Mesenchymal stem cell in venous leg ulcer: An intoxicating therapy.

Venous leg ulcers (VLU) are a prevalent and reoccurring type of complicated wound, turning as a considerable public healthcare issue, with critical social and economic concern. There are both medical and surgical therapies to treat venous leg ulcers; however, a cure does not yet exist. Mesenchymal stem cells (MSC) are capable and proved of accelerating wound healing in vivo and their study with human chronic wounds is currently awaited. MSCs are a promising source of adult progenitor cells for cellular therapy and have been demonstrated to differentiate into various mesenchymal cell lineages. They have a crucial and integral role in native wound healing by regulating immune response and inflammation. Improved understanding of the cellular and molecular mechanisms at work in delayed wound healing compels to the development of cellular therapy in VLU. This review focuses on the current treatment option of VLU and further emphasizing the role of MSCs in accelerating the healing process. With further understanding of the mechanism of action of these cells in wound improvement and, the involvement of cytokines can also be revealed that could be used for the therapeutic purpose for VLU healing. Clinical uses of MSCs have been started already, and induced MSCs are surely a promising tool or compelling therapy for VLU.

Structure elucidation and in silico docking studies of a novel furopyrimidine antibiotics synthesized by endolithic bacterium Actinomadura sp. AL2.

On screening of endolithic actinobacteria from a granite rock sample of Meghalaya for antibacterial compound, a novel antibacterial compound CCp1 was isolated from the fermentation broth of Actinomadura sp. AL2. On purification of the compound based on chromatographic techniques followed by characterization with FT-IR, UV-visible, 1H NMR, 13C NMR and mass spectrometry, the molecular formula of the compound was generated as C20H17N3O2, a furopyrimidine derivative. In vitro antibacterial activity of the compound was evaluated against both Gram positive and negative bacteria by agar well diffusion assay. The compound had lowest MIC (2.00 µg/ml) for Bacillus subtilis and highest MIC (> 64 µg/ml) for Staphylococcus epidermidis and Pseudomonas aeruginosa. The study revealed that the compound has potential antibacterial activity. The mode of action of the antibacterial compound was evaluated through in silico studies for its ability to bind DNA gyrase, 30S RNA molecules, OmpF porins and N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU). The antibacterial compound demonstrated more favorable docking with DNA gyrase, 30S RNA molecules and OmpF porins than GlmU which support the antibacterial compound CCp1 can be as a promising broad spectrum antibiotic agent with "multitarget" characteristics.

Short-duration judgment in young Indian subjects under 30 h constant wakefulness.

The present study aimed to investigate probability of a possible endogenous circadian rhythm in human cognitive attribute to estimate short intervals. Apparently healthy young males and females were selected for our study. Eight subjects prospectively produced the short-time intervals 10 s and 60 s at 2 hourly intervals in 30 h constant routine (CR) study conducted in spring (CR-1). The study was repeated again in autumn (CR-2) in the remaining eight subjects. The established circadian markers, namely serum cortisol, salivary melatonin levels and tympanic temperature were also measured either in CR-1 or CR-2. Oral temperature was measured simultaneously. Circadian rhythms were validated in serum cortisol, salivary melatonin, oral, and tympanic temperatures. Circadian rhythm in 60 s estimates was observed in a few subjects and in all males at group level in CR-1. The cognitive attribute to perceive short intervals vary as function of season. The results provide evidence in support of interaction among the interval, circadian and circannual timing systems in human.

Association of Inflammatory Biomarker C-Reactive Protein, Lipid Peroxidation and Antioxidant Capacity Marker with HbF Level in Sickle Cell Disease Patients from Chattisgarh.

