Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication.

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2α, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

Cellular HSF1 expression is induced during HIV-1 infection by activation of its promoter mediated through the cooperative interaction of HSF1 and viral Nef protein.

The Human Immunodeficiency Virus-1 (HIV-1) tends to activate cellular promoters driving expression of pro-viral genes by complex host-virus interactions for productive infection. We have previously demonstrated that expression of such a positive host factor HSF1 (heat shock factor 1) is elevated during HIV-1 infection; however, the mechanism remains to be elucidated. In the present study, we therefore examined whether HSF1 promoter is induced during HIV-1 infection leading to up-regulation of hsf1 gene expression. We mapped the putative transcription start site (TSS) predicted by Eukaryotic promoter database and deletion constructs of the predicted promoter region were tested through luciferase assay to identify the active promoter. The 347 bp upstream to 153 bp downstream region around the putative TSS displayed the highest activity and both Sp1 (stimulating protein 1) and HSF1 itself were identified to be important for its basal activation. Activity of HSF1 promoter was further stimulated during HIV-1 infection in CD4+ T cells, where interestingly the HSF1-site itself seems to play a major role. In addition, HIV-1 protein Nef (negative factor) was also observed to be responsible for the virus-mediated induction of hsf1 gene expression. Chromatin-immunoprecipitation assays further demonstrate that Nef and HSF1 are co-recruited to the HSF1-binding site and cooperatively act on this promoter. The interplay between host HSF1 and viral Nef on HSF1 promoter eventually leads to increase in HSF1 expression during HIV-1 infection. Understanding the mechanism of HSF1 up-regulation during HIV-1 infection might contribute to future antiviral strategies as HSF1 is a positive regulator of virus replication.

HIV-1 replication requires optimal activation of the unfolded protein response.

Several human diseases including viral infections activate the unfolded protein response (UPR) due to abnormal accumulation of unfolded/misfolded proteins. However, UPR modulation and its functional relevance in HIV-1 infection lack comprehensive elucidation. This study reveals that HIV-1 activates IRE1, PERK, and ATF6 signaling pathways of UPR. The knockdown of PERK and ATF6 reduces HIV-1 long terminal repeat (LTR)-driven gene expression, whereas the endoplasmic reticulum (ER) chaperone HSPA5 prevents proteasomal degradation of HIV-1 p24 through its chaperone activity. Interestingly, overstimulation of UPR by a chemical inducer leads to anti-HIV activity through an enhanced type-1 interferon response. Also, treatment with a chemical ER stress inhibitor reduces HIV-1 replication. These findings suggest that an optimal UPR activation is crucial for effective viral replication, as either overstimulating UPR or inhibiting ER stress leads to viral suppression.

DNAJB8 facilitates autophagic-lysosomal degradation of viral Vif protein and restricts HIV-1 virion infectivity by rescuing APOBEC3G expression in host cells.

HSP40/DNAJ family of proteins is the most diverse chaperone family, comprising about 49 isoforms in humans. Several reports have demonstrated the functional role of a few of these isoforms in the pathogenesis of various viruses, including HIV-1. Our earlier study has shown that several isoforms of HSP40 get significantly modulated at the mRNA level during HIV-1 infection in T cells. To explore the biological role of these significantly modulated isoforms, we analyzed their effect on HIV-1 gene expression and virus production using knockdown and overexpression studies. Among these isoforms, DNAJA3, DNAJB1, DNAJB7, DNAJC4, DNAJC5B, DNAJC5G, DNAJC6, DNAJC22, and DNAJC30 seem to positively regulate virus replication, whereas DNAJB3, DNAJB6, DNAJB8, and DNAJC5 negatively regulate virus replication. Further investigation on the infectivity of the progeny virion demonstrated that only DNAJB8 negatively regulates the progeny virion infectivity. It was further identified that DNAJB8 protein is involved in the downregulation of Vif protein, required for the infectivity of HIV-1 virions. DNAJB8 seems to direct Vif protein for autophagic-lysosomal degradation, leading to rescue of the cellular restriction factor APOBEC3G from Vif-mediated proteasomal degradation, resulting in enhanced packaging of APOBEC3G in budding virions and release of less infective progeny virion particles. Finally, our results also indicate that during the early stage of HIV-1 infection, enhanced expression of DNAJB8 promotes the production of less infective progeny virions, but at the later stage or at the peak of infection, reduced expression of DNJAB8 protein allows the HIV-1 to replicate and produce more infective progeny virion particles.

