An anionic ligand snap-locks a long-range interaction in a magnesium-folded riboswitch

The archetypical transcriptional crcB fluoride riboswitch from Bacillus cereus is an intricately structured non-coding RNA element enhancing gene expression in response to toxic levels of fluoride. Here, we used single molecule FRET to uncover three dynamically interconverting conformations appearing along the transcription process: two distinct undocked states and one pseudoknotted docked state. We find that the fluoride anion specifically snap-locks the magnesium-induced, dynamically docked state. The long-range, nesting, single base pair A40-U48 acts as the main linchpin, rather than the multiple base pairs comprising the pseudoknot. We observe that the proximally paused RNA polymerase further fine-tunes the free energy to promote riboswitch docking. Finally, we show that fluoride binding at short transcript lengths is an early step toward partitioning folding into the docked conformation. These results reveal how the anionic fluoride ion cooperates with the magnesium-associated RNA to govern regulation of downstream genes needed for fluoride detoxification of the cell.

Targeting Mechano-Transcription Process as Therapeutic Intervention in Gastrointestinal Disorders

Mechano-transcription is a process whereby mechanical stress alters gene expression. The gastrointestinal (GI) tract is composed of a series of hollow organs, often encountered by transient or persistent mechanical stress. Recent studies have revealed that persistent mechanical stress is present in obstructive, functional, and inflammatory disorders and alters gene transcription in these conditions. Mechano-transcription of inflammatory molecules, pain mediators, pro-fibrotic and growth factors has been shown to play a key role in the development of motility dysfunction, visceral hypersensitivity, inflammation, and fibrosis in the gut. In particular, mechanical stress-induced cyclooxygenase-2 (COX-2) and certain pro-inflammatory mediators in gut smooth muscle cells are responsible for motility dysfunction and inflammatory process. Mechano-transcription of pain mediators such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) may lead to visceral hypersensitivity. Emerging evidence suggests that mechanical stress in the gut also leads to up-regulation of certain proliferative and pro-fibrotic mediators such as connective tissue growth factor (CTGF) and osteopontin (OPN), which may contribute to fibrostenotic Crohn’s disease. In this review, we will discuss the pathophysiological significance of mechanical stress-induced expression of pro-inflammatory molecules, pain mediators, pro-fibrotic and growth factors in obstructive, inflammatory, and functional bowel disorders. We will also evaluate potential therapeutic targets of mechano-transcription process for the management of these disorders.

Nanopore-Based Direct RNA-Sequencing Reveals a High-Resolution Transcriptional Landscape of Porcine Reproductive and Respiratory Syndrome Virus

The TRS-mediated discontinuous transcription process is a hallmark of Arteriviruses. Precise assessment of the intricate subgenomic RNA (sg mRNA) populations is required to understand the kinetics of viral transcription. It is difficult to reconstruct and comprehensively quantify splicing events using short-read sequencing, making the identification of transcription-regulatory sequences (TRS) particularly problematic. Here, we applied long-read direct RNA sequencing to characterize the recombined RNA molecules produced in porcine alveolar macrophages during early passage infection of porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequencing two PRRSV isolates, namely XM-2020 and GD, we revealed a high-resolution and diverse transcriptional landscape in PRRSV. The data revealed intriguing differences in subgenomic recombination types between the two PRRSVs while also demonstrating TRS-independent heterogeneous subpopulation not previously observed in Arteriviruses. We find that TRS usage is a regulated process and share the common preferred TRS in both strains. This study also identified a substantial number of TRS-mediated transcript variants, including alternative-sg mRNAs encoding the same annotated ORF, as well as putative sg mRNAs encoded nested internal ORFs, implying that the genetic information encoded in PRRSV may be more intensively expressed. Epigenetic modifications have emerged as an essential regulatory layer in gene expression. Here, we gained a deeper understanding of m5C modification in poly(A) RNA, elucidating a potential link between methylation and transcriptional regulation. Collectively, our findings provided meaningful insights for redefining the transcriptome complexity of PRRSV. This will assist in filling the research gaps and developing strategies for better control of the PRRS.

Mathematical Modelling of Gene Regulatory Networks

This research includes the use of an artificial intelligence algorithm, which is one of the algorithms of biological systems which is the algorithm of genetic regulatory networks (GRNs), which is a dynamic system for a group of variables representing space within time. To construct this biological system, we use (ODEs) and to analyze the stationarity of the model we use Euler's method. And through the factors that affect the process of gene expression in terms of inhibition and activation of the transcription process on DNA, we will use TF transcription factors. The current research aims to use the latest methods of the artificial intelligence algorithm. To apply Gene Regulation Networks (GRNs), we used a program (MATLAB2020), which provides facilitation to the most important biological concepts for building this biological interaction

Intrapreneurship and technological innovation in optimizing qualitative research as evidenced at Infectious Diseases Institute, Uganda

