A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

Single-cell profiling of Anopheles gambiae spermatogenesis defines the onset of meiotic silencing and premeiotic overexpression of the X chromosome.

Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell-types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes.

Microbial genome (Illumina MiSeq) sequencing of drinking water treatment residuals to evaluate compatibility with environmental applications.

The clarification of drinking water leads to the production of large quantities of water treatment residuals (WTRs). DNA was extracted from six WTR samples collected from water treatment plants within the UK to compare their bacterial communities and examine whether factors such as coagulant usage (aluminium versus iron salt), the type of water source (reservoir or river), or leachable chemical composition influence these communities. Bacterial 16S variable region 4 (V4) was amplified and sequenced using Illumina MiSeq sequencing. The most abundant phyla in WTR samples were Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes, collectively representing 92.77-97.8% of the total bacterial sequences. Statistical analysis of microbial profiles indicated that water source played a significant role in microbial community structure, diversity, and richness, however coagulant type did not. PERMANOVA analysis showed that no single chemical variable (pH, organic matter, or extractable element concentration) influenced microbial composition significantly; however, canonical correspondence analysis of WTR microbiomes yielded a model using all these variables that could be used to explain variations in microbial community structures of WTRs (p < 0.05). No common, potentially toxic cyanobacteria, or related pathogens of concern were found. Analysis with PICRUSt showed that WTRs all had similar predicted microbial functional profiles. Overall, the results indicate that WTRs analysed in this study are unlikely to pose any threat to soil microbial community structure when applied to land as a soil conditioner or enhancer and may help to enhance the soil microbial community.

Extracellular vesicles derived from umbilical cord mesenchymal stromal cells show enhanced anti-inflammatory properties via upregulation of miRNAs after pro-inflammatory priming.

Autoimmune conditions, such as rheumatoid arthritis, are characterised by a loss of immune tolerance, whereby the immune cells attack self-antigens causing pain and inflammation. These conditions can be brought into remission using pharmaceutical treatments, but often have adverse side effects and some patients do not respond favourably to them. Human umbilical cord mesenchymal stromal cells (UCMSCs) present a promising alternative therapeutic due to their innate anti-inflammatory properties which can be strengthened using pro-inflammatory conditions. Their therapeutic mechanism of action has been attributed to paracrine signalling, by which nanosized acellular particles called 'extracellular vesicles' (EVs) are one of the essential components. Therefore, this research analysed the anti-inflammatory properties of UCMSC-EVs 'primed' with pro-inflammatory cytokines and at baseline with no inflammatory cytokines (control). Both control and primed EVs were co-cultured with un-pooled peripheral blood mononuclear cells (PBMCs; n = 6) from healthy donors. Neither control nor primed EVs exerted a pro-inflammatory effect on PBMCs. Instead, the primed EVs showed the immunosuppressive potential by increasing the expression of the anti-inflammatory protein FoxP3 in PBMCs. This may be attributed to the upregulated miRNAs identified in primed EVs in comparison to control EVs (miR-139-5p, miR-140-5p, miR-214-5p). These findings aid in understanding how UCMSC-EVs mediate immunosuppression and support their potential use in treating autoimmune conditions.

Platelets bridging the gap between gut dysbiosis and neuroinflammation in stress-linked disorders: A narrative review.

