Sequencing Of Fragments Research Articles

Genome-wide survey reveals the genetic background of Xinjiang Brown cattle in China.

Introduction: Xinjiang Brown cattle are a famous dual-purpose (dairy-beef) cultivated breed in China that occupy a pivotal position within the cattle breeding industry in Xinjiang, China. However, little information is available on the genetic background of this breed. To fill this research gap, we conducted a whole-genome screen using specific-locus amplified fragment sequencing to examine the genetic structure and diversity of 130 Xinjiang Brown cattle-grazing type (XBG, traditional type) cattle. Methods: A subsequent joint analysis incorporating two ancestral breeds, specifically 19 Brown Swiss (BS) foreign and nine Kazakh (KZ) Chinese cattle, as well as 20 Xinjiang Brown cattle-housing type (XBH) cattle, was used to explore the genetic background of the Xinjiang Brown cattle. Results: The results showed that, after nearly a century of crossbreeding, XBG cattle formed a single population with a stable genetic performance. The genetic structure, genetic diversity, and selection signature analysis of the two ancestral types showed highly different results compared to that of XBH cattle. Local ancestry inference showed that the average proportions of XGB cattle within the BS and KZ cattle lineages were 37.22% and 62.78%, respectively, whereas the average proportions of XBH cattle within the BS and KZ cattle lineages were 95.14% and 4.86%, respectively. Thus, XGB cattle are more representative of all Xinjiang Brown cattle, in line with their breeding history, which involves crossbreeding. Two complementary approaches, fixation index and mean nucleotide diversity, were used to detect selection signals in the four aforementioned cattle breeds. Finally, the analysis of 26 candidate genes in Xinjiang Brown cattle revealed significant enrichment in 19 Gene Ontology terms, and seven candidate genes were enriched in three pathways related to disease resistance (CDH4, SIRPB1, and SIRPα) and the endocrine system (ADCY5, ABCC8, KCNJ11, and KCNMA1). Finally, development of the core SNPs in XBG cattle yielded 8,379 loci. Conclusion: The results of this study detail the evolutionary process of crossbreeding in Xinjiang Brown cattle and provide guidance for selecting and breeding new strains of this species.

Multi-Omics Analyses Unravel Genetic Relationship of Chinese Coffee Germplasm Resources

The genetic relationships between Coffea arabica resources were analyzed via specific length amplified fragment sequencing (SLAF-seq) and transcriptome sequencing to provide the theoretical basis for breeding new varieties. Twenty C. arabica accessions were used to analyze genetic diversity on the basis of SNPs identified in SLAFs and the transcriptome data. For the SLAF-seq analysis of 20 C. arabica accessions, two Coffea canephora accessions, one Coffea liberica accession, and one Coffea racemosa accession, the number of reads ranged from 2,665,424 to 7,210,310, with a GC content of 38.49%–40.91% and a Q30 value of 94.99%–96.36%. A total of 3,347,069 SLAF tags were obtained, with an average sequencing depth of 13.90×. Moreover, the 1,048,575 SNPs identified in the polymorphic SLAFs were filtered, then the remaining 198,955 SNPs were used to construct a phylogenetic tree, perform a principal component analysis, and characterize the population structure. For the transcriptome analysis, 128.50 Gb clean reads were generated for the 20 C. arabica accessions, with a GC content of 44.36%–51.09% and a Q30 value of 94.55%–95.40%. Furthermore, 25,872 genes’ expression levels were used for the correlation analysis. The phylogenetic relationships as well as the results of the principal component analysis, population structure analysis, and correlation analysis clearly distinguished C. arabica Typica-type accessions from the C. arabica Bourbon-type accessions. Notably, several C. arabica local selections with unknown genetic backgrounds were classified according to all four clustering results.

Direct and Detailed Site-Specific Glycopeptide Characterization by Higher-Energy Electron-Activated Dissociation Tandem Mass Spectrometry.

