Nucleotide Sequence Research Articles

A novel dual CRISPR-Cas assay for detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp without false positives from its endogenous viral elements (EVEs)

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus posing significant economic challenges in shrimp aquaculture. Depending on the shrimp species, IHHNV infections can lead to mass mortalities, deformities, or growth retardation, all of which impact production output. Timely identification of IHHNV, particularly using nucleic acid detection methods, is paramount for effective prevention. However, PCR and methods relying on nucleic acid probes face challenges due to the presence of chromosome-integrated, endogenous viral elements (EVEs) of IHHNV sharing identical sequences to their infectious counterparts. While long-range PCR assays, such as LongAmp PCR (LA-PCR), have shown promise in minimizing false positive results, their applicability in the field is limited by their long turnaround times and reliance on costly equipment. To address these limitations, we have developed a rapid and potentially field-deployable platform termed Cas9-RPA-Cas12a (“CRPAC”) capable of differentiating the infectious form of IHHNV from EVEs, capitalizing on the fact that the genome of IHHNV is single-stranded, while EVEs are double-stranded DNA. This method employs Cas9 endonuclease to deplete EVEs, thereby enriching for single-stranded genomic DNA. This enriched DNA is then subjected to recombinase polymerase amplification (RPA) coupled with the Cas12a diagnostic assay. Here, we show that CRPAC gives positive results exclusively for infectious IHHNV, appearing as strong fluorescence or a distinct pattern on lateral flow strips that facilitates naked-eye evaluation. In field sample testing, CRPAC demonstrated excellent agreement with LA-PCR, identifying 8 out of 16 samples containing infectious IHHNV, while the WOAH-recommended PCR assays gave false positive results to all samples containing EVEs. Collectively, our findings underscore the utility of CRPAC as a robust and field-adaptable tool for distinguishing between infectious IHHNV and EVEs, irrespective of their nucleotide sequence similarities.

Colistin Resistance Mechanisms and Molecular Epidemiology of Enterobacter cloacae Complex Isolated from a Tertiary Hospital in Shandong, China.

Enterobacter cloacae complex (ECC), which includes major nosocomial pathogens, causes urinary, respiratory, and bloodstream infections in humans, for which colistin is one of the last-line drugs. This study aimed to analyse the epidemiology and resistance mechanisms of colistin-resistant Enterobacter cloacae complex (ECC) strains isolated from Shandong, China. Two hundred non-repetitive ECC strains were collected from a tertiary hospital in Shandong Province, China, from June 2020 to June 2022. Whole-genome sequencing and bioinformatics analyses were performed to understand the molecular epidemiology of the colistin-resistant ECC strains. The nucleotide sequences of heat shock protein (hsp60) were analyzed by using BLAST search to classify ECC. The gene expression levels of ramA, soxS, acrA, acrB, phoP, and phoQ were assessed using RT-qPCR. MALDI-TOF MS was used to analyse the modification of lipid A. Twenty-three colistin-resistant strains were detected among the 200 ECC clinical strains (11.5%). The hsp60 cluster analysis revealed that 20 of the 23 ECC strains belonged to heterogeneous resistance clusters. Variants of mgrB, phoPQ, and pmrAB, particularly phoQ and pmrB, were detected in the 23 ECC strains. The soxS and acrA genes were significantly overexpressed in all 23 colistin-resistant ECC strains (P < 0.05). Additionally, all 23 ECC strains contained modified lipid A related to colistin resistance, which showed five ion peaks at m/z 1876, 1920, 1955, 2114, and 2158. Among the 23 ECC strains, 6 strains possessed a phosphoethanolamine (pETN) moiety, 16 strains possessed a 4-amino-4-deoxy-L-arabinose (-L-Ara4N) moiety, and one strain had both pETN and -L-Ara4N moieties. This study suggests that diverse colistin resistance existed in ECC, including unknown resistance mechanisms, exist in ECC. Mechanistic investigations of colistin resistance are warranted to optimise colistin use in clinical settings and minimise the emergence of resistance.

