13S mRNA Research Articles

Edible Plant-Derived Extracellular Vesicles for Oral mRNA Vaccine Delivery.

Nucleic acid delivery through extracellular vesicles (EVs) is a well-preserved evolutionary mechanism in all life kingdoms including eukaryotes, prokaryotes, and plants. EVs naturally allow horizontal transfer of native as well as exogenous functional mRNAs, which once incorporated in EVs are protected from enzymatic degradation. This observation has prompted researchers to investigate whether EVs from different sources, including plants, could be used for vaccine delivery. Several studies using human or bacterial EVs expressing mRNA or recombinant SARS-CoV-2 proteins showed induction of a humoral and cell mediated immune response. Moreover, EV-based vaccines presenting the natural configuration of viral antigens have demonstrated advantages in conferring long-lasting immunization and lower toxicity than synthetic nanoparticles. Edible plant-derived EVs were shown to be an alternative to human EVs for vaccine delivery, especially via oral administration. EVs obtained from orange juice (oEVs) loaded with SARS-CoV-2 mRNAs protected their cargo from enzymatic degradation, were stable at room temperature for one year, and were able to trigger a SARS-CoV-2 immune response in mice. Lyophilized oEVs containing the S1 mRNA administered to rats via gavage induced a specific humoral immune response with generation of blocking antibodies, including IgA and Th1 lymphocyte activation. In conclusion, mRNA-containing oEVs could be used for developing new oral vaccines due to optimal mucosal absorption, resistance to stress conditions, and ability to stimulate a humoral and cellular immune response.

Acute effects of the environmental probiotics Rhodobacter sphaeroides on intestinal bacteria and transcriptome in shrimp Penaeusvannamei

In recent years, a substantial number of studies have been dedicated to exploring the potential benefits of probiotics in aquaculture. Rhodobacter sphaeroides can be used in aquaculture-related environmental bioremediation, and its protein is also used as a feed additive in Penaeus vannamei culture. To investigate the effects of releasing R. sphaeroides as environmental probiotics on P. vannamei, we employed 16S rRNA gene and mRNA transcriptome sequencing. Our study focused on assessing alterations in intestinal bacteria and intestinal gene expression in P. vannamei, establishing correlations between them. Our findings revealed a significant increase in the relative abundances of Rhodobacter, Paracoccus, Sulfitobacter, and other bacterial OTUs within the intestinal bacterial community. Additionally, we observed enhanced complexity and stability in the intestinal bacterial correlation network, indicating improved synergy among bacteria and reduced competition. Moreover, the introduction of R. sphaeroides resulted in the down-regulation of certain immune genes and the up-regulation of genes linked to growth and metabolism in the intestinal tissues of P. vannamei. Importantly, we identified a noteworthy correlation between the changes in intestinal bacteria and these alterations in intestinal tissue gene expressions. By conducting analyses of the intestinal bacterial community and intestinal tissue transcriptome, this study revealed the effects of releasing R. sphaeroides as sediment probiotics in P. vannamei culture water. These results serve as vital scientific references for the application of R. sphaeroides in P. vannamei aquaculture.

The co-regulation of the gut microbiome and host genes might play essential roles in metformin gastrointestinal intolerance

Metformin is commonly used, but approximately 20% of patients experience gastrointestinal intolerance, leading to medication discontinuation for unclear reasons and a lack of effective management strategies.In this study, the 18 fecal and blood samples were analyzed using 16S rRNA and mRNA transcriptome, respectively. These samples included 3 fecal and 4 blood from metformin-tolerant T2D patients before and after metformin treatment (T and Ta), 3 fecal and 5 blood from metformin-intolerant T2D patients before and after treatment (TS and TSa), and 6 fecal samples from healthy controls.The results showed that certain anti-inflammatory gut bacteria and gene, such as Barnesiella (p = 0.046), Parabacteroides goldsteinii (p = 0.016), and the gene JUND (p = 0.0002), exhibited higher levels in metformin-intolerant patients, and which decreased after metformin treatment (p < 0.05). This potentially invalidates patients' anti-inflammatory effect and intestinal mucus barrier protection, which may lead to alterations in intestinal permeability, decreased gut barrier function, and gastrointestinal symptoms, including diarrhea, bloating, and nausea. After metformin treatment, primary bile acids (PBAs) production species: Weissella confusa, Weissella paramesenteroides, Lactobacillus brevis, and Lactobacillus plantarum increased (p < 0.05). The species converting PBAs to secondary bile acids (SBAs): Parabacteroides distasonis decreased (p < 0.05). This might result in accumulation of PBAs, which also may lead to anti-inflammatory gene JUND and SQSTM1 downregulated.In conclusion, this study suggests that metformin intolerance may be attributed to a decrease in anti-inflammatory-related flora and genes, and also alterations in PBAs accumulation-related flora. These findings open up possibilities for future research targeting gut flora and host genes to prevent metformin intolerance.

