Anti-thrombotic poly(AAm-co-NaAMPS)-xanthan hydrogel-expanded polytetrafluoroethylene (ePTFE) vascular grafts with enhanced endothelialization and hemocompatibility properties.

Cardiovascular diseases (CVDs) are the leading cause of death among all non-communicable diseases globally. Although expanded polytetrafluoroethylene (ePTFE) has been widely used for larger-diameter vascular graft transplantation, the persistent thrombus formation and intimal hyperplasia of small-diameter vascular grafts (SDVGs) made of ePTFE to treat severe CVDs remain the biggest challenges due to lack of biocompatibility and endothelium. In this study, bi-layered poly(acrylamide-co-2-Acrylamido-2-methyl-1-propanesulfonic acid sodium)-xanthan hydrogel-ePTFE (poly(AAm-co-NaAMPS)-xanthan hydrogel-ePTFE) vascular grafts capable of promoting endothelialization and prohibiting thrombosis were synthesized and fabricated. While the external ePTFE layer of the vascular grafts provided the mechanical stability, the inner hydrogel layer offered much-needed cytocompatibility, hemocompatibility, and endothelialization functions. The interface morphology between the inner hydrogel layer and the outer ePTFE layer was observed by scanning electron microscope (SEM), which revealed that the hydrogel was well attached to the porous ePTFE through mechanical interlocking. Among all the hydrogel compositions tested with cell culture using human umbilical vein endothelial cells (HUVECs), the hydrogel with the molar ratio of 40:60 (NaAMPS/AAm) composition (i.e., Hydrogel 40:60) exhibited the best endothelialization function, as it produced the largest endothelialization area that was three times more than of that of plain ePTFE on day 14, maintained the highest average cell viability, and had the best cell morphology. Hydrogel 40:60 also showed excellent hemocompatibility, prolonged activated partial thromboplastin time (aPTT), and good mechanical properties. Overall, bi-layered poly(AAm-co-NaAMPS)-xanthan hydrogel-ePTFE vascular grafts with the Hydrogel 40:60 composition could potentially solve the critical challenge of thrombus formation in vascular graft transplantation applications.

Multiple Cholecystoenteric Fistulae With Bouveret Syndrome and Acute Pancreatitis: A Rare Combination.

Multiple cholecystoenteric fistulae, Bouveret syndrome (a form of gallstone ileus), and acute pancreatitis occurring together is very rare. Diagnosis is seldom clinical and is mostly based on computerised tomography (CT) or magnetic resonance imaging (MRI). Endoscopy and minimally invasive surgery have revolutionised the treatment of Bouveret syndrome and cholecystoenteric fistula, respectively, over the last two decades. Laparoscopic repair of cholecystoenteric fistula followed by cholecystectomy is successful on a consistent basis with skilled laparoscopic suturing and advanced laparoscopy. In patients with Bouveret syndrome, when the stone is <4cm and is in the proximal duodenum, it is usually amenable for endoscopic extraction with snares, nets, forceps and lithotripsy. When endoscopy is unavailable or fails, laparoscopic surgery is suitable for these patients. However, stones >4 cm, located in the distal duodenum, multiple fistulae, and associated acute pancreatitis may necessitate open surgery. We present here a case of a 65-year-old Indian female with multiple cholecystoenteric fistulae and Bouveret syndrome with acute pancreatitis with a 6.5 cm gallstone diagnosed on CT scan and MRI and treated successfully by open surgery. We also review the current literature on the management of this complex problem.

Dithiolethiones D3T and ACDT Protect Against Iron Overload-Induced Cytotoxicity and Serve as Ferroptosis Inhibitors in U-87 MG Cells.

