INTEGRATIVE MORPHO-MOLECULAR ANALYSIS OF Papilio polytes, Papilio polymnestor, AND Euploea core FROM JHARKHAND (INDIA) USING ADVANCED BIOTECHNOLOGICAL AND BIOINFORMATIC APPROACHES

Dragonflies and damselflies (Odonates) are important biological indicators in freshwater ecosystems. However, identification among Odonates is often challenging due to their similar morphological features. Therefore, the incorporation of morphological identification by taxonomists and validation using mitochondrial barcodes such as cytochrome c oxidase subunit I (COI) can be a more reliable approach to enhance the accuracy in species identification. In this study, four COI barcodes for Malaysian dragonflies (Neurothemis fluctuans) and damselflies (Neurobasis chinensis, Aristocypha fenestrella and Sundacypha petiolata) were generated. Three of the generated barcodes (D2 COI, D4 COI and D5 COI) supported the species identified by taxonomists meanwhile D3 COI deduced that the damselfly species was misidentified due to the very similar morphology between the same genus of damselfly. All of the COI barcodes are now available in the GenBank with the accession numbers of MT266926.1 (D2 COI), MT266925.1 (D3 COI), MT269676.1 (D4 COI) and MT266924.1 (D5 COI).

Familial adenomatous polyposis (FAP) is an Autosomal dominant inherited disorder and a rare form‌ of colorectal cancer (CRC) that is characterized by the development of hundreds to thousands of adenomas in the rectum and colon. Mostly, cancers develop after the advent of the polyps. It appears in both sexes evenly, and the occurrence of the disease is in the second decade of life. Mitochondrial genome mutations have been reported with a variety of Tumors, but the precise role of these mutations in the pathogenicity and tumor progression is not exactly clear. Cytochrome c oxidase subunit I (COX1) is the terminal enzyme of the mitochondrial respiratory chain. The present study aims at assessing the occurrence of mtDNA mutations in COX1 gene in FAP patients and attempts to find out the cause and effect relationship between mitochondrial mutations and tumor progression. In this study, 56 FAP patients were investigated for the presence of the mutations in mitochondrial COX1 coding gene by PCR and sequencing analysis. All sequences that differed from the revised Cambridge Reference Sequence (rCRS) were classified as missense/ nonsense or silent mutations. Functional genomic studies using Bio-informatics tools were performed on the founded mutations to understand the downstream alterations in structure and function of protein. We identified 38 changes in the COX1 gene in patients with FAP symptoms. Most of them were heteroplasmic changes of missense type (25/38). Tree of the changes (G6145A, C6988A, and T7306G) were nonsense mutations and had not been reported in the literature before. Our results of bioinformatics predictions showed that the identified mutations can affect mitochondrial functions, especially if the conservative domain of the protein is concerned. Our findings indicate a high frequency of mtDNA mutations in all of the FAP cases compared to matched controls. These data significantly enhance our understanding of how such mutations contribute to cancer pathologies and develop the cancer treatment methods by new diagnostic biomarkers, and new drugs for gene therapy.

Seed-borne fungi are fungi that are associated with seeds and can be a source of plant disease in the field. Therefore, this study aims to utilize DNA barcoding to identify seed-borne fungi isolates from Tanjung Morawa B village. This investigation took place at Tangerang's genetic science laboratory in Tangerang was the site of this investigation. Sequence and phylogenic tree building, PCR amplification with MyTaq HS Red Mix, 2X (Bioline, BIO-25048), and genomic DNA extraction with the Quick-DNA Magbead Plus Kit (Zymo Research, D4082) were the research methods used. The electrophoresis results showed that the DNA made from PCR using Primers ITS-1 and ITS-4 was of high quality. This was clear from the electrophorograms for both the TMIE isolate sample and the control. Using ITS-1 and ITS-4 for DNA amplification produced favorable results, with virtually no smears detected. The Basic Local Alignment and Search Tool (BLAST) program, which is connected to the Genebank database, was employed to analyze the sequence results in the form of a base sequence with a length of 581 base pairs. Based on the genetic similarity results of the isolate's DNA with Genebank, we determined that the TMIE isolate was 100% similar to Curvularia eragrostidis.

