Human Pannexin 1 channel: Insight in structure-function mechanism and its potential physiological roles.

Pannexins, large non-gap junction super family exists in vertebrates, play multiple roles in different cellular functions through their ATP release. Panx1-mediated adenosine 5'-triphosphate (ATP) release plays a vital role in physiological and pathophysiological conditions and is known major extracellular molecule in purinergic signaling. To modulate their function in vivo, a proper regulation of channel is necessary. Post-translational modifications are considered to be some regulating mechanisms for PANX1, while PANX2, PANX3 have been uncharacterized to date. Through their significant evidences, PANXs exclude from gap junction and conduits ATP release and other cellular molecules from cells by various mechanisms. PANX1 is most extensive characterized and implicated in ATP signaling and inflammatory processes. Despite the constant advances, much significance of PANX1 in physiological processes remains elusive. Recently, various research groups along with our group have reported the Cryo-EM structure of Panx1 channel and uncovered the hidden functions in structure-function mechanism as well as to provide the clear understanding in physiological and pathophysiological roles. These research groups reported the novel heptameric structure with contains 4 transmembrane helices (TM), two extracellular loops and one intracellular loop with N and C terminus located at the intracellular side. In addition, the structure contains a large pore of which an inhibitor CBX act as a plug that blocking the passage of substrate. In this context, this review will present current mechanistic understanding in structure and function together with significant physiological roles particularly ATP release in health and disease. As such, this review emphasizes on recent functional properties associated with novel heptameric channel and demystifies channel-mediated ATP release function.

Human CALHM5: Insight in large pore lipid gating ATP channel and associated neurological pathologies.

Recently calcium homeostasis modulators (CALHMs) are identified as ATP release channels play crucial role in functioning of neurons including gustatory signaling and neuronal excitability. Pathologies of Alzheimer's disease and depression have been associated with the dysfunction of CALHMs. Recently, CALHMs has been emerged as an important therapeutic research particularly in neurobiological studies. CALHM1 is most extensively studied among CALHMs and is an ATP and ion channel that is activated by membrane depolarization or removal of extracellular Ca2+. Despite the emerged role of CALHM5 shown by an recently assembled data; however, the neuronal function remains obscure until the first Cryo-EM structure of CALHM5 was recently solved by various research group which acts as a template to study the hidden functional properties of the CALHM5 protein based on structure function mechanism. It provides insight in some of the different pathophysiological roles. CALHM5 structure showed an abnormally large pore channel structure assembled as an undecamer with four transmembrane helices (TM1-TM4), an N-terminal helix (NTH), an extracellular loop region and an intracellular C-terminal domain (CTD) that consists of three α-helices CH1-3. The TM1 and NTH were always poorly defined among all CALHMs; however, these regions were well defined in CALHM5 channel structure. In this context, this review will provide insight in structure, function and mechanism to understand its significant role in pathological diseases particularly in Alzheimer's disease. Moreover, it focuses on CALHM5 structure and recent associated properties based on Cryo-EM research.

Artemisia amygdalina Upregulates Nrf2 and Protects Neurons Against Oxidative Stress in Alzheimer Disease.

Alzheimer disease is a complex neurodegenerative disorder. It is the common form of dementia in elderly people. The etiology of this disease is multifactorial, pathologically it is accompanied with accumulation of amyloid beta and neurofibrillary tangles. Accumulation of amyloid beta and mitochondrial dysfunction leads to oxidative stress. In this study, neuroprotective effect of Artemisiaamygdalina against H2O2-induced death was studied in differentiated N2a and SH-SY5Ycells. Cells were treated with H2O2 to induce toxicity which was attenuated by Artemisia amygdalina. The nuclear factor erythroid 2-related factor 2 (Nrf2) is an emerging regulator of cellular resistance to oxidants. It controls the basal and induced expression of antioxidant response element-dependent genes. Further, we demonstrated that Artemisia amygdalina protects neurons through upregulation of Nrf2 pathway. Moreover, reactive oxygen species and mitochondrial membrane potential loss formed by H2O2 was attenuated by Artemisia amygdalina. Thus, Artemisia amygdalina may have the possibility to be a therapeutic agent for Alzheimer disease.

Insights into the structure, functional perspective, and pathogenesis of ZIKV: an updated review.

