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: Pedicled flaps are still the workhorse flaps for reconstruction of upper limb soft tissue defects in many centers across the world. They are lifeboat options for coverage in vessel deplete wounds. In spite of their popularity existing algorithms are limited to a particular region of upper limb; a general algorithm involving entire upper limb which helps in clinical decision making is lacking. We attempt to propose one for the day to day clinical practice. METHODS: A retrospective analysis of patients who underwent pedicled flaps for coverage of post-traumatic upper extremity (arm, elbow, forearm, wrist & hand) soft tissue defects within the period of January 2016 to October 2017 was performed. Patients were divided into groups according to the anatomical location of the defects. The flaps performed for different anatomical regions were enlisted. Demographic data and complications were recorded. An algorithm was proposed based on our experience, with a particular emphasis made to approach to clinical decision making. RESULTS: Two hundred and twelve patients were included in the study. Mean age was 27.3 years (range: 1-80 years), 180 were male, and 32 were female. Overall flap success rate was 98%, the following complications were noted marginal flap necrosis requiring no additional procedure other than local wound care in 32 patients (15%), partial flap necrosis requiring flap advancement or extra flap in 15 patients (7%), surgical site infection in 11 patients (5%), flap dehiscence requiring re-suturing in 5 patients (2.4%), total flap necrosis 4 patients (2%). CONCLUSION The proposed algorithm allows a reliable and consistent method for addressing diverse soft tissue defects in the upper limb with high success rate.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Algorithms ; Child ; Child, Preschool ; Clinical Decision-Making ; Female ; Humans ; Infant ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; methods ; Retrospective Studies ; Soft Tissue Injuries ; surgery ; Surgical Flaps ; Treatment Outcome ; Upper Extremity ; Young Adult