In this narrative review, we examine the association between gut dysbiosis, neuroinflammation, and stress-linked disorders, including depression, anxiety, and post-traumatic stress disorder (PTSD), and investigate whether tryptophan (TRP) metabolism and platelets play a role in this association. The mechanisms underlying the aetiology of stress-linked disorders are complex and not yet completely understood. However, a potential link between chronic inflammation and these disorders may potentially be found in TRP metabolism and platelets. By critically analysing existing literature on platelets, the gut microbiome, and stress-linked disorders, we hope to elicit the role of platelets in mediating the effects on serotonin (5-HT) levels and neuroinflammation. We have included studies specifically investigating platelets and TRP metabolism in relation to inflammation, neuroinflammation and neuropsychiatric disorders. Alteration in microbial composition due to stress could contribute to increased intestinal permeability, facilitating the translocation of microbial products, and triggering the release of pro-inflammatory cytokines. This causes platelets to become hyperactive and secrete 5-HT into the plasma. Increased levels of pro-inflammatory cytokines may also lead to increased permeability of the blood-brain barrier (BBB), allowing inflammatory mediators entry into the brain, affecting the balance of TRP metabolism products, such as 5-HT, kynurenic acid (KYNA), and quinolinic acid (QUIN). These alterations may contribute to neuroinflammation and possible neurological damage. Furthermore, platelets can cross the compromised BBB and interact with astrocytes and neurons, leading to the secretion of 5-HT and pro-inflammatory factors, exacerbating inflammatory conditions in the brain. The mechanisms underlying neuroinflammation resulting from peripheral inflammation are still unclear, but the connection between the brain and gut through the bloodstream could be significant. Identifying peripheral biomarkers and mechanisms in the plasma that reflect neuroinflammation may be important. This review serves as a foundation for further research on the association between the gut microbiome, blood microbiome, and neuropsychiatric disorders. The integration of these findings with protein and metabolite markers in the blood may expand our understanding of the subject.

The 2022 dengue outbreak in Bangladesh: hypotheses for the late resurgence of cases and fatalities.

Bangladesh reported the highest number of annual deaths (n = 281) related to dengue virus infection in 2022 since the virus reappeared in the country in 2000. Earlier studies showed that >92% of the annual cases occurred between the months of August and September. The 2022 outbreak is characterized by late onset of dengue cases with unusually higher deaths in colder months, that is, October-December. Here we present possible hypotheses and explanations for this late resurgence of dengue cases. First, in 2022, the rainfall started late in the season. Compared to the monthly average rainfall for September and October between 2003 and 2021, there was 137 mm of additional monthly rainfall recorded in September and October 2022. Furthermore, the year 2022 was relatively warmer with a 0.71°C increased temperature than the mean annual temperature of the past 20 yr. Second, a new dengue virus serotype, DENV-4, had recently reintroduced/reappeared in 2022 and become the dominant serotype in the country for a large naïve population. Third, the post-pandemic return of normalcy after 2 yr of nonpharmaceutical social measures facilitates extra mosquito breeding habitats, especially in construction sites. Community engagement and regular monitoring and destruction of Aedes mosquitoes' habitats should be prioritized to control dengue virus outbreaks in Bangladesh.

New insights into the functional role of protein phosphatase 4 regulatory subunit PP4R3A/SMEK1 in the regulation of leukemic cell fate.

The serine/threonine protein phosphatase 4 holoenzyme consists of a PP4 catalytic subunit (PP4c), which interacts with four different regulatory subunits. Previous studies have shown that PP4c acts as a tumour suppressor. Emerging evidence suggests that the protein phosphatase 4 regulatory subunits might regulate cell fate independently of PP4c. To this end, we investigated the role of PP4R3A (SMEK1) in Jurkat and CEM-C7 leukemic cell lines. SMEK1 overexpression decreased cell growth, increased spontaneous apoptosis, and reduced the colony forming ability of leukemic cells. Conversely, siRNA-mediated silencing of SMEK1 led to increased short and long-term survival in these cells. Phospho-protein arrays revealed that increased expression of SMEK1 affected the phosphorylation of key proteins involved in MAPK3, AKT, JAK/STAT, NFκB and TGFß signalling pathways. These proteins include transcription factors such as NFκB, STAT3, c-JUN, SMAD1, and SMAD5, suggesting a role for SMEK1 in the regulation of gene expression. RNA sequencing confirmed the role of SMEK1 in the regulation of gene expression. RNA sequencing also confirmed the tumour suppressor role of SMEK1. Taken together, this study shows that SMEK1 regulates leukemic T cell survival, indicating that SMEK1 dysfunction may be important in the development and progression of leukemia.