Zika virus (ZIKV) poses a serious threat to the entire world. The rapid spread of ZIKV and recent outbreaks since 2007 have caused worldwide concern about the virus. Diagnosis is complicated because of the cross-reactivity of the virus with other viral antibodies. Currently, the virus is diagnosed by molecular techniques such as RT-PCR and IgM-linked enzyme immunoassays (MAC-ELISA). Recently, outbreaks and epidemics have been caused by ZIKV, and severe clinical symptoms and congenital malformations have also been associated with the virus. Although most ZIKV infections present with a subclinical or moderate flu-like course of illness, severe symptoms such as Guillain-Barre syndrome in adults and microcephaly in children of infected mothers have also been reported. Because there is no reliable cure for ZIKV and no vaccine is available, the public health response has focused primarily on preventing infection, particularly in pregnant women. A comprehensive approach is urgently needed to combat this infection and stop its spread and imminent threat. In view of this, this review aims to present the current structural and functional viewpoints, structure, etiology, clinical prognosis, and measures to prevent this transmission based on the literature and current knowledge. Moreover, we provide thorough description of the current understanding about ZIKV interaction with receptors, and a comparative examination of its similarities and differences with other viruses.

Current advancement of exosomes as biomarkers for cancer diagnosis and forecasting.

Exosomes are normal vesicles produced in the late endosomes of a cell. They are secreted by cells and play a role in cell-to-cell contact. They are an invaluable aid in cancer diagnosis as they include miRNA, proteins and lncRNAs. Depending on the function of these constituents in cancer, the expression of exosome constituents can be upregulated or downregulated in cancer. Exosomes provide high concentration and protective environment for their cargo, thereby making them superior targets for cancer diagnosis. It has recently been documented that exosomes modulate cell-cell connectivity by molecules included in the exosomes, leading to the maintenance of tissue homeostasis. In addition, exosomes released from cancer cells are implicated in the development of cancer. Data on the role of exosomes in cancer will thus enhance the effectiveness of new diagnostic and therapeutic approaches. In particular, exosomes are useful sources for biomarkers due to selective cargo loading and similarity to their parental cells. In this review, we summarize the recent findings to use exosomes as cancer biomarkers for early detection, diagnosis, and therapy selection.

In vitro complex formation of human PYRIN domain-only protein 3 prevented by self-oligomerization of ASC PYD domain.

The formation of inflammasome complexes contributes inactivation of inflammatory caspases viz caspase 1, which is generally considered essential for the innate response. Three proteins constituted this inflammasome complex, such as Nod-like receptors (NLRP or AIM2), ASC possessing caspase-recruiting domain, and caspase-1. The ASC proteins comprise two domains, the N-terminal PYD domain responsible for the interaction of various proteins, including PYD only protein 3 (POP3), and the CARD domain for association with other proteins. The PYRIN Domain-Only Protein POP3 negatively regulates responses to DNA virus infection by preventing the ALR inflammasome formation. POP3 directly interacts with ASC, therefore inhibiting ASC recruitment to AIM2-like receptors (ALRs). In the current study, we designed various constructs of the PYRIN Domain-Only Protein 3 (POP3) and ASC PYD domain to find the best-overexpressed construct for biochemical characterization as well as our complex studies. We cloned, purified, and characterized the PYD domain of pyrin only protein 3 and ASC PYD domain under physiological conditions. Our in vitro study clearly shows that the ASC PYD domain of corresponding amino acid 1-96 aa with ease self-oligomerization in physiological buffer conditions, and complex formation of POP3 PYD (1-83 aa) was inhibited by ASC PYD domain. Besides, we purified the PYD of POP3 protein in low and high salt conditions and different pH values for their biochemical characterization. Our results showed that POP3 formed a dimer under normal physiological conditions and was stable under normal buffer conditions; however, the purification in extremely low pH (pH5.0) conditions shows unstable behavior, the high salt conditions (500 mM NaCl) influence the protein aggregation. SDS PAGE arbitrated the homogeneity of the PYD domain of pyrin only protein 3 and ASC PYD domain of corresponding amino acids 1-83 and 1-96, respectively. Furthermore, our native PAGE shows the PYD domain of pyrin; only protein 3 did not form a complex with ASC PYD domain because of oligomerization mediated by the PYD domain.

In vitro assembly complex formation of TRAIP CC and RAP 80 zinc finger motif revealed by our study.

BACKGROUND: Tumor necrosis factor interacting protein (TRAIP/TRIP) is an important cell-signaling molecule that prevents the TNF-induced-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation via direct interaction with TRAF 2 protein. TRAIP is a crucial downstream signaling molecule, implicated in several signaling pathways. Due to these multifunctional effects, TRAIP is more related to cellular mitosis, chromosome segregation, and DNA damage response. Tumor necrosis factor interacting protein is a downstream signaling molecule that contains a RING domain with E3 ubiquitin ligase activity at the N terminal side followed by coiled-coil and C terminal leucine zipper domain. Human TRAIP is constituted of 469 amino acids with 76% sequence similarity with the mouse TRAIP protein. Although, the main inhibitory function of TRAIP has been known for decades, however, in vitro interaction of TRAIPCC domain with RAP80 Zinc finger motif has not been reported yet. Besides, RAP80, the binding partner of TRAIPCC protein has been implicated in DNA damage response. RESULTS: Our in vitro study shows that the TRAIP CC (64-166) associates with the RAP80 zinc finger of corresponding amino acid 490-584. However, TRAIP CCLZ (66-260) and TRAIP RINGCC (1 = 157) failed to interact with the RAP80 zinc finger of corresponding amino acid 490-584. The current study reinforces TRAIP CC (64-166) and RAP80 zinc finger of corresponding amino acid 490-584 associates to form a complex. Moreover, SDS PAGE arbitrated the homogeneity of RAP80 Zinc finger and TRAIP CC of corresponding amino acid 490-584 and 64-166, respectively. CONCLUSION: In vitro, a specific interaction was observed between the TRAIP CC (64-166) and the RAP80 zinc finger of the corresponding amino acid 490-584 and a specific binding area of the RAP80 zinc finger motif were investigated. The TRAIPCC region is required for the complex to bind to the RAP80-Zn finger motif. This strategy may be necessary for the RAP80 zinc finger activity to the TRAIP CC protein.