Glycosylation is widely recognized as the most complex post-translational modification due to the widespread presence of macro- and microheterogeneities, wherein its biological consequence is closely related to both the glycosylation sites and the glycan fine structures. Yet, efficient site-specific detailed glycan characterization remains a significant analytical challenge. Here, utilizing an Orbitrap-Omnitrap platform, higher-energy electron-activated dissociation (heExD) tandem mass spectrometry (MS/MS) revealed extraordinary efficacy for the structural characterization of intact glycopeptides. HeExD produced extensive fragmentation within both the glycan and the peptide, including A-/B-/C-/Y-/Z-/X-ions from the glycan motif and a-/b-/c-/x-/y-/z-type peptide fragments (with or without the glycan). The intensity of cross-ring cleavage and backbone fragments retaining the intact glycan was highly dependent on the electron energy. Among the four electron energy levels investigated, electronic excitation dissociation (EED) provided the most comprehensive structural information, yielding a complete series of glycosidic fragments for accurate glycan topology determination, a wealth of cross-ring fragments for linkage definition, and the most extensive peptide backbone fragments for accurate peptide sequencing and glycosylation site localization. The glycan fragments observed in the EED spectrum correlated well with the fragmentation patterns observed in EED MS/MS of the released glycans. The advantages of EED over higher-energy collisional dissociation (HCD), stepped collision energy HCD (sceHCD), and electron-transfer/higher-energy collisional dissociation (EThcD) were demonstrated for the characterization of a glycopeptide bearing a biantennary disialylated glycan. EED can produce a complete peptide backbone and glycan sequence coverage even for doubly protonated precursors. The exceptional performance of heExD MS/MS, particularly EED MS/MS, in site-specific detailed glycan characterization on an Orbitrap-Omnitrap hybrid instrument presents a novel option for in-depth glycosylation analysis.

DsRNA sequencing revealed a previously missed terminal sequence of a +ssRNA virus that infects dinoflagellate Heterocapsa circularisquama.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus cultured. However, only two strains of HcRNAV have been registered with complete genome sequences (strains 34 and 109 for UA and CY types, respectively). To extend the genomic information of HcRNAV, we performed full-genome sequencing of an unsequenced strain of HcRNAV (strain A8) using the fragmented and primer-ligated double-stranded RNA (dsRNA) sequencing (FLDS) method. The complete genome of HcRNAV A8 with 4457 nucleotides (nt) was successfully determined, and sequence alignment of the major capsid protein gene suggested that A8 was a UA-type strain, consistent with its intraspecific host specificity. The complete sequence was found to be 80 nt longer at the 5' terminus than the registered sequences of HcRNAV strains (34 and 109), suggesting that FLDS is more reliable for determining the terminal sequence than conventional methods (5' Rapid Amplification of cDNA End). Our study contributes to a better understanding of dinoflagellate-infecting viruses with limited sequence data.

Integration of Rubus yellow net virus in the raspberry genome: A story centuries in the making

Rubus yellow net virus (RYNV) belongs to genus Badnavirus. Badnaviruses are found in plants as endogenous, inactive or activatable sequences, and/or in episomal (infectious and active) forms. To assess the state of RYNV in Rubus germplasm, we sequenced the genomes of various cultivars and mined eight raspberry whole genome datasets. Bioinformatics analysis revealed the presence of a diverse array of endogenous RYNV (endoRYNV) sequences that differ significantly in their structure; some lineages have nearly complete, yet non-functional genomes whereas others have rudimentary, short sequence fragments. We developed assays to genotype the main lineages as well as the only known episomal lineage present in the United States. This study discloses the widespread presence of endoRYNVs in commercial raspberries, likely because breeding efforts have focused on a limited pool of germplasm that harbored endoRYNVs.