Background Discrepancies between what is transcribed and the actual interview recordings were noticed in qualitative research reports. This study aimed at the development of a new transcription software (Jiegnote), and the evaluation of its effectiveness in the optimization of the transcription process, to minimize transcription completion time, and errors in qualitative research. Methods The study was a mixed methods project implemented from September to November 2020. The qualitative aspect of the study was phenomenological in perspective whereas the quantitative consisted of a randomized controlled trial (RCT) with a parallel design. Results At the time of the study, the Jiegnote software was a working prototype. We enrolled a total of 26 participants; 14 participants had their data analyzed in the RCT part of the study, 13 participated in the in-depth interviews, and 22 in the answering of Semi Structured Questionnaires. Upon the execution of an independent t test, results showed that, there was no statistical significance between the intervention and control means. On considering the total average transcription completion time and the type of language in which an audio case was recorded, the effect size evaluation implied that the Jiegnote software had a small impact (Hedges' g = 0.413438) in reducing the total average time taken to translate and transcribe audio cases that were recorded in a local language (Luganda), and a large impact (Hedges' g = 1.190919) in reducing the total average time taken to transcribe audio cases that were recorded in a foreign language (English). On considering the total average number of transcription errors and the type of language in which an audio case is recorded, the effect size evaluation implied that the Jiegnote software had a small impact (Hedges' g = 0.213258) in reducing the total average time taken to translate and transcribe audio cases that were recorded in a local language (Luganda). This was further observed (Hedges' g = 0.039928) in the transcription of cases that were recorded in a foreign language (English). On considering the in-depth interview data outcomes, participants responded that the Jiegnote software media looping functions (algorithm) enabled them to accomplish their transcription tasks in a shorter time and with fewer errors compared to the traditional methods. Conclusion The study demonstrates utilities associated with intrapreneurship and technological innovation in an organization setting whereby, the Jiegnote technology that was developed by the researchers, had some impact on the optimization of the qualitative research value chain. This was observed through the effect size (impact) evaluations that were conducted to investigate the superiority of the Jiegnote software against the traditional transcription methods, in minimizing the average number of errors committed, and time taken to complete a transcription process.

A Qualitative Study on Secondary School Teacher’s Perceptions of Stunting in Majene District, West Sulawesi Province

Background: Stunting remains a major public health problem in Majene, Indonesia. School-based nutrition education is an effective strategy to reduce the prevalence of stunting in all settings. The teachers are the key to implementing the strategy in order to improve the students’ behavior and nutritional status.Purpose: The study aimed to explore the teachers’ perceptions about stunting.Method: A qualitative case-study approach was employed using two focus group discussions in four secondary schools. The study participants were teachers of biology, physical education and health science, and religious subjects or supervisor of school extracurricular activities. A total of 22 teachers were interviewed following the guidelines and were recorded using a camera and tape recorder. The transcription process was done using an inductive-interactive model.Result: The perceptions of teachers about stunting are varied. It might be due to teachers’ knowledge, value, and experiences, which also diverged. One teacher perceived that stunting is a mismatch between the child's weight and age, while another perceived genetics as the main cause. Interestingly, a teacher perceived that stunting is related to religion. Prayer has function as a spirit in creating a mindset towards food and drinks. Likewise, the prevention of stunting is also through worship and prayer during pregnancy. Low cognitive skill and productivity, illness, and detrimental to the state are the common impacts of stunting. In addition, teachers also mentioned that there are several agencies involved in the stunting intervention programs, including BPOM.Conclusion: There were variations of teacher perceptions about stunting definition, causes, impacts, prevention, and implementers of stunting programs.

Tracking the Transcription Kinetic of SARS-CoV-2 in Human Cells by Reverse Transcription-Droplet Digital PCR

Viral transcription is an essential step of SARS-CoV-2 infection after invasion into the target cells. Antiviral drugs such as remdesivir, which is used to treat COVID-19 patients, targets the viral RNA synthesis. Understanding the mechanism of viral transcription may help to develop new therapeutic treatment by perturbing virus replication. In this study, we established 28 ddPCR assays and designed specific primers/probe sets to detect the RNA levels of 15 NSP, 9 ORF, and 4 structural genes of SARS-CoV-2. The transcriptional kinetics of these viral genes were determined longitudinally from the beginning of infection to 12 hours postinfection in Caco-2 cells. We found that SARS-CoV-2 takes around 6 hours to hijack the cells before the initiation of viral transcription process in human cells. Our results may contribute to a deeper understanding of the mechanisms of SARS-CoV-2 infection.

