Region Sequences Research Articles

The updates of the ACMG variant interpretation guidelines affect the pathogenicity determination of OTOF gene variations in patients with auditory neuropathy

Objective: To compare the differences between the variation interpretation standards and guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015 (The 2015ACMG/AMP guideline) and the Deafness Specialist Group of the Clinical Genome Resource (ClinGen) in 2018 for hereditary hearing loss (Healing loss, HL) issued the expert specification of the variation interpretation guide (The 2018 HL-EP guideline) in evaluating the pathogenicity of OTOF gene variation in patients with auditory neuropathy. Methods: Thirty-eight auditory neuropathy patients with OTOF gene variant were selected as the study subjects (23 males and 15 females, aged 0.3-25.9 years). Using whole-genome sequencing, whole exome sequencing or target region sequencing (Panel) combined with Sanger sequencing, 38 cases were found to carry more than two OTOF mutation sites. A total of 59 candidate variants were independently interpreted based on the 2015 ACMG/AMP guideline and 2018 HL-EP guideline. Compared with the judgment results in 2015 ACMG/AMP guideline, the variants interpreted as lower pathogenic classifications in the 2018 HL-EP guideline were defined as downgraded variants, and the variants regarded as higher pathogenic classifications were defined as upgraded variants. Statistical analysis was conducted using SPSS 20.0. Results: The concordance rate of variant classification between the guidelines was 72.9%(43/59). The 13.6%(8/59) of variants were upgraded and 13.6% (8/59) of variants downgraded in the classifications of the 2018 HL-EP guideline. A couple of rules saw significant differences between the guidelines (PVS1, PM3, PP2, PP3 and PP5). The distribution of pathogenicity of splicing mutation was statistically different (P=0.013). Conclusions: The 2018 HL-EP guideline is inconsistent with the 2015 ACMG/AMP guideline, when judging the pathogenicity of OTOF gene variants in patients with auditory neuropathy. Through the deletion and refinement of evidence and the breaking of solidification thinking, the 2018 HL-EP guideline makes the pathogenicity grading more traceable and improves the credibility.

Evolutionarily conserved 12-oxophytodienoate reductase trans-lncRNA pair affects disease resistance in tea (Camellia sinensis) via the jasmonic acid signaling pathway.

Long non-coding RNAs (lncRNAs) have gathered significant attention due to their pivotal role in plant growth, development, and biotic and abiotic stress resistance. Despite this, there is still little understanding regarding the functions of lncRNA in these domains in the tea plant (Camellia sinensis), mainly attributable to the insufficiencies in gene manipulation techniques for tea plants. In this study, we designed a novel strategy to identify evolutionarily conserved trans-lncRNA (ECT-lncRNA) pairs in plants. We used highly consistent base sequences in the exon-overlapping region between trans-lncRNAs and their target gene transcripts. Based on this method, we successfully screened 24 ECT-lncRNA pairs from at least two or more plant species. In tea, as observed in model plants such as Arabidopsis, alfalfa, potatoes, and rice, there exists a trans-lncRNA capable of forming an ECT-lncRNA pair with transcripts of the 12-oxophytodienoate reductase (OPR) family, denoted as the OPRL/OPR pair. Considering evolutionary perspectives, the OPRL gene cluster in each species likely originates from a replication event of the OPR gene cluster. Gene manipulation and gene expression analysis revealed that CsOPRL influences disease resistance by regulating CsOPR expression in tea plants. Furthermore, the knockout of StOPRL1 in Solanum tuberosum led to aberrant growth characteristics and strong resistance to fungal infection. This study provides insights into a strategy for the screening and functional verification of ECT-lncRNA pairs.

