Star-PAP controlled alternative polyadenylation coupled poly(A) tail length regulates protein expression in hypertrophic heart.

Alternative polyadenylation (APA)-mediated 3'-untranslated region (UTR) shortening is known to increase protein expression due to the loss of miRNA regulatory sites. Yet, mRNAs with longer 3'-UTR also show enhanced protein expression. Here, we identify a mechanism by which longer transcripts generated by the distal-most APA site leads to increased protein expression compared to the shorter transcripts and the longer transcripts are positioned to regulate heart failure (HF). A Star-PAP target gene, NQO1 has three poly(A) sites (PA-sites) at the terminal exon on the pre-mRNA. Star-PAP selects the distal-most site that results in the expression of the longest isoform. We show that the NQO1 distal-specific mRNA isoform accounts for the majority of cellular NQO1 protein. Star-PAP control of the distal-specific isoform is stimulated by oxidative stress and the toxin dioxin. The longest NQO1 transcript has increased poly(A) tail (PA-tail) length that accounts for the difference in translation potentials of the three NQO1 isoforms. This mechanism is involved in the regulation of cardiac hypertrophy (CH), an antecedent condition to HF where NQO1 downregulation stems from the loss of the distal-specific transcript. The loss of NQO1 during hypertrophy was rescued by ectopic expression of the distal- but not the proximal- or middle-specific NQO1 mRNA isoforms in the presence of Star-PAP expression, and reverses molecular events of hypertrophy in cardiomyocytes.

Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3'-end processing.

Targeted proteomics of cerebrospinal fluid in treatment naïve multiple sclerosis patients identifies immune biomarkers of clinical phenotypes.

Multiple sclerosis (MS) is an inflammatory demyelinating disease with heterogeneous clinical presentations and variable long-term disability accumulation. There are currently no standard criteria to accurately predict disease outcomes. In this study we investigated the cross-sectional relationship between disease phenotype and immune-modulating cytokines and chemokines in cerebrospinal fluid (CSF). We analyzed CSF from 20 DMT-naïve MS patients using Olink Proteomics' Target 96 Inflammation panel and correlated the resulting analytes with respect to (1) disease subtype, (2) patient age and sex, (3) extent of clinical disability, and (4) MRI segmental brain volumes. We found that intrathecal IL-4 correlated with higher Expanded Disability Status Scale (EDSS) scores and longer 25-foot walk times, and CD8A correlated with decreased thalamic volumes and longer 9-hole peg test times. Male sex was associated with higher FGF-19 expression, and Tumefactive MS with elevated CCL4. Several inflammatory markers were correlated with older age at the time of LP. Finally, higher intrathecal IL-33 correlated with increased MS lesion burden and multi-compartment brain atrophy. This study confirms immune heterogeneity underlying CSF profiles in MS, but also identifies several inflammatory protein biomarkers that may be of use for predicting clinical outcomes in future algorithms.

Tyrosine phosphorylation controlled poly(A) polymerase I activity regulates general stress response in bacteria.

RNA 3'-end polyadenylation that marks transcripts for degradation is implicated in general stress response in Escherichia coli Yet, the mechanism and regulation of poly(A) polymerase I (PAPI) in stress response are obscure. We show that pcnB (that encodes PAPI)-null mutation widely stabilises stress response mRNAs and imparts cellular tolerance to multiple stresses, whereas PAPI ectopic expression renders cells stress-sensitive. We demonstrate that there is a substantial loss of PAPI activity on stress exposure that functionally phenocopies pcnB-null mutation stabilising target mRNAs. We identify PAPI tyrosine phosphorylation at the 202 residue (Y202) that is enormously enhanced on stress exposure. This phosphorylation inhibits PAPI polyadenylation activity under stress. Consequentially, PAPI phosphodeficient mutation (tyrosine 202 to phenylalanine, Y202F) fails to stimulate mRNA expression rendering cells stress-sensitive. Bacterial tyrosine kinase Wzc phosphorylates PAPI-Y202 residue, and that wzc-null mutation renders cells stress-sensitive. Accordingly, wzc-null mutation has no effect on stress sensitivity in the presence of pcnB-null or pcnB-Y202F mutation. We also establish that PAPI phosphorylation-dependent stress tolerance mechanism is distinct and operates downstream of the primary stress regulator RpoS.

Transgenesis of mammalian PABP reveals mRNA polyadenylation as a general stress response mechanism in bacteria.