Lactobacillus rhamnosus Modulates Lung Inflammation and Mitigates Gut Dysbiosis in a Murine Model of Asthma-COPD Overlap Syndrome.

The asthma-COPD overlap syndrome (ACOS) presents lung inflammation similar to both asthma and chronic obstructive pulmonary disease (COPD). Due to the immune response between the lung and gut, it is possible that ACOS individuals present gut dysbiosis. Due to therapeutic limitations in ACOS, Lactobacillus rhamnosus (Lr) have received attention once Lr has been effective in asthma and COPD. However, there is no data about the Lr effect on both lung inflammation and gut dysbiosis in ACOS. Thus, our study investigated the Lr effect on lung inflammation, bronchoconstriction, airway remodeling, and gut dysbiosis in the murine ACOS model. Treated mice with Lr were exposed to HDM and cigarette smoke to induce ACOS. Sixty days after ACOS induction, mice were euthanized. Lung inflammation was evaluated in leukocytes in bronchoalveolar lavage fluid (BALF), airway remodeling, cytokine secretion, and transcription factor expression in the lung. The gut microbiota was assayed by 16S mRNA sequencing from a fecal sample. Leukocyte population, bronchial hyperreactivity, pro-inflammatory cytokines, and airway remodeling were attenuated in Lr-treated ACOS mice. Likewise, IL-4, IL-5, and IL-13, STAT6 and GATA3, as well as IL-17, IL-21, IL-22, STAT3, and RORɣt were reduced after Lr. In addition, IL-2, IL-12, IFN-γ, STAT1, and T-bet as well as IL-10, TGF-β, STAT5, and Foxp3 were restored after the Lr. Firmicutes was reduced, while Deferribacteres was increased after Lr. Likewise, Lr decreased Staphylococcus and increased Mucispirillum in ACOS mice. Lr improves fecal bacterial β-diversity. Our findings show for the first time the Lr effect on lung inflammation and gut dysbiosis in murine ACOS.

Regulation of coronavirus nsp15 cleavage specificity by RNA structure.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, has had an enduring impact on global public health. However, SARS-CoV-2 is only one of multiple pathogenic human coronaviruses (CoVs) to have emerged since the turn of the century. CoVs encode for several nonstructural proteins (nsps) that are essential for viral replication and pathogenesis. Among them is nsp15, a uridine-specific viral endonuclease that is important in evading the host immune response and promoting viral replication. Despite the established endonuclease function of nsp15, little is known about other determinants of its cleavage specificity. In this study we investigate the role of RNA secondary structure in SARS-CoV-2 nsp15 endonuclease activity. Using a series of in vitro endonuclease assays, we observed that thermodynamically stable RNA structures were protected from nsp15 cleavage relative to RNAs lacking stable structure. We leveraged the s2m RNA from the SARS-CoV-1 3'UTR as a model for our structural studies as it adopts a well-defined structure with several uridines, two of which are unpaired and thus highly probable targets for nsp15 cleavage. We found that SARS-CoV-2 nsp15 specifically cleaves s2m at the unpaired uridine within the GNRNA pentaloop of the RNA. Further investigation revealed that the position of uridine within the pentaloop also impacted nsp15 cleavage efficiency suggesting that positioning within the pentaloop is necessary for optimal presentation of the scissile uridine and alignment within the nsp15 catalytic pocket. Our findings indicate that RNA secondary structure is an important determinant of nsp15 cleavage and provides insight into the molecular mechanisms of RNA recognition by nsp15.