Iron overload-induced oxidative stress is implicated in various neurodegenerative disorders. Given the numerous adverse effects associated with current iron chelators, natural antioxidants are being explored as alternative therapeutic options. Dithiolethiones found in cruciferous vegetables have emerged as promising candidates against a wide range of toxicants owing to their lipophilic and cytoprotective properties. Here, we test the dithiolethiones 3H-1,2-dithiole-3-thione (D3T) and 5-amino-3-thioxo-3H-(1,2) dithiole-4-carboxylic acid ethyl ester (ACDT) against ferric ammonium citrate (FAC)-induced toxicity in U-87 MG astrocytoma cells. Exposure to 15 mM FAC for 24 h resulted in 54% cell death. A 24-h pretreatment with 50 µM D3T and ACDT prevented this cytotoxicity. Both dithiolethiones exhibited antioxidant effects by activating the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor and upregulating levels of intracellular glutathione (GSH). This resulted in the successful inhibition of FAC-induced reactive oxygen species, lipid peroxidation, and cell death. Additionally, D3T and ACDT upregulated expression of the Nrf2-mediated iron storage protein ferritin which consequently reduced the total labile iron pool. A 24-h pretreatment with D3T and ACDT also prevented cell death induced by the ferroptosis inducer erastin by upregulating the transmembrane cystine/glutamate antiporter (xCT) expression. The resulting increase in intracellular GSH and alleviation of lipid peroxidation was comparable to that caused by ferrostatin-1, a specific ferroptosis inhibitor. Collectively, our findings demonstrate that dithiolethiones may show promise as potential therapeutic options for the treatment of iron overload disorders.

DSM-5-TR diagnosis as a guide to suicide risk assessment.

A specific mental disorder can itself constitute a risk factor for a completed suicide. Even more important, the disorder is typically a modifiable risk factor which informs its own treatment. Recent editions of the DSM have included "suicide subsections" for specific mental disorders and conditions in which the risks of suicidal thoughts and behaviors for the disorder are noted in the literature. The DSM-5-TR can therefore serve as a compendium to be referred to for initial guidance as to whether a specific disorder could contribute to the risk. Adding completed suicides and suicide attempts, also addressed in these subsections, the sections were examined individually for the four parameters of suicidality. Accordingly, the four parameters of suicidality examined here are: suicide, suicidal thoughts, suicidal behavior, and suicide attempts. After providing interpretive comments for each, the parameters for all disorders with a suicide subsection were tabulated for ease of reference. Because specific medical disorders are also associated with elevated rates of suicide, these disorders and the supporting research are tabulated and briefly acknowledged. Allowing for the limitations of the suicide subsections and their analysis, this exegesis is proposed to contribute to training in risk assessment for forensic psychiatry and psychology fellows and to highlight the potential referential value of the DSM-5-TR's suicide subsections for clinical practitioners and those who pursue research on suicide.

Transcriptomic analysis of the human placenta reveals trophoblast dysfunction and augmented Wnt signalling associated with spontaneous preterm birth.

Preterm birth (PTB) is the leading cause of death in under-five children. Worldwide, annually, over 15 million babies are born preterm and 1 million of them die. The triggers and mechanisms of spontaneous PTB remain largely unknown. Most current therapies are ineffective and there is a paucity of reliable predictive biomarkers. Understanding the molecular mechanisms of spontaneous PTB is crucial for developing better diagnostics and therapeutics. To address this need, we conducted RNA-seq transcriptomic analysis, qRT-PCR and ELISA on fresh placental villous tissue from 20 spontaneous preterm and 20 spontaneous term deliveries, to identify genes and signalling pathways involved in the pathogenesis of PTB. Our differential gene expression, gene ontology and pathway analysis revealed several dysregulated genes (including OCLN, OPTN, KRT7, WNT7A, RSPO4, BAMBI, NFATC4, SLC6A13, SLC6A17, SLC26A8 and KLF8) associated with altered trophoblast functions. We identified dysregulated Wnt, oxytocin and cellular senescence signalling pathways in preterm placentas, where augmented Wnt signalling could play a pivotal role in the pathogenesis of PTB due to its diverse biological functions. We also reported two novel targets (ITPR2 and MYLK2) in the oxytocin signalling pathways for further study. Through bioinformatics analysis on DEGs, we identified four key miRNAs, - miR-524-5p, miR-520d-5p, miR-15a-5p and miR-424-5p - which were significantly downregulated in preterm placentas. These miRNAs may have regulatory roles in the aberrant gene expressions that we have observed in preterm placentas. We provide fresh molecular insight into the pathogenesis of spontaneous PTB which may drive further studies to develop new predictive biomarkers and therapeutics.