Molecular Detection and Identification of Candida Species Isolates from Oral by RFLP-PCR

In this study, 10 local isolates from a total of 50 samples of Candida sp. were collected from oral swabs of patients with oral infections in Mosul hospitals. The isolates were diagnosed based on culturing, microscopic and biochemical characteristics, and then molecular methods. The first diagnosis by culturing, microscopic and biochemical tests found the isolates were identified as Candida sp. The ITS region was amplified using universal primers (ITS4-ITS5), The PCR product was size (510-721) bp. Performing RFLP-PCR using MspI, HhaI,and EcoRI, restriction enzyme to detect and identify Candida species, the results showed the presence of the cutting sequence of MspI and HhaI enzymes in the genomic DNA content of local isolate and the absence of the sequences for the EcoRI restriction enzyme. Two Candida species were identified (C. krusei and C. the basis of size and fragment sequences then compared with sequences of standard strains from the gene bank in previous studies. Therefore, it can be observed that there is a genetic variation between the local isolates and that there are different genotypes of rDNA 5.8S have been diagnosed in 10 isolates after the cutting process with three restriction enzymes. We conclude from this study that the RFLP-PCR technique was the best in diagnosing and identifying Candida species compared with traditional methods. and we are d the genetic variation between local isolates.

Metagenomic assembly is the main bottleneck in the identification of mobile genetic elements.

Antimicrobial resistance genes (ARG) are commonly found on acquired mobile genetic elements (MGEs) such as plasmids or transposons. Understanding the spread of resistance genes associated with mobile elements (mARGs) across different hosts and environments requires linking ARGs to the existing mobile reservoir within bacterial communities. However, reconstructing mARGs in metagenomic data from diverse ecosystems poses computational challenges, including genome fragment reconstruction (assembly), high-throughput annotation of MGEs, and identification of their association with ARGs. Recently, several bioinformatics tools have been developed to identify assembled fragments of plasmids, phages, and insertion sequence (IS) elements in metagenomic data. These methods can help in understanding the dissemination of mARGs. To streamline the process of identifying mARGs in multiple samples, we combined these tools in an automated high-throughput open-source pipeline, MetaMobilePicker, that identifies ARGs associated with plasmids, IS elements and phages, starting from short metagenomic sequencing reads. This pipeline was used to identify these three elements on a simplified simulated metagenome dataset, comprising whole genome sequences from seven clinically relevant bacterial species containing 55 ARGs, nine plasmids and five phages. The results demonstrated moderate precision for the identification of plasmids (0.57) and phages (0.71), and moderate sensitivity of identification of IS elements (0.58) and ARGs (0.70). In this study, we aim to assess the main causes of this moderate performance of the MGE prediction tools in a comprehensive manner. We conducted a systematic benchmark, considering metagenomic read coverage, contig length cutoffs and investigating the performance of the classification algorithms. Our analysis revealed that the metagenomic assembly process is the primary bottleneck when linking ARGs to identified MGEs in short-read metagenomics sequencing experiments rather than ARGs and MGEs identification by the different tools.

The Diversity and Growth-Promoting Potential of the Endophytic Fungi of Neuwiedia singapureana (Orchidaceae) in China

Neuwiedia singapureana is a rare and endangered plant of the Apostasioideae subfamily. The Apostasioideae subfamily has a unique evolutionary status, as it is considered to be the most primitive group forming the base of the Orchidaceae evolutionary tree. Therefore, N. singapureana has high scientific research and conservation value. The endophytic fungal communities associated with orchids are rich and diverse, but few studies have investigated the endophytic fungi of Neuwiedia orchid plants. In the present study, the aim was to examine the endophytic fungal community structures associated with wild N. singapureana rhizomes and normal roots in the ground and with bare prop roots in the air at two sampling sites in China. High-throughput sequencing of nuclear ribosomal DNA fragments of the internal transcribed spacer regions was conducted, and cultivable methods were adopted. A total of 2161 endophytic fungal operational taxonomic units (OTUs) were obtained at a 97% sequence similarity threshold. The endophytic fungal diversity differed among the samples but not significantly. There were many more non-mycorrhizal endophytic fungal than orchid mycorrhizal (OM) fungal species detected in the N. singapureana orchid, about 98.33% OTUs of non-mycorrhizal fungi contrasting with 1.67% OTUs of potential orchid mycorrhizal fungi, among which Ceratobasidiaceae, Russulaceae, and Thelephoraceae were the dominant orchid mycorrhizal fungi. One culturable OM fungal Epulorhiza sp. isolated from the rhizome was capable of significantly promoting the seed germination and seedling growth of Dendrobium officinale and Epidendrum secundum orchids, respectively, with different efficiencies. These endophytic fungal strains with growth-promoting functions will provide materials for orchid conservation and for the study of the mechanisms underlying orchid symbiotic associations.

