Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased, and cost-effective method for the isolation of high molecular weight (>23 kb) metagenomic DNA (260/280 ratio >1.8) with a good yield (55.8 ± 3.8 ng/mg of feces). We also confirm that there is very low human genomic DNA contamination (eubacterial:human genomic DNA marker genes=227.9:1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16S rRNA gene sequencing using 454 FLX+. Moreover, 16S rRNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richness and does not show microbial diversity biases, which is further confirmed by qPCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality, amount, user-friendliness, and cost effectiveness for its suitability toward usage for cultureindependent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.

PURPOSE: The p38 mitogen-activated protein kinase (MAPKs) play a crucial role in the production of pro-inflammatory cytokines and over-expression of it increase cytokines which promote cancer. Among four isoforms, p38α has been well studied in head and neck squamous cell carcinoma (HNSCC) and other cancers as a therapeutic target. p38δ has recently emerged as a potential disease-specific drug target. Elevated serum p38α level in HNSCC was reported earlier from our lab. This study aims to estimate the levels of p38 MAPK-isoforms in the serum of HNSCC and design peptide inhibitor targeting the same. MATERIALS AND METHODS: Levels of p38 MAPK isoforms in the serum of HNSCC and healthy controls were quantified by surface plasmon resonance technology. The peptide inhibitor for p38 MAPK was designed by molecular modeling using Grid-based Ligand Docking with Energetics tools and compared with known specific inhibitors. RESULTS: We have observed highly elevated levels of all four isoforms of p38 MAPK in serum of HNSCC patients compared to the control group. Further, serum p38α, p38β, and p38δ levels were down regulated after therapy in follow-up patients, while p38γ showed no response to the therapy. Present study screened designed peptide WFYH as a specific inhibitor against p38δ. The specific inhibitor of p38δ was found to have no effect on p38α due to great structural difference at ATP binding pocket. CONCLUSION: In this study, first time estimated the levels of p38 MAPK isoforms in the serum of HNSCC. It can be concluded that p38 MAPK isoforms can be a diagnostic and prognostic marker for HNSCC and p38δ as a therapeutic target.
Adenosine Triphosphate ; Carcinoma, Squamous Cell* ; Cytokines ; Epithelial Cells* ; Follow-Up Studies ; Head* ; Humans ; Models, Molecular ; Neck* ; p38 Mitogen-Activated Protein Kinases ; Protein Isoforms* ; Protein Kinases ; Surface Plasmon Resonance

BACKGROUND: Stem cell units (SCUs) that are cryopreserved prior to both autologous and allogeneic hematopoietic stem cell transplants (for donor lymphocyte infusion) remain unused or partially used several times, and become an increased burden to blood banks/SCU repositories. Because of the scarcity of data regarding the duration for which the storage is useful, there is no general consensus regarding disposal of SCUs. METHODS: We conducted a retrospective audit of SCU utilization in 435 patients who planned to undergo either autologous stem cell transplantation (auto-SCT) (N=239) or allogeneic stem cell transplantation (allo-SCT) (N=196) at a tertiary cancer care center between November 2007 to January 2015. RESULTS: Our cohort consisted of 1,728 SCUs stored for conducting auto-SCT and 729 SCUs stored for conducting donor lymphocyte infusions (DLIs) after allo-SCT. Stem cells were not infused in 12.5% of patients who had planned to undergo auto-SCT, and 80% of patients who underwent allo-SCT never received DLI. Forty-one percent of SCUs intended for use in auto-SCT remained unutilized, with a second auto-SCT being performed only in 4 patients. Ninety-four percent of SCUs intended for carrying out DLIs remained unused, with only minimal usage observed one year after undergoing allo-SCT. CONCLUSION: The duration of storage of unused SCUs needs to be debated upon, so that a consensus can be reached regarding the ethical disposal of SCU.
Cohort Studies ; Consensus ; Cryopreservation ; Developing Countries* ; Hematopoietic Stem Cell Transplantation* ; Hematopoietic Stem Cells* ; Humans ; Lymphocytes ; Retrospective Studies ; Stem Cell Transplantation ; Stem Cells* ; Tissue Donors

PURPOSE: Defects around the distal one third of the leg and ankle are difficult to manage by conservative measures or simple split thickness skin graft. Distally based peroneus brevis muscle flap is a well described flap for such defects. METHODS: This is a retrospective analysis conducted on 25 patients with soft tissue and bony defects of distal third of lower leg and ankle, which were treated using distally based peroneus brevis muscle flap from January 2013 to January 2018. Information regarding patient demographics, etiology, size and location of defects and complications were collected. All patients were followed up for at least 3 months after surgery. RESULTS: There were 21 males and 4 females with the mean age of 39 (5-76) years. The most common cause of injuries was road traffic accident, followed by complicated open injury. The average size of defects was 20 (4-50) cm. The mean operating time was 75 (60-90) min for flap harvest and inset. We had no patient with complete loss of the flap. Five patients (20%) had marginal necrosis of the flap and two patients have graft loss due to underlying hematoma and required secondary split thickness skin grafting. CONCLUSION The distally based peroneus brevis muscle flap is a safe option with reliable anatomy for small to moderate sized defects following low velocity injury around the ankle. The commonest complication encountered is skin graft loss which can be reduced by primary delayed grafting.
Adolescent ; Adult ; Aged ; Ankle Injuries ; surgery ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Humans ; Leg Injuries ; surgery ; Male ; Middle Aged ; Muscle, Skeletal ; Operative Time ; Retrospective Studies ; Surgical Flaps ; Tissue and Organ Harvesting ; Treatment Outcome ; Young Adult