Ribosomal Ribonucleic Acid Research Articles

The complete chloroplast genomes of Polygonatum hunanense, P. verticillatum, and P. caulialatum and their phylogenetic positions

Polygonatum hunanense H.H. Liu & B.Z. Wang (2021) and P. verticillatum (L.) All. (1875) have been widely used as foods and as folk medicines in China and India, and P. caulialatum S. R. Yi (2021) has recently been described as a new medical plant in China. There is at present a lack of genome information regarding the species. Hence, this study reports the complete chloroplast genomes of the three species. The genomes of P. hunanense, P. verticillatum, and P. caulialatum were 155,583 bp, 155,650 bp, and 155,352 bp in length, respectively. They contained large single-copy (LSC) regions of 84,412 bp, 84,404 bp, and 84,285 bp, small single-copy (SSC) regions of 18,427 bp, 18,416 bp, and 18,463 bp, and a pair of inverted repeats of 26,372 bp, 26,415 bp, and 26,302 bp, respectively. The chloroplast genomes of P. hunanense, P. verticillatum, and P. caulialatum had 133 (103 unique) genes, consisting of 87 protein-coding genes, 38 ribosomal ribonucleic acid (RNA) genes, and eight transfer RNA genes, respectively. A maximum-likelihood phylogenetic tree showed that P. kingianum Coll. et Hemsl. var. grandifolium D.M. Liu & W.Z. Zeng (1991) was closer to P. cyrtonema Hua (1892) rather than to P. kingianum Coll. et Hemsl. (1890), further supporting its status as a unique species of the genus. Moreover, P. verticillatum was separated from the easily confused herb P. cirrhifolium (Wall.) Royle (1839), while P. caulialatum was closest to P. humile Fisch. ex Maxim. (1859). This research provides a foundation for further study of these herbs.

Pathological investigation and morpho-molecular characterisation of Lasiodiplodia theobromae causing fruit rot diseases on Hydnocarpus longipedunculatus – A novel host record from India

Hydnocarpus longipedunculatus, a medically significant tree species, is threatened and has a narrow distribution, being endemic to the Southern Western Ghats of India. A case of fruit rot disease was observed in a solitary tree of H. longipedunculatus in the Kulamavu forest areas of Kerala, India. The causative fungus, Lasiodiplodia theobromae, was isolated and identified through a combination of morpho-cultural characteristics and molecular sequence analysis involving the internal transcribed spacer (ITS), partial translation elongation factor-1-α (TEF1-α), partial β-tubulin (TUB2), and large subunit ribosomal ribonucleic acid (LSU) regions. Additionally, concatenated multigene (ITS-TEF1-α-TUB2) phylogenetic analysis was done. Pathogenicity tests were conducted in vitro using mycelial disc methods and confirmed the pathogenic nature of the fungus through re-isolation and morphocultural analysis, satisfying Koch's postulates. Previous studies neither have reported H. longipedunculatus as a host for L. theobromae, nor has any genus within Hydnocarpus. Therefore, this represents the first documented instance of H. longipedunculatus as a novel host record for L. theobromae from India.

Comparing Oral Microbiota Changes after Pulpotomy/Pulpectomy in Pediatric Patients.

Pediatric dental procedures like pulpotomy and pulpectomy are commonly performed to treat pulp-related dental issues in primary teeth. The choice of instrumentation, such as traditional rotary instruments or laser technology, can impact the oral microbiota. This study aims to compare the changes in oral microbiota following pulpotomy/pulpectomy in pediatric patients treated with traditional rotary instruments versus laser technology. A total of 60 pediatric patients, aged 4-10 years, requiring pulpotomy or pulpectomy were randomly assigned to two groups: group A (traditional) and group B (laser). In group A, traditional rotary instruments were used for pulp removal, while in group B, a dental laser was utilized. Saliva and plaque samples were collected before the procedure (baseline), immediately after the procedure, and at the one-month follow-up. Microbial deoxyribonucleic acid (DNA) was extracted and analyzed using 16S ribosomal ribonucleic acid (rRNA) sequencing to assess changes in the oral microbiota. At baseline, both groups exhibited similar microbial diversity and composition. However, immediate post-procedure results showed a significant decrease in microbial diversity in both groups, with group B (laser) demonstrating a more pronounced reduction. At the one-month follow-up, group A (traditional) showed a partial recovery of microbial diversity, while group B (laser) continued to exhibit lower microbial diversity compared to baseline. Notably, group B (laser) displayed a shift in microbial composition with a decrease in pathogenic species and an increase in beneficial bacteria compared to group A. This study highlights that both traditional rotary instruments and laser technology have a significant impact on the oral microbiota of pediatric patients following pulpotomy/pulpectomy. While both methods initially reduce microbial diversity, laser treatment appears to have a more sustained effect, favoring the growth of beneficial bacteria over time. Further research is needed to assess the long-term clinical implications of these microbiota changes for the oral health of pediatric patients.