Detection of Internal Transcribed Spacer 1 and hsp70 Genetic Markers Using Restriction Fragment Length Polymorphisms and Sequencing in Identification of Leishmania Species Causing Tegumentary Leishmaniasis in Brazil.

The identification of Leishmania species that cause tegumentary leishmaniasis (TL) is important for taxonomic and prognostic purposes. Molecular analysis using different Leishmania genomic targets is the most useful method for identifying Leishmania species. Therefore, we evaluated the performance of ribosomal RNA internal transcribed spacer 1 (ITS1) and heat shock protein (hsp70) genetic markers by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism analysis (RFLP) and sequencing, for identification of Leishmania species. Samples from 84 Brazilian patients were amplified. Internal transcribed spacer 1 PCR followed by RFLP (HaeIII) [ITS1-RFLP (HaeIII)] identified 46.4% (39/84) of the samples as compatible with the Viannia subgenus. Internal transcribed spacer 1 PCR followed by sequencing (ITS1-sequencing) identified Leishmania (Viannia) braziliensis in 91.7% (77/84) of the TL samples, Leishmania (Leishmania) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 2.4% (2/84), and L. (L.) infantum in 1.2% (1/84). One of the samples showed the same proportion of similarity with L. (V.) guyanensis and L. (V.) panamensis. hsp70 nested PCR followed by RFLP (HaeIII) [nested hsp70-RFLP (HaeIII)] identified 91.7% (77/84) of the samples as compatible with L. (V.) braziliensis/L. (V.) naiffi, 3.6% (3/84) with L. (L.) amazonensis, 1.2% (1/84) with L. (L.) infantum, and 3.6% (3/84) with L. (V.) guyanensis. hsp70 PCR followed by sequencing (hsp70-sequencing) identified L. (V.) braziliensis in 91.7% (77/84) of the TL samples, L. (L.) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 3.6% (3/84), and L. (L.) infantum in 1.2% (1/84). Our findings clearly showed that nested hsp70-RFLP (HaeIII) is better than ITS1-RFLP (HaeIII) and that ITS1 or hsp70 PCR followed by sequencing was adequate for identifying Leishmania species. We also found that Leishmania (Viannia) braziliensis is the most common species causing TL in Brazil. Therefore, sequencing multiple target genes such as ITS1 and hsp 70 is more accurate than RFLP for identifying Leishmania species.

Integrating rapid pathogen identification and antimicrobial susceptibility testing through multiplex TaqMan qPCR assay

