NSAIDs, nisin, N-acetylcysteine, vitamin-C) along with their possible role in modulating the antibiotic resistance of MDR isolates was also investigated. A total of 768 goat milk samples were subjected to California mastitis test for SCM followed by bacteriological and molecular characterization of S. aureus. Moreover, in-vitro susceptibility of resistant antibiotics, non-antibiotics, and their combination against MDR S. aureus were conducted through well diffusion and broth microdilution assays. The results depicted that 55.47% and 26.82% of milk samples were positive for SCM and S. aureus, respectively. The molecular assay confirmed 35.92% of isolates as MRSA, 45.63% as BRSA, 50.49% as ARSA, and 32.52% but no isolate was confirmed as FRSA on molecular basis. The multidrug resistance was observed in 62.13% and 47.09% isolates, respectively. Molecular characterized MDR S. aureus revealed high homology of study isolates with the isolates of neighboring countries like India, Korea, Iran, and China. Antimicrobial susceptibility trials on well diffusion assay showed higher efficacy of different non-antibiotics with resistant antibiotics as penicillin with ketoprofen and gentamicin with flunixin meglumine while oxytetracycline with N-acetylcystiene. The synergy testing by checkerboard assay revealed synergistic activity of penicillin with ketoprofen, gentamicin with flunixin meglumine, and oxytetracycline with N-acetylcysteine. The current study highlighted the emergence and spread of AMR S. aureus strains from goat SCM and provided insights into possible drug repurposing of various non-antibiotics to modulate the multidrug resistance of S. aureus which will be helpful in devising the therapeutic options and control measures for this pathogen.

Interferon Regulatory Factors (IRF1, IRF4, IRF5, IRF7 and IRF9) in Sichuan taimen (Hucho bleekeri): Identification and Functional Characterization

Background/Objectives: Interferon regulatory factors (IRFs) are multifunctional transcription factors that play important roles in the transcriptional regulation of interferons and in the immune response to pathogens. Therefore, studying the interferon system in fish is highly relevant in the prevention and treatment of viral diseases. Methods: In this study, five IRF genes (IRF1, IRF4, IRF5, IRF7 and IRF9) were identified and characterized in Hucho bleekeri, and their expression profiles were determined after LPS and Poly(I:C) treatment. Results: These IRFs have typical DNA-binding domains and IRF-association domains. Amino acid sequence comparison revealed high homology between these IRFs and those of other vertebrates, with the highest homology being with other salmonid fish. Phylogenetic analysis revealed that these IRFs are divided into four subfamilies (IRF1, IRF3, IRF4 and IRF5), with both IRF4 and IRF9 belonging to the IRF4 subfamily. IRF genes were widely expressed in all of the tested tissues, with IRF1, IRF4 and IRF9 being highly expressed in the spleen and kidney and IRF5 and IRF7 highly expressed in the gonads. IRF1, IRF4 and IRF5 expression was induced at different time points post-LPS challenge. IRF7 and IRF9 expression in the spleen and head kidney was not significantly altered by LPS induction. Poly(I:C) treatment altered IRF expression more significantly than LPS treatment. Poly(I:C) significantly altered the spleen and head kidney expression of all five IRFs. Conclusions: These findings reveal the potential role of IRFs in the antiviral response of H. bleekeri and provide a reference for examining signal transduction pathways in the interferon system in fish.

Engineered odorant receptors illuminate the basis of odour discrimination.

How the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations remains poorly understood. Vertebrate animals perceive odours through G protein-coupled odorant receptors (ORs)1. In humans, around 400 ORs enable the sense of smell. The OR family comprises two main classes: class I ORs are tuned to carboxylic acids whereas class II ORs, which represent most of the human repertoire, respond to a wide variety of odorants2. A fundamental challenge in understanding olfaction is the inability to visualize odorant binding to ORs. Here we uncover molecular properties of odorant-OR interactions by using engineered ORs crafted using a consensus protein design strategy3. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modelling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled the determination of four cryogenic electron microscopy structures of distinct consORs with specific ligand recognition properties. The structure of a class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and generated a homology model of a related member of the human OR51 family with high predictive power. Structures of three class II consORs revealed distinct modes of odorant-binding and activation mechanisms between class I and class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