BackgroundAdvances in automated DNA sequencing technology have accelerated the generation of metagenomic DNA sequences, especially environmental ribosomal RNA gene (rDNA) sequences. As the scale of rDNA-based studies of microbial ecology has expanded, need has arisen for software that is capable of managing, annotating, and analyzing the plethora of diverse data accumulated in these projects.ResultsXplorSeq is a software package that facilitates the compilation, management and phylogenetic analysis of DNA sequences. XplorSeq was developed for, but is not limited to, high-throughput analysis of environmental rRNA gene sequences. XplorSeq integrates and extends several commonly used UNIX-based analysis tools by use of a Macintosh OS-X-based graphical user interface (GUI). Through this GUI, users may perform basic sequence import and assembly steps (base-calling, vector/primer trimming, contig assembly), perform BLAST (Basic Local Alignment and Search Tool; [1-3]) searches of NCBI and local databases, create multiple sequence alignments, build phylogenetic trees, assemble Operational Taxonomic Units, estimate biodiversity indices, and summarize data in a variety of formats. Furthermore, sequences may be annotated with user-specified meta-data, which then can be used to sort data and organize analyses and reports. A document-based architecture permits parallel analysis of sequence data from multiple clones or amplicons, with sequences and other data stored in a single file.ConclusionXplorSeq should benefit researchers who are engaged in analyses of environmental sequence data, especially those with little experience using bioinformatics software. Although XplorSeq was developed for management of rDNA sequence data, it can be applied to most any sequencing project. The application is available free of charge for non-commercial use at .

Blastocystis sp. is a commonly reported enteric protistan parasite in faecal specimens with a worldwide distribution afflicting both humans and a wide range of animals. The aim of this study is to characterize the subtypes (STs) of Blastocystis sp. isolates from asymptomatic individuals in an urban community in Pateros, Metro Manila, Philippines. The 600-bp small subunit ribosomal RNA (SSU rRNA) barcoding region of Blastocystis sp. isolates was amplified and sequenced using the primers RD5 and BhRDr. Subtypes were identified by uploading the sequences onto the Basic Local Alignment and Search Tool (BLAST) websites, the Blastocystis Subtype (18S) and Sequence Typing (MLST) Database and by construction of a phylogenetic tree. Twenty-nine (29) out of 35 individuals were detected positive for Blastocystis sp. ST3 is the most common among the three STs detected (65.5%), followed by ST1 (31.0%) and ST4 (3.44%). This study showed that DNA barcoding can serve as a helpful tool to investigate the diversity of Blastocystis sp. in the Philippines.

Fungus growing termites is a unique group of termites that can grow mutualistic fungi from Basidiomycota: Termitomyces inside their nest. This group of termites has an important ecological role as a major decomposer. However, comprehensive study of fungus-growing termites is limited in Thailand and the complex termites-fungus relationship is a challenging issue to study. This research was aimed to identify the species of termites and fungal symbionts using DNA barcoding, to compare the molecular results with morphological identification, and to gain insights into the co-evolutionary relationship by constructing co-cladogenesis analysis from the termites and the fungal symbionts. Four termite species (Macrotermes annandalei, Odontotermes feae, Ancistrotermes pakistanicus and Ancistrotermes sp.) from four Sakaerat and two Chulabhorn colonies were identified based on the BLAST-N of cytochrome oxidase sub-unit I (COI) sequence and morphological identification. The partial COI sequence of O. feae identified in this study is the first record of this species deposited into the NCBI nucleotide database. Molecular identification of the fungi were conducted by using nuclear ribosomal internal transcribed spacer (ITS) region. The results showed that those six termite colonies grew specifically the fungal genus Termitomyces. Three genetic groups of Termitomyces i.e. Group 1, Group 2 (T. cylindricus), and Group 6, were present among the six colonies. Molecular identification was confidently supported by morphological identification of termite samples, at least up to the genus level. High host-symbionts specificity is considerably observed within some clades on paired termite-fungus phylogeny. However, more sampling is needed for the more confident result.

