Isolation and Characterization of Bacillus Sp. Capable of Degradating Alkali Lignin

Alkali lignin-degrading Bacillus were isolated from forest soils in China and were identified as Bacillus subtilis TR-03 and Bacillus cereus TR-25 by 16S rDNA sequence analysis. Wherein TR-03 displayed optimal 26.72% alkali lignin (2 g/L) degradation at 7 days and 71.23% of Azure-B (0.01%) decolorization at 36 h of cultivation at 37°C. Ligninolytic enzyme analysis revealed that TR-03 was capable of depolymerizing alkali lignin effectively by the producing of lignin peroxidase and laccase, wherein higher laccase activity was cell-associated. At last, the physical and chemical changes of lignin via SEM and FTIR analysis was further observed to prove the lignin degradation by Bacillus subtilis TR-03.

Clostridium perfringens – antibiotic-associated and sporadic diarrhea in children: age, anamnestic and clinical and laboratory features

The article presents the data of the research on the problem of two clinical forms of enteroproducing Clostridium perfringens (C. perfringens) – associated infection in the children who are not connected with food – antibiotic-associated and sporadic forms. Within the period of 2019–2021 a retrospective analysis of the results of complex clinical and laboratory study of children aged from 2 months to 18 years with diarrhea (acute intestinal infection, n = 406 children) was carried out within the framework of the algorithm developed by Pediatric Research and Clinical Center for Infectious Diseases (Russia, Saint Petersburg). The material for the study included stool tests, coprofiltrates and strains of the microorganisms determinating microbiocoenosis of the intestines including enterotoxigenic strains of C. perfringens. The main group included children with diarrhea (n = 38 of 406 children with diarrhea) who at admission presented C. perfringens enterotoxin in coprofiltrates found by immune-enzyme analysis and there were identified the cultures of enterotoxigenic strains of C. perfringens by tests of intestinal contents. The first group included children (n = 30 of 406 children; 7 %) with antibiotic-associated diarrhea (AAD). The second group included children (n = 8 of 406 children; 2 %) with sporadic diarrhea. An overwhelming majority of patients with ААD (n = 20; 92.3 %) of the first 8 (100 %) years of life presented intensity of diarrhea syndrome in direct dependence on colonization activity of enterotoxigenic C perfringens (colony-forming units/g feces) (r = 0.78). The disease proceeded mainly with the syndromes of gastroenteritis, enterocolitis and hemorrhagic colitis. There was noted a reliable high frequency of severe forms of both ААD, and sporadic diarrhea (р < 0.05) caused by enterotoxigenic strains of C perfringens in children of the first year of life. Deep decompensated disorders of large intestine microbiota associated with Klebsiella spp., Staphylococcus aureus and Candida albicans, were characterized by severity and prolonged character of the course (р < 0.05). The received results determine the tactics of differential diagnosis and treatment of AAD and sporadic diarrhea caused by enteroproducing strains of C. perfringens.

Whole-genome based strain identification of fowlpox virus directly from cutaneous tissue and propagated virus

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn’t compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.

A simple method for detection of mutations in amino acid 452 of the Spike protein of SARS-CoV-2 using restriction enzyme analysis

Variants of Concern or Interest of SARS-CoV-2 (VOC or VOI), the coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 452 of the Spike protein are particularly important and associated with some of these variants: L452R, present in Delta VOC, and L452Q, present in Lambda VOI. These mutations have been associated with both increased infectivity and evasion of protective immune response. A search on GISAID to detect the number of sequences harboring the L452R mutation and the frequency of Delta VOC among them, showed that since August 2021, most of these sequences belong to the Delta VOC. Restriction enzyme analysis is proposed as a rapid method to detect L452R. A small amplicon from the Spike gene was digested with MspI. A 100% concordance was observed between digestion and sequencing results. The mutation L452Q can also be detected by restriction analysis, allowing the identification of putative Lambda VOIs. The proposed methodology, which allows screening of a great number of samples, could provide a faster information on the prevalence of Delta VOC cases.