Identification of 5' upstream sequence involved in HSPBP1 gene transcription and its downregulation during HIV-1 infection.

The Human Immunodeficiency Virus-1 (HIV-1) is known to modulate the host environment for successful replication and propagation like other viruses. The virus utilises its proteins to interact with or modulate host factors and host signalling pathways that may otherwise restrict the virus. A previous study from our lab has shown that the host heat shock protein 70 (HSP70) binding protein (HSPBP1) is a co-chaperone that inhibits viral replication. We have also shown that the virus downregulates HSPBP1 during infection. However, the mechanism of downregulation remains to be elucidated. In the present study, we hypothesized that the HSPBP1 promoter may be repressed during infection leading to its downmodulation at the RNA and protein levels. The 5' upstream region of the HSPBP1 gene was first mapped and it was identified that a fragment comprising of a ∼600 bp upstream region of the transcription start site show the highest promoter-like activity. Further, the Sp1 transcription factor was shown to be essential for normal promoter activation. Our results further demonstrate that HIV-1 downregulates the activity of the identified promoter. It was seen that the viral transactivator protein, Tat, was responsible for the downmodulation of the HSPBP1 promoter. HIV-1 Tat is known to bind and regulate several cellular promoters during infection, thereby making the environment conducive for establishment of the virus. Our results further show that Tat is recruited to the HSPBP1 promoter and in the presence of Tat, recruitment of Sp1 on HSPBP1 promoter was decreased, which explains the suppression of HSPBP1 during HIV-1 infection. Therefore, this study further adds to the list of cellular promoters that are modulated by Tat during HIV-1 infection either directly or indirectly.

Zipper interacting protein kinase (ZIPK) is a negative regulator of HIV-1 replication that is restricted by viral Nef protein through proteasomal degradation.

Human immunodeficiency virus-1 (HIV-1) infection leads to the development of acquired immunodeficiency syndrome (AIDS). To establish a productive infection, HIV-1 hijacks the cellular machinery and modulates various physiological processes to propagate itself. The pathways altered by HIV-1 include cell cycle, autophagy, apoptosis, cell stress pathways, immune response, antiviral response, etc. Zipper interacting protein kinase (ZIPK) is a member of the death-associated protein kinase (DAPK) family of proteins, known to be one of the key regulators of cell death and cell survival pathways. ZIPK is also involved in regulating many cellular processes that are altered during HIV-1 infection; thus, we have explored the functional role of ZIPK in HIV-1 infection. Our results show that ZIPK protein expression is downregulated during HIV-1 infection in Nef dependent manner. Overexpression of ZIPK leads to downregulation in LTR-driven gene expression and virus production, whereas ZIPK knockdown induces viral gene expression and replication. HIV-1 promoter activity is reportedly enhanced by Nef-mediated activation of some transcription factors like NFκB and STAT3. ZIPK is reported to inhibit the STAT3 activity by phosphorylating it at ser-727. Our results show that STAT3 (ser-727) phosphorylation is decreased upon overexpression of Nef with simultaneous downregulation of ZIPK expression. We finally show that HIV-1 Nef interacts with ZIPK and induces its proteasomal degradation. Overall, our data suggests that Nef is involved in downregulation of ZIPK thereby increasing the virus production through rescue of STAT3 activity.

Long term influence of groundwater preservation policy on stubble burning and air pollution over North-West India.

Stubble burning (SB) has been a major source of seasonal aerosol loading and pollution over northern India. The aftereffects of groundwater preservation act i.e., post 2010 era (2011-2020) has seen delay in crop harvesting thereby shifting the peak SB to May (Wheat SB) and to November (Paddy SB) by 8-10 and 10-12 days compared to pre-2010. Groundwater storage depletion rate of 29.2 mm yr-1 was observed over the region. Post 2010 era shows an increase of 1.4% in wheat SB and 21% in Paddy SB fires over Punjab and Haryana with 70% of PM2.5 air mass clusters (high probability > 0.8) advecting to the downwind regions leading to 23-26% increase in PM2.5 and 4-6% in aerosol loading over National Capital Region (NCR). Although the objective of water conservation policy was supposed to preserve the groundwater by delaying the paddy transplantation and sowing, on the contrary the implementation of this policy has seen groundwater storage after 2013 depleting at a rate of 29.2 mmyr-1 over these regions. Post policy implementation has led to shift and shrinking of harvest window with increased occurrences in SB fires which also increase associated particulate matter pollution over North India.