The novel HLA-A*24:617 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-A*24:617 differs from HLA-A*24:02:01:01 by one nucleotide in exon 4.

The novel HLA-C*15:279 allele, identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

The novel HLA-C*15:279 allele differs from HLA-C*15:02:01:01 by five nucleotide substitutions in exons 4 and 5.

Genetic Alterations Associated with Colistin Resistance Development in Escherichia coli.

Background: The increased incidence of infections due to multidrug-resistant Gram-negative bacteria has led to the renewed interest in the use of 'forgotten' antibiotics such as colistin. In this work, we studied the chromosomal colistin resistance mechanisms among laboratory-induced colistin-resistant Escherichia coli isolates. Methods: Three colistin-susceptible (ColS) clinical isolates of E. coli assigning to ST131, ST405, and ST361 were exposed to successively increasing concentrations of colistin. The nucleotide sequences of pmrA, pmrB, pmrD, phoP, phoQ, and mgrB genes were determined. The fitness burden associated with colistin resistance acquisition was determined by measuring the in vitro growth rate. Results: Colistin resistance induction resulted in 16-64 times increase in colistin MICs in mutants (n = 8) compared with parental isolates. Analysis of chromosomal genes in colistin-resistant mutants compared with those of ColS ancestors revealed genetic alterations confined to PmrAB two-component system and included PmrA G53R/R81S/L105P and PmrB E121K/E121A/A159P/A159V/G302E changes. The PmrB E121 was found as a critical position for colistin resistance development being altered in three mutants with different ancestors. The acquired colistin-resistance phenotype was stable following 10 consecutive passages in the absence of selective pressure of colistin and it did not alter the susceptibility of mutants to other antimicrobial agents. All mutants exhibited growth rates similar to their respective ColS ancestors, except for one isolate, which revealed a significant growth defect. Conclusion: Our results revealed that colistin resistance in E. coli was more related to PmrAB alterations, which did not impose a fitness cost in most cases.

DESCRIPTIONS AND PHYLOGENETIC AFFINITIES OF TWO NEW SPECIES OF RHABDIAS STILES AND HASSALL, 1905 (NEMATODA: RHABDIASIDAE) FROM NORTH AMERICAN FROGS: ARE WE STILL ONLY SCRATCHING THE SURFACE?

Two new species of lung-dwelling nematodes are described from North American frogs: Rhabdias aurorae n. sp. from Rana aurora and Rhabdias conni n. sp. from Rana clamitans and Rana catesbeiana from Arkansas; the latter species was also found in Oklahoma and Georgia. Rhabdias aurorae n. sp. differs from other Nearctic congeners in the combination of the following characteristics: buccal capsule 22-25 μm wide, elongated tail covered with inflated cuticle, esophagus with prominent dilatation in anterior part and 6 small circumoral lips. Rhabdias conni n. sp. is morphologically closest to Rhabdias ranae Walton, 1929 and Rhabdias joaquinensisIngles, 1936; it differs from them in the shape of lateral pseudolabia, the dimensions of the body, and the egg size. Both new species were found to be significantly different from the Nearctic congeners in the nucleotide sequences of nuclear ribosomal DNA (18S-ITS-28S region), 12S, and CO1 mitochondrial genes. The 2 new species differ from other currently sequenced Nearctic congeners by 1.1-2.7% of nucleotide positions in the nuclear rDNA region, 1.3-3.4% in the 12S gene, and 3.4-9.4% in CO1 gene. Molecular phylogenetic analysis based on nuclear ribosomal DNA sequences placed both new species into the clade consisting of Nearctic and Neotropical Rhabdias spp. The position of Rh. aurorae n. sp. within the clade is uncertain because of a polytomy, but Rh. conni n. sp. is nested within the "Rh. joaquinensis complex" related to Rh. ranae and Rhabdias tarichaeKuzmin, Tkach, and Snyder, 2003. The phylogenetic analysis based on nuclear ribosomal DNA sequences has revealed 3 evolutionary host-switching events from anuran to caudatan hosts among Rhabdias spp. that occurred in the Nearctic and Palearctic. The molecular phylogeny also suggests that Rhabdias may have originally evolved in what is now Africa.

