Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

Effects of Withania somnifera and Tinospora cordifolia Extracts on the Side Population Phenotype of Human Epithelial Cancer Cells: Toward Targeting Multidrug Resistance in Cancer

Recent reports suggest the existence of a subpopulation of stem-like cancer cells, termed as cancer stem cells (CSCs), which bear functional and phenotypic resemblance with the adult, tissue-resident stem cells. Side population (SP) assay based on differential efflux of Hoechst 33342 has been effectively used for the isolation of CSCs. The drug resistance properties of SP cells are typically due to the increased expression of ABC transporters leading to drug efflux. Conventionally used chemotherapeutic drugs may often leads to an enrichment of SP, revealing their inability to target the drug-resistant SP and CSCs. Thus, identification of agents that can reduce the SP phenotype is currently in vogue in cancer therapeutics. Withania somnifera (WS) and Tinospora cordifolia (TC) have been used in Ayurveda for treating various diseases, including cancer. In the current study, we have investigated the effects of ethanolic (ET) extracts of WS and TC on the cancer SP phenotype. Interestingly, we found significant decrease in SP on treatment with TC-ET, but not with WS-ET. The SP-inhibitory TC-ET was further fractionated into petroleum ether (TC-PET), dichloromethane (TC-DCM), and n-butyl alcohol (TC-nBT) fractions using bioactivity-guided fractionation. Our data revealed that TC-PET and TC-DCM, but not TC-nBT, significantly inhibited SP in a dose-dependent manner. Furthermore, flow cytometry-based functional assays revealed that TC-PET and TC-DCM significantly inhibited ABC-B1 and ABC-G2 transporters and sensitized cancer cells toward chemotherapeutic drug-mediated cytotoxicity. Thus, the TC-PET and TC-DCM may harbor phytochemicals with the potential to reverse the drug-resistant phenotype, thus improving the efficacy of cancer chemotherapy.

Biomechanical Properties of Human Ascending Thoracic Aortic Dissections

Thoracic aortic dissections are associated with a significant risk of morbidity and mortality, and currently challenge our understanding of the biomechanical factors leading to their initiation and propagation. We quantified the biaxial mechanical properties of human type A dissections (n = 16) and modeled the stress-strain data using a microstructurally motivated form of strain energy function. Our results show significantly higher stiffness for dissected tissues as compared to control aorta without arterial disease. Higher stiffness of dissected tissues did not, however, correlate with greater aortic diameter measured prior to surgery nor were there any age dependent differences in the tissue properties.

New dimeric carbazole-benzimidazole mixed ligands for the stabilization of human telomeric G-quadruplex DNA and as telomerase inhibitors. A remarkable influence of the spacer

for selectively targeting cancer cells. Herein, we report the design and evolution of a new kind of carbazole-based benzimidazole dimers for their efficient telomerase inhibition activity. Spectroscopic titrations reveal the ligands high affinity toward the G4 DNA with significantly higher selectivity over duplex-DNA. The electrophoretic mobility shift assay shows that the ligands efficiently promote the formation of 04 DNA even at a lower concentration of the stabilizing K+ ions. The TRAP-LIG assay demonstrates the ligand's potential telomerase inhibition activity and also establishes that the activity proceeds via G4 DNA stabilization. An efficient nuclear internalization of the ligands in several common cancer cells (HeLa, HT1080, and A549) also enabled differentiation between normal HFF cells in co-cultures of cancer and normal ones. The ligands induce significant apoptotic response and antiproliferative activity toward cancer cells selectively when compared to the normal cells.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.

The putative role of some conserved water molecules in the structure and function of human transthyretin

Human transthyretin (hTTR) is a multifunctional protein that is involved in several neurodegenerative diseases. Besides the transportation of thyroxin and vitamin A, it is also involved in the proteolysis of apolipoprotein A1 and A beta peptide. Extensive analyses of 32 high-resolution X-ray and neutron diffraction structures of hTTR followed by molecular-dynamics simulation studies using a set of 15 selected structures affirmed the presence of 44 conserved water molecules in its dimeric structure. They are found to play several important roles in the structure and function of the protein. Eight water molecules stabilize the dimeric structure through an extensive hydrogen-bonding network. The absence of some of these water molecules in highly acidic conditions (pH <= 4.0) severely affects the interfacial hydrogen-bond network, which may destabilize the native tetrameric structure, leading to its dissociation. Three pairs of conserved water molecules contribute to maintaining the geometry of the ligand-binding cavities. Some other water molecules control the orientation and dynamics of different structural elements of hTTR. This systematic study of the location, absence, networking and interactions of the conserved water molecules may shed some light on various structural and functional aspects of the protein. The present study may also provide some rational clues about the conserved water-mediated architecture and stability of hTTR.