This study was undertaken to determine the association of inflammatory biomarker, oxidative stress and antioxidant capacity marker with fetal haemoglobin (HbF) level among sickle cell trait and sickle cell disease (SCD) patients in Chattisgarh. The study group consisted of 51 SCD (SS) patients with painful episode, 49 SCD (SS) patients with steady state, 50 sickle cell trait (AS) and 50 controls. Malondialdehyde (MDA), CRP, total antioxidant power (FARP), total thiol and HbF levels were quantified. We found a significant positive (p < 0.0001) association between CRP and MDA levels and its inverse association with HbF level in SS patients. We also observed that antioxidant capacity had significantly positively (p < 0.0001) associated with HbF level. The protective effect of HbF was found, because the increase in HbF levels resulted in decrease in lipid peroxidation and inflammation in SCD patients. A decrease in the HbF level and its antioxidant capacity has been associated with the pathogenesis of SCD. These finding may explain the high level of HbF is ameliorating oxidative stress and inflammation in SCD patients.

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

Enhanced chondrogenesis of mesenchymal stem cells over silk fibroin/chitosan-chondroitin sulfate three dimensional scaffold in dynamic culture condition.

Chondroitin sulfate (Ch) is one of the main structural components of cartilage tissue, therefore, its presence in tissue engineered scaffold is expected to enhance cartilage regeneration. Previously, silk fibroin/chitosan (SF/CS) blend was proven to be a potential biomaterial for tissue development. In this study, the effect of Ch on physicochemical and biological properties of SF/CS blend was investigated and scaffolds with 0.8 wt% Ch was found to be favorable. The scaffolds possess pore size of 37-212 µm, contact angle 46.2-50.3°, showed controlled swelling and biodegradation. The biocompatibility of scaffold was confirmed by subcutaneous implantation in mouse. Human mesenchymal stem cells (hMSCs) seeded scaffolds cultured under spinner flask bioreactor promoted cell attachment, proliferation, distribution, and metabolic activity in vitro. The histology and immunofluorescence studies revealed that combined effect of Ch and dynamic condition resulted in higher glycosaminoglycan secretion and native cartilage type matrix synthesis in comparison to SF/CS scaffolds used as control. Higher expression of collagen-II, Sox9, aggrecan and decrease in collagen-I expression represented by quantitative polymerase chain reaction study confirmed the progression of chondrogenic differentiation. This study successfully demonstrates the potentiality of SF/CS-Ch scaffold for hMSCs recruitment and redirecting cartilage tissue regeneration with enhanced chondrogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2576-2587, 2018.

Assessment of Serum Vascular Endothelial Growth Factor Levels in Pregnancy-Induced Hypertension Patients.

Objective. The objective of the study was to assess the serum vascular endothelial growth factor (VEGF) levels in peripheral blood of patients with pregnancy-induced hypertension (PIH) and find association between serum VEGF levels and PIH. Methods. Thirty-five PIH subjects, 35 normal pregnant females, and 20 normal healthy females were included in the study. Detailed history, clinical examination, and relevant biochemical parameters were assessed; serum VEGF levels were estimated using Double-antibody enzyme-linked immunosorbent assay. Results. The study groups were found to be age matched (p = 0.38). VEGF level in the pregnancy-induced hypertensive group (median = 109.19 (3.38 ± 619)) was significantly higher than the normal pregnant (median = 20.82 (1.7-619)) and control (median = 4.92 (1.13-13.07)) group and the difference between these three groups was significant (p < 0.0001). The 3 groups are found to be significantly different in terms of RBS (p = 0.01), urea (p < 0.0001), creatinine (p = 0.0005), AST (p = 0.0032), ALT (p = 0.0007), total protein (p = 0.0004), albumin (p < 0.0001), calcium (p = 0.001), and sodium (p = 0.02), while no statistically significant difference was found between total bilirubin (p = 0.167), direct bilirubin (p = 0.07), uric acid (p = 0.16), and potassium (p = 0.14). Conclusion. Significantly higher levels of serum VEGF were noted in PIH subjects compared to normal pregnant and control subjects.