Protective effect of vanillic acid in hydrogen peroxide-induced oxidative stress in D.Mel-2 cell line.

The overproduction of reactive oxygen species (ROS) causes oxidative stress, such as Hydrogen peroxide (H2O2). Acute oxidative stress is one of the main reasons for cell death. In this study, the antioxidant properties of vanillic acid- a polyphenolic compound was evaluated. Therefore, this study aims to check the effectiveness of vanillic acid in H2O2-induced oxidative stress in D. Mel-2 cell line. The efficacy was determined by biochemical tests to check the ROS production. The cytotoxicity of H2O2 and vanillic acid was checked by MTT assay. The DNA fragmentation was visualized by gel electrophoresis. Protein biomarkers of oxidative stress were analyzed by western blotting. The results depict a promising antioxidant effect of vanillic acid. The IC50 value of vanillic acid and H2O2 was found 250 µg/ml and 125 µg/ml, respectively. The catalase activity, SOF, GPx, and PC was seen less in H2O2 treated group compared with the control and vanillic acid treated group. However, the TBRAS activity was hight in H2O2 treated group. The effect of H2O2 on DNA fragmentation was high as compared with vanillic acid-treated cells. The protein expression of Hsp70, IL-6 and iNOS was seen significant in a vanillic acid-treated group as compared with H2O2 treated group. These results reinforce that at low concentration, vanillic acid could be used as an antioxidant agent in the food and pharmaceutical industries.

SARS-CoV-2: Insight in genome structure, pathogenesis and viral receptor binding analysis - An updated review.

The novel coronavirus disease (COVID-19) a global pandemic outbreak is an emerging new virus accountable for respiratory illness caused by SARS-CoV-2, originated in Wuhan city, Hubei province China, urgently calls to adopt prevention and intervention strategies. Several viral epidemics such as severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 to 2003 and H1N1 influenza in 2009 were reported since last two decades. Moreover, the Saudi Arabia was the epicenter for Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012. The CoVs are large family with single-stranded RNA viruses (+ssRNA). Genome sequence of 2019-nCoV, shows relatively different homology from other coronavirus subtypes, categorized in betacoronavirus and possibly found from strain of bats. The COVID-19 composed of exposed densely glycosylated spike protein (S) determines virus binding and infiltrate into host cells as well as initiate protective host immune response. Recently published reviews on the emerging SARS-CoV-2 have mainly focused on its structure, development of the outbreak, relevant precautions and management trials. Currently, there is an urgency of pharmacological intervention to combat this deadly infectious disease. Elucidation of molecular mechanism of COVID-19 becomes necessary. Based on the current literature and understanding, the aim of this review is to provide current genome structure, etiology, clinical prognosis as well as to explore the viral receptor binding together functional insight of SARS-CoV-2 infection (COVID-19) with treatment and preventive measures.

Promoting the accumulation of scopolamine and hyoscyamine in Hyoscyamus niger L. through EMS based mutagenesis.

The overexploitation of medicinal plants is depleting gene pool at an alarming rate. In this scenario inducing the genetic variability through targeted mutations could be beneficial in generating varieties with increased content of active compounds. The present study aimed to develop a reproducible protocol for in vitro multiplication and mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6ß-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway. In vitro raised callus were treated with different concentrations (0.01% - 0.1%) of Ethyl Methane Sulfonate (EMS). Emerging multiple shoots and roots were obtained on the MS media supplemented with cytokinins and auxins. Significant effects on morphological characteristics were observed following exposure to different concentrations of EMS. EMS at a concentration of 0.03% was seen to be effective in enhancing the average shoot and root number from 14.5±0.30 to 22.2 ±0.77 and 7.2±0.12 to 8.8±0.72, respectively. The lethal dose (LD50) dose was calculated at 0.08% EMS. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The transcripts of PMT and H6H were significantly upregulated at 0.03-0.05% EMS compared to control. EMS treated explants showed increased accumulation of scopolamine (0.639 µg/g) and hyoscyamine (0.0344µg/g) compared to untreated.