Unveiling the gut microbiota and metabolite profiles in guinea pigs with form deprivation myopia through 16S rRNA gene sequencing and untargeted metabolomics

AimThe aim of this study was to confirm the presence of the form deprivation myopia (FDM) guinea pig eye-gut axis and investigate the relationship between serum vasoactive intestinal peptide (VIP), lipopolysaccharides (LPS), specific gut microbiota and their metabolites. Method20 specific-pathogen-free (SPF) guinea pigs were divided into the FDM and the control(Con) group. Following model induction, serum levels of VIP and LPS were quantified. A combination of 16S ribosomal ribosomal Ribonucleic Acid (rRNA) gene sequencing, non-targeted metabolomics and bioinformatics analysis were employed to identify disparities in gut microbiota and metabolites between the two groups of guinea pigs. ResultCompared to the control group, FDM guinea pigs exhibited a significant trend towards myopia, along with significantly elevated concentrations of LPS and VIP (p < 0.0001). Furthermore, Ruminococcus_albus emerged as the predominant bacterial community enriched in FDM (p < 0.05), and demonstrated positive correlations with 10 metabolites, including l-Glutamic acid, Additionally, Ruminococcus_albus exhibited positive correlations with VIP and LPS levels (p < 0.05). ConclusionThe findings suggest that the Ruminococcus_Albus and glutamate metabolic pathways play a significant role in myopia development, leading to concurrent alterations in serum VIP and LPS levels in FDM guinea pigs. This underscores the potential of specific gut microbiota and their metabolites as pivotal biomarkers involved in the pathogenesis of myopia.

Genetic Diversity of Blastocystis in Diarrheal Cases: Identification of Subtypes and Alleles

Blastocystis spp. are the most common intestinal protozoan parasites detected in human stool samples. While identified long before today, its pathogenicity remains controversial. It is generally asymptomatic but in symptomatic cases, many gastrointestinal symptoms, especially diarrhea, have been associated with Blastocystis infection. In recent years, the relationship between the symptoms observed in cases and Blastocystis subtypes (ST) has been reported. The aim of this study was to detect Blastocystis in diarrheal cases admitted to the Aydın Adnan Menderes University Faculty of Medicine, Department of Parasitology Laboratory, to determine subtypes and allele diversity and to investigate its relationship with clinical symptoms. For this purpose, diarrheal stool samples of 200 cases were included in the study and their demographic characteristics (age, gender, residence) and clinical findings (abdominal pain, dyspepsia, nausea-vomiting, weakness, weight loss, anal itching, rash, urticaria) were recorded. Blastocystis was detected by direct microscope method (DM) and by molecular analyses which were performed with polymerase chain reaction (PCR). Subtype diversity was determined based on DNA sequence analysis by PCR targeting the Blastocystis ribosomal ribonucleic acid small subunit (SSU rRNA) gene. In addition, alleles related to Blastocystis subtypes were determined and statistically compared between all data and clinical findings. In the current study, Blastocystis was detected in 31 (15.5%) samples by DM and in 35 (17.5%) samples by PCR specific to the Blastocystis SSU rRNA gene among 200 diarrheal stool samples. No statistical difference was detected between Blastocystis and demographic characteristics. Dyspepsia and nausea-vomiting symptoms differed significantly in cases with Blastocystis compared to negative ones (p= 0.0025, p= 0.0498). Blastocystis subtype was detected in 33 samples by SSU rRNA sequence analysis, and the subtype distribution was ST1 (n= 10, 30.3%), ST2 (n= 4, 12.1%) and ST3 (n= 19, 57.6%). In the statistical evaluation between clinical findings and Blastocystis subtypes, a relationship was found between dyspepsia and Blastocystis ST3 (p= 0.0039). The allele diversity of Blastocystis subtypes was determined as allele 4 (10/10) in all ST1, allele 11 (2/4) and 12 (2/4) in ST2, allele 34 (14/19), 36 (4/19), and 38 (1/19) in ST3. In conclusion, our study provides important data on the molecular epidemiological characteristics of the Blastocystis by determining positivity, subtypes and alleles in diarrheal cases. Therefore, within the scope of the one health approach, comprehensive molecular epidemiological studies are required to determine the presence and genotypes of Blastocystis in human, animal and environmental samples.

