Culture-Dependent and Metagenomic Profiling of Eukaryal Diversity in Petroleum Hydrocarbon-Polluted Soil from B-Dere, Gokana, Rivers State, Nigeria

B-Dere is one of the communities in Ogoniland and a major oil producing area in Rivers State where oil exploration and production activities commenced 50 years ago and is now characterized by oil fields and installations that have remained dormant for several decades. Past spills, lack of maintenance, oil trapping and damage to oil infrastructures have been common sight in this region and the environment has been without remediation over the years. B-Dere community has suffered surface water pollution throughout its creeks with massive hydrocarbons for years without remediation. The aim of this study was to determine the Culture- dependent and Metagenomic studies of fungal diversity in petroleum polluted soils in B-Dere community in Gokana LGA of Rivers State, Nigeria. This is to profile fungal communities through next-generation techniques by shotgun sequencing of total DNA isolates directly from the oil polluted environment. Soil samples were collected aseptically with hand auger at a depths of 0-15 and 15-30 cm and made up to a composite sample and transported to the laboratory for analysis using standard microbiological methods for culture- dependent analysis while the Metagenomic studies was carried out at the Microbial Insights, Incorporated; United State of America. In this study next-generation sequencing techniques by shotgun sequencing of total DNA methods were used for identification of fungal isolates from the crude oil polluted soils in B-Dere. Deoxyribonucleic acid (DNA) extraction from crude oil polluted soil samples was performed using ZymoBiomics DNA extraction kits (Zymo Research, Inc; USA). DNA sequencing was performed by the next generation sequencing technique to determine the nucleotide sequence of all eukaryal community structure present in the polluted soil sample using ITS region. Results of the culture-dependent technique showed that only two fungal genera namely Penicillium sp and Aspergillus sp were isolated and identified while the soil was mainly dominated by the genera Penicillium (73.33%), followed by the Rhodotorula (6.54%), Dactylellina(5.09%), Kalmanozyma(2.56%), Fereydounia(1.89%), Xerochrysium(1.36%), Arthrobotrys (1.14%) and Diutina (0.77%) by the metagenomic analysis. However the three major groups were classified as Ascomycota, Basidiomycota and Mucoromycota with Ascomycota having the highest taxonomic reads of 86.76%. However, a total of 60 eukaryal species were identified, in the metagenomic study. In conclusion, these fungal strains can be used in bioremediation process and oil pollution reduction in soil ecosystems because of their high activity in aliphatic hydrocarbon degradation and cell surface hydrophobicity. The next-generation techniques by shotgun sequencing assays appear to be suitable alternatives for rapid identification of the above mentioned fungal isolates.

Whole-Genome Sequence of Oryctes rhinoceros Nudivirus from Riau Province, Indonesia

A double-stranded DNA virus, Oryctes rhinoceros nudivirus (OrNV), was detected in the total DNA of diseased larvae of O. rhinoceros in Riau Province, Indonesia. The complete genome sequence was 124,926 bp long and encodes 123 open reading frames (ORFs). This strain belongs to the family Nudiviridae and was designated LiboV.

Proteus mirabilis carrying NTEKPC-IId, blaNDM-1, blaOXA-10, aph(3')-VI, qnrD1 and IncQ and Col3M plasmids from a hospital in Recife-PE, Brazil

The present study objective to characterize the clinical aspects of a patient infected with two strains of P. mirabilis and the presence of resistance determinants in the two isolates from a patient at a public hospital in Recife-PE, Brazil. The total DNA of the isolates was extracted and submitted to PCR and amplicon sequencing for the investigation of resistance genes, blaKPC, blaOXA-10, blaOXA-23, blaOXA-48, blaOXA-58, blaVIM, blaIMP, blaSPM, blaGES, blaNDM, qnrD and aac(6')-Ib). Isolate P21-A2 harbored the aac(6')-Ib, blaOXA-10 and qnrD genes. One of the isolates, P20-A2, was selected for plasmid DNA sequencing. The results showed that the patient developed multiple infections with various pathogens including two strains of P. mirabilis. The patient was hospitalized for 103 days, had septic shock of skin, abdominal, pulmonary and ulcer focus, and died. Isolate P20-A2 harbored the genes blaNDM, qnrD, aph(3')-VI, blaKPC and blaOXA-10, and plasmids IncQ and Col3M, together with NTEKPC-IId. To our knowledge, this is the first report of P. mirabilis harboring NTEKPC-IId. Although P. mirabilis is standing out as a cause of nosocomial infections and a resistant multidrug pathogen, this species is still neglected, the emergence of these P. mirabilis isolates harboring aforementioned resistance determinants and the plasmids IncQ and Col3M demonstrate the potential for dissemination of important resistance genes, mainly in the case of P. mirabilis.

