Biophysical characterization of mycobacterial model membranes and their interaction with rifabutin: Towards lipid-guided drug screening in tuberculosis

Lipid structure critically dictates the molecular interactions of drugs with membranes influencing passive diffusion, drug partitioning and accumulation, thereby underpinning a lipid-composition specific interplay. Spurring selective passive drug diffusion and uptake through membranes is an obvious solution to combat growing antibiotic resistance with minimized toxicities. However, the spectrum of complex mycobacterial lipids and lack thereof of suitable membrane platforms limits the understanding of mechanisms underlying drug-membrane interactions in tuberculosis. Herein, we developed membrane scaffolds specific to mycobacterial outer membrane and demonstrate them as improvised research platforms for investigating anti-tubercular drug interactions. Combined spectroscopy and microscopy results reveal an enhanced partitioning of model drug Rifabutin in trehalose dimycolate-containing mycobacterial membrane systems. These effects are apportioned to specific changes in membrane structure, order and fluidity leading to enhanced drug interaction. These findings on the membrane biophysical consequences of drug interactions will offer valuable insights for guiding the design of more effective antibiotic drugs coupled with tuned toxicity profiles.     

Combinatorial screening algorithm to engineer multiepitope subunit vaccine targeting human T-lymphotropic virus-1 infection

Human T-lymphotropic virus (HTLV), the first human retrovirus has been discovered which is known to cause the age-old assassinating disease HTLV-1 associated myelopathy. Cancer caused by this virus is adult T cell leukemia/lymphoma which targets 10–20 million throughout the world. The effect of this virus extends to the fact that it causes chronic disease to the spinal cord resulting in loss of sensation and further causes blood cancer. So, to overcome the complications, we designed a subunit vaccine by the assimilation of B-cell, cytotoxic T-lymphocyte , and helper T-lymphocyte epitopes. The epitopes were joined together along with adjuvant and linkers and a vaccine was fabricated which was further subjected to 3D modeling. The physiochemical properties, allergenicity, and antigenicity were evaluated. Molecular docking and dynamics were performed with the obtained 3D model against toll like receptor (TLR-3) immune receptor. Lastly, in silico cloning was performed to ensure the expression of the designed vaccine in pET28a(+) expression vector. The future prospects of the study entailed the in vitro and in vivo experimental analysis for evaluating the immune response of the designed vaccine construct.     

Iron deficiency influences NtcA‐dependent regulation of fatty acid desaturation and heterocyte envelop formation in Anabaena sp. PCC 7120

In Anabaena sp. PCC 7120, iron is an essential trace element and its availability determines proper functioning of several kinds of metabolisms. Iron deficiency leads to several unavoidable consequences including membrane damage. In the present study, we dealt with the impact of iron deficiency on NtcA (global nitrogen regulator)‐dependent regulation of two important processes, i.e. fatty acid desaturation and heterocyte envelop formation in cyanobacterium Anabaena sp. PCC 7120. In Anabaena sp. PCC 7120, NtcA regulates fatty acid desaturation by regulating enzyme fatty acid desaturases. The NtcA‐based regulation of fatty acid desaturation may be direct or indirect. Furthermore, the expression of genes involved in the heterocyte envelope polysaccharide (HEP) layer formation (hepABCK) and heterocyte‐specific glycolipids (HGLs) synthesis (devH, hglE_A, prpJ and devB) were also under the control of NtcA and reduced under iron deficiency background. The enhanced expression of furA and early downregulation of ntcA under iron deficiency is responsible for reduction in fatty acid desaturation as well as decrease in the expression of genes involved in HEP layer formation and HGL synthesis. Overall results confirmed that iron deficiency influences the NtcA‐based regulation of fatty acid desaturation and heterocyte envelop formation in Anabaena sp. PCC 7120.     

Valuation of ecosystem services in alternative bioenergy landscape scenarios

Agricultural land in the Midwest is a source of food and fuel, as well as biodiversity. It is also a cause of excess nutrients that make their way to the Mississippi River and the Gulf of Mexico. To address unsustainable changes to biogeochemical cycles and ecosystem functions, a multidisciplinary approach involving social science, natural science, and engineering is often effective. Given the potential of second‐generation biofuels, and capitalizing on the deep‐rooted perennial bioenergy crops capable of thriving in poor soils, we demonstrated an integrated socio‐environmental analysis of the impacts of growing switchgrass within row‐crop landscapes in Illinois. In this study, we model land use scenarios that incorporate switchgrass as a biofuel crop in a Midwest corn‐belt watershed using the Soil Water Assessment Tool coupled with an economic analysis for the Vermilion Basin in Illinois. We estimated the values of ecosystem services under an alternative bioenergy landscape, including commodity and bioenergy crops, changes in biogeochemistry, and recreational services. The estimated annual values of nitrate and sediment reduction attributed to bioenergy crops range from $38 million to $97 million and $16,000 to $197,000, respectively. The annual value of carbon dioxide emission reduction ranges from $1.8 million to $6.1 million based on the initial crop rotation pattern. Estimated average annual values for wildlife viewing, water‐based recreation, and pheasant hunting are $1.24 million, $0.17 million, and $0.3 million, respectively. To our knowledge, this study represents the first effort to comprehensively quantify ecosystem services using a process‐based model, and estimate their value in an alternative bioenergy landscape. The information we generate could aid in understanding the potential for biomass production from marginal land and the total economic value of the landscape at various spatial scales. The framework is useful in fostering alternative bioenergy landscapes with synergies in a food, energy, and conservation nexus.     

Radiation dose measurements in a dental orthopantogram unit using indigenously developed optically stimulated luminescence dosimeters

Dental orthopantogram (OPG)/cone beam computed tomography (CBCT) scanners are gaining popularity due to their 3D imaging with multiplanar view that provides clinical benefits over conventional dental radiography systems. Dental OPG/CBCT provides optimal visualization of adjacent overlaying anatomical structures that will be superpositioned in any single projection. The characteristics of indigenously developed optically stimulated luminescence dosimeters, namely, aluminium oxide doped with carbon (Al₂O₃:C), lithium magnesium phosphate doped with terbium and boron (LiMgPO₄:Tb,B) and lithium calcium aluminium fluoride doped with europium and yttrium (LiCaAlF₆:Eu,Y) were evaluated for their use in dental dosimetry. The dose?response of these dosimeters was studied at X‐ray energies 60 kV, 70 kV and 81 kV. Radiation doses were also measured using Gafchromic film for comparison. Radiation dose was measured at eight different locations of a polymethyl methacrylate (PMMA) head phantom including eyes. The optically stimulated luminescence (OSL) sensitivity of LiMgPO₄:Tb,B is about 1.5 times and LiCaAlF₆:Eu, is about 20 times higher than the sensitivity of Al₂O₃:C. It was found that measured radiation doses by the three optically stimulated luminescence dosimeters (OSLDs) and Gafchromic film in the occipital region (back side) of a PMMA phantom, were consistent but variations in dose at other locations were significantly higher. The three OSLDs used in this study were found to be suitable for radiation dose measurement in dental units.     

Simplified high-throughput screening of AOX1-expressed laccase enzyme in Pichia pastoris

The heterologous protein expression in Pichia pastoris under the control of alcohol oxidase (AOX1)promoter comprises two steps, the growth and induction phases, which are time-consuming and technically demanding. Here, we describe an alternate method where expression is carried out directly in the methanol-containing medium. Using this method, we were successful in screening high-activity laccase clones from a library of laccase mutants generated by random mutagenesis. This simplified method not only saves time but also is highly efficient and can be used for screening a large number of clones.     

In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy

Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.