High-fat diet-induced gut microbiota alteration promotes lipogenesis by butyric acid/miR-204/ACSS2 axis in chickens

The gut microbiota is known to have significant involvement in the regulation of lipogenesis and adipogenesis, yet the mechanisms responsible for this relationship remain poorly understood. The current study aims to provide insight into the potential mechanisms by which the gut microbiota modulates lipogenesis in chickens. Using chickens fed with a normal-fat diet (NFD, n=5) and high-fat diet (HFD, n=5), we analyzed the correlation between gut microbiota, cecal metabolomics, and lipogenesis by 16s rRNA sequencing, miRNA and mRNA sequencing as well as targeted metabolomics analysis. The potential metabolite/miRNA/mRNA axis regulated by gut microbiota was identified using chickens treated with antibiotics (ABX, n=5). The possible mechanism of gut microbiota regulating chicken lipogenesis was confirmed by fecal microbiota transplantation (FMT) from chickens fed with NFD to chickens fed with HFD (n=5). The results showed that HFD significantly altered gut microbiota composition and enhanced chicken lipogenesis, with a significant correlation between 3. Furthermore, HFD significantly altered the hepatic miRNA expression profiles and reduced the abundance of hepatic butyric acid. Procrustes analysis indicated that the HFD-induced dysbiosis of the gut microbiota might affect the expression profiles of hepatic miRNA. Specifically, HFD-induced gut microbiota dysbiosis may reduce the abundance of butyric acid and downregulate the expression of miR-204 in the liver. Multiomics analysis identified ACSS2 as a target gene of miR-204. Gut microbiota depletion by an antibiotic cocktail (ABX) showed a gut microbiota-dependent manner in the abundance of butyric acid and the expression of miR-204/ACSS2, which have been observed to be significantly correlated. Fecal microbiota transplantation from NFD chickens into HFD chickens effectively attenuated the HFD-induced excessive lipogenesis, elevated the abundance of butyric acid and the relative expression of miR-204, and reduced the expression of ACSS2 in the liver. Mechanistically, our results showed that the gut microbiota plays an antiobesity role by regulating the butyric acid/miR-204/ACSS2 axis in chickens. This work contributed to a better understanding of the functions of gut microbiota in regulating chicken lipogenesis.

Plant-Derived Extracellular Vesicles as a Delivery Platform for RNA-Based Vaccine: Feasibility Study of an Oral and Intranasal SARS-CoV-2 Vaccine.

Plant-derived extracellular vesicles (EVs) may represent a platform for the delivery of RNA-based vaccines, exploiting their natural membrane envelope to protect and deliver nucleic acids. Here, EVs extracted from orange (Citrus sinensis) juice (oEVs) were investigated as carriers for oral and intranasal SARS-CoV-2 mRNA vaccine. oEVs were efficiently loaded with different mRNA molecules (coding N, subunit 1 and full S proteins) and the mRNA was protected from degrading stress (including RNase and simulated gastric fluid), delivered to target cells and translated into protein. APC cells stimulated with oEVs loaded with mRNAs induced T lymphocyte activation in vitro. The immunization of mice with oEVs loaded with S1 mRNA via different routes of administration including intramuscular, oral and intranasal stimulated a humoral immune response with production of specific IgM and IgG blocking antibodies and a T cell immune response, as suggested by IFN-γ production by spleen lymphocytes stimulated with S peptide. Oral and intranasal administration also triggered the production of specific IgA, the mucosal barrier in the adaptive immune response. In conclusion, plant-derived EVs represent a useful platform for mRNA-based vaccines administered not only parentally but also orally and intranasally.

Multiomics profiling of the impact of an angiotensin (1-7)-expressing probiotic combined with exercise training in aged male rats.

Angiotensin (1-7) [Ang (1-7)] is an active heptapeptide of the noncanonical arm of the renin-angiotensin system that modulates molecular signaling pathways associated with vascular and cellular inflammation, vasoconstriction, and fibrosis. Preclinical evidence suggests that Ang (1-7) is a promising therapeutic target that may ameliorate physical and cognitive function in late life. However, treatment pharmacodynamics limits its clinical applicability. Therefore, this study explored the underlying mechanisms altered by a genetically modified probiotic (GMP) that expresses Ang (1-7) combined with and without exercise training in an aging male rat model as a potential adjunct strategy to exercise training to counteract the decline of physical and cognitive function. We evaluated cross-tissue (prefrontal cortex, hippocampus, colon, liver, and skeletal muscle) multi-omics responses. After 12 wk of intervention, the 16S mRNA microbiome analysis revealed a main effect of probiotic treatment within- and between groups. The probiotic treatment enhanced α diversity (Inverse Simpson (F[2,56] = 4.44; P = 0.02); Shannon-Wiener (F[2,56] = 4.27; P = 0.02)) and β-diversity (F[2,56] = 2.66; P = 0.01) among rats receiving our GMP. The analysis of microbes' composition revealed three genera altered by our GMP (Enterorhabdus, Muribaculaceae unclassified, and Faecalitalea). The mRNA multi-tissue data analysis showed that our combined intervention upregulated neuroremodeling pathways on prefrontal cortex (i.e., 140 genes), inflammation gene expression in the liver (i.e., 63 genes), and circadian rhythm signaling on skeletal muscle. Finally, the integrative network analysis detected different communities of tightly (|r| > 0.8 and P < 0.05) correlated metabolites, genera, and genes in these tissues.NEW & NOTEWORTHY This manuscript uses a multiomics approach (i.e., microbiome, metabolomics, and transcriptomics) to explore the underlying mechanisms driven by a genetically modified probiotic (GMP) designed to express angiotensin (1-7) combined with moderate exercise training in an aged male rat model. After 12 wk of intervention, our findings suggest that our GMP enhanced gut microbial diversity while exercise training altered the transcriptional response in relevant neuroremodeling genes, inflammation, and circadian rhythm signaling pathways in an aging animal model.