Molecular characterisation of the synovial fluid microbiome in rheumatoid arthritis patients and healthy control subjects.

METHODS: The presence and identity of bacterial and fungal DNA in the synovial fluid of rheumatoid arthritis (RA) patients and healthy control subjects was investigated through amplification and sequencing of the bacterial 16S rRNA gene and fungal internal transcribed spacer region 2 respectively. Synovial fluid concentrations of the cytokines IL-6, IL-17A, IL22 and IL-23 were determined by ELISA. RESULTS: Bacterial 16S rRNA genes were detected in 87.5% RA patients, and all healthy control subjects. At the phylum level, the microbiome was predominated by Proteobacteria (Control = 83.5%, RA = 79.3%) and Firmicutes (Control = 16.1%, RA = 20.3%), and to a much lesser extent, Actinobacteria (Control = 0.2%, RA = 0.3%) and Bacteroidetes (Control = 0.1%, RA = 0.1%). Fungal DNA was identified in 75% RA samples, and 88.8% healthy controls. At the phylum level, synovial fluid was predominated by members of the Basidiomycota (Control = 53.9%, RA = 46.9%) and Ascomycota (Control = 35.1%, RA = 50.8%) phyla. Statistical analysis revealed key taxa that were differentially present or abundant dependent on disease status. CONCLUSIONS: This study reports the presence of a synovial fluid microbiome, and determines that this is modulated by disease status (RA) as are other classical microbiome niches.

RNA sequencing reveals a key role for the long non-coding RNA MIAT in regulating neuroblastoma and glioblastoma cell fate.

Myocardial Infarction Associated Transcript (MIAT) is a subnuclear lncRNA that interferes with alternative splicing and is associated with increased risk of various heart conditions and nervous system tumours. The current study aims to elucidate the role of MIAT in cell survival, apoptosis and migration in neuroblastoma and glioblastoma multiforme. To this end, MIAT was silenced by MIAT-specific siRNAs in neuroblastoma and glioblastoma cell lines, and RNA sequencing together with a series of functional assays were performed. The RNA sequencing has revealed that the expression of an outstanding number of genes is altered, including genes involved in cancer-related processes, such as cell growth and survival, apoptosis, reactive oxygen species (ROS) production and migration. Furthermore, the functional studies have confirmed the RNA sequencing leads, with our key findings suggesting that MIAT knockdown eliminates long-term survival and migration and increases basal apoptosis in neuroblastoma and glioblastoma cell lines. Taken together with the recent demonstration of the involvement of MIAT in glioblastoma, our observations suggest that MIAT could possess tumour-promoting properties, thereby acting as an oncogene, and has the potential to be used as a reliable biomarker for neuroblastoma and glioblastoma and be employed for prognostic, predictive and, potentially, therapeutic purposes for these cancers.

Molecular Characterization of Circulating Microbiome Signatures in Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) has been increasingly associated with perturbations to the microbial communities that reside in and on the body (the microbiome), in both human and animal studies. To date, such studies have mainly focused on the microbial communities that inhabit the gut and oral cavity. Mounting evidence suggests that microbial DNA can be detected in the blood circulation using a range of molecular methods. This DNA may represent an untapped pool of biomarkers that have the potential to report on changes to the microbiome of distant sites (e.g., example, the gut and oral cavity). To this end, through amplification and sequencing of the bacterial 16S rRNA variable region four, we evaluated the presence and identity of microbial DNA in blood samples obtained from RA patients (both prior to and 3 months following the instigation of treatment) in comparison to a small number of healthy control subjects and samples obtained from patients with ankylosing spondylitis (AS) and psoriatic arthritis (PA). Bacterial-derived DNA was identified in the majority of our patient samples. Taxonomic classification revealed that the microbiome community in RA was distinct from AS, PA, and the healthy state. Through analysis of paired patient samples obtained prior to and 3 months following treatment (V0 vs. V3), we found the microbiome to be modulated by treatment, and in many cases, this shift reduced the distance between these samples and the healthy control samples, suggesting a partial normalization following treatment in some patients. This effect was especially evident in seronegative arthritis patients. Herein, we provide further evidence for the existence of a blood microbiome in health and identify specific taxa modulated in disease and following treatment. These blood-derived signatures may have significant utility as disease biomarkers and suggest this area warrants further investigation.