Autophagy Activation Alleviates Amyloid-ß-Induced Oxidative Stress, Apoptosis and Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Autophagy is an evolutionary conserved catabolic process that ensures continuous removal of damaged cell organelles and long-lived protein aggregates to maintain cellular homeostasis. Although autophagy has been implicated in amyloid-ß (Aß) production and deposition, its role in pathogenesis of Alzheimer's disease remains elusive. Thus, the present study was undertaken to assess the cytoprotective and neuroprotective potential of autophagy on Aß-induced oxidative stress, apoptosis and neurotoxicity in human neuroblastoma SH-SY5Y cells. The treatment of Aß1-42 impaired the cell growth and redox balance, and induced apoptosis and neurotoxicity in SH-SY5Y cells. Next, the treatment of rapamycin (RAP) significantly elevated the expression of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), sequestosome-1/p62, Beclin-1, and unc-51-like kinase-1 (ULK1) in SH-SY5Y cells. RAP-induced activation of autophagy notably alleviated the Aß1-42-induced impairment of redox balance by decreasing the levels of pro-oxidants such as reactive oxygen species, lipid peroxidation and Ca2+ influx, and concurrently increasing the levels of antioxidant enzymes such as superoxide dismutase and catalase. The RAP-induced autophagy also ameliorated Aß1-42-induced loss of mitochondrial membrane potential and apoptosis. Additionally, the activated autophagy provided significant neuroprotection against Aß1-42-induced neurotoxicity by elevating the expression of neuronal markers such as synapsin-I, PSD95, NCAM, and CREB. However, 3-methyladenine treatment significantly exacerbated the neurotoxic effects of Aß1-42. Taken together, our study demonstrated that the activation of autophagy provided possible neuroprotection against Aß-induced cytotoxicity, oxidative stress, apoptosis, and neurotoxicity in SH-SY5Y neuronal cells.

Assessment of renal function in Indian patients with sickle cell disease.

Sickle cell disease (SCD) and its variants are genetic disorders resulting from the presence of a mutated form of hemoglobin. Renal disease is one of the most frequent complications, and kidney damage starts very early and progresses throughout life causing severe complications. The present study is aimed to analyze creatinine-based estimated glomerular filtration rate (eGFR) in 616 SCD patients (507 HbSS and 109 HbSB+), receiving medical care at outpatient wing of Sickle Cell Institute, Chhattisgarh. Glomerular filtration rate (GFR) estimated using the Modification of Diet in Renal Disease (MDRD), Cockcroft-Gault, chronic kidney disease epidemiology collaboration (CKD-EPI) (<17 years analyzed with Schwartz), and SCD specific Jamaica Sickle Cell Cohort Study (JSCCS)-GFR equations were compared. Further, eGFR calculated using the CKD-EPI and Schwartz equations was used to define various stages of kidney function and compared with clinical and hematological variables. The mean age of patients was 15.8 years. Comparison of eGFR using various formulas revealed that MDRD and JSCCS formulas overestimated the GFR. Among SCD patients, prevalence of glomerular hyperfiltration (GHF) is high followed by renal insufficiency (RI) and renal failure (RF). However, no differences were found in hematological profiling among different functional stages of kidney. Age and body surface area are significantly more in SCD individuals with normal kidney function and GHF. Participants with RF showed a higher level of blood urea and fetal hemoglobin. In summary, this is the first study to analyze different functional stages of kidney among SCD patients of India. Our study revealed that the GHF and RI are the important indicators of kidney damage.

Crosstalk between Substrates and Rho-Associated Kinase Inhibitors in Cryopreservation of Tissue-Engineered Constructs.