The effects of esketamine on the intestinal microenvironment and intestinal microbiota in mice.

This study aimed to investigate the impact of esketamine on the intestinal flora and microenvironment in mice using mRNA transcriptome sequencing and 16S rRNA sequencing. Ten female mice were randomly assigned to two groups. One group received daily intramuscular injections of sterile water, while the other group received esketamine. After 24days, the mice were sacrificed, and their intestinal tissues and contents were collected for 16S rRNA sequencing and mRNA transcriptome sequencing. The intergroup differences in the mouse intestinal flora were analyzed. Differentially expressed genes were utilized to construct ceRNA networks and transcription factor regulatory networks to assess the effects of esketamine on the intestinal flora and intestinal tissue genes. Esketamine significantly altered the abundance of intestinal microbiota, including Adlercreutzia equolifaciens and Akkermansia muciniphila. Differential expression analysis revealed 301 significantly upregulated genes and 106 significantly downregulated genes. The ceRNA regulatory network consisted of 6 lncRNAs, 44 miRNAs, and 113 mRNAs, while the regulatory factor network included 13 transcription factors and 53 target genes. Gene Ontology enrichment analysis indicated that the differentially expressed genes were primarily associated with immunity, including B-cell activation and humoral immune response mediation. The biological processes in the ceRNA regulatory network primarily involved transport, such as organic anion transport and monocarboxylic acid transport. The functional annotation of target genes in the TF network was mainly related to epithelial cells, including epithelial cell proliferation and regulation. Esketamine induces changes in gut microbiota and the intestinal microenvironment, impacting the immune environment and transport modes.

Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

Comparative analysis of intestinal microbiota composition and transcriptome in diploid and triploid Carassius auratus

Polyploidy and the microbiome are crucial factors in how a host organism responds to disease. However, little is known about how triploidization and microbiome affect the immune response and disease resistance in the fish host. Therefore, this study aims to identify the relationship between intestinal microbiota composition, transcriptome changes, and disease resistance in triploid Carassius auratus (3nCC). In China’s central Dongting lake water system, diploid (2nCC) and triploid Carassius auratus were collected, then 16S rRNA and mRNA sequencing were used to examine the microbes and gene expression in the intestines. 16S rRNA sequencing demonstrated that triploidization altered intestinal richness, as well as the diversity of commensal bacteria in 3nCC. In addition, the abundance of the genus Vibrio in 3nCC was increased compared to 2nCC (P < 0.05). Furthermore, differential expression analysis of 3nCC revealed profound up-regulation of 293 transcripts, while 324 were down-regulated. Several differentially expressed transcripts were related to the immune response pathway in 3nCC, including NLRP3, LY9, PNMA1, MR1, PELI1, NOTCH2, NFIL3, and NLRC4. Taken together, triploidization can alter bacteria composition and abundance, which can in turn result in changes in expression of genes. This study offers an opportunity for deciphering the molecular mechanism underlying disease resistance after triploidization.