Selective bioaccumulation of polystyrene nanoplastics in fetal rat brain and damage to myelin development

Micro(nano)plastic, as a new type of environmental pollutant, have become a potential threat to the life and health of various stages of biology. However, it is not yet clear whether they will affect brain development in the fetal stage. Therefore, this study aims to explore the potential effects of nanoplastics on the development of fetal rat brains. To assess the allocation of NPs (25 nm and 50 nm) in various regions of the fetal brain, pregnant rats were exposed to concentrations (50, 10, 2.5, and 0.5 mg/kg) of PS-NPs. Our results provided evidence of the transplacental transfer of PS-NPs to the fetal brain, with a prominent presence observed in several cerebral regions, notably the cerebellum, hippocampus, striatum, and prefrontal cortex. This distribution bias might be linked to the developmental sequence of each brain region. Additionally, we explored the influence of prenatal exposure on the myelin development of the cerebellum, given its the highest PS-NP accumulation in offspring. Compared with control rats, PS-NPs exposure caused a significant reduction in myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) expression, a decrease in myelin thickness, an increase in cell apoptosis, and a decline in the oligodendrocyte population. These effects gave rise to motor deficits. In conclusion, our results identified the specific distribution of NPs in the fetal brain following prenatal exposure and revealed that prenatal exposure to PS-NPs can suppress myelin formation in the cerebellum of the fetus.

Addressing the Concern of Orange-Yellow Fungus Growth on Palm Kernel Cake: Safeguarding Dairy Cattle Diets for Mycotoxin-Producing Fungi.

Palm kernel cake (PKC), a byproduct of palm oil extraction, serves an important role in Ecuador's animal feed industry. The emergence of yellow-orange fungal growth in PKC on some cattle farms in Ecuador sparked concerns within the cattle industry regarding a potential mycotoxin-producing fungus on this substrate. Due to the limited availability of analytical chemistry techniques in Ecuador for mycotoxin detection, we chose to isolate and identify the fungus to determine its association with mycotoxin-producing genera. Through molecular identification via ITS region sequencing, we identified the yellow-orange fungus as the yeast Candida ethanolica. Furthermore, we isolated two other fungi-the yeast Pichia kudriavzevii, and the fungus Geotrichum candidum. Molecular identification confirmed that all three species are not classified as mycotoxin-producing fungi but in contrast, the literature indicates that all three have demonstrated antifungal activity against Aspergillus and Penicillium species, genera associated with mycotoxin production. This suggests their potential use in biocontrol to counter the colonization of harmful fungi. We discuss preventive measures against the fungal invasion of PKC and emphasize the importance of promptly identifying fungi on this substrate. Rapid recognition of mycotoxin-producing and pathogenic genera holds the promise of mitigating cattle intoxication and the dissemination of mycotoxins throughout the food chain.

First record of the genus Haritalodes Warren, 1890 and H. derogata (Fabricius, 1775) (Lepidoptera: Crambidae: Spilomelinae) from Türkiye and its morphological and molecular identification

The genus Haritalodes Warren, 1890 is represented by 12 species worldwide except in the Nearctic and Neotropical regions. Haritalodes derogata (Fabricius, 1775), commonly known as the cotton leaf roller moth, is the most widely distributed species of the genus and is common in the West Palaearctic Region. This species is one of the most serious pests of Malvaceae. The larvae primarily feed on the leaves, and severe population levels may lead to significant yield loss in the primary host, cotton. In a survey in 2023, the shrubs of the Hibiscus sp. were found to be infested by a group of larvae with a very dense population feeding on the leaves in Trabzon Province, Türkiye. The larvae were reared for pupation and adult emergence. Morphological identification according to male and female genitalia as well as sequencing of the mitochondrial cytochrome oxidase region I (COI) from larvae and adults revealed that the species is H. derogata. This is the first report of the genus Haritalodes and Haritalodes derogata in Türkiye.