Gut Microbiota in Patients with Prediabetes.

Prediabetes is characterized by abnormal glycemic levels below the type 2 diabetes threshold, and effective control of blood glucose may prevent the progression to type 2 diabetes. While the association between the gut microbiota, glucose metabolism, and insulin resistance in diabetic patients has been established in previous studies, there is a lack of research regarding these aspects in prediabetic patients in Asia. We aim to investigate the composition of the gut microbiota in prediabetic patients and their differences compared to healthy individuals. In total, 57 prediabetic patients and 60 healthy adult individuals aged 18 to 65 years old were included in this study. Biochemistry data, fecal samples, and 3 days of food records were collected. Deoxyribonucleic acid extraction and next-generation sequencing via 16S ribosomal ribonucleic acid metagenomic sequencing were conducted to analyze the relationship between the gut microbiota and dietary habits. Prediabetic patients showed a lower microbial diversity than healthy individuals, with 9 bacterial genera being less abundant and 14 others more abundant. Prediabetic patients who consumed a low-carbohydrate (LC) diet exhibited higher diversity in the gut microbiota than those who consumed a high-carbohydrate diet. A higher abundance of Coprococcus was observed in the prediabetic patients on an LC diet. Compared to healthy individuals, the gut microbiota of prediabetic patients was significantly different, and adopting an LC diet with high dietary fiber consumption may positively impact the gut microbiota. Future studies should aim to understand the relationship between the gut microbiota and glycemic control in the Asian population.

The First Case of Phaeohyphomycosis Caused by Exophiala xenobiotica in an Immunocompetent Patient in Korea

Phaeohyphomycosis is a rare fungal infection caused by dematiaceous fungi commonly associated with immunocompromised patients. Exophiala (E.) species are linked to phaeohyphomycosis, and in Korea, E. dermatitidis, E. jeanselmei, and E. salmonis were previously isolated from affected patients. Here, we describe a unique instance of phaeohyphomycosis in an immunocompetent patient attributed to E. xenobiotica. A 75-year-old female presented with an asymptomatic erythematous crusted subcutaneous nodule on the dorsum of her left-hand. Her past medical history was negative for immunosuppression or trauma to the affected area. Histopathological examination revealed suppurative granulomatous infiltration, with septate hyphae and spores detected by periodic acid-Schiff staining in the dermis. A fungal culture made from a tissue sample revealed E. xenobiotica, with subsequent confirmation through phylogenetic analyses of the internal transcribed spacer (ITS) and large subunit regions (LSU) of ribosomal ribonucleic acid (rRNA). The patient was diagnosed with phaeohyphomycosis and underwent a 3-week course of terbinafine, followed by 7 weeks of fluconazole. These treatments significantly improved the lesion. To our knowledge, this is the first reported case of phaeohyphomycosis caused by E. xenobiotica in Korea.