Timely bacterial identification (ID) and antimicrobial susceptibility testing (AST) are of significance for therapy of bacteria-infected patients. In the present study, we developed a multiplex TaqMan qPCR assay for rapid and accurate ID and AST of three common hospital acquired pneumonia species, namely Acinetobacter baumannii, Klebsiella pneumoniae and Staphylococcus aureus. In this assay, DNA extraction and bacterial co-incubation with antibiotics are accomplished based on a common PCR instrument. ID of three bacteria is based on specific conserved DNA sequence fragment (gltA for A. baumannii, phoE for K. pneumoniae and nuc for S. aureus) detection through multiplex TaqMan qPCR assay within 80 min. AST of three bacteria could be acquired within 200 min based on genomic DNA fold change detection after 2 h of antibiotic exposure. Testing of 23 bronchoalveolar lavage fluid samples spiked by different A. baumannii isolates, 20 bronchoalveolar lavage fluid samples spiked by different K. pneumoniae isolates, and 14 bronchoalveolar lavage fluid samples spiked by different S. aureus isolates showed that the multiplex TaqMan qPCR assay had 100% (95% CI: 85.69–100), 100% (95% CI: 83.89–100) and 100% (95% CI:78.47–100) identification agreement with the initial spiked bacteria. Subsequent AST results compared with the standard broth microdilution method showed an overall agreement of 91.30% (95% CI: 73.20 to 97.58) for A. baumannii, 90% (95% CI: 69.90 to 97.21) for K. pneumoniae and 92.86% (95% CI: 68.53 to 98.73) for S. aureus based on the current multiplex TaqMan assay. Due to the high rapidity, good agreement, simplicity, and high throughput, this multiplex TaqMan assay could be helpful for ID and broad-spectrum AST in A. baumannii, K. pneumoniae and S. aureus, as well as potentially applicable for other clinical bacteria by changing the primers and probes.

Polarization- and CGR-based binary representations as identifiers of the nucleotide sequences in bioinformatics

Purpose of this work is the comparative analysis of two approaches to the synthesis of two-dimensional binary identifiers of nucleotide sequences obtained using DNA sequencing of biological objects. Methods. One of the approaches is based on modeling the polarization-dependent diffraction of a coherent readout beam on a two-dimensional phase-modulating structure (phase screen) associated with the symbolic sequence obtained as a result of DNA sequencing. Another approach uses a two-dimensional representation of the symbolic sequence using a chaos game representation (CGR). To obtain a finite-element CGR mapping, it is fragmented into a given number of cells, ensuring acceptable sensitivity of the synthesized binary identifier to structural changes in the displayed sequence. Results. The comparative analysis was carried out using fragments of symbol sequences corresponding to various strains (Wuhan, Delta, Omicron) of the SarSCoV2 virus. In the course of the analysis, the correlation coefficients between the binary identifiers corresponding to various strains were obtained and compared with each other. Conclusion. It has been established that binary identifiers synthesized using the polarization encoding technique are characterized by significantly higher sensitivity to structural changes in the analyzed sequences and smaller sizes compared to CGR binary identifiers.

Ultramicrobacteria and filterable bacteria in the plankton of Lake Baikal

In Lake Baikal, the number, diversity, and structure of femtoplankton – bacteria passing through filters with a 0.2 μm pore size – were studied for the first time using a complex of methods. The bacterial abundance in the femtoplankton fraction was 7×104 cells/mL in the 0-50 m water layer, as measured by epifluorescence microscopy, and their contribution to the total bacterial number reached an average of 4.4%. High-throughput sequencing of 16S rRNA gene fragments revealed a significant genetic and taxonomic diversity of femtobacterioplankton in the pelagic and littoral zones of the lake. Dominant and minor phyla, orders, families, genera, and phylotypes of bacteria were identified in two fractions of Lake Baikal bacterioplankton: larger and smaller than 0.2 µm. We determined the contribution of ultra-small bacteria to the taxonomic composition of microbial communities in different parts of the lake. The microbiomes of bacterioplankton and femtobacterioplankton fractions differed significantly, and we described the peculiarities of ultramicrobacteria composition. The results demonstrated an important role of ultra-small bacteria in Lake Baikal’s ecosystem.

Entomological inferences highlight the risk of Leishmania transmission in the urban area of Porto Velho, Rondônia, Brazil.