Beyond 16S sequencing for microbiome research: Nanopore sequencing resource at The University of Toledo Microbiome Consortium

Objective: The recognition that humans are holobionts has opened a new research area to examine the contributions of the microbiome to health and disease. This requires a full understanding of the identities of microbiota at the taxonomical level of the species. Introduction: The University of Toledo Microbiome Consortium currently offers the second-generation Illumina sequencing platform for 16S sequencing to determine microbiota composition. 16S sequencing of variable regions of the 16S operon, such as V3, V4, and the combination of V3 to V4, has long been the main stay for analyzing the composition of microbiota. The advantage of this approach is the ability to screen large numbers of samples at a lower cost per sample. However, the major drawback of this approach is a lower resolution whereby taxonomical identities of microbiota are limited to the genus level. To overcome this disadvantage, we introduced the third-generation sequencing technology from Nanopore into our pipeline. The known advantages of this approach are longer reads and the ability to sequence DNA in the native form without PCR-induced bias. Here we sought to conduct a comparative study of the relative effciencies of the 16S versus the nanopore technology for metagenomics. Methods: Genomic DNA was isolated from control and experimental rat fecal samples provided by an end-user. Libraries were prepared using Ligation sequencing DNA V14 (SqK-LSK114) on a Flow cell R10.4.1and Lambda DNA was used as a sequencing control. The native Libraries were run for 72hr, and high accuracy base calling was performed. The Fastq files (passed) generated by GridION were run on cloud-based CZ-ID pipeline with host and human read filtering, the results were normalized to one million bases sequenced from reads of each sample. Results and Conclusions: The 16S sequencing data highlighted the relative abundance of different taxa up to genus level such as Lactobacillus and Erysipelatoclostridium in the experimental group where as Stepcococcus and prevotellaceaeGa6A1group in the control group. However, Nanopore sequencing further identified twenty different species of Lactobacillus and , four different species of Erysipelatoclostridium. Similarly, nanopore sequencing was able to profile diverse species belonging to genera prevotellaceaeGa6A1group and Streptococcus. This species level resolution will greatly help in focusing on specific species and the pathways that a researcher intends to pursue. Therefore, the nanopore sequencing presents an opportunity to explore sequencing with a small footprint in terms of instrumentation and associated costs that greatly helps even a smaller laboratory to adopt and reap the benefits of species level resolution of metagenomic data. Summary: This study correlated well with 16S sequencing results at the genus level, while offering the added benefit of species level Identification and classification. Authors acknowledge the National Institutes of Health for funding support to Bina Joe (R01HL1430820). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pathological, morphological, and molecular characterization of the infection by Brachycladium goliath in humpback whale Megaptera novaeangliae.

The humpback whale (Megaptera novaeangliae) is a cosmopolitan migratory, seasonal mysticete that frequents the Brazilian coast. Strands of specimens may occur during the migratory stay in the country. In 2021 and 2022, three live humpback whales stranded on the coast of Rio Grande do Sul and Santa Catarina states in southern Brazil. After euthanasia, specimens were necropsied, and organs were thoroughly examined for lesions. Grossly, in all three cases, the liver exhibited multifocal, irregular, firm, white areas on the hepatic capsule, which extended into the parenchyma. On the cut surface, the livers were yellow to pale brown with orangish to greenish areas, the bile ducts were prominent, thickened, and severely dilated, and leaf-shaped flukes were found inside of them. Additionally, one case showed moderate atrophy of the right hepatic lobe. The histological findings included dilation of bile ducts, hyperplasia of the bile duct epithelium, marked inflammatory infiltration of lymphocytes, plasma cells, and eosinophils, and portal fibrosis. The parasite Brachycladium goliath was both morphologically and molecularly identified based on diagnostic key for trematodes and the original description of the species, and the amplification and sequencing of the ITS-2 region, respectively. Even though hepatic injury was not the primary cause of stranding, it may have contributed to the debilitation of the whales. To the authors' knowledge, this is the first study that reports M. novaeangliae as a definitive host of B. goliath and that describes the lesions caused by the parasite in cetaceans.

