Biology subjects

Karim, A.

Publications and source records attributed to Karim, A..

3 recordsLinked to original sources

bioRxiv2020

Signature molecular changes in the skeletal muscle of hindlimb unloaded mice

Hind-limb unloaded (HU) mouse is a well-recognized model of muscle atrophy; however the molecular changes in the skeletal muscle during unloading are poorly characterized. We have used Fourier transform infrared (FTIR) and Raman spectroscopy to evaluate the structure and behavior of signature molecules involved in regulating muscle structural and functional health. The FTIR and the Raman spectroscopic analysis of gastrocnemius muscles was compared between 16-18 weeks old HU c57Bl/6J mice and ground-based controls. The molecular components of the samples were identified by using the FTIR spectra from the control and the unloaded samples. The Raman spectra showed that the signals for asparagine and glutamine were reduced in HU mice, possibly indicating increased catabolism. The peaks for hydroxyproline and proline were split, pointing towards molecular breakdown and reduced tendon repair. We also report a consistently increased intensity in > 1300 cm-1 range in the Raman spectra along with a shift towards higher frequencies in the HU mice, indicating activation of sarcoplasmic reticulum (SR) stress during HU. SIGNIFICANCEMouse model of hindlimb unloading recapitulates many features of disuse muscle atrophy due to spaceflight and prolonged bed rest. However, a thorough understanding of molecular changes underlying muscle detriment in disuse partly remains elusive. We have applied the spectroscopic and density functional techniques conjointly to characterize the molecular changes in the skeletal muscle of hindlimb unloaded mice. A number of conformational changes and the breakdown of the molecular bonds in the skeletal muscle are observed, which can potentially dictate loss of muscle mass and strength during mechanical unloading. Our reporting of the signature spectral changes in the unloaded skeletal muscle can be a useful step towards the therapeutic interventions targeting specific molecules.

biophysics↗

bioRxiv2020

Modelling early thermal injury using an ex vivo human skin model of contact burns

BackgroundEarly mechanisms underlying the progressive tissue death and the regenerative capability of burn wounds are understudied in human skin. A clinically relevant, reproducible model for human burn wound healing is needed to elucidate the early changes in the human burn wound environment. This study reports a reproducible contact burn model on human skin that explores the extent of tissue injury and healing over time, and defines the inter-individual variability in human skin to enable use in mechanistic studies on burn wound progression and healing. MethodsUsing a customized burn device, contact burns of various depths were created on human skin by two operators and were evaluated for histologic depth by three raters to determine reproducibility. Early burn wound progression and wound healing were also evaluated histologically after the thermally injured human skin was cultured ex vivo for up to 14 days. ResultsBurn depths were reproducibly generated on human skin in a temperature- or time-dependent manner. No significant difference in operator-created or rater-determined depth was observed within each patient sample. However, significant inter-individual variation was identified in burn depth in ten patient samples. Burn-injured ex vivo human skin placed into culture demonstrated differential progression of cell death and collagen denaturation for high and low temperature contact burns, while re-epithelialization was observed in superficial burn wounds over a period of 14 days. ConclusionThis model represents an invaluable tool to evaluate the inter-individual variability in early burn wound progression and wound healing to complement current animal models and enhance the translation of preclinical research to improvements in patient care.

cell biology↗

bioRxiv2020

Modular cell-free expression plasmids to accelerate biological design in cells

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms--organisms that are easy to use like Escherichia coli--often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have the desired metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has emerged as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institutes DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for C. autoethanogenum expression with efficiencies ranging from 68-90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

synthetic biology↗