Effects of Cadmium Sulfate on the Brown Garden Snail Cornu aspersum: Implications for DNA Methylation

An extensive literature exists regarding the cellular, physiological, and genetic effects of cadmium (Cd)—A highly toxic, but commonly used trace metal in modern industry. However, limited data are available on its epigenetic effects, especially for terrestrial sentinel invertebrates. We determined Cd retention, total DNA methylation, and the methylation status of 5′ end of the Cd-MT gene in the hepatopancreas of the brown garden snail, Cornu aspersum, fed Cd sulfate for four weeks. Bodyweight changes and survival were also measured. Hepatopancreas cadmium increased in a dose-dependent manner from the third-lowest dose onward, with very large amounts being found for the highest treatment group. However, no mortalities occurred, irrespective of dietary Cd dose. We identified significant genome-wide hypermethylation in specimens given the highest dose, which overlapped with a significant bodyweight decrease. The Cd-MT gene showed an unmethylated 5′ end of the Cd-MT gene and this status was not affected by cadmium exposure. Hepatopancreas DNA methylation is as sensitive as bodyweight to non-lethal concentrations of dietary Cd given as cadmium sulfate but less responsive than tissue accumulation. Such an exposure event, by contrast, does not affect the methylation status of the Cd-MT gene 5′ end.

Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection

Verifying the authenticity of food products is essential due to the recent increase in counterfeit meat-containing food products. The existing methods of detection have a number of disadvantages. Therefore, simple, cheap, and sensitive methods for detecting various types of meat are required. In this study, we propose a rapid full-cycle technique to control the chicken or pig adulteration of meat products, including 3 min of crude DNA extraction, 20 min of recombinase polymerase amplification (RPA) at 39 °C, and 10 min of lateral flow assay (LFA) detection. The cytochrome B gene was used in the developed RPA-based test for chicken and pig identification. The selected primers provided specific RPA without DNA nuclease and an additional oligonucleotide probe. As a result, RPA–LFA, based on designed fluorescein- and biotin-labeled primers, detected up to 0.2 pg total DNA per μL, which provided up to 0.001% w/w identification of the target meat component in the composite meat. The RPA–LFA of the chicken and pig meat identification was successfully applied to processed meat products and to meat after heating. The results were confirmed by real-time PCR. Ultimately, the developed analysis is specific and enables the detection of pork and chicken impurities with high accuracy in raw and processed meat mixtures. The proposed rapid full-cycle technique could be adopted for the authentication of other meat products.

Total DNA extraction from microalgae strain samples using NucleoSpin Plant modified kit (Macherey Nagel) v1

This DNA extraction protocol allows to get both eukaryotic and prokaryotic DNA from microalgae strains, so the microbiome diversity can be studied in cultures by using this protocol.

Perfusion-Decellularized Larynx as a Natural 3D Scaffold in a Rabbit Model

<b><i>Introduction:</i></b> Decellularized larynges could be used as scaffolds to regenerate the larynx. The purpose of this study was to establish a perfusion decellularization protocol to produce a 3-dimensional whole laryngeal extracellular matrix (ECM) scaffold in a rabbit model. <b><i>Methods:</i></b> The larynges of 20 rabbits assigned to the study group were harvested and decellularized using a perfusion decellularization protocol, while the larynges of 10 rabbits in the control group were harvested and untreated. Macroscopic and microscopic morphological analyses, a molecular analysis, a cellular content analysis, and scanning electron microscopy were performed. <b><i>Results:</i></b> A histological analysis showed the absence of cellular components, the presence of the ECM, and an intact cartilage structure filled with chondrocytes. The mean total DNA amounts of the native larynx, decellularized larynx, and decellularized cartilage-free larynx were 1,826.40, 434.70, and 41.40 μg/µL, respectively; those for the decellularized larynx and decellularized cartilage-free larynx were significantly lower (<i>p</i> &#x3c; 0.001 and <i>p</i> &#x3c; 0.001, respectively). The total amount of DNA in the decellularized sample was significantly lower compared to that in the native sample, at 57.2% in cartilage (<i>p</i> &#x3c; 0.001), 2.4% in the thyroid gland (<i>p</i> &#x3c; 0.001), 2.7% in muscle (<i>p</i> &#x3c; 0.001), 1.6% in vessels (<i>p</i> &#x3c; 0.001), and 4.8% in the vocal cords (<i>p</i> &#x3c; 0.001). <b><i>Conclusion:</i></b> Our perfusion decellularization protocol is feasible and reproducible to produce a 3-dimensional whole laryngeal ECM scaffold in a rabbit.