TPMGD: A genomic database for the traditional medicines in Pakistan

ObjectiveIn Pakistan, traditional medicines are an important component of the medical system, with numerous varieties and great demands. However, due to the scattered resources and the lack of systematic collection and collation, adulteration of traditional Pakistani medicine (TPM) is common, which severely affects the safety of their medicinal use and the import and export trades. Therefore, it is urgent to systematically organize and unify the management of TPM and establish a set of standards and operable methods for the identification of TPM. MethodsWe collected and organized the information on 128 TPMs with regard to their medicinal parts, efficacy, usage, and genetic material, based on Pakistan Hamdard Pharmacopoeia of Eastern Medicine: Pharmaceutical Codex. The genetic information of TPM is summarized from national center for biotechnology information (NCBI) and global pharmacopoeia genome database (GPGD). Furthermore, we utilized bioinformatics technology to supplement the chloroplast genome (cp-genome) data of 12 TPMs. To build the web server, we used the Linux + Apache + MySQL + PHP (LAMP) system and constructed the webpage on a PHP: Hypertext Preprocessor (PHP) model view controller (MVC) framework. ResultsWe constructed a new genomic database, the traditional Pakistani medicine genomic database (TPMGD). This database comprises five entries, namely homepage, medicinal species, species identification, basic local alignment search tool (BLAST), and download. Currently, TPMGD contains basic profiles of 128 TPMs and genetic information of 102 TPMs, including 140 cytochrome c oxidase subunit I (COI) sequences and 119 mitochondrial genome sequences from Bombyx mori, 1 396 internal transcribed spacer 2 (ITS2) sequences and 1 074 intergenic region (psbA-trnH) sequences specific to 92 and 83 plant species, respectively. Additionally, TPMGD includes 199 cp-genome sequences of 82 TPMs. ConclusionTPMGD is a multifunctional database that integrates species description, functional information inquiry, genetic information storage, molecular identification of TPM, etc. The database not only provides convenience for TPM information queries but also establishes the scientific basis for the medication safety, species identification, and resource protection of TPM.

New records of Trichinella patagoniensis from Argentina

Trichinella spiralis was long considered the sole Trichinella species in Argentina. However, since 2004, various Trichinella species, including the encapsulated Trichinella patagoniensis and Trichinella britovi, as well as the unencapsulated Trichinella pseudospiralis, have been detected in the country. The present study aimed to identify Trichinella ML at the species level from cougars naturally infected from Argentina. To this end, muscle tissue samples from one cougar each from Córdoba, Neuquén, and Santa Cruz Provinces were individually analysed using the artificial digestion technique. DNA extraction and molecular identification of Trichinella species were conducted on individual muscle larvae by PCR amplification of the ESV region and subsequent PCR amplification and sequencing of the COI gene. Morphological analysis revealed muscle larvae with characteristics consistent with Trichinella genus. PCR revealed a single band of approximately 127 bp for each individual muscle larva. PCR amplification of the COI gene from each isolate generated a 309 bp band. Sequencing of the mitochondrial COI gene confirmed the identity of the parasite as T. patagoniensis. The present study documents new occurrences of T. patagoniensis in Puma concolor from Argentina, including the first detection of T. patagoniensis in Puma concolor from Córdoba and Neuquén Province. These findings expand the limited knowledge of T. patagoniensis distribution in Argentina.

Features and evaluation of mucormycosis in COVID-19 patients from two referral hospitals in Iran

ObjectivesThe present study aimed to assess the features, clinical characteristics, and species diversity among patients admitted to referral Hospitals for SARS-CoV-2 pneumonia and mucormycosis in Tehran, Iran, and the relationship between seasonal and species diversity was considered. MethodsConfirmed COVID-19 patients with a positive reverse-transcriptase real-time (rRT-PCR) test for SARS-CoV2 were primarily included based on clinically suspected mucormycosis infection and confirmed by histopathology and mycology examination of biopsy specimens. The PCR technique was performed by the amplification of the high-affinity iron permease 1 (FTR1) gene for identification and discrimination between Rhizopus arrhizus and non- Rhizopus arrhizus isolates. In contrast, species identification of non-Rhizopus arrhizus was performed by sequencing of ITS rDNA region. ResultsRhino-sino-orbital mucormycosis was identified in the majority of cases (n = 33), with 66 % and 34 % of the cases involving male and female patients, respectively. Rhizopus arrhizus was found to be the most prevalent (84.6 %), followed by Mucor circinelloides (7.6 %). Rhizopus arrhizus was the most prevalent species and present in all the seasons; however, Mucor circinelloides was only present in the autumn. The overall mortality of the total population was 24.6 % (16/ 65); the mortality rates occurring in patients diagnosed with rhino-sino-orbital infection and rhino-sinusal form were 21.4 % and 25 %, respectively. ConclusionCAM can be a serious complication of severe COVID-19, especially in patients with uncontrolled diabetes. It is important to monitor the epidemiology of mucormycosis to raise awareness of the disease and improve diagnosis, treatment and prognosis, particularly in the setting of pandemic.

