Illegal wildlife trade is a great threat to the conservation efforts made worldwide to save wildlife species and their parts. Use of molecular methods, including DNA barcoding, is gaining acceptance to detect cross-border movement of endangered species. Here we report the utility of DNA barcoding in the detection of smuggling of an endangered turtle species from Pakistan. The consignment labeled as “fish meat” was intercepted at a Pakistani port and was tested for its source using DNA Barcoding with fish-specific primers. Sequences from the samples from this consignment matched (99%) with those from Lissemys punctata (Indian flap-shelled turtle), a species listed by the Convention on International Trade in Endangered Species (CITES). This report highlights the problem of smuggling protected species under false pretenses and the importance of DNA barcoding in stopping such illegal trade.
Human hair follicles are miniature hair growing organs. A common human hair disorder is androgenetic alopecia (AGA), which becomes a medical problem only when the hair loss is subjectively seen as excessive, premature, and distressing on the scalp. The objectives of the present study were to culture the hair stem cells in vitro and to study the morphology of the cultured cells for the treatment of AGA. The present study proposes that plucked human hairs are a cheap source to treat male baldness and in vitro culturing of hair follicle cells is the potential method to apply the cultured cells back into the balding scalp. It may be possible to create thousands of hair follicles from that original follicle. In this study human hair follicle cells of normal and AGA male groups were taken by plucking the hair follicle cells. The hair follicles cells of normal and AGA were cultured in vitro without a feeder layer, as the feeder layer has many drawbacks. The plucked hair follicles which were in the anagen stage were selected from both groups. These hair follicles were digested by enzymatic disaggregation using trypsin/EDTA. Then these cells were cultured in a FAD medium (Dulbecco's Modified Eagle Medium and Ham's F 12 medium, 3:1) plus a 17% serum and incubated in a CO2 Incubator at 37 °C in 5% CO2 without a feeder layer. The whole procedure was performed under sterile conditions. The morphology of cultured and subcultured cells was observed daily under a phase contrast microscope for 14 days. The viable cultured cells of both groups refracted light, while dead cells appeared black. Keratinocytes appeared after 24 hours, Melanocytes appeared at 48 hours, and stem cells appeared in 7 to 10 days. Shelf life of cultured and subcultured cells of normal and AGA group was 12 and 7 days, respectively. Live cell counting was done by using an improved Neubauer chamber. DNA extraction and optical density (OD) assay of cultured and subcultured cells was undertaken using the plucked hair follicles of normal and AGA human male subject. The plucked human hair follicles cells were harvested and cultured successfully without a feeder layer. Their genomic DNA was extracted successfully. This hair cloning technique is an alternative to the usual method of hair transplantation. The most positive aspect of the new technique compared to hair transplantation is the preservation of the 'donor hair area'. This technique will be cheaper and more 'patient friendly'.
Cytochrome c oxidase subunit I or COI in the mitochondrial genome (DNA Barcode) of goats was used to identify and differentiate two common breeds (beetal and berberi) and crossbreeds sampled in Punjab, Pakistan. This is the first study on the molecular taxonomy of the goat breeds of Pakistan. Sequencing of DNA barcode of the beetal goat showed a 99% similarity to Capra hircus isolate LS16 cytochrome c oxidase subunit 1 (CO1) gene. Beriberi goats showed a 99% similarity to Capra hircus breed Jining Qing goat mitochondrion. Identification of goat breeds via DNA barcoding may help in local genetic improvement and conservation programs, especially in pre-screening important breeds that can be considered for conservation in their pure form. However, more COI sequences should be determined from the native goat populations of Pakistan to improve reliability of using DNA barcodes to differentiate them from their exotic counterparts. Thus, the present study concluded that DNA barcoding can be used to confirm the species or breed origin of an unknown specimen and it is a reliable and practical tool to protect the local biodiversity of livestock genetic resources. Our results validated the effectiveness of barcoding for the identification of goat breeds.
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