In eukaryotes, mRNA 3'-polyadenylation triggers poly(A) binding protein (PABP) recruitment and stabilization. In a stark contrast, polyadenylation marks mRNAs for degradation in bacteria. To study this difference, we trans-express the mammalian nuclear PABPN1 chromosomally and extra-chromosomally in Escherichia coli. Expression of PABPN1 but not the mutant PABPN1 stabilizes polyadenylated mRNAs and improves their half-lives. In the presence of PABPN1, 3'-exonuclease PNPase is not detected on PA-tailed mRNAs compromising the degradation. We show that PABPN1 trans-expression phenocopies pcnB (that encodes poly(A) polymerase, PAPI) mutation and regulates plasmid copy number. Genome-wide RNA-seq analysis shows a general up-regulation of polyadenylated mRNAs on PABPN1 expression, the largest subset of which are those involved in general stress response. However, major global stress regulators are unaffected on PABPN1 expression. Concomitantly, PABPN1 expression or pcnB mutation imparts cellular tolerance to multiple stresses. This study establishes mRNA 3'-polyadenylation as a general stress response mechanism in E. coli.

Comparative evaluation of therapeutic efficacy of intra-articular oxaceprol with conventional modalities in osteoarthritis animal model.

The duration and dose-dependent side effects of conventional intra-articular corticosteroid treatment in osteoarthritis (OA) like cartilage damage and chondrocyte toxicity warrant the search for alternative therapeutics. Oxaceprol, a recognized oral therapeutic agent for osteoarthritis, is yet to be explored for its intra-articular route of administration confirming better safety profile. In this study, a comparative evaluation of intra-articular oxaceprol and corticosteroid is carried out in osteoarthritis rabbit model. Osteoarthritis was induced by monosodium iodoacetate in rabbits. After randomization into three groups of five animals each: OA with intra-articular injection of saline, OA with intra-articular injection of oxaceprol, and OA with intra-articular injection of corticosteroids, treatment efficacy was analyzed by evaluation of inflammation through knee swelling, pain assessment by wire walking, and hot plate method. Further biopsies were collected for histological characterization. Intra-articular oxaceprol and corticosteroids reduced 20.5 and 24.5% knee swelling respectively within 4 weeks compared to those in control osteoarthritic rabbits. Oxaceprol exhibited analgesic action in visual analogue scoring of wire walking method. Hot plate test further confirmed drastic minimization of pain in oxaceprol intervention. Histological investigation suggested that application of oxaceprol has the abilities to protect articular cartilages from degenerative changes that occur in osteoarthritis. Marked improvement both in bone and cellular matrixes was observed in oxaceprol-treated group while gross lesions were visible and consisted of a well-demarcated area of cartilage erosion in control group. Intra-articular injection of oxaceprol showed remarkable improvement of articular cartilage in chemically induced osteoarthritic rabbits.

Rising trend of diabetes mellitus amongst the undernourished: State -of- the -art review.

Diabetes mellitus is prevailing in the malnourished populations congruently in well-nourished ones with an escalating trend in the former group regardless of the absence of obesity as an etiologic determining factor as per the studies in underprivileged sectors of the population. Chronic undernutrition across a lifetime may be an imperative stimulator of diabetes in an individual either by progressively reducing beta cell function alongside islet cell volume and increasing the individual predisposition to other genetic or environmental diabetogenic influences with modifying influence on the course of clinical syndrome. Ketosis resistant insulinopenia is irreversible to the sustained vigorous nutritional convalescence in a substantial fraction of malnourished subjects. It also debunks a latent diabetic stage with insulin resistance reflected by greater insulin requirement in comparison to the patients with type I diabetes with the same beta cell failure fraction and obese type II diabetic patients with equivalent glycemic control gauged by HbA1c levels. Current tendency warrants the replacement of conventional therapy by community oriented theranostic approaches and health programs to curb the epidemic.

Radiopaque Hemocompatible Ruminant-Sourced Gut Material with Antimicrobial Physiognomies for Biomedical Applications in Diabetics.

This study comprises the fabrication of a radiopaque gut material with its mechanical properties conforming to the US Pharmacopeia guidelines giving an antimicrobial advantage for suture application, especially in conditions such as diabetes mellitus, which has a high wound infection rate. Schiff base cross-linking iodination of the material is evinced by the spectroscopic studies, and antimicrobial properties owing to released iodine are evinced through in vitro studies. Modified gut sutures demonstrated favorable physicomechanical features such as appropriate tensile strength (440 ± 20 MPa) and knot strength (270 ± 20) alongside a mean radiopacity value of 139.0 ± 10 in comparison with that of the femoral shaft with 160 ± 10. The diabetic model showed absence of clinical signs of infection, supported by wound swab culture and the absence of necrosis in histology. Hemocompatibility studies evinced the absence of contact platelet activation and hemolysis alongside the customary coagulation response. These promising results highlight the stimulating potential of the process in the development of biomedical applications, necessitating persistent studies for its evidence-based applicability, particularly in diabetic patients.