Pea, (Pisum sativum, Leguminaceae), also called garden pea is herbaceous annual plant grown worldwide for its edible seeds. Several studies reported that about 20 insect pest attack this crop and cause substantial yield loss. The pineapple mealybug, Dysmicoccus brevipes Cockerell (Hemiptera: Pseudococcidae) is reported for the first time from garden pea from Jharkhand state of India. The nymph and adult mealybugs were collected from four different locations of garden pea fields and identified through morphological characters and further their identity confirmed with molecular characterization using partial (658bp) mitochondrial cytochrome oxidase 1 (mtCO1) gene. Nymphs and adults of mealybugs suck the sap from the roots as well as stem base near the soil surface. The mealybug infested roots were observed with dark circles and plants showing wilting symptoms. The infestation of D. brevipes was observed in the month of November, 2022 with 5% plant infestation in the early crop growth stage, where as the maximum of 45% infestation was recorded in the month of August, 2022. This study confirms that garden pea as the new host for D. brevipes and its damage symptoms.

The aim of this study was to identify potential markers of atherosclerosis development in familial hypercholesterolemia (FH) patients. GSE13985 microarray data, generated using blood samples from 5 FH patients and 5 matched controls, was downloaded from the Gene Expression Omnibus. Differentially expressed genes (DEGs) between FH and controls were identified and a protein-protein interaction (PPI) network was constructed. Module and hub proteins were screened in this network. The module genes were subjected to a gene ontology (GO) analysis, and a Kyoto Encyclopedia of Genes and Genomes enrichment analysis was also performed. A total of 394 genes, including 125 up- and 269 down-regulated genes, were differentially expressed. Ribosomal proteins L9 (RPL9), L35 (RPL35), and S7 (RPS7) were designated as hub nodes in the PPI network. The DEGs were found to be significantly enriched in ribosomal and oxidative phosphorylation pathways. Ribosomal protein genes were found to be involved in the ribosomal pathway. The cytochrome-c oxidase (COX) genes COX subunit VIIa polypeptide 2 (COX7A2), COX subunit VIIb (COX7B), COX subunit VIIc (COX7C), and COX subunit VIc (COX6C) were enriched in the oxidative phosphorylation pathway. Module analysis and GO enrichment analysis identified ribosomal proteins as important regulators of FH. Ribosomal and oxidative phosphorylation pathways may be closely associated with atherosclerosis development. Ribosomal protein genes and cytochrome-coxidase genes may be potential therapeutic targets for atherosclerosis.

A fast-growing number of non-coding RNA structures have been resolved and deposited in Protein Data Bank (PDB). In contrast to the wide range of global alignment and motif search tools, there is still a lack of local alignment tools. Among all the global alignment tools for RNA 3D structures, STAR3D has become a valuable tool for its unprecedented speed and accuracy. STAR3D compares the 3D structures of RNA molecules using consecutive base-pairs (stacks) as anchors and generates an optimal global alignment. In this article, we developed a local RNA 3D structural alignment tool, named LocalSTAR3D, which was extended from STAR3D and designed to report multiple local alignments between two RNAs. The benchmarking results show that LocalSTAR3D has better accuracy and coverage than other local alignment tools. Furthermore, the utility of this tool has been demonstrated by rediscovering kink-turn motif instances, conserved domains in group II intron RNAs, and the tRNA mimicry of IRES RNAs.

For DNA differential diagnosis of human Taenia cestodes, a base excision sequence scanning thymine-base method using the cytochrome c oxidase subunit I and cytochrome b genes as targets was used. The characteristic thymine-base peak profiles provide four distinct types, unique for T. saginata, T. asiatica, and two genotypes of T. solium. This approach provides a useful tool for the identification and diagnosis of human taeniid cestodes without DNA sequencing if nucleotide sequence databases are available.