Multi-Method Characterization of the Human Circulating Microbiome.

The term microbiome describes the genetic material encoding the various microbial populations that inhabit our body. Whilst colonization of various body niches (e.g., the gut) by dynamic communities of microorganisms is now universally accepted, the existence of microbial populations in other "classically sterile" locations, including the blood, is a relatively new concept. The presence of bacteria-specific DNA in the blood has been reported in the literature for some time, yet the true origin of this is still the subject of much deliberation. The aim of this study was to investigate the phenomenon of a "blood microbiome" by providing a comprehensive description of bacterially derived nucleic acids using a range of complementary molecular and classical microbiological techniques. For this purpose we utilized a set of plasma samples from healthy subjects (n = 5) and asthmatic subjects (n = 5). DNA-level analyses involved the amplification and sequencing of the 16S rRNA gene. RNA-level analyses were based upon the de novo assembly of unmapped mRNA reads and subsequent taxonomic identification. Molecular studies were complemented by viability data from classical aerobic and anaerobic microbial culture experiments. At the phylum level, the blood microbiome was predominated by Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The key phyla detected were consistent irrespective of molecular method (DNA vs. RNA), and consistent with the results of other published studies. In silico comparison of our data with that of the Human Microbiome Project revealed that members of the blood microbiome were most likely to have originated from the oral or skin communities. To our surprise, aerobic and anaerobic cultures were positive in eight of out the ten donor samples investigated, and we reflect upon their source. Our data provide further evidence of a core blood microbiome, and provide insight into the potential source of the bacterial DNA/RNA detected in the blood. Further, data reveal the importance of robust experimental procedures, and identify areas for future consideration.

Amplicon-based metagenomic analysis of mixed fungal samples using proton release amplicon sequencing.

Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific "conserved" primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.

The role of microRNAs in the pathogenesis of MMPi-induced skin fibrodysplasia.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes involved in extracellular matrix (ECM) homeostasis. MMPs have been an attractive pharmacological target for a number of indications. However, development has been hampered by the propensity of compounds targeting these enzymes to cause connective-tissue pathologies. The broad-spectrum MMP-inhibitor (MMPi) AZM551248 has been shown to induce such effects in the dog. Histopathological changes were consistent with fibrodysplasia (FD), characterised by fibroblast proliferation and the deposition of collagen in the subcutaneous tissues. We conducted a time-course study administering 20mg/kg/day AZM551248 between 4 and 17 days. Cervical subcutaneous tissue and plasma were sampled during the time-course. miRNA expression profiles in subcutaneous skin specimens following the administration of AZM551248 were determined by high-throughput-sequencing. RESULTS: An increasing number of miRNAs were differentially expressed compared with vehicle treated control animals as the study progressed. Several of these were members of the miR-200 family and were significantly attenuated in response to MMPi. As the severity of FD increased at the later time-points, other miRNAs associated with TGFß synthesis and regulation of the acute inflammatory response were modulated. Evidence indicative of epithelial to mesenchymal transition was present at all study time points. Receiver operator curve (ROC) analysis revealed that miR-21 expression in the cervical subcutaneous tissue was a sensitive and specific biomarker of FD incidence. CONCLUSIONS: Our data reveal significant perturbations in canine skin miRNA expression in response to MMPi administration. Furthermore, we have identified dysregulated miRNAs that are associated with processes relevant to the key histopathological events of MMPi-induced FD.