Molecular approach to the characterization of lipase encoding genes from Moraxella sp. SBE01

Lipase from Moraxella sp. SBE01 is an expression of the gene encoding lipase. Detection and characterization of the Moraxella sp. SBE01 lipase coding gene is necessary for large-scale lipase production through genetic engineering. This study aimed to observe the molecular weight, amino acid sequence, length, and conserved amino acids in the DNA encoding the lipase gene, with the goal of identifying and characterizing the lipase-coding gene from Moraxella sp. SBE01. The primer design process was conducted to amplify the lipase gene from Moraxella sp. SBE01 using specialized software for sequence alignment and phylogenetic analysis. Amplification was carried out using PCR with the designed primer, forward primer (GTC ATG ATG TAC TTC CAY GGN GGN GG), reverse primer (GGT TGC CGC CGG CDS WRT CNC C). PCR was carried out under pre-denatured conditions at 95°C (3 minutes), followed by 30 cycles of denaturation at 95°C, annealing at 66°C (30 seconds), 70°C elongations (1 minute) and final elongation of 70°C (10 minutes). The PCR results were electrophoresed using 1% agarose gel with a 1 kb DNA marker. The PCR results were sequenced and analyzed for gene and amino acid sequences and the type of lipase expressed. Sequencing resulted in 387 bp of the nucleotide sequence. The gene and amino acid sequences from Moraxella sp. SBE01 had high homology with the gene and amino acid sequences from Moraxella sp. strain TA144. The lipase gene encodes a protein consisting of 129 amino acids and contains a conserved HGG (His-Gly-Gly) motif, which is characteristic of lipases in family IV, also known as the hormone-sensitive lipase (HSL) family. This conserved sequence suggests that the lipase shares structural and functional similarities with other enzymes in the HSL family, playing a key role in lipid metabolism.

Genome-wide analysis of the SWEET gene family and its response to powdery mildew and leaf spot infection in the common oat (Avena sativa L.)

The nutritional quality and yield of oats (Avena sativa) are often compromised by plant diseases such as red leaf, powdery mildew, and leaf spot. Sugars Will Eventually be Exported Transporters (SWEETs) are newly identified sugar transporters involved in regulating plant growth and stress responses. However, the roles of SWEET genes in biotic stress responses remain uncharacterized in oats. In this study, 13 AsSWEET genes were identified across nine chromosomes of the oat genome, all of which were predicted to contain seven transmembrane regions. Phylogenetic analysis revealed four clades of AsSWEET proteins, with high homology to SWEET proteins in the Poaceae family. Collinearity analysis demonstrated strong relationships between oat and Zea mays SWEETs. Using subcellular localization prediction tools, AsSWEET proteins were predicted to localize to the plasma membrane. Promoter analysis revealed cis-acting elements associated with light response, growth, and stress regulation. Six AsSWEET proteins were predicted to interact in a network centered on AsSWEET1a and AsSWEET11. Gene expression analysis of two oat varieties, ‘ForagePlus’ and ‘Molasses’, indicated significant expression differences in several AsSWEET genes following infection with powdery mildew or leaf spot, including AsSWEET1a, AsSWEET1b, AsSWEET2b, AsSWEET3a, AsSWEET11, and AsSWEET16. These SWEET genes are potential candidates for disease resistance in oats. This study provides a foundation for understanding the regulatory mechanisms of AsSWEET genes, particularly in response to powdery mildew and leaf spot, and offers insights for enhancing oat molecular breeding.

RHO subfamily of small GTPases in the development and function of hematopoietic cells.

RHOA, RHOB, and RHOC comprise a subfamily of RHO GTPase proteins famed for controlling cytoskeletal dynamics. RHO proteins operate downstream of multiple signals emerging from the microenvironment, leading to diverse cell responses, such as proliferation, adhesion, and migration. Therefore, RHO signaling has been centrally placed in the regulation of blood cells. Despite their high homology, unique roles of RHOA, RHOB, and RHOC have been described in hematopoietic cells. In this article, we overview the contribution of RHO proteins in the development and function of each blood cell lineage. Additionally, we highlight the aberrations of the RHO signaling pathways found in hematological malignancies, providing clues for the identification of new therapeutic targets.