Host Variation in Interferon, MHC Class I, Glycosylation, and Viral Transcription Genes Predict HIV Persistence

Background: Prior host genomewide association studies have failed to observe an association with the HIV reservoir. Methods: Custom whole exome sequencing and direct HLA typing were performed from 202 HIV+ ART-suppressed individuals and associated with 4 measures of the circulating CD4+ T cell reservoir: total DNA, unspliced (us)RNA, RNA/DNA, and intact DNA. Common variant, gene-based, and HLA analyses were performed using linear mixed models adjusted for sex, timing of ART initiation, nadir CD4 count, input cell count, and ancestry. Results: HIV total DNA was associated with variants in genes involved in type I interferon (MX1, PPP1CB, DDX3X) and MHC class I peptide recognition (LRMP), while HIV usRNA was associated with a variant related to lymphocyte lymph node migration (PLVAP; this SNP was also <30kb from BST2, which encodes tetherin, an HIV host restriction factor). Gene-based analyses demonstrated significant associations with type I interferon (intact DNA), glycosylation (total DNA), and retroviral transcription (usRNA). HLA "protective" B*57:01 and "risk" C*07 alleles, as well as CCR5Δ32, were associated with HIV usRNA and total DNA, but not intact DNA. Conclusions: Host genetic variation in type I interferon, MHC class I, glycosylation, residual viral transcription, and lymphocyte lymph node migration may influence the circulating HIV CD4+ T cell reservoir.

Optimization of annealing temperature for amplification of EhoscnOla locus in pranajiwa (Euchresta horsfieldii) plant collected from mountains, urban and coastal areas in Bali

Pranajiwa plant is a medicinal plant that grows wildly and is classified as a rare plant. Currently, its existence is increasingly threatened. Pranajiwa grows around Indonesia and is known with several scientific names and morphological features due to unclear identification. Molecular identification is recommended to clarify its species. DNA Barcoding is considered the suitable method to identify pranajiwa plant molecularly. The purpose of this study was to optimized the PCR annealing temperature of EhcSnOla locus barcoding marker of pranajiwa plants collected from the coastal (Jimbaran), urban (Renon), and mountain (Bedugul) areas, representing three different areas in Bali. Research procedures include total DNA extraction, PCR procedure, and electrophoresis. The primers used in this study were EhoScnOla forward primer and EhoscnOla reverse primer. Five different temperatures were used for annealing temperature optimization: 51°C, 52°C, 55°C, 57°C, and 60°C. The result showed that all temperatures produced a clear, thick, and single electrophoresis band, indicating that all temperatures were suitable for the annealing temperature and the most optimal temperature is in the Mountains sample (Bedugul) which is 60°C. The Jimbaran, Renon, and Bedugul samples produced 882, 820, and 889 bp, respectively. EhcSnOla locus can be used as the barcoding marker to identify pranajiwa molecularly.

Intestinal Dominance by Serratia marcescens and Serratia ureilytica among Neonates in the Setting of an Outbreak

(1) Background: We determined the relevance of intestinal dominance by Serratia spp. during a neonatal outbreak over 13 weeks. (2) Methods: Rectal swabs (n = 110) were obtained from 42 neonates. Serratia spp. was cultured from swabs obtained from 13 neonates (Group 1), while the other 29 neonates were culture-negative (Group 2). Total DNA was extracted from rectal swabs, and quantitative PCRs (qPCRs) using Serratia- and 16SrRNA-gene-specific primers were performed. relative intestinal loads (RLs) were determined using ΔΔCt. Clonality was investigated by random amplified polymorphic DNA analysis and whole-genome sequencing. (3) Results: The outbreak was caused by Serratia marcescens during the first eight weeks and Serratia ureilytica during the remaining five weeks. Serratia spp. were detected by qPCR in all Group 1 neonates and eleven Group 2 neonates. RLs of Serratia spp. were higher in Group 1 as compared to Group 2 (6.31% vs. 0.09%, p < 0.05) and in the first swab compared to the last (26.9% vs. 4.37%, p < 0.05). Nine neonates had extraintestinal detection of Serratia spp.; eight of them were infected. RLs of the patients with extraintestinal spread were higher than the rest (2.79% vs. 0.29%, p < 0.05). (4) Conclusions: Intestinal dominance by Serratia spp. plays a role in outbreaks and extraintestinal spread.