Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

Evaluation of reverse transcription yield of RNA standards and forensic samples based on droplet digital PCR.

RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 µg/µL - 0.24 ng/µL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 µL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.

Nucleic acid-based electrochemical biosensor for detection of influenza B by gold nanoparticles.

The influenza virus is a pervasive pathogen that exhibits increased prevalence during colder seasons, resulting in a significant annual occurrence of infections. Notably, pharmaceutical interventions effective against influenza A strains often exhibit limited efficacy against influenza B variants. Against this backdrop, the need for innovative approaches to accurately and swiftly differentiate and detect influenza B becomes evident. Biosensors play a pivotal role in this detection process, offering rapid, specific, and sensitive identification of the virus, facilitating timely intervention and containment efforts. Oligonucleotide sequences targeting the conserved B/Victoria/2/87 influenza virus NP region were designed. Nasopharyngeal swabs were collected from patients suspected of influenza virus infection, and viral RNA was extracted. RNA quality was assessed through one-step PCR. cDNA synthesis was performed using random hexamers, and real-time PCR quantified the influenza genome. Gold nanoparticles were immobilized on a surface to immobilize the specific DNA probe, and electrochemical hybridization was electrochemically followed. The biosensor exhibited high selectivity and effective distinction of complementary sequences from mismatches and influenza virus cDNA genome. The biosensor successfully detected the influenza B virus genome in real samples. Non-influenza samples yielded no significant hybridization signals. The comparison between the results obtained from the biosensor and real-time PCR revealed full agreement of these methods. The biosensor utilized electrochemical detection of hybridization and proved effective in detecting the influenza B virus genome with high specificity, sensitivity, and selectivity. Comparative analysis with real-time PCR underscored the accuracy and potential applicability of the biosensor in rapid and specific virus detection. This innovative approach holds promise for future diagnostic and epidemiological applications in detecting influenza B virus and other pathogens.

Substrate profiling of human transglutaminase 1 using cDNA display and next-generation sequencing.

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.

Hypermethylation and down-regulation of vitamin D receptor (VDR) as contributing factors for polycystic ovary syndrome (PCOS): a case-control study from Kashmir, North India.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a prevailing endocrinopathy affecting a significant population of women of reproductive age across the globe. A myriad set of complex intertwined factors ranging from etiological, genetic, and epigenetic reasons cause this disorder. Out of the different factors, vitamin D shows an imperative aspect in health and fertility of women with polycystic ovary syndrome (PCOS). The importance of vitamin D is facilitated by vitamin D receptor (VDR), a ligand-dependent transcription factor in the steroid/ thyroid hormone receptor superfamily that controls the pleiotropic biological properties of vitamin D. PURPOSE: The purpose of this study was to evaluate the role of promoter methylation of the VDR gene, a transcription factor with numerous biological utilities, with its relative expression and clinico-pathological findings and outcomes. METHODOLOGY: A total of 200 blood samples were collected, 100 from PCOS case subjects, and 100 from the normal healthy controls respectively, which were assessed by qRT-PCR for determining the expression summary. MS-PCR technique was used for analyzing the promoter methylation status of the VDR gene. Blood samples were withdrawn, respectively, for each case and the control study separately experimented for different stages for the given study, of which estimation of vitamin D was also a part. RESULTS: In this test-versus-control study, first, the promoter methylation status of VDR gene was identified which was found more prominent i.e., hyper-methylation of the VDR gene was identified in 84 cases (84%), and in the normal healthy controls, it was found (62%). The promoter methylation status of the VDR gene has remarkably shown the results with a significant difference (p value < 0.0001*). Second, the expression analysis of VDR gene was found to be strongly downregulated in majority (64%) of PCOS case samples analyzed by means fold change of 0.8743 (± 0.06466) (p value 0.0054**). This result is, therefore, indicative of VDR gene role in PCOS pathogenesis as the said gene is downregulated. Moreover, compared to the vitamin D parameter, hyper-methylation and expression analysis of the VDR promoter gene were found to correspond to some associations with PCOS. Certain case-and-control study analyses showed that patients with normal vitamin D levels showed less indicative effects of PCOS and vice versa. CONCLUSION: Our study, being exclusive from Kashmir, one of the foremost specified that VDR confirms anomalous methylation configuration in PCOS with subsequent downregulation in the gene expression i.e., there is an inverse correlation among VDR gene expression (downregulated) and methylation status (hyper-methylated) from the conclusion of our PCOS case-versus-control study.