The influence of nucleotide context on non-specific amplification of DNA with Bst exo- DNA polymerase

In recent years, nucleic acid amplification methods that proceed in isothermal mode and require the use of DNA polymerases with strand-displacement activity have become increasingly widespread. Among these, the most popular is the Bst exo- polymerase, but it tends to carry out nonspecific DNA synthesis through multimerization. This study shows the influence of the nucleotide sequence on the binding of Bst exo- with DNA and on the efficiency of multimerization initiation. On single-stranded trinucleotides (sst) and dinucleotide duplexes (dst), molecular docking revealed the preference for binding of the “closed” form of Bst exo- to purine-rich sequences, especially those containing dG at the 3′ end of the synthesized strand. The data obtainedin silicowere confirmed in experiments using oligonucleotide templates that differ in the structure of the 3′- and 5′-terminal motifs. It has been shown that templates with an oligopurine 3′-terminal fragment and an oligopyrimidine 5′-terminal part contribute to an earlier start of multimerization. The obtained data can be used at the stage of selecting optimal nucleotide sequences for isothermal amplification, ensuring more reliable results. Thus, to avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

Molecular identification and eco-friendly management of rice brown planthoppers in Bangladesh

Infestation by various insect pests is the main constraint for growing rice where rice brown planthopper (Nilaparvata lugens Stål) can severely damage rice plants directly through feeding. Therefore, the study aims to detect rice brown planthoppers (BPH) and provide environment-friendly management tactics to mitigate the problem which caused by brown planthoppers. The BPH samples were collected from rice fields of different locations in the Patuakhali of Bangladesh for molecular identification. A molecularly single species of rice brown planthopper, Nilaparavata lugens was identified using mitochondrial cytochrome oxidase subunit I (mtCOI) universal marker. The nucleotide sequences of collected samples were compared with other nucleotide sequences from the GenBank database of NCBI, which make single clades in the phylogenetic tree at an insignificant distance. Moreover, brown planthopper management observations were recorded in laboratory conditions after providing an artificial diet with different treatments of plant-based insecticides Neem oil (1 %, 5 %, and 10 %), Castor oil (1 %, 5 %, and 10 %) where only 20 % sucrose solution was used as negative control and Abamectin (1 %, 5 % and 10 %) were also used as a positive control for comparing the efficacy of plant-based insecticides on rice brown planthoppers. The results showed the highest mortality (100 %) of rice brown planthoppers was recorded by Abamectin 10 %, followed by Abamectin 5 %. Neem 10 % performed better than Abamectin 1 % during 1st hour. Initial after exposure of 2nd hour for Abamectin 1 % revealed greater mortality (59 %) than Neem 10 %. Neem 5 % showed less effect on mortality in brown planthopper than Neem 10 % but was higher than Neem 1 % during 6 h of observation. The Castor oil of 10 % caused higher mortality than the Castor of 5 % but not up to the marks of Abamectin and different concentrations of Neem oil. Castor oil of 1 % and control have shown no mortality of brown planthopper for 6 h of observation.

First report of Tomato interveinal chlorosis virus infecting Rhynchosia minima plants in Brazil.