On the detection of human influence in extreme precipitation over India

Climate change is expected to influence extreme precipitation which in turn might affect risks of pluvial flooding. Recent studies on extreme rainfall over India vary in their definition of extremes, scales of analyses and conclusions about nature of changes in such extremes. Fingerprint-based detection and attribution (D&A) offer a formal way of investigating the presence of anthropogenic signals in hydroclimatic observations. There have been recent efforts to quantify human effects in the components of the hydrologic cycle at large scales, including precipitation extremes. This study conducts a D&A analysis on precipitation extremes over India, considering both univariate and multivariate fingerprints, using a standardized probability-based index (SPI) from annual maximum one-day (RX1D) and five-day accumulated (RX5D) rainfall. The pattern-correlation based fingerprint method is used for the D&A analysis. Transformation of annual extreme values to SPI and subsequent interpolation to coarser grids are carried out to facilitate comparison between observations and model simulations. Our results show that in spite of employing these methods to address scale and physical processes mismatch between observed and model simulated extremes, attributing changes in regional extreme precipitation to anthropogenic climate change is difficult. At very high (95%) confidence, no signals are detected for RX1D, while for the RX5D and multivariate cases only the anthropogenic (ANT) signal is detected, though the fingerprints are in general found to be noisy. The findings indicate that model simulations may underestimate regional climate system responses to increasing human forcings for extremes, and though anthropogenic factors may have a role to play in causing changes in extreme precipitation, their detection is difficult at regional scales and not statistically significant. (C) 2015 Elsevier B.V. All rights reserved.

Primates, Provisioning and Plants: Impacts of Human Cultural Behaviours on Primate Ecological Functions

Human provisioning of wildlife with food is a widespread global practice that occurs in multiple socio-cultural circumstances. Provisioning may indirectly alter ecosystem functioning through changes in the eco-ethology of animals, but few studies have quantified this aspect. Provisioning of primates by humans is known to impact their activity budgets, diets and ranging patterns. Primates are also keystone species in tropical forests through their role as seed dispersers; yet there is no information on how provisioning might affect primate ecological functions. The rhesus macaque is a major human-commensal species but is also an important seed disperser in the wild. In this study, we investigated the potential impacts of provisioning on the role of rhesus macaques as seed dispersers in the Buxa Tiger Reserve, India. We studied a troop of macaques which were provisioned for a part of the year and were dependent on natural resources for the rest. We observed feeding behaviour, seed handling techniques and ranging patterns of the macaques and monitored availability of wild fruits. Irrespective of fruit availability, frugivory and seed dispersal activities decreased when the macaques were provisioned. Provisioned macaques also had shortened daily ranges implying shorter dispersal distances. Finally, during provisioning periods, seeds were deposited on tarmac roads that were unconducive for germination. Provisioning promotes human-primate conflict, as commensal primates are often involved in aggressive encounters with humans over resources, leading to negative consequences for both parties involved. Preventing or curbing provisioning is not an easy task as feeding wild animals is a socio-cultural tradition across much of South and South-East Asia, including India. We recommend the initiation of literacy programmes that educate lay citizens about the ill-effects of provisioning and strongly caution them against the practice.

Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-alpha and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-alpha treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs - LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival. (C) 2015 The Authors. Published by Elsevier B.V.

Biochemical and Behavioral Evaluation of Human MAPT Mutations in Transgenic Drosophila melanogaster

Mutations in the human microtubule-associated protein tau (hMAPT) gene including R406W and V337M result in autosomal dominant neurodegenerative disorder. These mutations lead to hyperphosphorylation and aggregation of Tau protein which is a known genetic factor underlying development of Alzheimer's disease (AD). In the present study, transgenic Drosophila models of AD expressing wild-type and mutant forms of hMAPT exhibit a progressive neurodegeneration which was manifested in the form of early death and impairment of cognitive ability. Moreover, they were also found to have significantly decreased activity of neurotransmitter enzymes accompanied by decreased cellular endogenous antioxidant profile. The extent of neurodegeneration, memory impairment, and biochemical profiles was different in the tau transgenic strains which indicate multiple molecular and cellular responses underlie each particular form of hMAPT.