Highly sensitive and homogeneous detection of unamplified RNA based on the light scattering properties of gold nanoparticle probes

RNA is a crucial diagnostic biomarker for several human diseases. Hence, a simple, rapid, sensitive, and cost-effective assay for detecting pathogen-derived RNAs (e.g., RNA viruses) in water is necessary for transcriptomic analysis. Here, we present a highly sensitive assay to detect unamplified RNA based on the light scattering properties of oligonucleotide-functionalized gold nanoparticle probes (Au-nanoprobes). In this assay, Au-nanoprobes recognize and hybridize to the target RNA. Subsequently, they scatter light when the sample solution is placed onto a quartz waveguide slide and is excited with an evanescent wave. This assay enables highly sensitive quantification (approximately 102 copies/μL) of bacterial 16S rRNA and mRNA without reverse transcription and cDNA amplification within 10 min of RNA extraction. Moreover, this assay was three orders of magnitude sensitive than previously reported assays for direct RNA quantification.

RNA Leaders Explore New Frontiers in mRNA-Based Medications

Genetic Engineering & Biotechnology NewsVol. 42, No. S2 mRNA Ready to Make Its MarkRNA Leaders Explore New Frontiers in mRNA-Based MedicationsEncouraged by the success of mRNA-based COVID-19 vaccines, developers advance new technologies for leveraging or targeting mRNAVivienne Raper, PhDVivienne Raper, PhDSearch for more papers by this authorPublished Online:9 Jun 2022https://doi.org/10.1089/gen.42.S2.03AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 42Issue S2Jun 2022 Information© 2022 by GEN PublishingTo cite this article:Vivienne Raper, PhD.RNA Leaders Explore New Frontiers in mRNA-Based Medications.Genetic Engineering & Biotechnology News.Jun 2022.12-16.http://doi.org/10.1089/gen.42.S2.03Published in Volume: 42 Issue S2: June 9, 2022PDF download

Targeting a conserved structural element from the SARS-CoV-2 genome using l-DNA aptamers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, has resulted in an ongoing global pandemic that has already claimed more than 4 million lives. While most antiviral efforts have focused on essential SARS-CoV-2 proteins, RNA structural elements within the viral genome are also compelling targets. In this study, we identified high-affinity l-DNA aptamers against a SARS-CoV-2 stem-loop II-like motif (s2m), a highly conserved RNA structure with promising diagnostic and therapeutic potential. Optimized l-C1t and l-C3t aptamers bind selectively to s2m RNA with Kd values in the nanomolar range, and are capable of differentiating the monomeric s2m stem-loop from the proposed homodimer duplex. This structure-specific mode of recognition also allows l-C1t and l-C3t to discriminate between s2m RNAs from SARS-CoV-2 and SARS-CoV-1, which differ by just two nucleotides. Finally, we show that l-C1t and l-C3t induce dramatic conformational changes in s2m structure upon binding, and thus, have the potential to block protein–s2m interactions. Overall, these results demonstrate the feasibility of targeting SARS-CoV-2 RNA using l-aptamers, which has important implications in the diagnosis and treatment of COVID-19. Moreover, the high affinity and selectivity of l-C1t and l-C3t, coupled with the intrinsic nuclease resistance of l-DNA, present an opportunity for generating new tools and probes for interrogating s2m function in SARS-CoV-2 and related viruses.

Targeting the Mitotic Checkpoint in Myeloma with OSU-13, a Novel Mps1/Ttk Inhibitor

