The Baseplate Wedges of Bacteriophage T4 Spontaneously Assemble into Hubless Baseplate-Like Structure In Vitro

The baseplate of phage T4 is an important model system in viral supramolecular assembly. The baseplate consists of six wedges surrounding the central hub. We report the first successful attempt at complete wedge assembly using an in vitro approach based on recombinant proteins. The cells expressing the individual wedge proteins were mixed in a combinatorial manner and then lysed. Using this approach, we could both reliably isolate the complete wedge along with a series of intermediate complexes as well as determine the exact sequence of assembly. The individual proteins and intermediate complexes at each step of the wedge assembly were successfully purified and characterized by sedimentation velocity and electron microscopy. Although our results mostly confirmed the hypothesized sequential wedge assembly pathway as established using phage mutants, interestingly, we also detected some protein interactions not following the specified order. It was found that association of gene product 53 to the immediate precursor complex induces spontaneous association of the wedges to form a six-fold star-shaped baseplate-like structure in the absence of the hub. The formation of the baseplate-like structure was facilitated by the addition of gene product 25. The complete wedge in the star-shaped supramolecular complex has a structure similar to the baseplate in the expanded “star” conformation found after infection. Based on the results of the present and previous studies, we assume that the strict order of wedge assembly is due to the induced conformational change caused by every new binding event. The significance of a 40-S star-shaped baseplate structure, which was previously reported and was also found in this study, is discussed in the light of a new paradigm for T4 baseplate assembly involving the star-shaped wedge ring and the central hub. Importantly, the methods described in this article suggest a novel methodology for future structural characterization of supramolecular protein assemblies.     

Assessing high conservation value areas for rare,endemic and threatened (RET) species: A study in high altitude Changthang landscape of India

The Forest Stewardship Council developed the concept of High Conservation Values (HCVs) as a criteria in the forest certification process in order to promote sustainable forest management. It has six major components or values and component one and two of HCVs deal with the habitat for viable populations of “rare, endemic and threatened (RET) species” using the IUCN Red List category and other national / regional / local lists. But a consistent robust methodology for identification of these areas, does not exist. The present study tried to develop for the first time, a straight forward inclusive methodology for identification of HCVAs for the RET species on a spatio-temporal scale. A total of 50 RET and other significant species (32 flora, 10 fauna and 8 avifauna) were identified after a thorough literature review, field surveys and consultations with experts. Occurrence data of the selected species was collected from different secondary sources, field surveys, institutes and scientists who have worked on them. A 10 km grid-based approach and stratified random sampling was used for the primary GPS field surveys conducted during 2018–2019. MaxEnt species distribution model (SDM) software was used based on the occurrence data and environmental variables for identification of potential suitable habitats for the selected species. Linear support vector machine (LSVM) model was used for assessing the performance of the SDMs. The performance of each SDM has been validated through Cohen's Kappa (KAPPA), true skill statistic (TSS) and receiver operating characteristics (ROC) models. The proposed methodology addresses the urgent need for a holistic and robust set of techniques to apply the HCV toolkit. This is key to identify and map HCVAs for RET species at the landscape level and can be easily adapted to and adopted at the national, regional, state or local level in India. The methods offer an efficient, reliable approach for the application of the HCV concept, elsewhere in the world.     

Liposomal co-delivery-based quantitative evaluation of chemosensitivity enhancement in breast cancer stem cells by knockdown of GRP78/CLU

Resistance to chemotherapy is a key factor in the inefficacy of various forms of treatments for cancer. In the present study, chemo-resistant proteins, including glucose-regulated protein 78 (GRP78)/clusterin (CLU) targeted 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) liposomes, were developed as a delivery system for co-delivery of camptothecin (CPT) and GRP78 siRNA/CLU siRNA. Their drug/gene co-deliveries were quantitatively assessed in cancer stem cells (CSC) and MCF-7 cells. DOTAP-CPT/siRNA were prepared via electrostatic interaction on GRP78 siRNA or CLU siRNA. The size and ζ-potential of liposomes and lipoplexes were measured by dynamic light scattering techniques and electrophoretic light scattering spectrophotometry. The lipoplexes formation was tested by using gel electrophoresis. Immunofluorescence analysis showed that the expression level of CLU and GRP78 were significantly elevated in CSC compared to MCF-7 cells. Transfection and drug-delivery efficiency of DOTAP-CPT/siRNA were quantitatively compared with Lipofectamine 2000. Compared to free CPT, DOTAP-CPT-siCLU delivery in CSC and MCF-7 cells increased transfection efficiency and chemo-sensitivity by 4.1- and 5.9-fold, respectively. On the other hand, DOTAP-CPT-siGRP78 delivery increased transfection efficiency and chemo sensitivity by 4.4- and 6.2-fold in CSC and MCF-7 cells, respectively, compared to free CPT. It is significant that 3?±?1.2-fold increase in transfection efficiency was achieved by lipofectamine. Consequently, an increase in anti-cancer/gene silencing efficacy was quantitatively observed as an effect of DOTAP-CPT/siRNA treatment, which was relatively higher than lipofectamine treatment. Conclusively, our experimental data quantitatively demonstrate that using DOTAP-CPT-siRNA specifically targeting (CSCs) chemo-resistant protein in vitro offers substantial potential for synergistic anti-cancer therapy.     

Purification of L-Isoleucyl t-rna Synthetase by Affinity Chromatography

L-Isoleucyl t-RNA synthetase was purified to 97% purity in a single step by affinity chromatography on a column carrying L-isoleucinyl 5′-adenylate as the insolubilized ligand. Recovery was 50–60%.     

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.     

Expression of Endogenous Retrovirus ev/J gp85 Gene and Analysis of Its Immunoreactivity in Comparison with Exogenous Viral Protein

The envelope gene gp85 of ev/J,a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian ieukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC<,10200> strain).     

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 10⁶ acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.     

IL-17A and IL-17F repair HIV-1 gp140 damaged Caco-2 cell barriers by upregulating tight junction genes

It is widely accepted that impairment of the intestinal epithelial barrier from HIV/AIDS contributes significantly to microbial translocation and systemic immune activation. Such factors present potential targets for novel treatments aimed toward a functional cure. However, the extracellular mechanisms of intestinal barrier repair are poorly understood. In the current study, we investigated the abilities of IL-17A and IL-17F to repair the damaged barrier caused by HIV-1 gp140 using Caco-2 monolayers. It was found that HIV-1 gp140 downregulated the expression of tight junction-associated genes and disrupted the barrier integrity of Caco-2 monolayers. However, IL-17A and IL-17F treatment reversed the HIV-1 gp140-induced barrier dysfunction by upregulating the expression of tight junction-associated genes, the combination of which resulted in a stronger induction of barrier repair. Furthermore, the effects of IL-17A and IL-17F were reduced by downregulation of Act1 with siRNA and inhibition of NF-κB and MAPK pathways with BAY11-7082 and U0126, respectively. These data indicated that the NF-κB and MAPK pathways are involved in the repair of barrier integrity mediated by IL-17A and IL-17F, and IL-17 pathways are potential targets for gut barrier restoration therapies during HIV/AIDS.