Tomato interveinal chlorosis virus (ToICV; Begomovirus solanumintervenae, genus Begomovirus, family Geminiviridae) has been described infecting tomato (Solanum lycopersicum) and Macroptilium lathyroides in Northeastern (NE) Brazil for more than a decade (Albuquerque et al., 2012; Silva et al., 2012). During a survey in 2020, plants of the leguminous weed Rhynchosia minima exhibiting virus-like symptoms such as mosaic and interveinal chlorosis were observed in the state of Alagoas, NE Brazil. Symptomatic leaf samples of R. minima were randomly collected (n=15; supplementary figure 1). Total DNA from each sample was used as a template for PCR amplification of partial begomoviral DNA-A sequences using the degenerate primer pair PAL1v1978 and PAR1c496, universal for geminiviruses (Rojas et al., 1993). Amplicons of ~1.2 kbp were observed from 12 samples, although this should not be considered as incidence since only symptomatic plants were collected. To identify the begomovirus associated with R. minima, viral genomes were amplified from PCR-positive samples using rolling circle amplification (RCA) (Inoue-Nagata et al., 2004). The RCA products were digested with HindIII, cloned into the pBluescript II KS+ plasmid vector and bidirectionally Sanger-sequenced (Macrogen Inc., Seoul). BLASTn searches indicated that the clones (n=4) reported here corresponded to a begomovirus DNA-A component, and pairwise comparisons showed that they shared the highest identity with ToICV, at 92.4-94.7% nucleotide sequence identity. Based on the species demarcation criteria of ≥91% nucleotide identity for the genus Begomovirus (Brown et al., 2015), the begomoviruses obtained from R. minima are new isolates of ToICV. The new DNA-A sequences of 2,619-2,623 nt in length were deposited in GenBank under accession numbers PP639092 to PP639095. Multiple nucleotide sequence alignments were prepared using the MUSCLE algorithm implemented in MEGA v.11 (Kumar et al., 2018), and a maximum likelihood (ML) tree was reconstructed in RaxML-NG (Kozlov et al., 2019), assuming a general time reversible (GTR) nucleotide substitution model with a gamma (G) model of rate heterogeneity and 1,000 bootstrap replicates. The DNA-A-based tree showed that the ToICV sequences clustered into a monophyletic group, additionally supporting these isolates as members of the species Begomovirus solanumintervenae. At least two independent interspecies recombination events were predicted among the ToICV isolates, with breakpoints located in the Rep-encoding region and ToICV (GenBank Accession JF803253), tomato mottle leaf curl virus (JF803248) and soybean blistering mosaic virus (MN486865) detected as putative parents. To the best of our knowledge, this is the first report of ToICV infecting R. minima worldwide, expanding the host range of this begomovirus. Non-cultivated plants such as R. minima play a crucial role as reservoirs and sources of inoculum for begomoviruses (Paz-Carrasco et al., 2014), reinforcing their relevance to socioeconomically important crops.

The complete chloroplast genome sequence of Lemna turionifera (Araceae)

Lemna turionifera is native to North America and northern Asia, with significant potential for industrial wastewater remediation. The complete nucleotide sequence of the L. turionifera chloroplast genome (cpDNA) was determined. The cpDNA is a circular molecule of 166,606 bp and containing a pair of inverted repeats (IRs) measuting 31,663 bp each. These IRs are flanked by a small single-copy region of 13,542 bp and a large single-copy region of 89,738 bp. The chloroplast genome of L. turionifera consisted of 112 unique genes, including 78 protein-encoding genes, 30 tRNA genes, and four rRNA genes. The phylogenetic analysis utilizing cpDNA provided a well-supported resolution of the relationships among subfamilies within the Araceae family. Our findings indicated a close relationship between L. turionifera and a clade consisting of L. minor, L. japonica, and L. gibba. The availability of the complete chloroplast genome sequence of L. turionifera presents valuable data for future phylogenetic investigations within the Lemnaceae family.

Genotype classification and pathogenicity of infectious bursal disease virus circulating in vaccinated broiler chicken farms.