Morphological, Histological Changes and Acetyl Cholinesterase Activity in Chick Embryos After Exposure to Abamectin Insecticide

Abamectin is a bio-insecticide, derived from the soil bacteria Streptomyces avermitilis. This insecticide is used in public health and agriculture to protect crops. Major adverse impacts of Abamectin are neurological symptoms acting on the peripheral nervous system. The aim of the current study is to reveal the toxic effects of Abamectin on chick embryo Ross 308 including morphological and histological changes and acetyl cholinesterase activity. 120 fresh fertilized eggs were divided into 6 groups; two of them were used as control. After 2 days of incubation, the eggs were injected with 100 µL of Abamectin solution (diluted at concentrations 360, 540, 900 and 1800 ppm) into the yolk sac. The Results showed that the mortality increased significantly in chicks treated with Abamectin, but had a lower weight in comparison to the control groups. Treated chicks started hatching at day 22 but were physically weak with drooping limbs, paralysis and then died after 24 hours of hatching. Some chicks did not normally hatch and needed assistance. They characterized by limb defects, failure retraction of yolk sac with bleeding. Histological examination of the liver showed hepatic cell degeneration, congestion in the central vein, infiltration of inflammatory cells and hepatocytes necrosis. Furthermore, the Acetyl cholinesterase enzyme analysis showed a significant decrease in the enzyme activity which leads to inhibition the activity of the body systems. It is concluded that low and high concentration of Abamectin has adverse impacts on chick embryo by changing some of morphological, histological characteristics and acetyl cholinesterase activity.

Heterodera amaranthusiae n. sp. (Nematoda: Heteroderidae), a new cyst nematode parasitising Amaranthus retroflexus L. in China

Summary A new species of cyst-forming nematode, Heterodera amaranthusiae n. sp., is described and illustrated from the weed, Amaranthus retroflexus, in a potato field in Yunnan Province, China. It is characterised by having canary to russet-brown and asymmetric lemon-shaped cyst, distinct neck, bifenestrate vulval cone, relatively short vulval slit of 29 (28-32) μm, bullae absent and underbridge absent or weak if present. Second-stage juveniles are characterised by a well-developed stylet of 23 (22-25) μm with robust shaft and basal knobs concave anteriorly, tail conoid, 51 (48-58) μm long and hyaline region comprising 48 (41-53)% of its length. Morphologically and morphometrically it most resembles H. vallicola in the Humuli group. The ITS, 28S and COI gene sequences of H. amaranthusiae n. sp. clearly differentiate it from other Heterodera species. For diagnostic purposes, restriction enzyme analysis of the ITS region and three restriction enzymes, AluI, BsuRI (HaeIII) and CfoI (HhaI), were selected, clearly distinguishing H. amaranthusiae n. sp. from representative species in the Humuli group. Phylogenetic relationships with other species of the genus, inferred from two ribosomal regions and the cytochrome oxidase c subunit 1 region, based on Bayesian analysis, consistently showed that H. amaranthusiae n. sp. clustered with high support with other Humuli group species but with separate species status.

A simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that develops during infancy and early childhood. The array-based international consensus definition of DNA methylation has recently classified patients with JMML into the following three groups: high methylation (HM), intermediate methylation (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML have been analyzed using the Digital Restriction Enzyme Analysis of Methylation (DREAM), which is a next-generation sequencing based methylation analysis. Unsupervised consensus clustering of the discovery cohort (n=99) using the DREAM data has identified HM and LM subgroups (HM_DREAM, n=35; LM_DREAM; n=64). Of the 98 cases that could be compared with the international consensus classification, 90 cases of HM (n=30) and LM (n=60) had 100% concordance with the DREAM clustering results. For the remaining eight cases classified as the IM group, four cases were classified into the HM_DREAM group and four cases into the LM_DREAM group. A machine-learning classifier has been successfully constructed using a Support Vector Machine (SVM), which divided the validation cohort (n=38) into HM (HM_SVM; n=18) and LM (LM_SVM; n=20) groups. Patients with the HM_SVM profile had a significantly poorer 5-year overall survival rate than those with the LM_SVM profile. In conclusion, a robust methylation test has been developed using the DREAM analysis for patients with JMML. This simple and straightforward test can be easily incorporated in diagnosis to generate a methylation classification for patients so that they can receive risk-adapted treatment in the context of future clinical trials.