Features of the vagina microbiotope in patients with adenomyosis

The objective: to assess the composition of the vaginal microbiota in women of reproductive age with adenomyosis.Materials and methods. A comprehensive clinical-laboratory and instrumental prospective examination of 224 patients aged 30 to 50 years (average age – 42.0±1.8 years) with adenomyosis, who were included in the main study group, was conducted. The patients of the main group were divided into 2 subgroups depending on the form of adenomyosis – I group (diffuse form) included 106 women, II group (nodular form) – 118 persons. The control group included 84 healthy women of reproductive age with a regular ovulatory menstrual cycle and no history of gynecological diseases.Patients underwent microscopic examination of vaginal secretions, bacterioscopic examination of vaginal smears, stained according to Gram. Lactobacillus cultures were identified by polymerase chain reaction (PCR) and 16S ribosomal ribonucleic acid analysis.​Results. During the microscopic examination of the vaginal secretion, more than 15 leukocytes in the field of view were detected in 84 (37.5±3.2%) patients of both groups: in 40 (47.9±3.3%) patients of the I group and in 44 (52.1±3.3%) – II group. During the bacterioscopic examination of vaginal swabs, stained according to Gram, no gonococci and trichomonads were found in the main group. Candida fungi were detected microscopically in 158 patients (70.5±3.1%) of the main group: in 71 (66.9±4.6%) of the I group and in 87 (33.1±4.3%) of II group.Genotyping results showed that lactobacilli isolated from women with adenomyosis belonged to seven species: Lactobacillus fermentum, Lactobacillus iners, Lactobacillus gasseri, Lactobacillus amnionii, Lactobacillus jensenii, Lactobacillus crispatus, and Lactobacillus delbrueskii. The majority of lactobacilli strains have high inhibitory activity against all test strains, except for Candida albicans (39 cases, 46.4%). High adhesion to human erythrocytes was determined by 54.7% (46 cases) of the studied cultures.All lactobacilli strains were resistant to metronidazole, 83.3% (70 cases) to kanamycin, 57.1% (48 cases) to vancomycin, all strains were sensitive to roxithromycin, amoxiclav, and ampicillin. Moderate sensitivity to gentamicin and cefazolin was found in 51.9% (43 cases) of cultures, peroxide-forming activity in 80.9% (68 cases).The use of quantitative diagnostic thresholds has led to significant improvements in diagnostic performance. PCR showed 88% sensitivity and 95% specificity for Gardnerella vaginalis and 96% sensitivity and 97% specificity for Atopobium vaginae.Conclusions. It has been established that bacterial vaginosis and microorganisms associated with bacterial vaginosis are independently associated with adenomyosis. The results of the study confirmed that vaginal microorganisms, which are usually diagnosed in women with adenomyosis, ascend to the endometrium from the vagina vertically, which leads to infection of the upper parts of the genital tract.

Short-Term Ingestion of Essential Amino Acid Based Nutritional Supplements or Whey Protein Improves the Physical Function of Older Adults Independently of Gut Microbiome.

Dietary proteins and essential amino acids (EAAs) are the major nutritional supplements that support the growth and activity of gut microbes contributing to the wellbeing of their host. This study hypothesizes that daily supplementation of the diet with either EAAs or whey protein for 12 weeks would improve the gut microbiome of older adults. The stool samples are processed and subjected to Illumina-based 16S ribosomal ribonucleic acid (rRNA)gene amplicon sequencing. In both groups, the most abundant families are found in order of relative abundance included: Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Rikenellaceae, Enterobacteriaceae, Oscillospiraceae, Tannerellaceae, and Akkermansiaceae, which indicate that these subjects are able to maintain a same healthy microbial diversity in their guts. A significant finding is a reduction of proinflammatory cytokine, interleukin-18 (IL-18) in the EAAs group. It also uses the standard 6-min walking test (6MWT) as a measure of cardiopulmonary fitness. At the end of the study, the subjects in the EAAs group perform significantly better in the 6MWT as compared to the whey group. It seems plausible that the improved physical performance and reduced proinflammatory cytokine, IL-18 seen in the EAAs group, are independent of changes in gut microbiota.