This study investigated the genotype classification and pathogenicity of infectious bursal disease virus (IBDV) circulating in vaccinated broiler chicken farms in Egypt. A total of 150 samples were collected from 30 vaccinated commercial broiler chicken farms and pooled into 30 working samples. IBDV was tested using reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the hypervariable region of the viral protein 2 (hvVP2) and the VP1 gene 5' extremity. Both RT-PCR fragments were sequenced from six samples, and then the obtained nucleotide sequences were analyzed. The IBDV genotypes were identified using nucleotide sequences. Five sequences of the six strains examined were classified as genotype A3B2 for the highly virulent segments A and B (vv-A/vv-B IBDV). Interestingly, this study identified and classified a novelsegment-reassortant strainas the A1B2 genotype. Specifically, it involved the segment reassortment of classical virulent segment A (cv-A) with vv-B producing cv-A/vv-B reassortant IBDV. Subsequently, we compared the pathogenicity of reassortant (cv-A/vv-B) IBDV and vvIBDV strains identified in this study. Both strains developed typical IBD clinical signs, postmortem lesions, histopathology, immunohistochemistry, and lesion scores, which were more severe in vvIBDV than reassortant IBDV. In conclusion, this is the first report of the genotype classification based on both genome segments (hvVP2 and VP1) with pathogenicity of IBDV circulating in vaccinated broiler chicken farms and this pathogenicity is more severe in vvIBDV strain than a novel reassortant IBDV strain.

Identification and biological control of bacterial leaf spot disease of cucurbits

Objective: The present study was conducted for isolation and detection of the phyto-pathogen responsible for bacterial leaf spot disease of cucurbits as well as evaluation of its biological control techniques. Methods: The pathogen of the disease was isolated from infected leaf of bitter gourd and cultured on Luria-Bertani (LB) growth medium. The morphological tests and biochemical tests revealed the isolated bacteria as gram negative. Furthermore, advanced molecular technique was applied to facilitate proper detection of the isolated bacteria. The PCR of the bacterial genomic DNA using specific primers generated clear band of approximately 1450 bp in gel electrophoresis. Results: In gram staining test, the bacterial strain was found to be rod shaped and pinkish in color and Gram-negative. All the biochemical test showed positive without mannitol salt agar. The nucleotide sequencing of 16S rDNA gene of the bacterial isolate showed 86.02% similarities with the original sequence of Xanthomonas cucurbitae. In antibiotic sensitivity assay, the antibiotic cefixime showed highest 28.0±0.7 mm diameter of inhibition zone against the tested bacteria. The highest antibacterial activity was 8.0±0.5 mm zone of inhibition by of Allium sativum. Conclusion: The present findings may be benevolent for the detection, characterization and development of biological control techniques to prevent the leaf spot disease of cucurbits crops in Bangladesh. It may help to find out the specific signaling pathway techniques for plant microbes relationship for disease formation in future.

Calreticulin from Apostichopus japonicus relieves endoplasmic reticulum stress induced by Vibrio splendidus through autophagy

When organisms are exposed to external stimuli, misfolded proteins accumulate continuously, resulting in endoplasmic reticulum (ER) stress. Autophagy is of great significance for eliminating aggregated proteins and maintaining cellular homeostasis. However, the molecular mechanism of activating autophagy in response to ER stress in sea cucumber is remain unclear. In the current study, we demonstrated that the pathogen Vibrio splendidus can cause ER stress in Apostichopus japonicus coelomocytes and identified a Ca2+ binding partner calreticulin (designated as AjCRT), which increased with the occurrence of ER stress. The nucleotide sequence analysis showed that the open reading frame of AjCRT was 1242 bp and encoded a 413-amino-acid residue polyprotein with calreticulin domains. The spatial expression analysis revealed that AjCRT was ubiquitously expressed in all examined tissues with large magnitude in the coelomocytes and was minimally expressed in muscle. Furthermore, silencing AjCRT in vivo could significantly exacerbate ER stress induced by V. splendidus and resulted in the significant reduction of coelomocyte autophagy. These findings indicate a calreticulin-based mechanism that positively regulates autophagy in response to ER stress induced by pathogen infection. The results will provide a basis for understanding the way of host alleviating ER stress through autophagy, and pharmacological approaches may have potential for managing ER stress induced by pathogen and related cellular disorders.