Outbreak of Contagious Ecthyma in Free-Ranging Iberian Ibex (Capra pyrenaica) in the Montgrí Massif Natural Park, Catalonia, Northeastern Spain.

Contagious ecthyma is a common, worldwide, and highly transmissible viral zoonotic skin disease caused by the orf virus (ORFV). It mainly affects farmed small ruminants, but it has also been described in a broad range of wild and domestic mammals, with Caprinae species most susceptible. Between November 2019 and January 2020, adults, juveniles, yearlings, and kids from an Iberian ibex (Capra pyrenaica) population established in the Montgrí Massif Natural Park (Catalonia, northeastern Spain) were observed with clinical signs and lesions compatible with ORFV infection. The carcass of an adult male with severe disease enabled sample collection for histopathology and molecular studies, confirming ORFV DNA in the skin lesions. Sequence analyses indicated that the ORFV strain detected had high homology (>98%) with strains previously obtained from other European wild ruminant species. The outbreak peaked in December 2019, with an estimated prevalence of 68.97% (95% confidence interval, 53.35-84.59). From February 2020 forward, no individuals with lesions were observed. The yearly counts for population monitoring corroborated the apparently negligible ORFV impact on the ibex population of the Montgrí Natural Park. There are no previous reports of contagious ecthyma in a free-ranging Iberian ibex population.

A Comparison of DNA-Methylation during Protoplast Culture of Ponkan Mandarin (Citrus reticulata Blanco) and Tobacco (Nicotiana tabacum L.)

The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (Citrus reticulata), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts.

Identification and Functional Characterization of the FATP1 Gene from Mud Crab, Scylla paramamosain.

In mammals, fatty acid transport protein 1 (FATP1) plays important roles in cellular uptake and activation of long-chain fatty acid (LCFA), especially in processes of transportation, oxidation and triacylglycerol synthesis. However, the role of FATP1 in invertebrates, especially decapod crustaceans, is still poorly understood. In this study, the cDNA of a FATP1 gene from a decapod crustacean, mud crab Scylla paramamosain, was cloned and functionally characterized. The FATP1 gene encoded a polypeptide consisting of 643 amino acids that exhibits all the typical features of the FATP family and shares high homology with the other FATP orthologs of crustaceans. The relative mRNA expression levels of FATP1 were observed to be higher in metabolically active tissues such as hepatopancreas, stomach and gill than in other crab parts. Knockdown of the FATP1 mRNA in vivo significantly reduced triacylglycerols and total lipid levels in the hepatopancreas, accompanied by an increase in the expression of genes related to fatty acid transportation, allocation and hydrolysis, including long-chain acyl-CoA synthetase 3/4 (ACSL3/4) and carnitine palmitoyl transferase 1 (CPT1), and a decrease in the expression of genes related to fatty acid synthesis such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the hepatopancreas. Furthermore, increased dietary n-3 long-chain polyunsaturated fatty acid (LC-PUFA) levels resulted in the up-regulation of the FATP1 expression in the hepatopancreas, accompanied by an increase in LC-PUFA content, especially eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), in both polar (PLs) and neutral lipids (NLs) in the hepatopancreas and muscles of crabs. These findings suggested that the FATP1 gene identified in S. paramamosain might play important roles in regulating long-chain fatty acid metabolism and deposition in crustaceans.

Genomic Analysis and Virulence Assessment of Hypervirulent Klebsiella pneumoniae K16-ST660 in Severe Cervical Necrotizing Fasciitis