Assessment of Insulin Sensitivity and its Convalescence with Dietary Rehabilitation in Undernourished Rural West Bengal Population.

INTRODUCTION: Metabolic syndrome involving diabetes is acquiring the eminence of prospective epidemic in India with dramatic rise in rural population prevalence where majority are undernourished and creates significant healthcare burden. AIM: To investigate the prevalence of the insulin resistance amongst the undernourished rural population, involving children, adolescents and adult volunteers of the West Bengal and the effect of nutritional supplementation in the parameters echoing insulin sensitivity. MATERIALS AND METHODS: A prospective interventional study was carried out on 120 volunteers in three age groups; Group 1 (5-12 years), Group 2 (13-18 years) and Group 3 (19-40 years), each with 40 subjects who fulfilled the inclusion criteria, under informed consent over six months. Baseline data regarding demography, family history including parental history of diabetes, medical history, physical, activity, anthropometrical status and blood parameters echoing insulin sensitivity were obtained from volunteers. Intervention included daily nutritional supplementation with eggs and banana. After six months of food supplementation, parameters reflecting insulin sensitivity were assessed. RESULTS: Initial low figures of fasting insulin and blood sugar levels improvised with the nutritional supplementation alongside the absence of wasting in Group 1. HOMA-IR and IRI values of the Group 2 improved from the initial figures (3.19, 2.87) to (2.204, 1.99) and that of Group 3 from (5.0, 4.52) to (4.08, 3.67) respectively over six months (p<.05). Average Fasting Blood Sugar (FBS) enhanced from 85.5±9.6 mg/dl to 78.4±7.3 mg/dl and from 90.1±10.2 mg/dl to 84.8±8.4 mg/dl in Group 2 and 3 respectively by the end of the supplementation study (p<.05). CONCLUSION: Six months study revealed the existence of insulin resistance and malnourishment in the selected rural population in three groups. Decrement in HOMA-IR, IRI, FBS and serum insulin alongside the improvement in the body mass, in comparison to that of initial values is evocative of restoration of insulin sensitivity with nutrition supplementation.

In Situ Iodination Cross-Linking of Silk for Radio-Opaque Antimicrobial Surgical Sutures.

The purpose of the study is to develop a method of imparting radio-opacity to the silk fibers by stepwise 2,5-dimethoxy-2,5-dihydro-furan (DMDF)-iodine cross-linking reaction for suture fabrication with mechanical properties abiding with U.S. pharmacopeia guidelines along with non invasive imaging advantage in postoperative follow-up. Silk fibers isolated from Bombyx mori were cross-linked with suitable concentration of DMDF linked with iodine under elevated temperature and pressure. Cross-linked fibers knitted into sutures were subjected to further testing.Computed tomography (CT) images on day 28 of in vivo studies showed mean radio-opacity value (MRV) of 213 ± 19.46 compared to the vertebral bone having value of 254.66 ± 0.51. Modified silk sutures demonstrated several advantages like high tensile strength (626 ± 23.3 MPa) and knot strength (388.6 ± 16.8 MPa) besides antimicrobial property. Encouraging preliminary in vitro and in vivo biocompatibility studies advocate the potential use of modified suture material in cardiac surgery, aneurysmal embolization surgeries, and arterio-venous occlusion surgeries.

Excavating the Role of Aloe Vera Wrapped Mesoporous Hydroxyapatite Frame Ornamentation in Newly Architectured Polyurethane Scaffolds for Osteogenesis and Guided Bone Regeneration with Microbial Protection.

Guided bone regeneration (GBR) scaffolds are unsuccessful in many clinical applications due to a high incidence of postoperative infection. The objective of this work is to fabricate GBR with an anti-infective electrospun scaffold by ornamenting segmented polyurethane (SPU) with two-dimensional Aloe vera wrapped mesoporous hydroxyapatite (Al-mHA) nanorods. The antimicrobial characteristic of the scaffold has been retrieved from the prepared Al-mHA frame with high aspect ratio (∼14.2) via biosynthesis route using Aloe vera (Aloe barbadensis miller) extract. The Al-mHA frame was introduced into an unprecedented SPU matrix (solution polymerized) based on combinatorial soft segments of poly(ε-caprolactone) (PCL), poly(ethylene carbonate) (PEC), and poly(dimethylsiloxane) (PDMS), by an in situ technique followed by electrospinning to fabricate scaffolds. For comparison, pristine mHA nanorods are also ornamented into it. An enzymatic ring-opening polymerization technique was adapted to synthesize soft segment of (PCL-PEC-b-PDMS). Structure elucidation of the synthesized polymers is established by nuclear magnetic resonance spectroscopy. Sparingly, Al-mHA ornamented scaffolds exhibit tremendous improvement (175%) in the mechanical properties with promising antimicrobial activity against various human pathogens. After confirmation of high osteoconductivity, improved biodegradation, and excellent biocompatibility against osteoblast-like MG63 cells (in vitro), the scaffolds were implanted in rabbits as an animal model by subcutaneous and intraosseous (tibial) sites. Improved in vivo biocompatibilities, biodegradation, osteoconductivity, and the ability to provide an adequate biomimetic environment for biomineralization for GBR of the scaffolds (SPU and ornamented SPUs) have been found from the various histological sections. Early cartilage formation, endochondral ossification, and rapid bone healing at 4 weeks were found in the defects filled with Al-mHA ornamented scaffold compared to pristine SPU scaffold. Organ toxicity studies further confirm the absence of appreciable tissue architecture abnormalities in the renal hepatic and cardiac tissue sections. The entire results of this study manifest the feasibility of fabricating a mechanically adequate tailored nanofibrous SPU scaffold based on combinatorial soft segments of PCL, PEC, and PDMS by a biomimetic approach and the advantages of an Aloe vera wrapped mHA frame in promoting osteoblast phenotype progression with microbial protection for potential GBR applications.