Costs of ribosomal RNA stabilization affect ribosome composition at maximum growth rate

Ribosomes are key to cellular self-fabrication and limit growth rate. While most enzymes are proteins, ribosomes consist of 1/3 protein and 2/3 ribonucleic acid (RNA) (in E. coli).Here, we develop a mechanistic model of a self-fabricating cell, validated across diverse growth conditions. Through resource balance analysis (RBA), we explore the variation in maximum growth rate with ribosome composition, assuming constant kinetic parameters.Our model highlights the importance of RNA instability. If we neglect it, RNA synthesis is always cheaper than protein synthesis, leading to an RNA-only ribosome at maximum growth rate. Upon accounting for RNA turnover, we find that a mixed ribosome composed of RNA and proteins maximizes growth rate. To account for RNA turnover, we explore two scenarios regarding the activity of RNases. In (a) degradation is proportional to RNA content. In (b) ribosomal proteins cooperatively mitigate RNA instability by protecting it from misfolding and subsequent degradation. In both cases, higher protein content elevates protein synthesis costs and simultaneously lowers RNA turnover expenses, resulting in mixed RNA-protein ribosomes. Only scenario (b) aligns qualitatively with experimental data across varied growth conditions.Our research provides fresh insights into ribosome biogenesis and evolution, paving the way for understanding protein-rich ribosomes in archaea and mitochondria.

Improvement of ribonucleic acid production in Cyberlindnera jadinii and optimization of fermentation medium

To enhance the ribonucleic acid (RNA) productivity for industrial applications, this study employed strain screening and medium optimization to improve the content of RNA in Cyberlindnera jadinii. A rapid screening method, combining atmospheric and room temperature plasma mutagenesis, 48-deep-well plates fermentation, and microplate reader detection, was developed. A mutant strain named WB15 with high RNA content was successfully obtained, exhibiting the RNA content of 156 ± 4.5 mg/g DCW, 1.4 times of the starting strain CCTCC AY 92020. Furthermore, Plackett–Burman design and response surface methodology were employed to identify three significant factors (yeast extract, soybean peptone, and KH2PO4) affecting the RNA content. By utilizing the optimal medium composed of 13.43 g/L yeast extract, 12.12 g/L soybean peptone and 2.78 g/L KH2PO4, the RNA content of WB15 further increased to 184 ± 4.9 mg/g DCW. Additionally, the mutant strain WB15 exhibited a greater cellular width compared to AY 92020, along with increased growth rate and single-cell RNA content by 22% and 48.9%, respectively. Perturbations in ribosome assembly, specifically a reduction in the ratio of ribosomal proteins to ribosomal RNA of the large subunit, might indirectly contribute to the higher RNA content in the WB15 strain. Overall, the combination of rapid screening with fermentation medium optimization proved to be an effective approach for improving the RNA content of C. jadinii, thus facilitating the industrial production of RNA.

Temporal gut microbiota variability and association with dietary patterns: From the one-year observational Diet, Cancer, and Health - Next Generations MAX study

BackgroundKnowledge about the variability of gut microbiota within an individual over time is important to allow meaningful investigations of the gut microbiota in relation to diet and health outcomes in observational studies. Plant-based dietary patterns have been associated with a lower risk of morbidity and mortality and may alter gut microbiota in a favorable direction. ObjectivesTo assess the gut microbiota variability during one year and investigate the association between adherence to diet indexes and the gut microbiota in a Danish population. MethodsFour hundred forty-four participants were included in the Diet, Cancer, and Health - Next Generations MAX study (DCH-NG MAX). Stool samples collected up to three times during a year were analyzed by 16S ribosomal ribonucleic acid gene sequencing. Diet was obtained by 24-hour dietary recalls. Intraclass correlation coefficient (ICC) was calculated to assess temporal microbial variability based on 214 individuals. Diet indexes (Nordic, Mediterranean, and plant-based diets) and food groups thereof were associated with gut microbiota using linear regression analyses. ResultsWe found that 91 out of 234 genera had an ICC >0.5. We identified three subgroups dominated by Bacteroides, Prevotella 9, and Ruminococcaceae and adherence to diet indexes differed between subgroups. Higher adherence to diet indexes was associated with the relative abundance of 22 genera. Across diet indexes, higher intakes of fruit, vegetables, whole grains/cereals, and nuts were most frequently associated with these genera. ConclusionsIn the DCH-NG MAX study, 39% of the genera had an ICC >0.5 over one year, suggesting that these genera could be studied with health outcomes in prospective analyses with acceptable precision. Adherence to the Nordic, Mediterranean, and plant-based diets differed between bacterial subgroups and was associated with a higher abundance of genera with fiber-degrading properties. Fruits, vegetables, whole grains/cereals, and nuts were frequently associated with these genera.