DNA barcoding is a molecular method that rapidly identifies an individual to a known taxon or its closest relative based on a 650-bp fragment of the cytochrome c oxidase subunit I (COI). In this study, DNA barcodes of members of the family Accipitridae, including Haliastur indus (brahminy kite), Haliaeetus leucogaster (white-bellied sea eagle), Ichthyophaga ichthyaetus (grey-headed fish eagle), Spilornis holospilus (crested serpent-eagle), Spizaetus philippensis (Philippine hawk-eagle), and Pithecophaga jefferyi (Philippine eagle), are reported for the first time. All individuals sampled are kept at the Philippine Eagle Center in Davao City, Philippines. Basic local alignment search tool results demonstrated that the COI sequences for these species were unique. The COI gene trees constructed using the maximum-likelihood and neighbour-joining (NJ) methods supported the monophyly of the booted eagles of the Aquilinae and the sea eagles of the Haliaeetinae but not the kites of the Milvinae.

Setaria marshalli is a mosquito-borne filarial nematode that causes infection in calves younger than two years old. In the present study, nematodes were obtained from a calf in Japan and morphologically identified as S. marshalli. Additionally, the partial cytochrome oxidase subunit I (COI) region (596 bp) was analyzed for the first time to establish a reliable DNA barcode. Nucleotide sequences of COI were identical among the seven worms obtained. The COI region can be a useful marker for species discrimination in the case of S. marshalli since nucleotide variations observed between the closest congener, Setaria cervi (51/596 bp), were sufficient to allow species discrimination. However, the phylogenetic relationship of S. marshalli with its congeners was unclear in a maximum likelihood tree. We found that the partial COI sequence of S. marshalli analyzed in the present study matched a relevant section of the complete mitochondrial genome of S. labiatopapillosa that was deposited in the International Nucleotide Sequence Database. This finding suggests that S. marshalli was misdiagnosed as S. labiatopapillosa in a previous study. It is crucial to conduct accurate morphological analyses to obtain reliable molecular information regarding Setaria nematodes.

Identifying parasitism genes encoding proteins secreted from nematodes is the key to understanding the molecular basis of nematode parasitism to insects. In this paper, a cDNA with two introns and three exons encoding a cysteine protease inhibitor was identified by screening a cDNA subtractive library constructed from the nematode, Steinernema carpocapsae, induced by Galleria mellonella hemolymph. The full-length cDNA contains an open reading frame encoding a 139-amino acid protein, designated Sc-cys, with a 19-residue signal peptide. The mature protein was predicted to have a molecular weight of 12,531.59 Da, a pI of 9.44, one disulfide bond, and three conserved domains believed to be important for the inhibition of cysteine proteases. In Basic Local Alignment and Search Tool analyses, the putative protein precursor displayed 26-42% identities to a multitude of cystatins or cystatin-like proteins. Phylogenetic analysis suggested the novel cystatin is likely a new member of the family 2 cystatins. Reverse northern blot, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and real-time RT-PCR analyses showed that the expression level of Sc-cys was upregulated substantially after induction by insect hemolymph. The specific analysis of genes encoding secretory proteins is providing a profile of putative parasitism genes expressed in S. carpocapsae throughout the parasitic cycle.

Different approaches are in progress to study phylogenetic relatedness and divergence among organisms. Genomic study is the most effective method for resolving phylogenetic relationships. Multiple genes are preferred over single gene sequence analysis and whole genome sequences are nowadays is the best nucleotide sequence-based approach to identify and classify the taxonomically complex group of organisms to deal with several phylogenetic issues. Various bioinformatics tools are providing valuable information to study significant evolutionary relationships such as next generation sequencing, proteomics etc. DNA barcoding technology is also recently used skill to study phylogeny and identify the morphologically ambiguous species by amplifying a small fragment of Cytochrome c Oxidase Subunit I (COI). We recently reported multiple gene phylogenies of Indian termites resulting in incongruent results. However, whole genome analysis will provide a significant outcome to resolve the diverse observations studied in these species.

DNA barcoding is a new perspective that uses gene sequence Cytochrome C oxidase subunit I (COI) for identification of unknown species by using "barcodes". The present study of” Labeo bata” specie was conducted for identification because it is important food specie in Pakistan. PCR products were analyized by bioinformatic tools. Evolutionary tree based on kimura 2 parameter indicate phylogenetic relationship among labeo species.A specimen is recognized if the barcode sequence of unknown specimen resembles the sequence in the barcode library or else, a new record is determined. It was concluded that DNA barcoding serves as milstone for identification at molecular level.