Origin of the neonatal gut microbiota and probiotic intervention: a randomized controlled trial.

This study aims to evaluate the origin of the neonatal gut microbiota on the 14th day and probiotic intervention in the third trimester. Samples were obtained from a total of 30 pregnant individuals and their offspring, divided into a control group with no intervention and a probiotic group with live combined Bifidobacterium and Lactobacillus tablets, analyzing by 16S rRNA amplicon sequencing of the V4 region to evaluate the composition of them. Non-metric multidimensional scaling and SourceTracker were used to evaluate the origin of neonatal gut microbiota. We found that the microbiota in the neonatal gut at different times correlated with that in the maternal microbiota. The placenta had more influence on meconium microbiota. Maternal gut had more influence on neonatal gut microbiota on the 3rd day and 14th day. We also found that the maternal gut, vaginal, and placenta microbiota at full term in the probiotic group did not have a significantly different abundance of Bifidobacterium, Lactobacillus, or Streptococcus. However, some other bacteria changed in the maternal gut and their neonatal gut in the probiotic group.

Three helmet-orchid species share abundant fungi of Serendipitaceae regardless of altitude

The tiny terrestrial orchids (plant height less than 8 cm) in the genus Corybas dependent on mycorrhizal fungal (OMF) partners for seed germination and seedling development. The OMF community of the Corybas remains poorly understood, although the relevant knowledge is very important for in situ and ex situ conservation of these orchids. In this study, we characterized OMF richness and compositions of three helmet-orchid species, i.e., Corybas geminigibbus, C. himalaicus, and C. shanlinshiensis, from their natural habitats by using Illumina sequencing of the ITS2 region. We found that fungal colonization was restricted in the rhizomes of the helmet-orchids instead of their tuberoids, and serendipitoid fungi were predominant while tulasnelloid were absent in the three investigated Corybas species regardless of their altitude. The three Corybas species shared 27 serendipitoid operational taxonomic units (OTUs) that are different to those of their related orchids, the genera of Cyrtostylis and Stigmatodactylus. Corybas shanlinshiensis alone had a range of ectomycorrhizal fungi (mainly russuloid and thelephoroid) broader than C. himalaicus and C. shanlinshiensis. Our study provides new information about terrestrial orchid-fungi associations and may further contribute to orchid conservation.

Does the Artificial Enhancement and Release Activity Affect the Genetic Diversity of Marbled Rockfish Sebastiscus marmoratus in Zhoushan Waters?

Artificial enhancement and release activity is an important method in the restoration of fishery resources. In order to understand the possible genetic effect of hatchery-released populations on wild populations during the artificial enhancement and release activities of Sebastiscus marmoratus in Zhoushan waters, we utilized mitochondrial DNA control region sequences to examine the genetic diversity in four S. marmoratus populations, including one farmed population, one released population and two wild populations. A total of 68 haplotypes from 123 individuals were detected, including 3 shared haplotypes. Haplotype diversity ranged from 0.944 to 0.980, with a mean of 0.966. The nucleotide diversity ranged from 0.020 to 0.025, with a mean of 0.022. Analysis of Molecular Variance (AMOVA) indicated that the primary genetic variation occurs within populations and the index of genetic differentiation between populations (FST) among the four populations showed no differentiation. The results indicate that the current artificial enhancement and release has not impacted the S. marmoratus population in Zhoushan waters. Continued long-term monitoring is essential to protect the high-quality germplasm resources of S. marmoratus.