Synthesis and anti-HIV activity of a new isoxazole containing disubstituted 1,2,4-oxadiazoles analogs.

Continuing on our antiviral drug discovery research, we intended to diversify our lead anti-HIV-1 inhibitor by non-classical isosteric replacement of amide to 1,2,4-oxadiazoles. The resulting molecules isoxazole-1,2,4-oxadiazole analogs were synthesized using mild bases in ethanol under microwave irradiation. The anti-HIV potential was checked in human CD4+ reporter cell lines, TZM-bl and CEM-GFP, at the highest non-cytotoxic concentration (HNC), demonstrating that 3-((3-(p-tolyl)isoxazol-5-yl)methyl)-1,2,4-oxadiazole and 3-((3-(4-chlorophenyl)isoxazol-5-yl)methyl)-1,2,4-oxadiazole inhibit HIV-1 replication significantly and could be considered as a new lead candidate against HIV-1.

Abdominoperineal Tunnel Crafted with Alken's Telescopic Dilators: A Novel Technique!

BACKGROUND: Abdomino-perineal pull through procedure needs perineal dissection and for that swap of supine to prone may be necessary. To avoid that as well as to avoid neuro-muscular damage; we are describing a simple minimal invasive procedure with help of Alken's telescopic dilators. PATIENTS & METHODS: We created abdomino-perineal tunnel with Alken's telescopic dilators to bring down the lumen of intestine in perineum in eight patients. RESULTS: Operative time happened to be less and procedure found to be less traumatic. All the eight patients had satisfactory outcome. CONCLUSIONS: Actually, we have repurposed the Alken's dilator for creation of abdomino-perineal tunnel or track to get benefit of minimal dissection of perineum during pull-through procedure as well as to avoid neuro-muscular damage.

Diversity in heat shock protein families: functional implications in virus infection with a comprehensive insight of their role in the HIV-1 life cycle.

Heat shock proteins (HSPs) are a group of cellular proteins that are induced during stress conditions such as heat stress, cold shock, UV irradiation and even pathogenic insult. They are classified into families based on molecular size like HSP27, 40, 70 and 90 etc, and many of them act as cellular chaperones that regulate protein folding and determine the fate of mis-folded or unfolded proteins. Studies have also shown multiple other functions of these proteins such as in cell signalling, transcription and immune response. Deregulation of these proteins leads to devastating consequences, such as cancer, Alzheimer's disease and other life threatening diseases suggesting their potential importance in life processes. HSPs exist in multiple isoforms, and their biochemical and functional characterization still remains a subject of active investigation. In case of viral infections, several HSP isoforms have been documented to play important roles with few showing pro-viral activity whereas others seem to have an anti-viral role. Earlier studies have demonstrated that HSP40 plays a pro-viral role whereas HSP70 inhibits HIV-1 replication; however, clear isoform-specific functional roles remain to be established. A detailed functional characterization of all the HSP isoforms will uncover their role in cellular homeostasis and also may highlight some of them as potential targets for therapeutic strategies against various viral infections. In this review, we have tried to comprehend the details about cellular HSPs and their isoforms, their role in cellular physiology and their isoform-specific functions in case of virus infection with a specific focus on HIV-1 biology.

Factors Accountable for Unabated Obstruction Following Pyeloplasty.