On-Demand Guided Bone Regeneration with Microbial Protection of Ornamented SPU Scaffold with Bismuth-Doped Single Crystalline Hydroxyapatite: Augmentation and Cartilage Formation.

Guided bone regeneration (GBR) scaffolds are futile in many clinical applications due to infection problems. In this work, we fabricated GBR with an anti-infective scaffold by ornamenting 2D single crystalline bismuth-doped nanohydroxyapatite (Bi-nHA) rods onto segmented polyurethane (SPU). Bi-nHA with high aspect ratio was prepared without any templates. Subsequently, it was introduced into an unprecedented synthesized SPU matrix based on dual soft segments (PCL-b-PDMS) of poly(ε-caprolactone) (PCL) and poly(dimethylsiloxane) (PDMS), by an in situ technique followed by electrospinning to fabricate scaffolds. For comparison, undoped pristine nHA rods were also ornamented into it. The enzymatic ring-opening polymerization technique was adapted to synthesize soft segments of PCL-b-PDMS copolymers of SPU. Structure elucidation of the synthesized polymers is done by nuclear magnetic resonance spectroscopy. Sparingly, Bi-nHA ornamented scaffolds exhibit tremendous improvement (155%) in the mechanical properties with excellent antimicrobial activity against various human pathogens. After confirmation of high osteoconductivity, improved biodegradation, and excellent biocompatibility against osteoblast cells (in vitro), the scaffolds were implanted in rabbits by subcutaneous and intraosseous (tibial) sites. Various histological sections reveal the signatures of early cartilage formation, endochondral ossification, and rapid bone healing at 4 weeks of the critical defects filled with ornamented scaffold compared to SPU scaffold. This implies osteogenic potential and ability to provide an adequate biomimetic microenvironment for mineralization for GBR of the scaffolds. Organ toxicity studies further confirm that no tissue architecture abnormalities were observed in hepatic, cardiac, and renal tissue sections. This finding manifests the feasibility of fabricating a mechanically adequate nanofibrous SPU scaffold by a biomimetic strategy and the advantages of Bi-nHA ornamentation in promoting osteoblast phenotype progression with microbial protection (on-demand) for GBR applications.

Covalent cross-links in polyampholytic chitosan fibers enhances bone regeneration in a rabbit model.

Chitosan fibers were prepared in citric acid bath, pH 7.4 and NaOH solution at pH 13, to form ionotropically cross-linked and uncross-linked fibers, respectively. The fibers formed in citric acid bath were further cross-linked via carbodiimide chemistry; wherein the pendant carboxyl moieties of citric acid were used for new amide bond formation. Moreover, upon covalent cross-linking in the ionically gelled citrate-chitosan fibers, incomplete conversion of the ion pairs to amide linkages took place resulting in the formation of a dual network structure. The dual cross-linked fibers displayed improved mechanical property, higher stability against enzymatic degradation, hydrophobicity and superior bio-mineralization compared to the uncross-linked and native citrate cross-linked fibers. Additionally, upon cyclic loading, the ion pairs in the dual cross-linked fibers dissociated by dissipating energy and reformed during the relaxation period. The twin property of elasticity and energy dissipation mechanism makes the dual cross-linked fiber unique under dynamic mechanical conditions. The differences in the physico-chemical characteristics were reflected in protein adsorption, which in turn influenced the cellular activities on the fibers. Compared to the uncross-linked and ionotropically cross-linked fibers, the dual cross-linked fibers demonstrated higher proliferation and osteogenic differentiation of the MSCs in vitro as well as better osseous tissue regeneration in a rabbit model.