Entomological investigations were conducted for the first time in urban forest remnants of Porto Velho, state of Rondônia, Brazil, to explore the transmission dynamics of Leishmania. Sand fly collections were carried out at ten sites, encompassing both canopy and ground strata, from October to December 2021. A total of 1,671 sand flies were collected, representing 42 species within 12 genera. Nyssomyia Antunesi (n = 384) and Psychodopygus davisi (n = 111) were the most abundant species. Molecular analyses targeting the V7V8 region (18S gene) unveiled the presence of sequences 100% identical to Leishmania infantum in females of Bichromomyia flaviscutellata (1), Nyssomyia Antunesi complex (6), Nyssomyia umbratilis (1), Nyssomyia sp. (1), Psychodopygus ayrozai (1), Ps. davisi (3), Psychodopygus paraensis (1), and Sciopemyia sordellii (1). Sequences 100% similar to Trypanosoma minasense were found in two samples of the Nyssomyia Antunesi complex, and two samples of Sc. sordellii presented 100% identity to a Trypanosoma sp. strain, previously identified in this same sand fly in Rondônia. Sequencing of Cytb fragment suggested Homo sapiens, Dasypus novemcinctus and Tamandua tetradactyla as the blood source for distinct sand flies. The identification of sequences similar to L. infantum in sand flies collected in urban forest fragments is noteworthy, correlating with the recent local and regional occurrence of autochthonous cases of human visceral leishmaniasis. However, further studies are imperative to ascertain the presence of hosts/reservoirs and evaluate the risk of L. infantum transmission to humans.

Biological diversity and structure of sub-ice algal-bacterial communities in the Maloye More Strait (Lake Baikal)

An analysis of the taxonomic structure and diversity of sub-ice algal-bacterial communities in the Maloye More Strait of Lake Baikal in two different ecotopes (the bottom surface of the ice and the water column) was carried out. According to high-throughput sequencing of the 18S rRNA fragment and microscopy, the dinoflagellates Gymnodinium baicalense and green algae Binuclearia lauterbornii predominated on the bottom surface of the ice, while the diatoms Ulnaria acus and Fragilaria radians and green algae of the genus Monoraphidium> and Koliela predominated in the water column. In the bacterial communities, the most abundant phyla were Actinobacteria, Proteobacteria and Bacteroidetes.

The Novel PTX3 Variant g.22645332G>T Is Strongly Related to Awassi and Hamdani Sheep Litter Size.

The detection of polymorphisms in genes that control livestock reproduction could be highly beneficial for identifying and enhancing economic traits. One of these genes is pentraxin 3 (PTX3), which affects the reproduction of sheep. Therefore, this study investigated whether the variability of the PTX3 gene was related to the litter size of Awassi and Hamdani ewes. A total of 200 ewes (130 Awassi and 70 Hamdani) were used for genomic DNA extraction. Polymerase chain reaction was used to amplify the sequence fragments of exons 1, 2, 3, and 4 from the PTX3 gene (Oar_v4.0; Chr 1, NC_056054.1), resulting in products of 254, 312, 302, and 253, respectively. Two genotypes, GG and GT, were identified for 302 bp amplicon. A novel mutation was discovered through sequence analysis in the GT genotype at position g.22645332G>T. The statistical analysis revealed a significant association between single nucleotide polymorphism (SNP g.22645332G>T; Oar_v4.0; Chr 1, NC_056054.1) and litter size. The presence of the SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1) genotype in ewes resulted in a significant difference compared to ewes with GG genotypes. The discrepancy became apparent in several aspects, including litter sizes, twinning rates, lambing rates, litter weight at birth, and days to lambing. There were fewer lambs born to ewes with the GG genotype than to ewes with the GT genotype. The variant SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1) has positive effects on the litter size of Awassi and Hamdani sheep. The SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1 has been associated with an increase in litter size and higher prolificacy in ewes.

Genetic variation of the freshwater snail Indoplanorbis exustus (Gastropoda: Planorbidae) in Thailand, inferred from 18S and 28S rDNA sequences.