The effect of diet composition on the diversity of active gut bacteria and on the growth of Spodoptera exigua (Lepidoptera: Noctuidae).

Gut microbiota plays a functional role in nutrition among several insects. However, the situation is unclear in Lepidoptera. Field studies suggest the microbiome may not be stable and is determined by diet, while in the laboratory, Lepidoptera are routinely reared on diet containing antibiotics with unknown effects on microbial communities. Furthermore, molecular approaches for the characterization of lepidopteran microbiomes rarely describe the metabolically active gut bacteria. The aim of this study was to evaluate how diet and antibiotics affect Spodoptera exigua (Hübner) growth and the diversity and activity of the gut bacteria community. We assessed how alfalfa and wheat germ-based diets affected larval growth, in the presence and absence of streptomycin. Alfalfa diet improved larval growth, pupal mass, and survival, but antibiotic was only beneficial in the wheat germ diet. We observed diet-driven changes in the gut bacterial communities. In the active community, the alfalfa colony was dominated by Enterococcus and Rhodococcus whereas in the wheat germ colony, only Enterococcus was present. In contrast, spore-forming Bacilli species were very common members of the DNA community. In both cases, streptomycin had a selective effect on the relative abundance of the taxa present. Our study highlights the importance of characterizing both the diversity and activity of the gut microbiota community. DNA-derived communities may include environmental DNA, spores, or non-viable bacteria, while RNA-derived communities are more likely to give an accurate representation of the diversity of active members that are potentially directly involved in the metabolic processes of the host.

The Current Situation and Development Prospect of Whole-Genome Screening.

High-throughput genetic screening is useful for discovering critical genes or gene sequences that trigger specific cell functions and/or phenotypes. Loss-of-function genetic screening is mainly achieved through RNA interference (RNAi), CRISPR knock-out (CRISPRko), and CRISPR interference (CRISPRi) technologies. Gain-of-function genetic screening mainly depends on the overexpression of a cDNA library and CRISPR activation (CRISPRa). Base editing can perform both gain- and loss-of-function genetic screening. This review discusses genetic screening techniques based on Cas9 nuclease, including Cas9-mediated genome knock-out and dCas9-based gene activation and interference. We compare these methods with previous genetic screening techniques based on RNAi and cDNA library overexpression and propose future prospects and applications for CRISPR screening.

Molecular Characterization and Pathogenicity of an Infectious cDNA Clone of Youcai Mosaic Virus on Solanum nigrum.

Virus infections cause devastative economic losses for various plant species, and early diagnosis and prevention are the most effective strategies to avoid the losses. Exploring virus genomic evolution and constructing virus infectious cDNA clones is essential to achieve a deeper understanding of the interaction between host plant and virus. Therefore, this work aims to guide people to better prevent, control, and utilize the youcai mosaic virus (YoMV). Here, the YoMV was found to infect the Solanum nigrum under natural conditions. Then, an infectious cDNA clone of YoMV was successfully constructed using triple-shuttling vector-based yeast recombination. Furthermore, we established phylogenetic trees based on the complete genomic sequences, the replicase gene, movement protein gene, and coat protein gene using the corresponding deposited sequences in NCBI. Simultaneously, the evolutionary relationship of the YoMV discovered on S. nigrum to others was determined and analyzed. Moreover, the constructed cDNA infectious clone of YoMV from S. nigrum could systematically infect the Nicotiana benthamiana and S. nigrum by agrobacterium-mediated infiltration. Our investigation supplied a reverse genetic tool for YoMV study, which will also contribute to in-depth study and profound understanding of the interaction between YoMV and host plant.

Purification, Characterization, cDNA Cloning, and Bioinformatic Analysis of Zinc-Binding Protein from Magallana hongkongensis.