Development and evaluation of Deferasirox loaded nanoparticles for the treatment of breast cancer: evaluation in-vitro and cell line study / Desarrollo y evaluación de nanopartículas cargadas de Deferasirox para el tratamiento del cáncer de mama: evaluación in-vitro y estudio de línea celular

The aim of the present study was to study the potential efficacy of antitumor activity of nanoparticles loaded with Deferasirox In the current research work, nanoparticle loaded with Deferasirox were developed and evaluated to study its efficacy using breast cancer cell line. Nanoparticles of Deferasirox were prepared by solvent evaporation method using EthocelTM as a polymer and Kolliphor® P 188 as a surfactant. 23 factorial design used with concentration of polymer, concentration of surfactant and rpm as independent variables and percentage of entrapment efficiency and percentage of drug release as dependent variables. All formulations were prepared and evaluated for particle size, spectral studies, thermal studies, drug entrapment efficiency and in-vitro drug dissolution studies. In-vitrocell viability study on breast cancer cell line (MCF-7) was performed by MTT assay Developed nanoparticles possess the particle size 428.3 nm and PDI 0.626. Optimized formulation showed 82.62±1.04 maximum drug release after 6 h in a controlled manner and entrapment efficiency 72.64±0.24. The FTIR spectral studies and DSC thermogram indicated that there was no interaction between the drug and polymers used. Thus, Deferasirox nanoparticle formulation showed the anti-tumoral activity against human breast cancer cell line (MCF7) as per outcome of cell line study Nanoparticles of Deferasirox were stable and could be promising drug delivery for cancerous cells. (AU)

Introducción: El objetivo del presente estudio fue estudiar la eficacia potencial de la actividad antitumoral de nanopartículas cargadas con Deferasirox En el trabajo de investigación actual, se desarrollaron y evaluaron nanopartículas cargadas con Deferasirox para estudiar su eficacia utilizando una línea celular de cáncer de mama. Las nanopartículas de Deferasirox se prepararon mediante el método de evaporación del disolvente usando EthocelTM como polímero y Kolliphor® P 188 como surfactante.23 diseño factorial usado con concentración de polímero, concentración de surfactante y rpm como variables independientes y porcentaje de eficiencia de atrapamiento y porcentaje de liberación de fármaco como variables dependientes. Las 9 formulaciones se prepararon y evaluaron en cuanto al tamaño de partícula, estudios espectrales, estudios térmicos, eficacia de atrapamiento de fármacos y estudios de disolución de fármacos in-vitro. El estudio de viabilidad celular in-vitro en la línea celular de cáncer de mama (MCF-7) se realizó mediante un ensayo MTT.Resultados:Las nanopartículas desarrolladas poseen un tamaño de partícula de 428,3 nm y un PDI de 0,626. La formulación optimizada mostró una liberación máxima de fármaco de 82,62 ± 1,04 después de 6 h de forma controlada y una eficiencia de atrapamiento de 72,64 ± 0,24. Los estudios espectrales FTIR y el termograma DSC indicaron que no hubo interacción entre el fármaco y los polímeros utilizados. Por lo tanto, la formulación de nanopartículas de Deferasirox mostró actividad antitumoral contra la línea celular de cáncer de mama humano (MCF7) según el resultado del estudio de la línea celular las nanopartículas de deferasirox fueron estables y podrían ser prometedoras para la administración de fármacos a las células cancerosas. (AU)

Effectiveness of Audio and Audio-Visual Distraction Aids for Management of Pain and Anxiety in Children and Adults Undergoing Dental Treatment- A Systematic Review And Meta-Analysis.