Identification and Full-Length Sequence Analysis of a Novel Recombinant Goat Astrovirus Genotype in Guangxi, China.

Astroviruses are single-stranded, positive-sense RNA viruses capable of infecting humans as well as a wide range of mammalian and avian species, with a length of approximately 6.6-7.7 kb. In this study, 139 goat fecal samples collected from the Guangxi province were used for the RT-PCR detection, and two of these were positive for goat astrovirus, with a positivity rate of 1.44% (2/139). The complete genome sequence of an astrovirus strain and the partial genome sequence of a strain astrovirus, named GX WZ 2023 and GX HC 2023, were amplified and sequenced, and their sequence lengths were 6284 nt and 6213 nt, respectively. Among them, the capsid protein of goat astrovirus GX HC 2023 showed the highest amino acid identity of 95.9% with ovine astrovirus GX, which belonged to the MAstV-2 genotype. However, the closest relative of the GX WZ 2023 strain was found to be the caprine astrovirus Sichuan, with a nucleotide sequence identity of 76.8%. The ORF1ab nonstructural protein of this strain showed the highest amino acid identities of 89.2 and 95.8% with the ovine astrovirus S5.1 and caprine astrovirus G5.1 strains, respectively. However, its ORF2 capsid protein has 68.4% amino acid identity with the bovine astrovirus (BAstV) 16 2021 CHN strain and only 21.9-64% amino acid identity with all available strains of goat astrovirus. The GX WZ 2023 strain was recombined with the Chinese (BAstV 16 2021 CHN) and Japanese bovine strains (BAstV JPN 2015) in the ORF2 region. Therefore, the goat astrovirus GX WZ 2023 is proposed as a new member of the family goat astroviridae based on the species classification criteria of the International Committee on Taxonomy of Viruses. These findings enhance our understanding of the prevalence and genetic evolution of goat astrovirus and provide a scientific basis for future studies of these viruses in other animals.

Comparative phylogenetic analysis of Siberian isolates of bovine pestiviruses

The results of comparative phylogenetic analysis of 52 isolates of bovine pestiviruses of three species circulating among highly productive dairy cattle in Siberia, as well as those present in the samples of commercial fetal serum, transfected cell culture lines and live vaccines on four genes: Npro, Erns, E1 and E2 are presented. The obtained data were compared with the results of the 5'UTR gene sequencing obtained earlier. The results confirmed the circulation in Siberia of eleven subtypes of BVDV-1 (a,b,c,d,f,g,i,j, k,p,r), three subtypes of BVDV-2 (a,b,c) and one BVDV -3(a). The genetic profiles of the isolates for the 5'UTR, Npro, Erns and E1 genes matched completely. The exceptions were three BVDV-1a isolates (R/FBS/96, N/MDBK/08 and SA/FBS/08) detected in infected cell cultures that clustered in a clade formed by BVDV-1j reference sequences. The remaining isolates previously shown to have 100% sequence identity of the 5'UTR had nucleotide sequence similarities of the Npro, Erns, E1, and E2 genes ranging from 96-99%, which also implies that they are closely related. The results of the BVDV-2 isolates sequencing showed a complete correspondence with the genome sequences identified in the previous studies. Comparative sequencing also confirmed the circulation of H Italian-Brazilian pestivirus isolates (BVBD-3a) in Siberia, but all the isolates were divided into two subclades. The first included the isolates isolated from fetal sera and cell cultures, while the second included those isolated from the vaccines and internal organs of diseased animals during outbreaks in farms. The genetic diversity of bovine pestiviruses, their origin and variability are discussed.