Oysters contain significant amounts of the zinc element, which may also be found in their proteins. In this study, a novel zinc-binding protein was purified from the mantle of the oyster Magallana hongkongensis using two kinds of gel filtration chromatograms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that its molecular weight was approximately 36 kDa. The protein identified by the Q-Exactive mass spectrometer shared the highest sequence identity with carbonic anhydrase derived from Crassostrea gigas concerning amino acid sequence similarity. Based on homologous cloning and RACE PCR, the full-length cDNA of carbonic anhydrase from Magallana hongkongensis (designated as MhCA) was cloned and sequenced. The cDNA of MhCA encodes a 315-amino-acid protein with 89.74% homology to carbonic anhydrase derived from Crassostrea gigas. Molecular docking revealed that the two zinc ions primarily form coordination bonds with histidine residues in the MhCA protein. These results strongly suggest that MhCA is a novel zinc-binding protein in Magallana hongkongensis.

Functional and Structural Properties of Type V Collagen from the Skin of the Shortbill Spearfish (Tetrapturus angustirostris).

Type V collagen is considered to be a crucial minor collagen in fish skin with unique physiological functions. In this research, the cDNAs of three procollagens (Tacol5a1, Tacol5a2, and Tacol5a3) in type V collagen were cloned from the skin of shortbill spearfish (Tetrapturus angustirostris). The open reading frames (ORFs) of Tacol5a1, Tacol5a2, and Tacol5a3 contained 5991, 4485, and 5607 bps, respectively, encoding 1997, 1495, and 1869 amino acid residues. Each of the deduced amino acid sequences of procollagens contained a signal peptide and a fibrillar collagen C-terminal domain (COLFI). A conserved thrombospondin-like N-terminal domain (TSPN) was found at the N-terminus of Tacol5a1 and 5a3 procollagens, whereas a von Willebrand factor (VWC) was found at the N-terminus of Tacol5a2 procollagen. Tacol5a1, Tacol5a2, and Tacol5a3 had their theoretical isoelectric points of 5.06, 6.75, and 5.76, respectively, and predicted molecular weights of 198,435.60, 145,058.48, and 189,171.18, respectively. The phylogenetic tree analysis revealed that Tacol5a1 of shortbill spearfish clustered with that of yellow perch (Perca flavescens) instead of broadbill swordfish (Xiphias gladius). In addition, type V collagen was extracted from the shortbill spearfish skin. The in silico method demonstrated that shortbill spearfish type V collagen has a high potential for angiotensin-converting enzyme (ACE) inhibition activity (79.50%), dipeptidyl peptidase IV inhibition (74.91%) activity, and antithrombotic activity (46.83%). The structural clarification and possible functional investigation in this study provide the foundation for the applications of exogenous type V collagen derived from fish sources.

Identification of Transcription Factors of Santalene Synthase Gene Promoters and SaSSY Cis-Elements through Yeast One-Hybrid Screening in Santalum album L.

The main components of sandalwood heartwood essential oil are terpenoids, approximately 80% of which are α-santalol and ß-santalol. In the synthesis of the main secondary metabolites of sandalwood heartwood, the key gene, santalene synthase (SaSSY), can produce α-santalene and ß-santalene by catalyzed (E, E)-FPP. Furthermore, santalene is catalyzed by the cytochrome monooxygenase SaCYP736A167 to form sandalwood essential oil, which then produces a fragrance. However, the upstream regulatory mechanism of the key gene santalene synthase remains unclear. In this study, SaSSY (Sal3G10690) promoter transcription factors and SaSSY cis-elements were screened. The results showed that the titer of the sandalwood cDNA library was 1.75 × 107 CFU/mL, 80% of the inserted fragments identified by PCR were over 750 bp in length, and the positivity rate of the library was greater than 90%. The promoter region of the SaSSY gene was shown to have the structural basis for potential regulatory factor binding. After sequencing and bioinformatics analysis, we successfully obtained 51 positive clones and identified four potential SaSSY transcriptional regulators. Sal6G03620 was annotated as the transcription factor MYB36-like, and Sal8G07920 was annotated as the small heat shock protein HSP20 in sandalwood. Sal1G00910 was annotated as a hypothetical protein of sandalwood. Sal4G10880 was annotated as a homeobox-leucine zipper protein (ATHB-15) in sandalwood. In this study, a cDNA library of sandalwood was successfully constructed using a yeast one-hybrid technique, and the transcription factors that might interact with SaSSY gene promoters were screened. This study provides a foundation for exploring the molecular regulatory mechanism involved in the formation of sandalwood heartwood.

A Flexible Mouse Model of Autoimmune Thyroiditis Induced by Immunization with an Adenovirus Containing Full-Length Thyroglobulin cDNA.