Despite recent advances, there is still a lack of treatment options for patients with high-risk multiple myeloma (MM), warranting the need for novel therapeutic targets. Monopolar spindle 1 (Mps1), also known as TTK protein kinase, is localized at the kinetochores and centromeres essential for the mitotic spindle checkpoint and centrosome duplication. In cancer cells, high levels of Mps1 help to support aneuploidy, a common malignant trait. In fact, high MPS1 expression correlates to unfavorable prognosis in colon and breast cancers. Here, we report the importance of Mps1 in MM and perform functional characterization of a novel Mps1 inhibitor, OSU-13.To assess the relevance of Mps1 in MM prognosis, we analyzed the CoMMpass database and examined the association of MPS1 expression with clinical outcome and genetic alterations in MM patients (n=769). Elevated MPS1 expression correlates with decreased overall survival (p-value = 0.0001) and decreased event-free survival (p-value < 0.0001). In addition, MPS1 expression is higher in high-risk MM with specific genetic alterations, such as deletion of 17p (p-value = 0.0003), Myc translocation t(8;14) (p-value = 0.02), and gain of 1q21 (p-value = 0.0001).We therefore compared MPS1 expression in eight different MM cell lines and primary CD138 - and CD138 + cells isolated from bone marrow (BM) of four recently diagnosed, untreated MM patients. Gene expression levels assessed by quantitative qPCR were normalized to 18S mRNA internal control, and relative quantification was performed using the ΔΔCt method. MPS1 expression was higher in all MM cell lines compared to the average expression level in primary CD138 + BM plasma cells, with a minimum of 3.2-fold increase in L363 and a maximum of 16.2-fold increase in MM1S. In addition, MPS1 expression was 2-fold higher in CD138 + MM cells compared to CD138 - counterparts from the BM of the same patient, suggesting its selectivity as a target.Next, we characterized the effects of OSU-13, a novel Mps1 inhibitor. In an Mps1 target engagement assay (NanoBRET™) in HEK293 cells, we measured the relative levels of OSU-13-mediated inhibition of Mps1-NanoLuc binding to a fluorescent tracer in comparison to MM clinical candidates. OSU-13 showed EC 50=10 nM, almost 10-fold lower than other agents tested.Endpoint measurement by MTT (3-[4,5- dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate cytotoxicity of OSU-13. MM cell lines were treated with increasing doses of OSU-13 for 72h and cell viability was measured. OPM-2 was the most sensitive cell line (IC 50 = 610 nM), followed by H929 (IC 50 = 1440 nM), RPMI-8226 (IC 50 = 4196 nM), and U266 (IC 50 = 5071 nM).To investigate the kinetics and the mechanisms used by OSU-13 to cause cell death, we treated H929 and OPM-2 cell lines with 500 nM of OSU-13 for 24h, 48h, 72h, 96h, and 120h and assessed both Annexin V-FITC and Zombie-Aqua staining by flow cytometry. Treatment with OSU-13 caused an increase in Annexin V staining as early as 24h in H929 (2.1-fold) and OPM-2 (1.6-fold). Maximum increase of apoptotic cells (Annexin V single positive) was observed after 72h, with a 5-fold increase in OPM-2 and 8.6-fold increase in H929 cell line. Cell death peaked at 120h, with Zombie staining 74% of OPM-2 and 60% of H929 cells. The activity of caspases 3/7 was further assessed in H929 myeloma cells at five time points after treatment with different concentrations of OSU-13. Caspases 3/7 activation started after 48h with 500nM OSU-13, and peaked after 72h post-treatment with the same dose, in agreement with the Annexin staining kinetics results.Finally, we evaluated the in vivo effect of OSU-13 in tumor growth in a H929 MM subcutaneous xenograft model in CB.17 SCID mice. Mice were treated with 5 mg/kg or 10 mg/kg daily dose of OSU-13 per oral gavage for 21 days. Treatment with 10 mg/kg daily dose of OSU-13 produced a 22% tumor growth delay (p<0.05).Collectively, we show here that elevated MPS1/TTK expression correlates with poorer prognosis in MM, and OSU-13, a novel Mps1/TTK inhibitor, induces apoptosis and cell death in MM cell lines, and decreases tumor growth in mice containing H929 cell xenografts. Together, our findings reveal that targeting the mitotic checkpoint using OSU-13 is a potential novel strategy, particularly for high risk MM. DisclosuresNo relevant conflicts of interest to declare.

Asymptomatic and sub-microscopic Plasmodium falciparum infection in children in the Mount Cameroon area: a cross-sectional study on altitudinal influence, haematological parameters and risk factors

BackgroundThe Mount Cameroon area has experienced a 57.2% decline in confirmed malaria cases between 2006 and 2013 with the implementation of different control measures but, the disease is still of public health concern. The objective of the study was to assess the burden of asymptomatic and sub-microscopic Plasmodium infection, altitudinal influence on it, their effect on haematological parameters as well as identify the risk factors of infection.MethodologyA cross-sectional community-based survey involving 1319 children of both sexes aged 6 months to 14 years was conducted between July 2017 and May 2018. Malaria parasitaemia was confirmed by Giemsa-stained microscopy, sub-microscopic Plasmodium infection by 18S mRNA using nested PCR and full blood count analysis was done using an auto haematology analyser.ResultsMalaria parasite, asymptomatic malaria parasitaemia and sub-microscopic Plasmodium infection and anaemia were prevalent in 36.4%, 34.0%, 43.8% and 62.3% of the children, respectively. The risk of having sub-microscopic Plasmodium infection was highest in children 5‒9 (OR = 3.13, P < 0.001) and 10‒14 years of age (OR = 8.18, P < 0.001), non-insecticide treated net users (OR = 1.69, P < 0.04) and those anaemic (OR = 9.01, P < 0.001). Children with sub-microscopic infection had a significantly lower mean haemoglobin (9.86 ± 1.7 g/dL, P < 0.001), red blood cell counts (4.48 ± 1.1 × 1012/L, P < 0.001), haematocrit (31.92%, P < 0.001), mean corpuscular haemoglobin concentration (313.25 ± 47.36, P = 0.035) and platelet counts (280.83 ± 112.62, P < 0.001) than their negative counterparts. Children < 5 years old (73.8%), having asymptomatic (69.8%) and sub-microscopic Plasmodium infection (78.3%) as well as resident in the middle belt (72.7%) had a higher prevalence of anaemia than their peers.ConclusionThe meaningful individual-level heterogeneity in the burden of asymptomatic and sub-microscopic Plasmodium infection in addition to its corollary on haematological variables among children in the different attitudinal sites of the Mount Cameroon Region accentuate the need for strategic context specific planning of malaria control and preventative measures.