Dentists have a wide variety of techniques available to them such as tell -show-do, relaxation, distraction, systematic desensitisation, modelling, audio analgesia, hypnosis, and behaviour rehearsal. There is no concrete research as systematic review and meta-analysis indicating which explains the most effective distraction technique. AIM: To summarize effectiveness of audio and audio-visual (AV) distraction aids for management of pain and anxiety in children undergoing dental treatment. STUDY DESIGN: Literature search: PubMed/MEDLINE, DOAJ, Science Direct from June - July 2020 with randomized control clinical trials conducted on children with audio and AV distraction aids as intervention and those which had anxiety and pain as outcomes were searched. Fifty articles were identified and relevance was determined. 14 studies were included for qualitative synthesis and 05 were eligible for meta-analysis. Cochrane handbook used to assess the risk of bias. The meta analysis conducted using review manager 5.3 software. RESULTS: Meta-analysis, cumulative mean difference for audio and AV distraction techniques was calculated with main outcomes as pulse rate, O2 level, Vehman's picture and clinical test. These findings showed significant difference favoring the intervention (audio and AV) group when compared with control but indicating more effectiveness of AV distractions. CONCLUSION: Different audio-visual aids assist in reducing pain and anxiety in children but using audio distraction aids when audio-visual aids are not available could be acceptable way for distracting and treating children.

Reversal of genetic brain iron accumulation by N,N'-bis(2-mercaptoethyl)isophthalamide, a lipophilic metal chelator, in mice.

N,N'-bis(2-mercaptoethyl)isophthalamide (NBMI) is a novel lipophilic metal chelator and antioxidant used in mercury poisoning. Recent studies have suggested that NBMI may also bind to other metals such as lead and iron. Since NBMI can enter the brain, we evaluated if NBMI removes excess iron from the iron-loaded brain and ameliorates iron-induced oxidative stress. First, NBMI exhibited preferential binding to ferrous (Fe2+) iron with a negligible binding affinity to ferric (Fe3+) iron, indicating a selective chelation of labile iron. Second, NBMI protected SH-SY5Y human neuroblastoma cells from the cytotoxic effects of high iron. NBMI also decreased cellular labile iron and lessened the production of iron-induced reactive oxygen species in these cells. Deferiprone (DFP), a commonly used oral iron chelator, failed to prevent iron-induced cytotoxicity or labile iron accumulation. Next, we validated the efficacy of NBMI in Hfe H67D mutant mice, a mouse model of brain iron accumulation (BIA). Oral gavage of NBMI for 6 weeks decreased iron accumulation in the brain as well as liver, whereas DFP showed iron chelation only in the liver, but not in the brain. Notably, depletion of brain copper and anemia were observed in BIA mice treated with DFP, but not with NBMI, suggesting a superior safety profile of NBMI over DFP for long-term use. Collectively, our study demonstrates that NBMI provides a neuroprotective effect against BIA and has therapeutic potential for neurodegenerative diseases associated with BIA.

In vitro and in vivo anti-inflammatory activity of Tetrastigma sulcatum leaf extract, pure compound and its derivatives.