The main challenge in the "post-GWAS" era is to determine the functional meaning of genetic variants and their contribution to disease pathogenesis. Development of suitable mouse models is critical because disease susceptibility is triggered by complex interactions between genetic, epigenetic, and environmental factors that cannot be modeled by in vitro models. Thyroglobulin (TG) is a key gene for autoimmune thyroid disease (AITD) and several single nucleotide polymorphisms (SNPs) in the TG coding region have been associated with AITD. The classical model of experimental autoimmune thyroiditis (EAT), based on immunization of genetically susceptible mouse strains with purified TG protein in adjuvant, does not allow testing the impact of TG sequence variants on the development of autoimmune thyroiditis. Here we describe a protocol for the induction of EAT by immunization of mice susceptible to thyroiditis with an adenovirus vector carrying full-length human TG cDNA (Ad-TG EAT). We also provide support protocols for evaluation of autoimmune thyroiditis including serological assessment of TG antibodies, in vitro splenocyte proliferation assay and cytokines secretion, thyroid histology, and evaluation of thyroid lymphocytic infiltration by immunostaining. This protocol for EAT induction allows manipulation of the TG cDNA to introduce variants associated with AITD, enabling the testing of the functional effects of susceptible variants and their haplotypes on the immunogenicity of TG. Furthermore, the Ad-TG EAT mouse model is a valuable model for studying the interactions of the TG variants with non-genetic factors influencing AITD development (e.g., cytokines, iodine exposure) or with variants of other susceptible genes (e.g., HLA-DRß1). © 2024 Wiley Periodicals LLC. Basic Protocol: Development of a mouse model of autoimmune thyroiditis induced by immunization with adenovirus containing full-length thyroglobulin cDNA Support Protocol 1: Splenocytes isolation Support Protocol 2: T cell stimulation and carboxyfluorescein diacetate succinimidyl ester (CFSE) based cell proliferation assay Support Protocol 3: Cytokine assays: measuring levels of interferon gamma (IFNγ) and interleukins IL-2, IL-4, and IL-10 in splenocyte supernatants Support Protocol 4: Evaluating thyroid histology and infiltration with immune cells: hematoxylin-eosin staining of mice thyroid glands Support Protocol 5: Immunohistochemistry of thyroid tissues: Immunofluorescence protocol of paraffin-embedded thyroid sections Support Protocol 6: Anti-thyroglobulin antibody measurement in mice sera by enzyme-linked immunosorbent assay (ELISA).

Construction and characterization of an infectious cDNA clone of human rhinovirus A89.

Rhinoviruses (RVs) are major causes of the common cold and are related to severe respiratory tract diseases, leading to a considerable economic burden and impacts on public health. Available and stable viral resources of rhinoviruses for laboratory use are important for promoting studies on rhinoviruses and further vaccine or therapeutic drug development. Reverse genetic technology can be useful to produce rhinoviruses and will help to promote studies on their pathogenesis and virulence. In this study, rhinovirus A89, an RV-A species that has been found to be highly involved in hospitalization triggered by RV infections, was selected to construct an infectious clone based on its sequence as a representative. The viral mRNA produced by a T7 RNA transcript system was transfected into H1-HeLa cells, and the rescued RV-A89 viruses were harvested and confirmed by sequencing. The rescued RV-A89 induced a similar cytopathic effect (CPE) and shared almost identical growth kinetics curves with parental RV-A89. Moreover, 9A7, a prescreened monoclonal antibody against the parental RV-A89, had a good and specific reaction with the rescued RV-A89, and further characterization showed almost the same morphology and protein composition of both viruses; thus, recombinant RV-A89 with similar biological characterization and virulence to the parental virus was obtained. In summary, the infectious clone of RV-A89 was successfully established, and the development of reverse genetic technology for rhinovirus will provide a framework for further studies on rhinoviruses.

Engineering of stable infectious cDNA constructs of a fluorescently tagged tomato chlorosis virus.