Effect of 15 days -6° head-down bed rest on microbial communities of supragingival plaque in young men.

The microgravity environment astronauts experience during spaceflight can lead to an increased risk of oral diseases and possible changes in oral microecology. In this study, we aimed to assess changes in the microbial community of supragingival plaques to explore the effects of spaceflight microgravity environment on oral microecology. Sixteen healthy male volunteers were recruited, and supragingival plaque samples were collected under -6° head-down bed rest (HDBR) at five-time points: day 1 before HDBR; days 5, 10, and 15 of HDBR; and day 6 of recovery. Bacterial genomic DNA was sequenced using gene sequencing technology with 16S ribosomal ribonucleic acid V3-V4 hypervariable region amplification and the obtained data were analyzed bioinformatically. Alpha diversity analysis showed a significant increase in species richness in supragingival plaque samples on day 15 of HDBR compared with that at pre-HDBR. Beta diversity analysis revealed that the community composition differed among the groups. Species distribution showed that, compared with those at pre-HDBR, the relative abundances of Corynebacterium and Aggregatibacter increased significantly during HDBR, while those of Veillonella, Streptococcus, and Lautropia decreased significantly. Moreover, compared with those at pre-HDBR, the relative abundance of Leptotrichia increased significantly on day 6 of recovery, whereas the relative abundances of Porphyromonas and Streptococcus decreased significantly. Network analysis showed that the interaction relationship between the dominant genera became simpler during HDBR, and the positive and negative correlations between them showed dynamic changes. Phylogenetic investigation of communities by reconstruction of unobserved states analysis showed that the amino acid metabolism function of plaque microorganisms was more enriched during HDBR. In summary, in a 15-day simulated microgravity environment, the diversity, species distribution, interaction relationship, and metabolic function of the supragingival plaque microbial community changed, which suggests that microgravity may affect the oral microecosystem by changing the balance of supragingival plaque microbial communities and further leading to the occurrence and development of oral diseases.

P334 The longitudinal analysis of fecal microbiota in response to ustekinumab treatment in patients with Crohn’s disease

Abstract Background Emerging evidence supports that treatment of Crohn’s disease (CD) can alter the intestinal microbiota, which can play a significant role in the pathogenesis and therapeutic outcomes of CD. Ustekinumab (UST) has also been reported to modify the intestinal microbiota during a short-term treatment period. However, the long-term impact of UST treatment on the intestinal microbiota has rarely been studied. Methods We prospectively enrolled patients with CD who were planning to start UST treatment and collected fecal samples before (baseline) and after UST administration at week 20 and week 52. Fecal bacterial taxonomic diversity and composition were analyzed through 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing. The difference in the intestinal microbiota between clinical remitters and non-remitters defined by Crohn’s disease activity index (CDAI) was investigated, along with the longitudinal changes in the intestinal microbiota. Results A total of 124 fecal samples acquired from 43 patients were analyzed (median age 30 years, male 76.7%, median CDAI at baseline 183.2). Clinical remission was observed in 34.9% (n=15/43), 61.0% (n=25/41), and 60.0% (n=24/40) at baseline, week 20, and week 52, respectively. Shannon’s alpha diversity index increased significantly at week 20 and week 52 compared to baseline (both P&amp;lt;0.001). Compared to non-remitters, a significant increase of Shannon’s alpha diversity index was also observed among remitters (P=0.044) across all time points, while no significant difference was noted at each time point. The beta diversity analysis based on the Bray-Curtis distance revealed a significant difference among remitters at week 20 (P=0.027), however, there were no significant differences at other time points. Compared to the baseline, the abundance of the Firmicutes phylum was higher at week 20 (P=0.048) and 52 (P=0.019). The Lactobacillus genus (P=0.042), and Lactobacillus gasseri group (P=0.016) were significantly less abundant in remitters, while the Lachnospiraceae family (P=0.017) was more abundant in remitters. At week 20, Bifidobacterium genus (P=0.027) and Bifidobacterium pseudolongum group (P=0.040) were significantly more abundant in remitter while the Lactobacillus genus was less abundant (P=0.009) in remitter (Figure 1). Conclusion The alteration of fecal microbiota after UST treatment may have the potential to serve as a biomarker for predicting clinical remission. Financial Support This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1A2C2095096), the grant (2020IT0012) from the Asan Institute for Life Sciences, Seoul, Korea and the grant (CR 2017-4) from CELLTRION PHARM, Inc., Chungcheongbuk-do, Korea.