Evidence of changes to skeletal muscle contractile properties during the initiation of disease in the ageing guinea pig model of osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is the most common joint disorder in the world and represents the leading cause of pain and disability in the elderly population. Advancing age remains the single greatest risk factor for OA. Several studies have characterised disease development in the guinea pig ageing model of OA in terms of its joint histopathology and inflammatory cytokine profile. However, the quadriceps muscle has yet to be studied in relation to age-related disease onset or early disease progression. Therefore, we examined whether the initiation of OA in the Dunkin Hartley guinea pig is associated with changes in the quadriceps skeletal muscle. Male Dunkin Hartley guinea pigs (N = 24) were group housed with free access to standard guinea pig chow and water. At 2, 3, 5 and 7 months of age, six animals were selected based on their proximity to the median weight of the cohort. OA severity was graded at each time point by the assessment of toluidine blue stained step coronal sections of the total knee joint. Serum CTX II was measured as a potential biomarker of OA severity. Myosin Heavy Chain (MHC) isoforms were determined by a validated real-time PCR assay. Oxidative and glycolytic potential was determined in quadriceps homogenates via the measurement of ICDH and LDH activity. RESULTS: Initiation of OA in the DH strain guinea pig occurred between 2 and 3 months of age and progressed until 7 months when the final analyses were conducted. Serum CTX II significantly decreased during this early period of OA initiation and levels were unrelated to the histopathological severity of knee OA at any of the time points assessed. MHC mRNA measurements revealed a significant elevation in MHC IIX mRNA (associated with fast-twitch skeletal muscle fibres) coincident with the initiation of OA at 3 months of age, with preliminary findings suggestive of a positive correlation to OA severity at this time point. CONCLUSIONS: These preliminary findings suggest that disease initiation in the ageing guinea pig model of OA is not associated with overt quadriceps muscle atrophy but instead is coincident with altered expression of mRNAs associated with quadriceps skeletal muscle contractile properties (specifically fast-twitch MHC IIX).

Characterisation of the sarcomeric myosin heavy chain multigene family in the laboratory guinea pig.

BACKGROUND: Several chronic conditions leading to skeletal muscle dysfunction are known to be associated with changes in the expression of myosin heavy chain (MHC) isoforms at both the mRNA and protein level. Many of these conditions are modelled, pre-clinically, in the guinea pig due to similar disease onset and progression to the human condition, and their generally well-characterised anatomy. MHC composition is amenable to determination by protein and mRNA based methodologies, the latter quantifying the expression of MHC isoform-specific gene transcripts allowing the detection of earlier, and more subtle changes. As such, the MHC mRNAs, and specific oligonucleotide primers of all common laboratory species have been available for some time. However, due to incomplete genomic annotation, assessment of guinea pig MHC mRNA expression has not been previously possible, precluding the full characterisation of early changes in skeletal muscle in response to disease and disease modulation.The purpose of this study was to characterise the multigenic structure of the sarcomeric MHC family in the guinea pig, and to design and validate specific oligonucleotide primers to enable the assessment of the predominant adult-muscle associated MHC mRNAs in relevant disease models. RESULTS: Using a combination of ligase-mediated rapid amplification of 5' and 3' cDNA ends (RACE) and bioinformatics, mRNAs to the four main skeletal-muscle isoforms of MHC were determined. Specific oligonucleotide primers were designed, and following verification of their specificity, found to successfully determine the expression of each MHC mRNA independently. CONCLUSIONS: Because of their utilisation in the in vivo modelling of disease, there is a requirement to develop molecular methods that accurately differentiate the different MHC mRNAs in the guinea pig to enable rapid profiling of muscle composition in appropriate disease models. The methods developed here are suitable for the characterisation of muscle MHC expression at the molecular level from animal tissue samples and biopsy material. The publication of these specific oligonucleotide primers for the guinea pig MHC variants will enable researchers to rapidly and accurately quantify acute changes in MHC mRNA expression in either developmental or in guinea pig disease models where a marker of altered skeletal muscle function is required.