Dipsacus inermis Wall. modulates inflammation by inhibiting NF-κB pathway: An in vitro and in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Dipsacus inermis Wall. is an edible Himalayan herb which is extensively used in traditional Ayurvedic system of medicine against various inflammation related disorders. AIM OF THE STUDY: This study was designed to evaluate the anti-inflammatory effects of Dipsacus inermis Wall. methanol extract (DIME) by using in vitro and in vivo models and to elucidate the underlying mechanism of action. MATERIALS AND METHODS: The in vitro anti-inflammatory potential of DIME was determined in LPS stimulated J774A.1 cells. The inhibitory effect of DIME on COX-2, PGE2 and inflammatory cytokines was determined by ELISA and RT-PCR. The suppression of ROS in response to DIME was determined by flow cytometry. Phosphorylation of NF-κBp65 and IκB degradation was determined by western blotting. RESULTS: Significant inhibition of NO, COX-2, PGE2 and pro-inflammatory cytokines including IL-1ß, TNF-α and IL-6 was found in response to DIME in LPS stimulated J774A.1 cells. The extract was found to down regulate the LPS induced expression of TNF-α, IL-6, iNOS and COX-2 along with inhibition of intracellular ROS. The in vivo studies carried on Wistar rats showed significant preventive effect of DIME against acetic acid induced increase in vascular permeability and carrageenan induced paw edema along with stabilization of histopathological alterations. CONCLUSION: The study demonstrated that DIME has significant in vitro and in vivo anti-inflammatory effect which is mediated by inhibiting the activation of NF-κB pathway. Our data opened a promising new pharmacological approach of designing anti-inflammatory drugs by studying individual fractions of the plant extract.

Molecular cloning, expression, overproduction and characterization of human TRAIP Leucine zipper protein.

The TRAIP interacting protein is known as a negative regulator of TNF-induced-nuclear factor, kappa-light-chain-enhancer of activated B cell (NF-κB) by direct interaction with the adaptor protein TRAF2, which inhibits the function of TRAF2 via the RINGCC domain protein. The TRAIP protein is composed of 469 amino acids with an N-terminal RING motif that is followed by a coiled coil (CC) and leucine zipper domain. TRAIP proteins are critical in programmed cell death, cell proliferation and differentiation, and embryonic development. The critical functions of TRAIP together with the molecular inhibitory mechanism effect of TRAIP have been reported by two different studies and have opened up new research into the field of TRAF biology. In this study, we designed different constructs of the Leucine zipper domain to find the over -expressed construct for further studies. We successfully cloned the C-terminal TRAIP containing the leucine zipper domain. In addition, we have over-expressed and purified the TRAIP LZ for their biochemical characterization.

Recognition of TRAIP with TRAFs: Current understanding and associated diseases.

TNF receptor proteins were primarily recognized as adaptor proteins that ligate with the tumor necrosis factor receptor (TNFR)-associated factor (TNFR) family to execute various signaling pathways. However, recent studies showed that they act as a signal-transducing molecules and are reported to have a functional role as a Toll/interleukin-1 receptor family member. Seven members of this family have been identified to date. Among TNF receptor family, TRAF7 does not share a common TRAF domain homology. The tumor necrosis factor receptor associated factor (TRAF) domain comprises of about 230 amino acid motif at the C-terminal region that has the capability to bind TNFR and execute different downstream signaling pathways. Moreover, N-terminal RING and ZINC finger constituted by the tumor necrosis factor associated protein 2 and tumor necrosis factor associated protein 6 are critical and execute various downstream signaling events. TRAF proteins have emerged as critical regulators that provide the cellular response to stress and lead to cell death. Nuclear factor kappa beta (NF-KB) and c-Jun N-terminal kinases (JNK) pathways are activated through tumor necrosis factor associated protein 2, tumor necrosis factor associated protein 5 and tumor necrosis factor associated protein 6 members. TRAF proteins in pathogenesis were observed from their abnormal expression in diseased tissue and in normal tissue, suggesting its important role in physiological processes. Recently, unique specificity of TRAF4 for glycoprotein Ibß (GPIbß) and glycoprotein VI (GPVI) in human platelets has been reported. The multifunctional effects of TRAIP (TNF) interacting protein in many cellular signaling pathways emerged as very important signaling molecule. Furthermore, the new insights into the structure of TRAF members along with new studies involved in health and disease prompted to explore their role particularly the TNF receptor associated proteins with novel inhibitor protein TRAIP (TNF) interacting protein and human diseases associated with it. As such, this review emphasis on tumor necrosis factor receptor associated proteins, present their current understanding with novel inhibitor protein TRAIP (TNF) interacting protein.