Transcriptome Analysis Provides Insights into Hepatic Responses to Trichloroisocyanuric Acid Exposure in Goldfish (Carassius auratus)

In this study, goldfish (Carassius auratus) were exposed to 0 (control group) and 0.81 mg/L TCCA for four consecutive days. The liver transcriptome, the molecular indices of oxidative stress, and gills histopathology were investigated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that energy metabolism-related pathways such as glycolysis/gluconeogenesis were significantly enriched, suggesting their perturbation in the liver of goldfish. Additionally, TCCA exposure also caused pathological damage in gills, which compromised physiological function and decreased oxygen intake capacity of gills, thus leading to the enhancement of anaerobic metabolism. This finding was confirmed by the significant upregulation of lactate dehydrogenase in the liver of goldfish. Moreover, many phase I and phase II metabolic enzymes might be activated to alleviate TCCA-induced toxicity in goldfish, and glutathione S-transferases (GSTs) and cytochrome P450s (CYPs) play a crucial role in the metabolism of TCCA in the liver of goldfish. Furthermore, the antioxidant enzyme analysis showed that TCCA exposure induced oxidative damage in the liver and partially impaired the antioxidant defense system of goldfish, evidenced by decreased superoxide dismutase (SOD) and catalase (CAT), and increased malondialdehyde (MDA) level. In summary, this study will improve our understanding of the molecular mechanisms of the TCCA-induced toxicity in goldfish.

Effect of CO2 exposure on the antioxidant enzymes activity and gill histology of adult zebrafish (Danio rerio)

Increase in carbon dioxide level is considered as a threat to all living organisms and in particular aquatic organisms are vulnerable. Although a number of studies were carried out to predict the effect of increased CO2 with marine organisms, fresh water fish were the least concerned ones. Therefore, the aim of this study was to study the effect of antioxidant enzymes in zebrafish exposed to acute concentration of CO2 (based on prediction for 2300). Three ranges of pH were selected below the range where no fatality was observed, 6.0 – 6.5, 5.5 – 6.0 and 5.0 – 5.5. Antioxidant enzyme analysis of superoxide dismutase (SOD), catalase (CAT), lipid peroxidation (LP), glutathione reductase (GR) and glutathione peroxidase (GPx) was performed for the tissues, liver, gills and muscles. Although a significant decrease in SOD activity was observed in all three tissues, only a slight variation in CAT activity was found in all the tissues. MDA content which is an indicator of lipid peroxidation showed an increasing pattern in all the tissues. A slight increase in GR activity was found in all the tissues; however, a significant decrease in GPx activity was noted. Further, the histology of gills exposed to CO2 showed marked damage and injury including lamellar fusion and necrosis. Our study concludes that exposure to CO2 has led to oxidative stress which has resulted in cellular injury and damage

A Comparative Study on the Efficiency of Two Mycobacterium avium subsp. paratuberculosis (MAP)-Derived Lipopeptides of L3P and L5P as Capture Antigens in an In-House Milk ELISA Test

Mycobacterium avium subsp. paratuberculosis (MAP) surface-exposed lipopeptides could be specific capture-antigen molecules targeting antibodies against MAP, in milk, through ELISA. Previous studies have revealed that MAP strains, isolated from sheep (S) or cow (C), could produce specific lipopeptides, L3P or L5P, respectively. In this study, we used L3P and L5P as capture antigens in an in-house milk ELISA (H-MELISA) to assess how these antigens perform, in comparison with other ELISA tests, on well-defined milk samples from MAP-infected sheep. The overall positivity rates of H-MELISA via L3P and L5P varied by the source of milk samples, in which, at bulk-tank-milk (BTM) level, the majority of positive cases (63.83%) reacted more against L5P, whereas a predominant number (69.14%) of milk samples were more responsive against L3P at the individual level. To clarify whether the positivity status of milk samples in H-MELISA L3P/L5P were predictive of MAP strain-types (S/C), strain-typing was carried out using PCR IS1311-restriction enzyme analysis. Although the presence of three MAP strains (S/C/bison types) was detected among the milk samples, the C-type (46.67%) and S-type (75%) MAP strains were detected with higher incidence among BTMs and individual milk samples, respectively. However, further examination on the H-MELISA L3P/L5P-positivity pattern of each C/S-type-MAP sample revealed that some samples had a reverse reactivity against both L3P and L5P. These results could be the consequence of either cross-reactivity between L3P and L5P (due to the similarity in the structures of the two epitopes) or simply a within-herd mixed infection with MAP strains of C and S types. These findings suggest that lipopeptide antigens could contribute a diagnostic test with optimal performance, considering the diversity of MAP strains.