It is documented that human mesenchymal stem cells (hMSCs) can be differentiated into various types of cells to present a tool for tissue engineering and regenerative medicine. Thus, the preservation of stem cells is a crucial factor for their effective long-term storage that further facilitates their continuous supply and transportation for application in regenerative medicine. Cryopreservation is the most important, practicable, and the only established mechanism for long-term preservation of cells, tissues, and organs, and engineered tissues; thus, it is the key step for the improvement of tissue engineering. A significant portion of MSCs loses cellular viability while freeze-thawing, which represents an important technical limitation to achieving sufficient viable cell numbers for maximum efficacy. Several natural and synthetic materials are extensively used as substrates for tissue engineering constructs and cryopreservation because they promote cell attachment and proliferation. Rho-associated kinase (ROCK) inhibitors can improve the physiological function and postthaw viability of cryopreserved MSCs. This review proposes a crosstalk between substrate topology and interaction of cells with ROCK inhibitors. It is shown that incorporation of ionic nanoparticles in the presence of an external electrical field improves the generation of ROCK inhibitors to safeguard cellular viability for the enhanced cryopreservation of engineered tissues.

Selaginella bryopteris Aqueous Extract Improves Stability and Function of Cryopreserved Human Mesenchymal Stem Cells.

The effective long-term cryopreservation of human mesenchymal stem cells (MSCs) is an essential prerequisite step and represents a critical approach for their sustained supply in basic research, regenerative medicine, and tissue engineering applications. Therefore, attempts have been made in the present investigation to formulate a freezing solution consisting of a combination of Selaginella bryopteris water-soluble extract with and without dimethyl sulfoxide (Me2SO) for the efficient long-term storage of human umbilical cord blood- (hUCB-) derived MSCs. The cryopreservation experiment using the formulated freezing solution was further performed with hUCB MSCs in a controlled rate freezer. A significant increase in postthaw cell viability and cell attachment of MSCs was achieved with freezing medium containing Selaginella bryopteris water extract along with 10% Me2SO as compared to the freezing medium containing Me2SO (10% v/v) alone. Furthermore, the decreasing apoptotic events and reactive oxygen species production along with increasing expression of heat shock proteins also confirmed the beneficial effect of Selaginella bryopteris water extract. The beneficial effect of Selaginella bryopteris water extract was validated by its ability to render postpreservation high cell viability. In conclusion, the formulated freezing solution has been demonstrated to be effective for the standardization of cryopreservation protocol for hMSCs.

Implementation of Indigenous Electronic Medical Record System to Facilitate Care of Sickle Cell Disease Patients in Chhattisgarh.

INTRODUCTION: Sickle cell disease (SCD) is prevalent in central India including Chhattisgarh. Screening for SCD is being carried out by Government of Chhattisgarh. Electronic Medical Record (EMR) system was developed and implemented in two phases. AIM: Aim was to use informatics techniques and indigenously develop EMR system to improve the care of SCD patients in Chhattisgarh. EMR systems had to be developed to store and manage: i) huge data generated through state wide screening for SCD; ii) clinical data for SCD patients attending the outpatient department (OPD) of institute. MATERIALS AND METHODS: 'State Wide Screening Data Interface' (SWSDI) was designed and implemented for storing and managing data generated through screening program. Further, 'Sickle Cell Patients Temporal Data Management System' (SCPTDMS) was developed and implemented for storing, managing and analysing sickle cell disease patients' data at OPD. Both systems were developed using VB.Net and MS SQL Server 2012. RESULTS: Till April 2015, SWSDI has data of 1294558 persons, out of which 121819 and 4087 persons are carriers and patients of sickle cell disease respectively. Similarly till June 2015, SCPTDMS has data of 3760 persons, of which 923 are sickle cell disease patients (SS) and 1355 are sickle cell carriers (AS). CONCLUSION: Both systems are proving to be useful in efficient storage, management and analysis of data for clinical and research purposes. The systems are an example of beneficial usage of medical informatics solutions for managing large data at community level.

Identification of the Rare, Four Repeat Allele of IL-4 Intron-3 VNTR Polymorphism in Indian Populations.