The severity and perseverance of inflammation have been demonstrated in many health conditions. The limitations of existing medications suggest the need for new alternative anti-inflammatory medications. In our earlier studies, we demonstrated the topical anti-inflammatory potential of the crude ethanolic extract of Tetrastigma sulcatum leaves and its fractions. In the present study, we further explored the anti-inflammatory activity of T. sulcatum extract, fractions, pure compound and its derivatives using in vitro and in vivo bioassay techniques. We attempted to isolate a pure compound from the leaf extract and identified it as a Friedelan-3ß-ol (CI). Furthermore, Friedelinol acetate (C II) and friedelinol methyl ether (C III), derivatives of Friedelan-3ß-ol (CI) were synthesised. LPS-induced inflammatory RAW 264.7 macrophages were used as in vitro model to study anti-inflammatory and anti-oxidative effects. Inflammation-induced oxidative damage was found to be restricted significantly (P < 0.001), with scavenging activity and increased SOD activity of crude extract and fractions. Treatment with crude extract (TSETOH) and fractions (TSHEX, TSTOL) significantly reduced (P < 0.001) the mRNA expression of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) and nitric oxide (NO) production in LPS-stimulated inflammation in RAW 264.7 cells in a dose-dependent manner. Likewise, compounds CI and CIII showed a similar pattern of significant inhibition (P < 0.001) of pro-inflammatory cytokines and NO production in a dose-dependent manner. An in vivo study in a carrageenan-induced mouse paw oedema model demonstrated reduced paw oedema and pro-inflammatory cytokines in a dose-dependent manner upon treatment with the extract, its fractions, pure compound (CI), and their derivatives (CII, and CIII). The present study confirmed the anti-inflammatory activity of T. sulcatum, suggesting that Friedelan-3ß-ol is an active component of the crude extract.

Islet Dysfunction in a Novel Transgenic Model of T Cell Insulitis.

The newly established CD3FLAG-mIR transgenic mouse model on a C57Bl/6 background has a FLAG tag on the mouse Insulin Receptor (mIR), specifically on T cells, as the FLAG-tagged mIR gene was engineered behind CD3 promoter and enhancer. The IR is a chemotactic molecule for insulin and the Flag-tagged mIR T cells in the BL/6-CD3FLAGmIR transgenic mice can migrate into the pancreas, as shown by immunofluorescent staining. While the transgenic mice do not become diabetic, there are phenotypic and metabolic changes in the islets. The transgenic islets become enlarged and disorganized by 15 weeks and those phenotypes continue out to 35 weeks of age. We examined the islets by RT-PCR for cell markers, ER stress markers, beta cell proliferation markers, and cytokines, as well as measuring serum insulin and insulin content in the pancreas at 15, 25, and 35 weeks of age. In transgenic mice, insulin in serum was increased at 15 weeks of age and glucose intolerance developed by 25 weeks of age. Passage of transgenic spleen cells into C57Bl/6 RAG-/- mice resulted in enlarged and disorganized islets with T infiltration by 4 to 5 weeks post-transfer, replicating the transgenic mouse studies. Therefore, migration of non-antigen-specific T cells into islets has ramifications for islet organization and function.

Neuroprotective effects of disubstituted dithiolethione ACDT against manganese-induced toxicity in SH-SY5Y cells.

Dithiolethiones are lipophilic, organosulfur compounds that activate the Nrf2 transcription factor causing an upregulation of various phase II antioxidant enzymes. A disubstituted dithiolethione 5-amino-3-thioxo-3H-(1,2) dithiole-4-carboxylic acid ethyl ester (ACDT) retains the functional pharmacophore while also containing modifiable functional groups. Neuroprotection against autoimmune encephalomyelitis in vivo and 6-hydroxy dopamine (a model for Parkinson's disease) in vitro have been previously reported with ACDT. Manganese (Mn) is a metal essential for metabolic processes at low concentrations. Overexposure and accumulation of Mn leads to a neurological condition called manganism which shares pathophysiological sequelae with parkinsonism. Here we hypothesized ACDT to be protective against manganese-induced cytotoxicity. SH-SY5Y human neuroblastoma cells exposed to 300 µM MnCl2 displayed approximately 50% cell death, and a 24-h pretreatment with 75 µM ACDT significantly reversed this cytotoxicity. ACDT pretreatment was also found to increase total GSH levels (2.18-fold) and the protein levels of NADPH:quinone oxidoreductase-1 (NQO1) enzyme (6.33-fold), indicating an overall increase in the cells' antioxidant defense stores. A corresponding 2.32-fold reduction in the level of Mn-induced reactive oxygen species was also observed in cells pretreated with ACDT. While no changes were observed in the protein levels of apoptotic markers Bax and Bcl-2, pretreatment with 75 µM ACDT led to a 2.09-fold downregulation of ZIP14 import transporter, indicating a potential reduction in the cellular uptake of Mn as an additional neuroprotective mechanism. These effects did not extend to other transporters like the divalent metal transporter 1 (DMT1) or ferroportin. Collectively, ACDT showed substantial neuroprotection against Mn-induced cytotoxicity, opening a path for dithiolethiones as a potential novel therapeutic option against heavy metal neurotoxicity.