Tomato chlorosis virus (ToCV) is an emerging pathogen that cause severe yellow leaf disorder syndrome in tomato plants. In this study, we aimed to generate a recombinant ToCV tagged with green fluorescent protein (GFP) to enable real-time monitoring of viral infection in living plants. Transformation of the full-length cDNA construct of ToCV RNA1 into Escherichia coli resulted in instability issues, which were successfully overcome by inserting a plant intron into RNA1. Subsequently, a GFP tag was engineered into a cDNA construct of ToCV RNA2. The resulting recombinant ToCV-GFP could systemically infect Nicotiana benthamiana plants, and GFP expression was observed along the major veins. Utilizing ToCV-GFP, we also showed that ToCV engages in antagonistic relationships with two different tomato-infecting viruses in mixed infections in N. benthamiana. This study demonstrates the potential of ToCV-GFP as a valuable tool for the visual tracking of infection and movement of criniviruses in living plants.

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

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

Gene expression analysis of potato drought-responsive genes under drought stress in potato (Solanum tuberosum L.) cultivars.

The potato (Solanum tuberosum L.), an important field crop consumed extensively worldwide, is adversely affected by abiotic stress factors especially drought. Therefore, it is vital to understand the genetic mechanism under drought stress to decrease loose of yield and quality . This trial aimed to screen drought-responsive gene expressions of potato and determine the drought-tolerant potato cultivar. The trial pattern is a completely randomized block design (CRBD) with four replications under greenhouse conditions. Four cultivars (Brooke, Orwell, Vr808, Shc909) were irrigated with four different water regimes (control and three stress conditions), and the gene expression levels of 10 potato genes were investigated. The stress treatments as follows: Control = 100% field capacity; slight drought = 75% field capacity; moderate drought = 50% field capacity, and severe drought 25% field capacity. To understand the gene expression under drought stress in potato genotypes, RT-qPCR analysis was performed and results showed that the genes most associated with drought tolerance were the StRD22 gene, MYB domain transcription factor, StERD7, Sucrose Synthase (SuSy), ABC Transporter, and StDHN1. The StHSP100 gene had the lowest genetic expression in all cultivars. Among the cultivars, the Orwell exhibited the highest expression of the StRD22 gene under drought stress. Overall, the cultivar with the highest gene expression was the Vr808, closely followed by the Brooke cultivar. As a result, it was determined that potato cultivars Orwell, Vr808, and Brooke could be used as parents in breeding programs to develop drought tolerant potato cultivars.

Molecular characterization and expression analysis of thyroid hormone receptors in protogynous rice field eel, Monopterus albus.

Thyroid hormones (THs) play important roles in growth, development, morphogenesis, reproduction, and so on. They are mainly meditated by binding to thyroid hormone receptors (TRs) in vertebrates. As important members of the nuclear receptor superfamily, TRs and their ligands are involved in many biological processes. To investigate the potential roles of TRs in the gonadal differentiation and sex change, we cloned and characterized the TRs genes in protogynous rice field eel (Monopterus albus). In this study, three types of TRs were obtained, which were TRαA, TRαB and TRß, encoding preproproteins of 336-, 409- and 415-amino acids, respectively. Multiple alignments of the three putative TRs protein sequences showed they had a higher similarity. Tissue expression analysis showed that TRαA mainly expressed in the gonad, while TRαB and TRß in the brain. During female-to-male sex reversal, the expression levels of all the three TRs showed a similar trend of increase followed by a decrease in the gonad. Intraperitoneal injection of triiodothyronine (T3) stimulated the expression of TRαA and TRαB, while it had no significant change on the expression of TRß in the ovary. Gonadotropin-releasing hormone analogue (GnRHa) injection also significantly upregulated the expression levels of TRαA and TRαB after 6 h, while it had no significant effect on TRß. These results demonstrated that TRs were involved in the gonadal differentiation and sex reversal, and TRα may play more important roles than TRß in reproduction by the regulation of GnRHa in rice field eel.

The Features of Shared Genes among Transcriptomes Probed in Atopic Dermatitis, Psoriasis, and Inflammatory Acne: S100A9 Selection as the Target Gene.