Reliable method for predicting the binding affinity of RNA-small molecule interactions using machine learning.

Ribonucleic acids (RNAs) play important roles in cellular regulation. Consequently, dysregulation of both coding and non-coding RNAs has been implicated in several disease conditions in the human body. In this regard, a growing interest has been observed to probe into the potential of RNAs to act as drug targets in disease conditions. To accelerate this search for disease-associated novel RNA targets and their small molecular inhibitors, machine learning models for binding affinity prediction were developed specific to six RNA subtypes namely, aptamers, miRNAs, repeats, ribosomal RNAs, riboswitches and viral RNAs. We found that differences in RNA sequence composition, flexibility and polar nature of RNA-binding ligands are important for predicting the binding affinity. Our method showed an average Pearson correlation (r) of 0.83 and a mean absolute error of 0.66 upon evaluation using the jack-knife test, indicating their reliability despite the low amount of data available for several RNA subtypes. Further, the models were validated with external blind test datasets, which outperform other existing quantitative structure-activity relationship (QSAR) models. We have developed a web server to host the models, RNA-Small molecule binding Affinity Predictor, which is freely available at: https://web.iitm.ac.in/bioinfo2/RSAPred/.

Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS.

The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC-MS/MS). LC-MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.

Cover crop monocultures and mixtures enhance bacterial abundance and functionality in the maize root zone.

Cover cropping is an effective method to protect agricultural soils from erosion, promote nutrient and moisture retention, encourage beneficial microbial activity, and maintain soil structure. Re-utilization of winter cover crop root channels by maize roots during summer allows the cash crop to extract resources from distal regions in the soil horizon. In this study, we investigated how cover cropping during winter followed by maize (Zea mays L.) during summer affects the spatiotemporal composition and function of the bacterial communities in the maize rhizosphere and surrounding soil samples using quantitative polymerase chain reaction (PCR), 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing, and metaproteomics. We found that the bacterial community differed significantly among cover crop species, soil depths, and maize growth stages. Bacterial abundance increased in reused root channels, and it continued to increase as cover crop diversity changed from monocultures to mixtures. Mixing Fabaceae with Brassicaceae or Poaceae enhanced the overall contributions of several steps of the bacterial carbon and nitrogen cycles, especially glycolysis and the pentose phosphate pathway. The deeper root channels of Fabaceae and Brassicaceae as compared to Poaceae corresponded to higher bacterial 16S rRNA gene copy numbers and improved community presence in the subsoil regimes, likely due to the increased availability of root exudates secreted by maize roots. In conclusion, root channel reuse improved the expression of metabolic pathways of the carbon and nitrogen cycles and the bacterial communities, which is beneficial to the soil and to the growing crops.

FEASIBILITY OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF METHICILLIN-SUSCEPTIBLE AND METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS IN TISSUE SAMPLES

To date, few studies have investigated the feasibility of the loop-mediated isothermal amplification (LAMP) assay for identifying pathogens in tissue samples. This study aimed to investigate the feasibility of LAMP for the rapid detection of methicillin-susceptible or methicillin-resistant Staphylococcus aureus (MSSA or MRSA) in tissue samples, using a bead-beating DNA extraction method. Twenty tissue samples infected with either MSSA (n = 10) or MRSA (n = 10) were obtained from patients who underwent orthopedic surgery for suspected musculoskeletal infection between December 2019 and September 2020. DNA was extracted from the infected tissue samples using the bead-beating method. A multiplex LAMP assay was conducted to identify MSSA and MRSA infections. To recognize the Staphylococcus genus, S. aureus, and methicillin resistance, 3 sets of 6 primers for the 16S ribosomal ribonucleic acid (rRNA) and the femA and mecA genes were used, respectively. The limit of detection and sensitivity (detection rate) of the LAMP assay for diagnosing MSSA and MRSA infection were analyzed. The results of this study suggest that the LAMP assay performed with tissue DNA samples can be a useful diagnostic method for the rapid detection of musculoskeletal infections caused by MSSA and MRSA.