Selenium intracanal dressing: effects on the periapical immune response.

To evaluate the selenium (Se) behavior when used as an endodontic dressing in teeth with pulp necrosis. Additionally, its effects was also compared with the calcium hydroxide (C.H.), which is used globally as a root canal dressing, and the combination of the C.H. with Se (C.H. + Se). The sample consisted of 60 patients requiring endodontic treatment who were divided into groups, i.e., without intracanal medication (empty) and with medications as follows: selenium (Se), calcium hydroxide (C.H.), and calcium hydroxide + selenium (C.H. + Se) (n = 15). After the coronary opening, three absorbent paper points were placed in the RCS and maintained for 2min for microbial evaluation. Following the cleaning and shaping procedures, new paper points were introduced into the root canal system, passing passively through the root apex (2mm) into the periapical tissues for 2min, for immune evaluation. The collections were performed again 15days later. Real-time PCR quantified the expression of the prokaryotic 16S ribosomal RNA. The 16S mRNA was evaluated before the cleaning and shaping procedures and 15days later in the groups treated with or without medication. A significant reduction in the microbial load was observed only in the groups that received endodontic dressing (p < 0.05). The cytokines IFN-γ, TNF-α, IL-1α, IL-17A, IL-10, IL-6 and MCP-1, were also quantified by real-time PCR. There was an increase in the gene expression level of the cytokines (T15) TNF-α and IL-10 in the C.H. group compared to the other groups (p < 0.05). The IFN-γ mRNA expression was reduced in the groups treated with the medications (Se, C.H., and C.H. + Se). The findings of the present study indicate that in the case of treatment over multiple sessions, the use of root canal dressing is essential to avoid the root canal system (RCS) microbial recolonization. Selenium potentiated the effects of calcium hydroxide inducing an anti-inflammatory response in periapical tissues. Se is a mineral essential for the formation of the amino acid selenocysteine, which is directly involved in the maintenance of the immune response. Selenium has been widely used in the medical field in the treatment of cancer, as an activator of bone metabolism, and as a stimulator of the immune system. In this study, it was shown that the incorporation of Se, whether as intracanal medication alone or in conjunction with other medications, may potentiate periapical tissue repair after RCS cleaning and shaping procedures.

Myosin Heavy Chain Isoform Mrna Expression In Low- And High-capacity Running Rats