BACKGROUND: Atopic dermatitis (AD), psoriasis (PS), and inflammatory acne (IA) are well-known as inflammatory skin diseases. Studies of the transcriptome with altered expression levels have reported a large number of dysregulated genes and gene clusters, particularly those involved in inflammatory skin diseases. OBJECTIVE: To identify genes commonly shared in AD, PS, and IA that are potential therapeutic targets, we have identified consistently dysregulated genes and disease modules that overlap with AD, PS, and IA. METHODS: Microarray data from AD, PS, and IA patients were downloaded from Gene Expression Omnibus (GEO), and identification of differentially expressed genes from microarrays of AD, PS, and IA was conducted. Subsequently, gene ontology and gene set enrichment analysis, detection of disease modules with known disease-associated genes, construction of the protein-protein interaction (PPI) network, and PPI sub-mapping analysis of shared genes were performed. Finally, the computational docking simulations between the selected target gene and inhibitors were conducted. RESULTS: We identified 50 shared genes (36 up-regulated and 14 down-regulated) and disease modules for each disease. Among the shared genes, 20 common genes in PPI network were detected such as LCK, DLGAP5, SELL, CEP55, CDC20, RRM2, S100A7, S100A9, MCM10, AURKA, CCNB1, CHEK1, BTC, IL1F7, AGTR1, HABP4, SERPINB13, RPS6KA4, GZMB, and TRIP13. Finally, S100A9 was selected as the target gene for therapeutics. Docking simulations between S100A9 and known inhibitors indicated several key binding residues, and based on this result, we suggested several cannabinoids such as WIN-55212-2, JZL184, GP1a, Nabilone, Ajulemic acid, and JWH-122 could be potential candidates for a clinical study for AD, PS, and IA via inhibition of S100A9-related pathway. CONCLUSION: Overall, our approach may become an effective strategy for discovering new disease candidate genes for inflammatory skin diseases with a reevaluation of clinical data.

Development and application of sugarcane streak mosaic virus vectors.

Sugarcane streak mosaic virus (SCSMV) is one of the major pathogens of sugarcane in the world. Molecular studies and disease management of SCSMV are hindered by the lack of efficient infectious clones. In this study, we successfully constructed Agrobacterium infiltration based infectious clone of SCSMV with different variants. Infectious clones of wild type SCSMV could efficiently infect Nicotiana benthamiana and sugarcane plants resulting in streak and mosaic symptoms on systemic leaves which were further confirmed with RT-PCR and serological assays. SCSMV variants of less adenylation displayed attenuated pathogenicity on N.benthamiana. SCSMV-based recombinant heterologous EGFP protein vector was also developed. The EGFP-tagged recombinant SCSMV could highly expressed in vegetative organs including roots. These infectious clones of SCSMV could be further developed for platform tools for both biotechnological studies and management of SCSMV disease.

Quail GHRL and LEAP2 gene cloning, polymorphism detection, phylogenetic analysis, tissue expression profiling and its association analysis with feed intake.

The GHRL, LEAP2, and GHSR system have recently been identified as important regulators of feed intake in mammals and chickens. However, the complete cloning of the quail GHRL (qGHRL) and quail LEAP2 (qLEAP2) genes, as well as their association with feed intake, remains unclear. This study cloned the entire qGHRL and qLEAP2 cDNA sequence in Chinese yellow quail (Coturnix japonica), including the 5' and 3' untranslated regions. Sanger sequencing analysis revealed no missense mutations in the coding region of qGHRL and qLEAP2. Subsequently, phylogenetic analysis and protein homology alignment were conducted on the qGHRL and qLEAP2 in major poultry species. The findings of this research indicated that the qGHRL and qLEAP2 sequences exhibit a high degree of similarity with those of chicken and turkey. Specifically, the N-terminal 6 amino acids of GHRL mature peptides and all the mature peptide sequence of LEAP2 exhibited consistent patterns across all species examined. The analysis of tissue gene expression profiles indicated that qGHRL was primarily expressed in the proventriculus and brain tissue, whereas qLEAP2 exhibited higher expression levels in the intestinal tissue, kidney, and liver tissue, differing slightly from previous studies conducted on chicken. It is necessary to investigate the significance of elevated expression of qGHRL in brain and qLEAP2 in kidney in the future. Further research has shown that the expression of qLEAP2 can quickly respond to changes in different energy states, whereas qGHRL does not exhibit the same capability. Overall, this study successfully cloned the complete cDNA sequences of qGHRL and qLEAP2, and conducted a comprehensive examination of their tissue expression profiles and gene expression levels in the main expressing organs across different energy states. Our current findings suggested that qLEAP2 is highly expressed in the liver, intestine, and kidney, and its expression level is regulated by feed intake.