PURPOSE: Split renal function (SFR) and frusemide washout (FWO) are assessed by the DTPA renogram to measure the renal parenchymal functions as well as the evidence of obstruction, both for diagnosis and to treat the pelviureteric junction obstruction. In good number of renal units, both these parameters remain unaltered even after surgery and cause anguish to parents and referring physicians and are usually attributed toward "defective pyeloplasty." In this study, we have tried to single out the bona fide responsible factor for the bad outcome; either the nonreversible kidney or the restenosis of pyeloplasty. PATIENTS AND METHODS: We studied file of 69 patients in whom a double "J" (DJ) stent was left in situ for internal drainage for a duration of 8 weeks in the postoperative period. DTPA scans were performed preoperative, at 8 weeks with a stent in place, and at 12 and 24 months postremoval of the stent to assess renal function. RESULTS: In our study, 45 patients (65.2%) showed improvement either in SRF or in FWO or in both after 8 weeks following pyeloplasty and 24 of 69 units (34.8%) did not show any change in renal function with DJ stent in place. Only in six units (8.7%), out of 69 units had deterioration of renal function after removal of DJ stent. CONCLUSIONS: In our opinion, no improvement of renal function found in 24 units (34.8%) even with internal drainage with DJ indicates irreversible renal damage. In 45 units (65.2%), renal function reversed after pyeloplasty and DJ stent. However, after the removal of the DJ, functions deteriorated in six units (8.7%) due to restenosis following pyeloplasties.

A comprehensive profile of genomic variations in the SARS-CoV-2 isolates from the state of Telangana, India.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 has rapidly turned into a pandemic, infecting millions and causing 1 157 509 (as of 27 October 2020) deaths across the globe. In addition to studying the mode of transmission and evasion of host immune system, analysing the viral mutational landscape constitutes an area under active research. The latter is expected to impart knowledge on the emergence of different clades, subclades, viral protein functions and protein-protein and protein-RNA interactions during replication/transcription cycle of virus and response to host immune checkpoints. In this study, we have attempted to bring forth the viral genomic variants defining the major clade(s) as identified from samples collected from the state of Telangana, India. We further report a comprehensive draft of all genomic variations (including unique mutations) present in SARS-CoV-2 strain in the state of Telangana. Our results reveal the presence of two mutually exclusive subgroups defined by specific variants within the dominant clade present in the population. This work attempts to bridge the critical gap regarding the genomic landscape and associate mutations in SARS-CoV-2 from a highly infected southern region of India, which was lacking to date.

Monitoring and predicting regional land use and land cover changes in an estuarine landscape of India.

Deciphering land use and land cover (LULC) change patterns, identifying the variables that act as the major driving forces of change, and predicting possible changes are necessary tools of decision support for policymakers. Estuarine landscapes world over are under extreme pressure of developmental activities because of their resources. The developmental activities lead to unforeseen changes in the traditional land use practices, making it necessary for investigation of the possible outcomes. The present study aims to study the changing pattern of LULC in the East Godavari River Estuarine Ecosystem (EGREE) landscape during 1977-2015 using temporal satellite data and to predict the possible LULC changes by 2029. Cellular Automata-Markov model (CAMM) with and without the multi-criteria evaluator (MCE) and the multi-layer perceptron (MLP) models were used for future LULC prediction. Between 1977 and 2015, mangroves were converted to aquaculture (5.81 km2) on the landward side and were also lost to submergence at the seaward side (15 km2). All of the coastal scrub (69 km2) was lost to beach clearing. Over this period, the aquaculture area rose to 177 km2. The CAMM with MCE was found to yield better predictions. A further rise was predicted in aquaculture (16%), built-up (30%), and Casuarina plantations (28%) by 2029. The study highlighted the LULC change patterns in EGREE, an important estuarine landscape of India. The information generated in this study can act as baseline information for the stakeholders and policy makers in decision-making of developmental projects, land acquisition, and diversion of agricultural land to non-agricultural purposes.

Comparative analysis of differential gene expression of HSP40 and HSP70 family isoforms during heat stress and HIV-1 infection in T-cells.