Phenotypic Characteristics, Pathogenicity, and Molecular Identification of Hypomyces perniciosus Causing Wet Bubble Disease of Edible Mushrooms

Abstract The research aimed to determine the phenotypic characteristic, pathogenicity and molecular characterization of Hypomyces perniciosus isolates responsible for wet bubble disease of the white button mushroom Agaricus bisporus. Phenotypic characteristics such as colony appearance, mycelium texture, and pigmentation were studied on four different agar media, i.e., potato dextrose agar (PDA), malt extract agar (MEA), Czapek-Dox yeast agar (CYA), and Sabouraud dextrose agar (SDA), after eight days of incubation. Additionally, the growth rate of the tested isolates was studied depending on the pH of the medium. Fungal isolates showed the highest mycelial growth on MEA and SDA at pH 6.0. However, on CYA at pH 7.0, the mycelium exhibited the worst growth. Isolate identification and genetic relationship analysis were carried out using internal transcribed spacer region sequencing and the random amplified polymorphic DNA method. The research confirmed that all Polish isolates belong to the species H. perniciosus, and the genetic diversity is relatively low. Phylogenetic analyses revealed three subgroups of H. perniciosus isolates. The first group included three genetically distinct isolates with a similarity coefficient in the range of 0.76–0.85 to isolates CBS 815.73 and CBS 322.52. The second group was divided into two subgroups and included 16 isolates with a genetic similarity range of 0.91 to 1.0 to CBS 815.73 and CBS 322.52 isolates. Furthermore, the eight genetically similar isolates exhibited the greatest pathogenicity towards A. bisporus.

Analyzing microbial communities and metabolic functions in estuaries affected by human activity to uncover bioelectrochemical system potential

In the context of bioelectrochemical systems (BES), a profound understanding of the taxonomic composition within indigenous microbial communities is crucial. Their metabolic versatility assumes a foundational role in dictating the functionality of BES, yielding diverse applications for bioenergy production and bioremediation in anthropogenically impacted estuaries. Employing high-throughput sequencing of the V3-V4 region of 16S rRNA and predictive metagenomic analyses of three different estuaries samples (eutrophicated urban reservoir, mud volcano impacted river, and its river mouth), this study delves into the intricate interplay between these communities and their metabolic functions in response to human-induced perturbations, with a specific focus on their applicability to BES. Our findings illuminate remarkable distinctions among the three samples, manifesting in variations in microbial composition and functional metabolic pathways within the respective ecosystems. The mud volcano-affected river, characterized by the highest microbial diversity, exhibited the most diverse array of functional metabolic processes, prominently dominated by chemoheterotrophy (67.43%). In contrast, the river mouth displayed a distinct penchant for autotrophy (33.33%). The eutrophicated urban reservoir showcased a balanced coexistence of chemoheterotrophy (41.18%) and autotrophy (35.29%). Regarding metabolic potential for BES application, most of metabolism potential were associated with anodic activity, while cathodic activity was restricted to autotrophy and its relatives. The results suggested that the microbial communities anthropogenically impacted estuaries were potentially used for anodic and cathodic reactions in BES as electrogens to generate bioelectricity and reduce of pollutants.

Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China.

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/μL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

Molecular phylogeny confirms the subspecies delineation of the Malayan Siamang (Symphalangussyndactyluscontinentis) and the Sumatran Siamang (Symphalangussyndactylussyndactylus) based on the hypervariable region of mitochondrial DNA.