Insulin Receptor-Expressing T Cells Appear in Individuals at Risk for Type 1 Diabetes and Can Move into the Pancreas in C57BL/6 Transgenic Mice.

Insulin receptor (IR) expression on the T cell surface can indicate an activated state; however, the IR is also chemotactic, enabling T cells with high IR expression to physically move toward insulin. In humans with type 1 diabetes (T1D) and the NOD mouse model, a T cell-mediated autoimmune destruction of insulin-producing pancreatic ß cells occurs. In previous work, when purified IR+ and IR- T cells were sorted from diabetic NOD mice and transferred into irradiated nondiabetic NOD mice, only those that received IR+ T cells developed insulitis and diabetes. In this study, peripheral blood samples from individuals with T1D (new onset to 14 y of duration), relatives at high-risk for T1D, defined by positivity for islet autoantibodies, and healthy controls were examined for frequency of IR+ T cells. High-risk individuals had significantly higher numbers of IR+ T cells as compared with those with T1D (p < 0.01) and controls (p < 0.001); however, the percentage of IR+ T cells in circulation did not differ significantly between T1D and control subjects. With the hypothesis that IR+ T cells traffic to the pancreas in T1D, we developed a (to our knowledge) novel mouse model exhibiting a FLAG-tagged mouse IR on T cells on the C57BL/6 background, which is not susceptible to developing T1D. Interestingly, these C57BL/6-CD3FLAGmIR/mfm mice showed evidence of increased IR+ T cell trafficking into the islets compared with C57BL/6 controls (p < 0.001). This transgenic animal model provides a (to our knowledge) novel platform for investigating the influence of IR expression on T cell trafficking and the development of insulitis.

Ballistic lobar trajectory outcomes in civilian firearm penetrating brain injury.

OBJECTIVE: Penetrating brain injury (PBI) is the most lethal of all firearm injuries, with reported survival rates of less than 20%. The projectile trajectory (PT) has been shown to impact mortality, but the significant lobar tracks have not been defined. The aim of this retrospective case-control study was to test for associations between distinct ballistic trajectories, missile types, and patient outcomes. METHODS: A total of 243 patients who presented with a PBI to the Saint Louis University emergency department from 2008 through 2019 were identified from the hospital registry. Conventional CT scans combined with 3D CT reconstructions and medical records were reviewed for each patient to identify distinct PTs. RESULTS: A total of 65 ballistic lobar trajectories were identified. Multivariable regression models were used, and the results were compared with those in the literature. Penetrating and perforating types of PBI associated with bitemporal (t-statistic = -2.283, p = 0.023) or frontal-to-contralateral parietal (t-statistic = -2.311, p = 0.025) projectile paths were universally found to be fatal. In the group in which the Glasgow Coma Scale (GCS) score at presentation was lower than 8, a favorable penetrating missile trajectory was one that involved a single frontal lobe (adjusted OR 0.02 [95% CI 0.00-0.38], p = 0.022) or parietal lobe (adjusted OR 0.15 [95% CI 0.02-0.97], p = 0.048). Expanding or fragmenting types of projectiles carry higher mortality rates (OR 2.53 [95% CI 1.32-4.83], p < 0.001) than do nondeformable missiles. Patient age was not associated with worse outcomes when controlled by other significant predictive factors. CONCLUSIONS: Patients with penetrating or perforating types of PBI associated with bitemporal or frontal-to-contralateral parietal PTs should be considered as potential donor candidates. Trauma patients with penetrating missile trajectories involving a single frontal or parietal lobe should be considered for early neurosurgical intervention, especially in the circumstances of a low GCS score (< 8). Surgeons should not base their decision-making solely on advanced patient age to defer further treatment. Patients with PBIs caused by nondeformable types of projectiles can survive multiple simultaneous intracranial missile trajectories.