On the host specificity and genetic diversity of Iodamoeba bütschlii: Observations from short amplicon-based next-generation sequencing

Iodamoeba is a single-celled intestinal parasite, which is common in humans in certain parts of the world, and also in pigs. For the first time, we provide DNA-based evidence of goat, dromedary, fallow deer, and donkey as hosts of Iodamoeba and show that Iodamoeba-specific nucleotide sequences from these four hosts do not appear to overlap with those of humans, unlike those from pigs. We moreover show that similar strains of Iodamoeba can be found in Madagascar, Western Sahara, and Ecuador and that intra-sample diversity is typically extensive across even small fragments of DNA in both human and non-human hosts.

Study of CaDreb2c and CaDreb2h Gene Sequences and Expression in Chickpea (Cicer arietinum L.) Cultivars Growing in Northern Kazakhstan under Drought.

Drought poses a significant challenge to plant growth and productivity, particularly in arid regions like northern Kazakhstan. Dehydration-responsive element-binding (DREB) transcription factors play an important role in plant response to drought and other abiotic stresses. In Arabidopsis thaliana, the DREB subfamily consists of six groups, designated DREB1 to DREB6. Among these, DREB2 is primarily associated with drought and salinity tolerance. In the chickpea genome, two DREB genes, CaDREB2c and CaDREB2h, have been identified, exhibiting high sequence similarity to Arabidopsis DREB2 genes. We investigated the nucleotide sequences of CaDREB2c and CaDREB2h genes in several chickpea cultivars commonly grown in northern Kazakhstan. Interestingly, the CaDREB2h gene sequence was identical across all varieties and corresponded to the sequence deposited in the GenBank. However, the CaDREB2c gene sequence exhibited variations among the studied varieties, categorized into three groups: the first group (I), comprising 20 cultivars, contained a CaDREB2c gene sequence identical to the GenBank (Indian cultivar CDC Frontier). The second group (II), consisting of 4 cultivars, had a single synonymous substitution (T to C) compared to the deposited CaDREB2c gene sequence. The third group, encompassing 5 cultivars, displayed one synonymous substitution (C to T) and two non-synonymous substitutions (G to T and G to A). Furthermore, we assessed the gene expression patterns of CaDREB2c and CaDREB2h in different chickpea varieties under drought conditions. Chickpea cultivars 8 (III), 37 (I), 6 (III), and 43 (I) exhibited the highest drought resistance. Our analysis revealed a strong positive correlation between drought resistance and CaDREB2h gene expression under drought stress. Our findings suggest that the chickpea's adaptive responses to water deprivation are associated with changes in CaDREB2 gene expression. To further elucidate the mechanisms underlying drought tolerance, we propose future research directions that will delve into the molecular interactions and downstream targets of CaDREB2 genes.

Generating information-dense promoter sequences with optimal string packing.

Dense arrangements of binding sites within nucleotide sequences can collectively influence downstream transcription rates or initiate biomolecular interactions. For example, natural promoter regions can harbor many overlapping transcription factor binding sites that influence the rate of transcription initiation. Despite the prevalence of overlapping binding sites in nature, rapid design of nucleotide sequences with many overlapping sites remains a challenge. Here, we show that this is an NP-hard problem, coined here as the nucleotide String Packing Problem (SPP). We then introduce a computational technique that efficiently assembles sets of DNA-protein binding sites into dense, contiguous stretches of double-stranded DNA. For the efficient design of nucleotide sequences spanning hundreds of base pairs, we reduce the SPP to an Orienteering Problem with integer distances, and then leverage modern integer linear programming solvers. Our method optimally packs sets of 20-100 binding sites into dense nucleotide arrays of 50-300 base pairs in 0.05-10 seconds. Unlike approximation algorithms or meta-heuristics, our approach finds provably optimal solutions. We demonstrate how our method can generate large sets of diverse sequences suitable for library generation, where the frequency of binding site usage across the returned sequences can be controlled by modulating the objective function. As an example, we then show how adding additional constraints, like the inclusion of sequence elements with fixed positions, allows for the design of bacterial promoters. The nucleotide string packing approach we present can accelerate the design of sequences with complex DNA-protein interactions. When used in combination with synthesis and high-throughput screening, this design strategy could help interrogate how complex binding site arrangements impact either gene expression or biomolecular mechanisms in varied cellular contexts.