BACKGROUND: Cytokines are cell signaling molecules which upon release by cells facilitate the recruitment of immune-modulatory cells towards the sites of inflammation. Genetic variations in cytokine genes are shown to regulate their production and affect the risk of infectious as well as autoimmune diseases. Intron-3 of interleukin-4 gene (IL-4) harbors 70-bp variable number of tandem repeats (VNTR) that may alter the expression level of IL-4 gene. OBJECTIVE: To determine the distribution of IL-4 70-bp VNTR polymorphism in seven genetically heterogeneous populations of Chhattisgarh, India and their comparison with the finding of other Indian and world populations. METHODS: A total of 371 healthy unrelated individuals from 5 caste and 2 tribal populations were included in the present study. The IL-4 70-bp VNTR genotyping was carried out using PCR and electrophoresis. RESULTS: Overall, 3 alleles of IL-4 70-bp VNTR (a2, a3 and a4) were detected. The results demonstrated the variability of the IL-4 70-bp VNTR polymorphism in Chhattisgarh populations. Allele a3 was the most common allele at the 70-bp VNTR locus in all populations followed by a2 allele. This study reports the presence four repeat allele a4 at a low frequency in the majority of the Chhattisgarh populations studied. Further, the frequency of the minor allele (a2) in Chhattisgarh populations showed similarity with the frequencies of European populations but not with the East Asian populations where the a2 allele is a major allele. CONCLUSIONS: Our study provides a baseline for future research into the role of the IL-4 locus in diseases linked to inflammation in Indian populations.

Enhanced cryopreservation of MSCs in microfluidic bioreactor by regulated shear flow.

Cell-matrix systems can be stored for longer period of time by means of cryopreservation. Cell-matrix and cell-cell interaction has been found to be critical in a number of basic biological processes. Tissue structure maintenance, cell secretary activity, cellular migration, and cell-cell communication all exist because of the presence of cell interactions. This complex and co-ordinated interaction between cellular constituents, extracellular matrix and adjacent cells has been identified as a significant contributor in the overall co-ordination of tissue. The prime objective of this investigation is to evaluate the effects of shear-stress and cell-substrate interaction in successful recovery of adherent human mesenchymal-stem-cells (hMSCs). A customized microfluidic bioreactor has been used for the purpose. We have measured the changes in focal-point-adhesion (FPAs) by changing induced shear stress inside the bioreactor. The findings indicate that with increase in shear stress, FPAs increases between substrate and MSCs. Further, experimental results show that increased FPAs (4e-3 µbar) enhances the cellular survivability of adherent MSCs. Probably, for the first time involvement of focal point interaction in the outcome of cryopreservation of MSCs has been clarified, and it proved a potentially new approach for modification of cryopreservation protocol by up-regulating focal point of cells to improve its clinical application.

Recent Advances and Future Direction in Lyophilisation and Desiccation of Mesenchymal Stem Cells.

Mesenchymal Stem Cells (MSCs) are a promising mammalian cell type as they can be used for the reconstruction of human tissues and organs. MSCs are shown to form bone, cartilage, fat, and muscle-like cells under specific cultivation conditions. Current technology of MSCs cryopreservation has significant disadvantages. Alternative technologies of mammalian cells preservation through lyophilisation or desiccation (air-drying) are among the upcoming domains of investigation in the field of cryobiology. Different protectants and their combinations were studied in this context. Loading of the protectant in the live cell can be a challenging issue but recent studies have shown encouraging results. This paper deals with a review of the protectants, methods of their delivery, and physical boundary conditions adopted for the desiccation and lyophilisation of mammalian cells, including MSCs. A hybrid technique combining both methods is also proposed as a promising way of MSCs dry preservation.

Fetal Haemoglobin and ß-globin Gene Cluster Haplotypes among Sickle Cell Patients in Chhattisgarh.