Molecular and In Vivo Characterization of the High Pathogenicity H7N6 Avian Influenza Virus That Emerged in South African Poultry in 2023

A high pathogenicity avian influenza (HPAI) subtype H7N6 virus emerged in South African poultry in 2023 and later spread to Mozambique, the first documented emergence of H7 HPAI in the African continent. A total of 6.82 million birds succumbed to the disease or were culled, representing about 20% of the South African egg‐laying flock and almost 30% of the broiler breeder flock. The complete genomes of 68 outbreak viruses were sequenced and analyzed, tracing the phylogenetic origins of the ancestral H7N6 virus to a reassortment of various subtypes that circulated in southern African wild birds. Molecular clock analysis determined that the virus emerged in the first week of May 2023, probably in a smallholder chicken flock, before spreading to commercial farms, where the disease was first reported in early June. The multibasic hemagglutinin protein cleavage site (HA0) was derived from a nonhomologous recombination event with chicken 28S ribosomal ribonucleic acid (RNA). Few genetic markers associated with an increased risk to humans were present in the translated viral proteins. The intravenous pathogenicity index (IVPI) value of the index case isolate was 1.67, reflecting that 50% of the specific pathogen‐free chickens died within 4 days of infection. Surviving birds showing mostly mild clinical signs and recovered by day 10 postinfection. Prior to death, chickens shed the virus primarily through the respiratory route, with lower amounts shed from the cloaca, but in the survivors, the virus was still being shed from the cloaca on day 10. Fomites were the likely source of disease spread between farms, and the amount of H7N6 HPAI virus per gram of feces was calculated at ~383,193 (5.58 log10) egg infectious dose 50 (EID50) equivalents, chicken feather follicles contained on average 739,712.43 (5.87 log10) EID50 equivalents, and 20 µg of feather dust contained 14,976.96 (4.175 log10) EID50 equivalents.

Evaluation of loop-mediated isothermal amplification method for efficient detection of the periodontopathic bacteria Porphyromonas gingivalis.

Periodontitis is a multifactorial, polymicrobial oral inflammatory illness brought on by oral pathogens. Porphyromonas gingivalis is a Gram-negative, obligatory anaerobic, black-pigmented coccobacillus and is regarded as a primary etiological factor in the progression of periodontitis. Rapid, highly senstitive and specific detection methods are emerging. The present study aimed to evaluate the loop-mediated isothermal amplification (LAMP) technique for efficiently detecting P. gingivalis from subgingival plaque samples of chronic periodontitis patients. This study included 50 subgingival plaque samples from patients suffering from chronic periodontitis. The DNA (Deoxyribonucleic acid) was extracted by the "modified proteinase K" method. A set of six primers, targeting the pepO gene of P. gingivalis, was used for conducting LAMP. The amplification was visualized by naked-eye detection and agarose electrophoresis. Conventional polymerase chain reaction (PCR) and real-time qantitative PCR (qPCR) were carried out by targeting the 16SrRNA (16S ribosomal ribonucleic acid) gene of P. gingivalis. The results showed that LAMP detected P. gingivalis in 40 out of 50 samples (80%). Whereas, qPCR and conventional PCR technique detected P. gingivalis in 38 (76%) and 33 (66%) samples respectively. The sensitivity and specificity of the LAMP method were 94.87% and 90.90%, respectively. With qPCR, the sensitivity and specificity were found to be 92.30% and 81.81%, respectively, whereas, with conventional PCR, it was found to be 76.92% and 72.72%, respectively. LAMP is an efficient technique for quick, accurate, and reliable identification of P. gingivalis from subgingival plaque samples. The technique needs to be validated analytically, and further studies can be conducted by taking saliva and/or gingival crevicular fluid samples from periodontitis patients.