Two promoters integrate multiple enhancer inputs to drive wild-type knirps expression in the D. melanogaster embryo

Proper development depends on precise spatiotemporal gene expression patterns. Most developmental genes are regulated by multiple enhancers and often by multiple core promoters that generate similar transcripts. We hypothesize that multiple promoters may be required either because enhancers prefer a specific promoter or because multiple promoters serve as a redundancy mechanism. To test these hypotheses, we studied the expression of the knirps locus in the early Drosophila melanogaster embryo, which is mediated by multiple enhancers and core promoters. We found that one of these promoters resembles a typical “sharp” developmental promoter, while the other resembles a “broad” promoter usually associated with housekeeping genes. Using synthetic reporter constructs, we found that some, but not all, enhancers in the locus show a preference for one promoter, indicating that promoters provide both redundancy and specificity. By analyzing the reporter dynamics, we identified specific burst properties during the transcription process, namely burst size and frequency, that are most strongly tuned by the combination of promoter and enhancer. Using locus-sized reporters, we discovered that enhancers with no promoter preference in a synthetic setting have a preference in the locus context. Our results suggest that the presence of multiple promoters in a locus is due both to enhancer preference and a need for redundancy and that “broad” promoters with dispersed transcription start sites are common among developmental genes. They also imply that it can be difficult to extrapolate expression measurements from synthetic reporters to the locus context, where other variables shape a gene’s overall expression pattern.

An overview of genes and mutations associated with Chlamydiae species’ resistance to antibiotics

Background Chlamydiae are intracellular bacteria that cause various severe diseases in humans and animals. The common treatment for chlamydia infections are antibiotics. However, when antibiotics are misused (overuse or self-medication), this may lead to resistance of a number of chlamydia species, causing a real public health problem worldwide. Materials and methods In the present work, a comprehensive literature search was conducted in the following databases: PubMed, Google Scholar, Cochrane Library, Science direct and Web of Science. The primary purpose is to analyse a set of data describing the genes and mutations involved in Chlamydiae resistance to antibiotic mechanisms. In addition, we proceeded to a filtration process among 704 retrieved articles, then finished by focusing on 24 studies to extract data that met our requirements. Results The present study revealed that Chlamydia trachomatis may develop resistance to macrolides via mutations in the 23S rRNA, rplD, rplV genes, to rifamycins via mutations in the rpoB gene, to fluoroquinolones via mutations in the gyrA, parC and ygeD genes, to tetracyclines via mutations in the rpoB gene, to fosfomycin via mutations in the murA gene, to MDQA via mutations in the secY gene. Whereas, Chlamydia pneumoniae may develop resistance to rifamycins via mutations in the rpoB gene, to fluoroquinolones via mutations in the gyrA gene. Furthermore, the extracted data revealed that Chlamydia psittaci may develop resistance to aminoglycosides via mutations in the 16S rRNA and rpoB genes, to macrolides via mutations in the 23S rRNA gene. Moreover, Chlamydia suis can become resistance to tetracyclines via mutations in the tet(C) gene. In addition, Chlamydia caviae may develop resistance to macrolides via variations in the 23S rRNA gene. The associated mechanisms of resistance are generally: the inhibition of bacteria’s protein synthesis, the inhibition of bacterial enzymes’ action and the inhibition of bacterial transcription process. Conclusion This literature review revealed the existence of diverse mutations associated with resistance to antibiotics using molecular tools and targeting chlamydia species’ genes. Furthermore, these mutations were shown to be associated with different mechanisms that led to resistance. In that regards, more mutations and information can be shown by a deep investigation using the whole genome sequencing. Certainly, this can help improving to handle chlamydia infections and healthcare improvement by decreasing diseases complications and medical costs.

SARS-CoV-2 spike protein-induced platelet activation: Mechanism for virus and vaccine-induced thrombotic thrombocytopenia

Covid-19 pandemic stimulated an extremely fast development of effective vaccines. Recent studies found platelet-activating antibodies against platelet factor 4 (PF4) in both clinically ill Covid-19 patients and vaccine-induced thrombotic thrombocytopenia (VITT) patients. Here, we use various tools to identify the binding reaction of the SARS-CoV-2 spike glycoprotein (SP) with PF4 that results in immunogenic platelet-activating PF4/SP complexes. This binding is evidenced by an increase in mass, optical intensity, and stable binding force observed by quartz crystal microbalance, enzyme immune assay, and force spectroscopy, respectively. The SP induced an increase in the size of PF4 and switched the surface zeta potential of the PF4 from positive to negative values as evaluated by dynamic light scattering. The SP-induced platelet aggregation was identified by functional assay and flow cytometry but in a concentration-dependent manner. Our results indicated that the formed PF4/SP complexes can, on one hand, trigger the formation of PF4-antibodies and on the other hand mediate/activate platelets followed by inducing thrombotic events, which is the mechanism for excessive procoagulant activity observed in Covid-19 patients. With vector-based vaccines, we suggest that soluble SP are produced during the transcription process, forming antigenic PF4/SP complexes that result in a high rate of clotting effects in vaccinated individuals with Ad26.COV2.S and ChAdOx1nCoV-19 vaccines. An additional consideration of PF4/SP complexes in the current guidelines for the diagnosis of VITT will improve the treatment in patients. Our results serve a high demand to develop an effective method to treat Covid-19 patients and improve the safety for Covid-19 vaccination.