BACKGROUND: Foetal Haemoglobin (HbF) is the best-known genetic modulator of sickle cell anaemia, which varies dramatically in concentration in the blood of these patients. The patients with SCA display a remarkable variability in the disease severity. High HbF levels and the ß-globin gene cluster haplotypes influence the clinical presentation of sickle cell disease. To identify the genetic modifiers which influence the disease severity, we conducted a ß-globin haplotype analysis in the sickle cell disease patients of Chhattisgarh. AIM: The foetal haemoglobin and the ß-globin gene haplotypes of the sickle cell trait and the sickle cell disease patients from Chhattisgarh were investigated. MATERIALS AND METHOD: A total of 100 sickle cell patients (SS), 50 sickle cell trait patients (AS) and 50 healthy control individuals were included in the present study. The distribution of the ß-globin gene haplotype was done by the PCR-RFLP method. RESULT: PCR-RFLP showed that the homozygous Arab-Indian haplotype (65%) was the most frequent one, followed by the heterozygous Arab-Indian haplotype (11%) in the sickle cell patients (SS), while the AS patients had a higher frequency of the heterozygous Arab-Indian haplotype (38%) in comparison to homozygous one (32%). Four atypical haplotypes, 3 Benin and 1 Cameroon were also observed, although they were in lower frequencies. In the present study, the HbF levels were higher in the AS and the SS patients, with one or two Arab-Indian haplotypes as compared to the other haplotypes. CONCLUSION: The presence of the Arab-Indian haplotype as the predominant haplotype might be suggestive of a gene flow to/from Saudi-Arabia or India and it was associated with higher HbF levels and a milder disease severity.

Association between XmnI Polymorphism and HbF Level in Sickle Cell Disease Patients from Chhattisgarh.

The γ(G)-158 (C→T) polymorphism plays important function in the disease severity of sickle cell anemia. The XmnI restriction site at -158 position of the γ(G)-gene is associated with increased expression of the γ(G)-globin gene and higher production of HbF. This study aims to determine the frequency of the different genotypes of the γ(G) Xmn I polymorphism in sickle cell anemia and sickle cell trait patients in Chhattisgarh and its association with high HbF level. The Xmn1 polymorphic site was determined by PCR-RFLP procedure. XmnI polymorphism were studied in 100 sickle cell patients (SS), 50 sickle cell trait (AS) and 50 controls individuals (AA). The presence of XmnI (+/+) site in SS and AS patients associated with the increase of HbF (P<0.0001) synthesis. we also find that presence of one XmnI (+/-) site in SS patients compared with XmnI-/- site had not shows difference in HbF level. Polymorphic association is found between presence and absence of XmnI site with HbF level, in AS and AA individuals.

Deep rooting in-situ expansion of mtDNA Haplogroup R8 in South Asia.

BACKGROUND: The phylogeny of the indigenous Indian-specific mitochondrial DNA (mtDNA) haplogroups have been determined and refined in previous reports. Similar to mtDNA superhaplogroups M and N, a profusion of reports are also available for superhaplogroup R. However, there is a dearth of information on South Asian subhaplogroups in particular, including R8. Therefore, we ought to access the genealogy and pre-historic expansion of haplogroup R8 which is considered one of the autochthonous lineages of South Asia. METHODOLOGY/PRINCIPAL FINDINGS: Upon screening the mtDNA of 5,836 individuals belonging to 104 distinct ethnic populations of the Indian subcontinent, we found 54 individuals with the HVS-I motif that defines the R8 haplogroup. Complete mtDNA sequencing of these 54 individuals revealed two deep-rooted subclades: R8a and R8b. Furthermore, these subclades split into several fine subclades. An isofrequency contour map detected the highest frequency of R8 in the state of Orissa. Spearman's rank correlation analysis suggests significant correlation of R8 occurrence with geography. CONCLUSIONS/SIGNIFICANCE: The coalescent age of newly-characterized subclades of R8, R8a (15.4+/-7.2 Kya) and R8b (25.7+/-10.2 Kya) indicates that the initial maternal colonization of this haplogroup occurred during the middle and upper Paleolithic period, roughly around 40 to 45 Kya. These results signify that the southern part of Orissa currently inhabited by Munda speakers is likely the origin of these autochthonous maternal deep-rooted haplogroups. Our high-resolution study on the genesis of R8 haplogroup provides ample evidence of its deep-rooted ancestry among the Orissa (Austro-Asiatic) tribes.