The Value of White Matter Tractography by Diffusion Tensor Imaging in Altering a Neurosurgeon's Operative Plan.

OBJECTIVE: To investigate if the implementation of white matter (WM) fiber tractography by diffusion tensor imaging in presurgical planning for supratentorial tumors proximal to eloquent WM tracts can alter a neurosurgeon's operative strategy. METHODS: A retrospective review was conducted of patients with supratentorial brain tumors within eloquent WM tracts who underwent diffusion tensor imaging (DTI) tractography as part of their preoperative assessment. These patients were classified into 3 different DTI groups per the radiology reports: group 1, intact WM tracts; group 2, deviated and/or displaced WM bundles; and group 3, patients with an established WM injury (interrupted and/or destroyed tracts). A blinded prospective behavioral study followed, in which 4 neurosurgeons reviewed the preoperative images at 2 different times (magnetic resonance imaging without DTI, followed by a review of the DTI). They provided estimations about the DTI group of each individual eloquent WM category in every patient, and their planned surgical approach. RESULTS: Fifteen patients (mean age, 58.3 years) were included in the study. The neurosurgeons provided a correct DTI group estimation in 53%, 60%, and 57% of the cases that involved motor/sensory pathway tracts, optic tracts, and language tracts, respectively. The neurosurgeons underestimated DTI group 3 in the motor category and in the optic category 75% of the time. DTI did not alter the planned surgical approach. CONCLUSIONS: DTI WM tractography helped neurosurgeons to correctly identify patients with interrupted motor and optic pathway tracts so they could be more aggressive with the extent of tumor resection, despite its inability to alter the operative approach.

Development of a Bioconjugate Platform for Modifying the Immune Response of Autoreactive Cytotoxic T Lymphocytes Involved in Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disorder characterized by autoimmune cell mediated destruction of pancreatic beta cells. Pancreatic beta cells are the only source of insulin in the body. T1D patients then have to depend on insulin injections for their lifetime. Insulin injection can modulate the blood sugar levels, but insulin has little effect on the autoimmune process. Altered peptide ligands (APL) derived from known autoantigens in T1D are able to induce tolerance in autoreactive cells in T1D animal models, but are currently unable to elicit this protection in humans. There is a need to improve immunogenicity of the APLs, as these short peptides can be easily degraded by enzymes in the blood. GAD546-554 is a dominant epitope recognized by autoreactive T cells in the nonobese diabetic (NOD) mouse model that can cause destruction of beta cells. Alanine substitution at the eighth position of GAD546-554 peptide (APL9) induced tolerance in a GAD546-554 specific cytotoxic T lymphocyte clone. To improve the antigen presentation and endosomal escape of APL9, we developed a bioconjugate platform that consists of a liposome containing a bioconjugate of APL9 and toll-like receptor 2 ligand Pam3CysSK4 as well as an antibody against macrophage protein F4/80. APL9 bioconjugate liposome with F4/80 antibody was able to induce tolerance in a GAD 546-554 specific clone. Diabetic NOD splenocytes pretreated with APL9 bioconjugate were also not able to transfer diabetes into prediabetic NOD recipient mice. This work is beneficial to prevent T1D as an immunotherapy strategy to render autoreactive immune cells more tolerant of beta cells.

Reproducible bioinformatics project: a community for reproducible bioinformatics analysis pipelines.