A Mendelian randomization study investigating causal links between gut microbiota or metabolites and chronic hepatitis B.

This study aimed to explore the potential causal relationship between the gut microbiota and/or its metabolites and the progression of chronic hepatitis B (CHB). The gut microbiota was used as the exposure factor. The training set exposure data were obtained from the China Nucleotide Sequence Archive (CNSA). Genome-wide association study (GWAS) data from Asia were used as the outcome variables. Outcome data for both the training and validation sets were sourced from the GWAS Catalog database. A dual-sample Mendelian randomization approach was used to analyze the causal relationships, with the inverse variance-weighted method serving as the main analytical strategy. Sensitivity analysis was conducted to assess the robustness of Mendelian randomization analysis results. In the training set database, analysis using the inverse variance-weighted method revealed a positive correlation between Fusobacterium varium and chronic hepatitis B [OR = 1.122, 95% CI (1.016, 1.240), p = 0.022]. Conversely, Veillonella parvula exhibited a negative correlation with chronic hepatitis B [OR = 0.917, 95% CI (0.852, 0.987), p = 0.021]. Sensitivity analysis revealed no evidence of pleiotropy and heterogeneity. No gut microbiota metabolites with a causal effect on chronic hepatitis B were identified. Additionally, no associations between the gut microbiota and the progression of chronic hepatitis B were found in the validation data from the European cohort. This study suggests that F. varium may facilitate the progression of chronic hepatitis B, whereas V. parvula may impede it. No causal relationships between gut microbiota metabolites and chronic hepatitis B were established.

Analysis of potential genes, immunological and antioxidant profiles associated with trypanosomiasis susceptibility in dromedary camels

Trypanosomiasis is associated with tissue damage and may trigger an immunological response. These tissue lesions are linked to metabolic issues and oxidative stress. The current study aimed to investigate the immunological, antioxidant, and metabolic changes that may be connected to camel trypanosomiasis. Blood samples were collected from 54 camels and allocated into two groups: The control group (35 camels) and the infected group (19 camels). The genes TLR2, TLR5, IL-17, MARCHF3, RASGRP1, EPS15L1, PPIE, ASB16, CMPK2, LPCAT1, FPGT, GPHN, TNNI3K, DIO3, keap1, and OXSR1 were significantly up-regulated in trypanosomiasis camels. However, down-regulation was observed for the genes Nrf2, PRDX6, and NDUFS5. PCR-DNA sequencing was used to identify nucleotide sequence polymorphisms in the immune (TLR2, TLR5, IL-17, MARCHF3, RASGRP1, and EPS15L1), metabolic (PPIE, ASB16, CMPK2, LPCAT1, FPGT, GPHN, TNNI3K, and DIO3), and antioxidant (Nrf2, Keap1, PRDX6, NDUFS5, and OXSR1) genes between healthy and trypanosomiasis-affected camels. Exploring the serum profile also showed a significant (P ˂ 0.05) increase in Hp, SAA, Cp, IL-1β, IL-6, IL 10, TNF-α, and MDA, with significant (P ˂ 0.05) reduction in the serum levels of CAT, SOD, GSH, T3, and T4 in diseased camels compared with healthy ones. Our findings confirm the significance of nucleotide variations, gene expression patterns, and the biochemical profile of the investigated markers as indicators for the susceptibility of trypanosomiasis in dromedary camels and may be utilized to create management strategies.