Differential expression of Myosin Heavy Chain (MHC) isoforms in skeletal muscle determines its metabolic and functional characteristics. Endurance exercise training is associated with fast-to-slow MHC isoform transition, and which is considered to mediate the beneficial effects of exercise on metabolic health. Rats genetically bred for low-capacity running (LCR) and high-capacity running (HCR) are characterized by the health benefits typically described after endurance exercise training. LCR and HCR rats differed in maximal running capacity 14 fold. PURPOSE: To quantify mRNA expression for slow (MHC-I) and fast (MCH-IIa, MCH-IIb) MHC isoforms in soleus, gastrocnemius and vastus skeletal muscle of LCR and HCR male adult rats, and determine if HCR rats have higher mRNA expression for slow, and lower mRNA expression for fast, MHC isoforms. METHODS: Muscle tissue from 5 HCR and 5 LCR rats was homogenized and mRNA was isolated. The mRNA content of MHC isoforms in muscle was quantified using reverse transcriptase polymerase chain reaction (RT-PCR), and standardized to the 18S mRNA content in muscle. Differences between groups were calculated using the comparative CT method (2-ΔΔCT). LCR were the control group. RESULTS: Expression of mRNA of the MHC isoforms differed between groups and the type of skeletal muscle analyzed. MHC-I mRNA expression was lower in the HCR compared to LCR across all muscle groups, but this difference (2-ΔΔCT) was more pronounced for the gastrocnemius muscle [LCR: 1.0 ± 0.6 (mean±SE); HCR: 0.3 ± 0.2; P = 0.08]. Although MHC-IIa mRNA expression appeared lower in soleus and vastus in the HCR, it was higher in the HCR compared to LCR in the gastrocnemius muscle [LCR: 1.0 ± 1.0; HCR: 4.6 ± 3.6; P = 0.07]. MHC-IIb mRNA expression was lower in the HCR compared to LCR across all muscle groups, but this difference was more pronounced for the soleus muscle [LCR: 1.0 ± 1.1; HCR: 0.2 ± 0.4; P = 0.08]. CONCLUSION: Genetic differences linked to the MHC transcriptome (i.e., decrease in MHC-I mRNA) in muscle do not explain the exercise performance observed in HCR rats. Changes in the muscle MHC transcriptome may not directly be responsible for the health benefits associated with exercise training. It remains to be determined on whether the differences we observed at the MHC mRNA level are translated to the MHC proteome level.

Sexual Dimorphism of Gut Microbiota Dictates Therapeutics Efficacy of Radiation Injuries.

Accidental or iatrogenic ionizing radiation exposure precipitates acute and chronic radiation injuries. The traditional paradigm of mitigating radiotherapy‐associated adverse side effects has ignored the gender‐specific dimorphism of patients' divergent responses. Here, the effects of sexual dimorphism on curative efficiencies of therapeutic agents is examined in murine models of irradiation injury. Oral gavage of simvastatin ameliorates radiation‐induced hematopoietic injury and gastrointestinal tract dysfunction in male mice, but adversely deteriorates these radiation syndromes in female animals. In a sharp contrast, feeding animals with high‐fat diet (HFD) elicites explicitly contrary results. High‐throughput sequencing of microbial 16S rRNA, host miRNA, and mRNA shows that simvastatin or HFD administration preventes radiation‐altered enteric bacterial taxonomic structure, preserves miRNA expression profile, and reprogrammes the spectrum of mRNA expression in small intestines of male or female mice, respectively. Notably, faecal microbiota transplantation of gut microbes from opposite sexual donors abrogates the curative effects of simvastatin or HFD in respective genders of animals. Together, these findings demonstrate that curative efficiencies of therapeutic strategies mitigating radiation toxicity might be dependent on the gender of patients, thus simvastatin or HFD might be specifically useful for fighting against radiation toxicity in a sex‐dependent fashion partly based on sex‐distinct gut microbiota composition in preclinical settings.

Functional dysbiosis within dental plaque microbiota in cleft lip and palate patients

BackgroundDental caries is a polymicrobial disease and prevalent among cleft lip and palate (CLP) patients, although their oral hygiene is well maintained. Dysbiosis, the state of imbalance within the dental plaque microbiota, may cause caries prevalence among these patients. However, little is known about how dysbiosis occurs and affects cariogenicity. To find dysbiotic signs, here we conducted a metatranscriptomic analysis for the plaque microbiota in six CLP patients and four controls.MethodsTotal bacterial RNA was extracted from each sample and sequenced. Bacterial composition and functional profiles were estimated from 16S rRNA and mRNA reads, respectively. The mRNA reads were further used for estimating bacterial composition. Species listed in both rRNA-based and mRNA-based bacterial composition were identified as viable taxa with in situ function (VTiF), and the VTiF with a high mRNA-to-rRNA ratio were considered to be transcriptionally active. A network was constructed for each group by connecting two VTiF if their mRNA abundances were positively correlated.ResultsThe bacterial composition and functional profiles themselves did not provide remarkable signs of dysbiosis in the CLP group. However, the group-specific active taxa were identified, including streptococcal and Prevotella species in the CLP group. Moreover, the network structure was different between groups; Actinomyces johnsonii and several species in the CLP group were the active taxa, which were connected based on positive correlations with statistical significance.ConclusionsFunctional dysbiosis within the plaque microbiota was observed such as difference of the network structure between groups, and may be associated with cariogenicity. The observed functional dysbiosis was an invisible change within the microbiota in the oral cavity of CLP patients. This may emphasize the importance of maintaining good oral hygiene of the patients with cleft anomalies.