Siamangs (Symphalangussyndactylus) are native to Peninsular Malaysia, Sumatra and southern Thailand and their taxonomical classification at subspecies level remains unclear. Morphologically, two subspecies were proposed as early as 1908 by Thomas namely Symphalangus s.syndactylus and Symphalanguss.continentis. Thus, this study aims to clarify the Siamang subspecies status, based on mtDNA D-loop sequences. Faecal samples were collected from wild Siamang populations at different localities in Peninsular Malaysia. A 600-bp sequence of the mitochondrial D-loop region was amplified from faecal DNA extracts and analysed along with GenBank sequences representing Symphalangus sp., Nomascus sp., Hylobates sp., Hoolock sp. and outgroups (Pongopygmaeus, Macacafascicularis and Papiopapio). The molecular phylogenetic analysis in this study revealed two distinct clades formed by S.s.syndactylus and S.s.continentis which supports the previous morphological delineation of the existence of two subspecies. Biogeographical analysis indicated that the Sumatran population lineage was split from the Peninsular Malaysian population lineage and a diversification occurrred in the Pliocene era (~ 3.12 MYA) through southward expansion. This postulation was supported by the molecular clock, which illustrated that the Peninsular Malaysian population (~ 1.92 MYA) diverged earlier than the Sumatran population (~ 1.85 MYA). This is the first study to use a molecular approach to validate the subspecies statuses of S.s.syndactylus and S.s.continentis. This finding will be useful for conservation management, for example, during Siamang translocation and investigations into illegal pet trade and forensics involving Malayan and Sumatran Siamangs.

Population Structure Using Mitochondrial DNA for the Conservation of Liobagrus geumgangensis (Siluriformes: Amblycipitidae), an Endemic Freshwater Fish in Korea

Liobagrus geumgangensis is a novel Korean fish species endemic to the Geumgang and Mangyeonggang River basins on the Korean Peninsula. During a survey of L. geumgangensis, the discovery of Liobagrus mediadiposalis as a potential threat prompted an investigation into L. geumgangensis genetic diversity and structure. Three populations of L. geumagangensis and one population of L. mediadiposalis were investigated using a 1024-bp sequence in the cytb region of mitochondrial DNA. The Mangyeonggang River of L. geumagangensis displayed the lowest haplotype diversity (Hd) within a range of 0.000–0.337, with one to two haplotypes (h). The Jecheon region of the Geumgang River for L. geumagangensis population had the highest nucleotide diversity (π) and was within the range of 0.00000–0.00066. The h of L. mediadiposalis population was 3, the range of Hd was 0.292, and π was 0.00231. Tajima’s D (D) and Fu’s Fs (F) were negative and non-significant in the LgGJ population. The genetic structure of L. geumgangensis had no shared haplotypes among the three populations. The discovery of L. mediadiposalis in the Geumgang River suggests the necessity of non-habitat conservation and population management of fish farms to conserve L. geumgangensis.

Feasibility of Direct Vitrectomy-Sparing Subretinal Injection for Gene Delivery in Large Animals

Purpose To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. Methods The experimental Liběchov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. Results Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. Conclusions This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.

Influence of tree mycorrhizal type, tree species identity, and diversity on forest root-associated mycobiomes.

Understanding the complex interactions between trees and fungi is crucial for forest ecosystem management, yet the influence of tree mycorrhizal types, species identity, and diversity on tree-tree interactions and their root-associated fungal communities remains poorly understood. Our study addresses this gap by investigating root-associated fungal communities of different arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) tree species pairs (TSPs) in a subtropical tree diversity experiment, spanning monospecific, two-species, and multi-species mixtures, utilizing Illumina sequencing of the ITS2 region. The study reveals that tree mycorrhizal type significantly impacts the alpha diversity of root-associated fungi in monospecific stands. Meanwhile, tree species identity's influence is modulated by overall tree diversity. Tree-related variables and spatial distance emerged as major drivers of variations in fungal community composition. Notably, in multi-species mixtures, compositional differences between root fungal communities of AM and EcM trees diminish, indicating a convergence of fungal communities irrespective of mycorrhizal type. Interestingly, dual mycorrhizal fungal communities were observed in these multi-species mixtures. This research underscores the pivotal role of mycorrhizal partnerships and the interplay of biotic and abiotic factors in shaping root fungal communities, particularly in varied tree diversity settings, and its implications for effective forest management and biodiversity conservation.