BACKGROUND: Reproducibility of a research is a key element in the modern science and it is mandatory for any industrial application. It represents the ability of replicating an experiment independently by the location and the operator. Therefore, a study can be considered reproducible only if all used data are available and the exploited computational analysis workflow is clearly described. However, today for reproducing a complex bioinformatics analysis, the raw data and the list of tools used in the workflow could be not enough to guarantee the reproducibility of the results obtained. Indeed, different releases of the same tools and/or of the system libraries (exploited by such tools) might lead to sneaky reproducibility issues. RESULTS: To address this challenge, we established the Reproducible Bioinformatics Project (RBP), which is a non-profit and open-source project, whose aim is to provide a schema and an infrastructure, based on docker images and R package, to provide reproducible results in Bioinformatics. One or more Docker images are then defined for a workflow (typically one for each task), while the workflow implementation is handled via R-functions embedded in a package available at github repository. Thus, a bioinformatician participating to the project has firstly to integrate her/his workflow modules into Docker image(s) exploiting an Ubuntu docker image developed ad hoc by RPB to make easier this task. Secondly, the workflow implementation must be realized in R according to an R-skeleton function made available by RPB to guarantee homogeneity and reusability among different RPB functions. Moreover she/he has to provide the R vignette explaining the package functionality together with an example dataset which can be used to improve the user confidence in the workflow utilization. CONCLUSIONS: Reproducible Bioinformatics Project provides a general schema and an infrastructure to distribute robust and reproducible workflows. Thus, it guarantees to final users the ability to repeat consistently any analysis independently by the used UNIX-like architecture.

In vivo efficacy of biocompatible silver nanoparticles cream for empirical wound healing.

Wound healing is a complicated process that begins at the onset of injury and a continued process till complete healing. The emergence of nanotechnology has provided a new therapeutic modality to silver nanoparticles in treatment of wounds. However, the safety of these silver nanoparticles in the process of wound healing is yet to be elucidated; nevertheless, biocompatibility is the primary concern. Biosynthesis of silver nanoparticles was synthesized using aqueous clove extract and silver nitrate solution under microwave and the obtained particles size were 30-60 nm and roughly spherical in shape. The present study focused on the efficacy of biocompatible silver nanoparticles in vivo wound healing process. Consequently, this study supported the incorporation of biosynthesized silver nanoparticles in wound dressings as a cream formulation for improved healthcare.

Small non-coding RNA profiling in human biofluids and surrogate tissues from healthy individuals: description of the diverse and most represented species.

The role of non-coding RNAs in different biological processes and diseases is continuously expanding. Next-generation sequencing together with the parallel improvement of bioinformatics analyses allows the accurate detection and quantification of an increasing number of RNA species. With the aim of exploring new potential biomarkers for disease classification, a clear overview of the expression levels of common/unique small RNA species among different biospecimens is necessary. However, except for miRNAs in plasma, there are no substantial indications about the pattern of expression of various small RNAs in multiple specimens among healthy humans. By analysing small RNA-sequencing data from 243 samples, we have identified and compared the most abundantly and uniformly expressed miRNAs and non-miRNA species of comparable size with the library preparation in four different specimens (plasma exosomes, stool, urine, and cervical scrapes). Eleven miRNAs were commonly detected among all different specimens while 231 miRNAs were globally unique across them. Classification analysis using these miRNAs provided an accuracy of 99.6% to recognize the sample types. piRNAs and tRNAs were the most represented non-miRNA small RNAs detected in all specimen types that were analysed, particularly in urine samples. With the present data, the most uniformly expressed small RNAs in each sample type were also identified. A signature of small RNAs for each specimen could represent a reference gene set in validation studies by RT-qPCR. Overall, the data reported hereby provide an insight of the constitution of the human miRNome and of other small non-coding RNAs in various specimens of healthy individuals.

Structural and biophysical properties of h-FANCI ARM repeat protein.

Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.