Indoplanorbis exustus, a freshwater pulmonate snail, is widely distributed in tropical and subtropical zones and plays a significant role as an intermediate host for trematode parasites. Various genetic markers have been used for species identification and phylogenetic studies of this snail. However, there are limited studies about their molecular genetics based on nuclear ribosomal DNA (rDNA) genes. A genetic analysis of I. exustus in Thailand was conducted based on the nuclear 18S rDNA (339 bp) and 28S rDNA (1036 bp) genes. Indoplanorbis snails were collected from 29 localities in 21 provinces covering six regions of Thailand. Nucleotide sequences from 44 snails together with sequences from the GenBank database were examined for phylogenetic relationships and genetic diversity. All sequences of the selected nucleotide regions exhibited a high level of similarity (99%) to the sequences of I. exustus in the GenBank database. The maximum likelihood tree based on the 18S and 28S rDNA fragment sequences of I. exustus in Thailand revealed only one group with clear separation from another genus in the family Planorbidae. The I. exustus 28S rDNA sequences showed intraspecific genetic divergence ranging from 0 to 0.78% and were classified into 8 different haplotypes. Conversely, the 18S rDNA data showed lower variation than the 28S rDNA data and revealed a single haplotype and intraspecific distances of zero among all sampled individuals. The haplotype network of 28S rDNA sequences of I. exustus in Thailand revealed six unique haplotypes and two haplotypes shared by at least two regions. Overall, both markers were successful in the identification of I. exustus. However, these markers, particularly the 18S rDNA, may not be suitable for genetic analysis within the species, particularly for population genetic studies, due to their limited variation as seen in this study. In summary, this study not only enhances understanding of genetic variation in I. exustus but is also useful for the selection of molecular markers in future genetic research.

A novel viral RNA detection method based on the combined use of trans-acting ribozymes and HCR-FRET analyses.

The diagnoses of retroviruses are essential for controlling the rapid spread of pandemics. However, the real-time Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR), which has been the gold standard for identifying viruses such as SARS-CoV-2 in the early stages of infection, is associated with high costs and logistical challenges. To innovate in viral RNA detection a novel molecular approach for detecting SARS-CoV-2 viral RNA, as a proof of concept, was developed. This method combines specific viral gene analysis, trans-acting ribozymes, and Fluorescence Resonance Energy Transfer (FRET)-based hybridization of fluorescent DNA hairpins. In this molecular mechanism, SARS-CoV-2 RNA is specifically recognized and cleaved by ribozymes, releasing an initiator fragment that triggers a hybridization chain reaction (HCR) with DNA hairpins containing fluorophores, leading to a FRET process. A consensus SARS-CoV-2 RNA target sequence was identified, and specific ribozymes were designed and transcribed in vitro to cleave the viral RNA into fragments. DNA hairpins labeled with Cy3/Cy5 fluorophores were then designed and synthesized for HCR-FRET assays targeting the RNA fragment sequences resulting from ribozyme cleavage. The results demonstrated that two of the three designed ribozymes effectively cleaved the target RNA within 10 minutes. Additionally, DNA hairpins labeled with Cy3/Cy5 pairs efficiently detected target RNA specifically and triggered detectable HCR-FRET reactions. This method is versatile and can be adapted for use with other viruses. Furthermore, the design and construction of a DIY photo-fluorometer prototype enabled us to explore the development of a simple and cost-effective point-of-care detection method based on digital image analysis.

The horizontal gene transfer of perchlorate reduction genomic island in three bacteria from an ecological niche.