ObjectiveTo investigate the source of infection in a patient with recurrent severe neck infections caused by Klebsiella pneumoniae and to analyze the virulence of isolates obtained from different sites of the patient. MethodsWe collected preoperative neck abscess puncture fluid, intraoperative neck drainage fluid, sputum, intestinal fecal specimens, and blood samples from a patient who visited Wuxi Second People's Hospital twice between 2017 and 2018. We conducted isolation, identification, drug sensitivity tests, and string tests on the isolates. Capsule serotyping and virulence gene analysis were performed using PCR. The genetic relationship of different isolates was assessed by Multilocus Sequence Typing and virulence was evaluated using the Galleria mellonella infection model. Additionally, whole-genome sequencing was used to analyze the chromosomal and plasmid genes of one isolate. ResultsKlebsiella pneumoniae was detected in the sputum and fecal specimens from both hospitalizations, as well as the preoperative ultrasound-guided puncture fluid and intraoperative drainage fluid from the first hospitalization, resulting in six isolates. These isolates were all K16 serotype, positive in the string test, and identified as ST660 by Multilocus Sequence Typing, indicating they belonged to the same clone. Virulence gene analysis showed that wcaG, iucB, iroNB, rmpA, rmpA2, Aer, kfuBC, ureA, fimH, mrkD, uge, and peg344 were positive, while allS, cf29a, and Wzy_K1 were negative. In the Galleria mellonella virulence assay, the lethality of different isolates was dose-dependent. The K16 group showed significantly higher larval mortality compared to other control groups (including K1, K2, K5, K20, and K57 groups). Genome sequencing revealed that plasmid p17388 carried numerous virulence genes and insertion sequences, particularly ISKPN74, and showed high homology with other Klebsiella plasmids. ConclusionThis study is the first to report severe cervical necrotizing fasciitis caused by the K16-ST660 Klebsiella pneumoniae Isolate. The high virulence of these isolates was confirmed by the Galleria mellonella virulence assay and the detection of numerous virulence genes. In-depth analysis of plasmid p17388 suggests that ISKPN74 may enhance stable integration of the plasmid into the bacterial chromosome through recombinases and transposases, thereby reducing the likelihood of plasmid loss and increasing bacterial virulence. Additionally, IS5 family insertion sequences may carry extra promoters or enhancers that, when inserted upstream of mucoviscosity-associated genes such as rmpA, may increase the transcription levels of downstream genes. This ISKPN74-mediated integration or insertion reveals a complex genetic mechanism that may contribute to the severity of infections caused by ST660 isolates. Our findings offer new insights into the virulence and structure of ST660-K16 Klebsiella pneumoniae, suggesting that further investigation into the specific mechanisms by which these insertion sequences enhance virulence could aid in developing novel infection management strategies.

Genome Survey of Male Rana dybowskii to Further Understand the Sex Determination Mechanism.

Rana dybowskii is one of the important aquaculture species in Northeast China. The fallopian tubes of female R. dybowskii are used to prepare oviductus ranae (an important traditional Chinese medicine). Therefore, R. dybowskii females have higher economical value than males. An increasing female R. dybowskii population can increase the benefits from R. dybowskii culture. However, the genome of amphibians is complex, making it difficult to investigate their sex determination mechanism. In this study, we analyzed the genome of male R. dybowskii using next-generation sequencing technology. A total of 200,046,452,400 bp of clean data were obtained, and the K-mer analysis indicated that the depth was 50×. The genome size of R. dybowskii was approximately 3585.05 M, with a heterozygosity rate, repeat sequence ratio, and genome GC content of 1.15%, 68.96%, and approximately 43.0%, respectively. In total, 270,785 contigs and 498 scaffolds were generated. The size of the contigs and scaffolds was 3,748,543,415 and 3,765,862,278 bp, respectively, with the N50 length of 31,988 and 336,385,783. The longest contig and scaffold were of the size 137,967,485 and 1,808,367,828 bp, respectively. The number of contigs and scaffolds > 10K nt was 99,620 and 451, respectively. Through annotation, 40,913 genes were obtained, including 156,609 CDS (i.e., 3.83 CDS per gene). Sequence alignment was performed with the assembled scaffolding genome in this study. Two and one fragment had high homology with two male-specific DNA molecular markers of R. dybowskii discovered previously (namely, MSM-222 and MSM-261, respectively). In addition, the Dmrt1 gene of R. dybowskii was obtained with a length of 18,893 bp by comparison and splicing. The forward primers amplifying MSM-222 and MSM-261 were located at 322-343 and 14,501-14,526 bp of Dmrt1, respectively. However, sequence alignment revealed that MSM-222 and MSM-261 were not located on Dmrt1, and only some homologous parts were observed. This indicated that in addition to Dmrt1, other important genes may play a crucial role in the sex determination mechanism of R. dybowskii. Our study provided a foundation for the subsequent high-quality genome construction and provided important genomic resources for future studies on R. dybowskii.