Three new strains of dissimilatory perchlorate-reducing bacteria (DPRB), QD19-16, QD1-5, and P3-1, were isolated from an active sludge. Phylogenetic trees based on 16S rRNA genes indicated that QD19-16, QD1-5, and P3-1 belonged to Brucella, Acidovorax, and Citrobacter, respectively, expanding the distribution of DPRB in the Proteobacteria. The three strains were gram-negative and facultative anaerobes with rod-shaped cells without flagella, which were 1.0-1.6μm long and 0.5-0.6μm wide. The three DPRB strains utilized similar broad spectrum of electron donors and acceptors and demonstrated a similar capability to reduce perchlorate within 6days. The enzyme activity of perchlorate reductase in QD19-16 toward chlorate was higher than that toward perchlorate. The high sequence similarity of the perchlorate reductase operon and chlorite dismutase genes in the perchlorate reduction genomic islands (PRI) of the three strains implied that they were monophyletic origin from a common ancestral PRI. Two transposase genes (tnp1 and tnp2) were found in the PRIs of strain QD19-16 and QD1-5, but were absent in the strain P3-1 PRI. The presence of fragments of IR sequences in the P3-1 PRI suggested that P3-1 PRI had previously contained these two tnp genes. Therefore, it is plausible to suggest that a common ancestral PRI transferred across the strains Brucella sp. QD19-16, Acidovorax sp. QD1-5, and Citrobacter sp. P3-1 through horizontal gene transfer, facilitated by transposases. These results provided a direct evidence of horizontal gene transfer of PRI that could jump across phylogenetically unrelated bacteria through transposase. KEY POINTS: • Three new DPRB strains can effectively remove high concentration of perchlorate. • The PRIs of three DPRB strains are acquired from a single ancestral PRI. • PRIs are incorporated into different bacteria genome through HGT by transposase.

Reconstructing the Evolutionary History of Pinna nobilis: New Genetic Signals from the Past of a Species on the Brink of Extinction.

Pinna nobilis, commonly known as the noble pen shell, is a marine bivalve endemic to the Mediterranean Sea. Unfortunately, due to a multifactorial disease that began affecting its populations in 2016, the species is currently facing the threat of extinction. To gain insights into the evolutionary history of P. nobilis before the mass mortality event (MME), and to obtain a comprehensive understanding of how evolutionary processes led to the adaptation of the species into the Mediterranean Sea, phylogenetic and phylogeographic analyses were carried out. The dataset analysed includes 469 sequences of COI gene fragment both from GenBank and the present study (100). The analysis performed evidenced that P. nobilis diverged about 2.5 mya, after the entrance of its ancestor into the Mediterranean Sea following the Zanclean flood (5.33 mya). Moreover, our results suggest that the starting point of colonisation was the central part of the western Mediterranean basin, with the eastern basin being populated subsequently. From a conservational viewpoint, these results provide important hints for present and future restocking plans, helping to reconstruct the pre-existing genetic variability in sites where the species became extinct.

Variations of Physiological Parameters and HSP70 and HSP90 Polymorphisms in Water Buffaloes in Taiwan During Cool and Warm Season

Background: This study examined the physiological parameters of water buffaloes in Taiwan in the cool (February) and warm (August) seasons of 2020 and 2021.
 Methods: Data was collected for a study in February, August 2020, and 2021. The ambitious temperature, humidity, water buffaloes’ rectal temperature (RT), and respiratory rate (RR) were recorded. The plasma expression levels of heat-shock protein (HSP)70 and HSP90 were examined using an ELISA kit. Furthermore, the HSP70 and HSP90 fragment genetic sequence variations were analyzed using the PCR method and MEGA6 software.
 Results: The results revealed that in the warm season, the rectal temperature (RT), respiratory rate (RR), and heat tolerance coefficient (HTC) were significantly higher compared to the cool season (all P < 0.05). Additionally, the temperature-humidity index (THI) had moderate to high correlations with RT (0.518), RR (0.744), and HTC (0.757). Plasma HSP70 expression levels were higher in the warm season than in the cool season (P < 0.05). The genetic sequences of HSP70 and HSP90 fragments were compared, and five single-nucleotide variation (SNV) sites were identified. However, each genotype showed no significant physiological difference between the cool and warm seasons.
 Conclusion: Temperature and humidity changes in Taiwan had a significant correlation with the physical condition of water buffaloes. This information can be valuable in improving the living conditions of these animals, leading to better animal welfare. Additionally, the HSP70 and HSP90 gene variations in water buffaloes in Taiwan could be used as a reference for future research on breeding and identifying molecular markers.