Heat shock proteins (HSPs) are a family of cellular proteins involved in a variety of biological functions including chaperone activity. HSPs are classified based on their molecular weight and each family has several isoforms in eukaryotes. HSP40 is the most diverse family acting as a co-chaperone for the highly conserved HSP70 family. Some of the isoforms are reported to be induced during heat stress. Few studies have also highlighted the diverse role of some isoforms in different stress conditions including viral infections. But till date, no study has comprehensively examined the expression profile of different HSP40 and 70 isoforms in either heat stress or HIV-1 infection, a virus that is responsible for the pandemic of AIDS. In the present study, we have compared the mRNA expression profile of HSP40 and HSP70 isoforms during heat stress and HIV-1 infection in a T-cell line and also validated the HIV-1 stress results in peripheral blood mononuclear cells. In case of HSP70, we observed that three isoforms (HSPA1A, HSPA1B, and HSPA6) are highly upregulated during heat stress, but these isoforms were found to be downregulated during the peak of HIV-1 infection. While in case of HSP40, we found that only DNAJA4, DNAJB1, and DNAJB4 showed significant upregulation during heat stress, whereas in HIV-1 infection, majority of the isoforms were induced significantly. Stress-dependent differential expression observed here indicates that different HSP40 and HSP70 isoforms may have specific roles during HIV-1 infection and thus could be important for future studies.

Source apportionment of carbon monoxide over India: a quantitative analysis using MOZART-4.

MOZART-4 chemistry transport model has been used to examine the contribution of carbon monoxide (CO) from different source regions/types by tagging their emissions in model simulations. These simulations are made using tagged tracer approach to estimate the relative contribution of different geographical regions and different emission sources, such as anthropogenic or biomass burning to the CO concentration at the surface, in the planetary boundary layer (PBL), and in the free troposphere (FT) over the Indian sub-continent. The CO budget analyses highlight the significant contribution of the Indian emissions on surface CO and influence of chemical production on the free tropospheric CO concentration. The total CO mixing ratio is estimated as 263 ± 139 parts per billion by volume (ppbv) for surface, 177 ± 71 ppbv for PBL, and 112 ± 14 ppbv for FT. The percentage contributions of primary sources are found to be 80%, 68%, and 53% at the surface, in the PBL, and in the FT, respectively. The sub-regional analysis of India shows that anthropogenic and photochemical processes contribute 41-75% and 15-46% CO, respectively, at the surface. Maximum percentage contribution of anthropogenic CO is observed over Indo-Gangetic Plain and Eastern India (75%). CO contribution from local anthropogenic and biomass burning emissions and transported from other global source regions are analyzed over the Indian region at the surface, in the PBL, and in the FT. The local anthropogenic sources contribute largest to the surface CO over India with 108 ppbv, followed by China with 98 ppbv, Europe with 55 ppbv, North America (NA) with 46 ppbv, and South-east Asia (SEA) and Middle East (ME) with 23 ppbv each. India's PBL (FT) CO is mostly influenced by China's anthropogenic emissions with 12 ppbv (8 ppbv) followed by SEA with 7 ppbv (6 ppbv). Surface biomass burning CO over India (6 ppbv) is much lower than in other regions such as SEA (32 ppbv), Africa (24 ppbv), and South America (11 ppbv). In the PBL (FT), SEA and Africa's BB emissions show major impact on CO over India with 6 ppbv (5 ppbv) and 5 ppbv (4 ppbv), respectively.

Insight into Posterior Urethral Valve from Our Experience: Paradigm Appended to Abate Renal Failure.

OBJECTIVE: The objective of the study was to share our experience of management of posterior urethral valve (PUV) and to suggest a paradigm to impede upstaging of chronic kidney disease (CKD) and prevent end-stage renal failure (ESRF). PATIENTS AND METHODS: We have treated 332 patient of PUV from March 2005 to April 2016, Of which 272 case records had adequate data to be analyzed. The mean age was 2.48 years (range: 1 day-18 years). We did primary fulguration in 231 patients, of which five patients needed bilateral ureterostomy for obstinate high creatinine level. The remaining 36 patients had primary fulguration done elsewhere. RESULTS: The mean duration of follow-up was 7.8 years (range 3-14 years). In the end of this study, 10 patients had down staging in CKD, 36 patients had up staging in CKD, and 9 patients ended in ESRF (3.8%). CONCLUSIONS: Detection of deterioration of renal function with creatinine clearance along with identifying the causes of deterioration and necessary interventions would help to arrest upstaging of CKD otherwise that might end in ESRF. From this study and reviewing the literature, we presume that the rhabdosphincter spasm underneath actually renders bladder outlet obstruction, and cusps of PUV, particularly in neonates, amplify the obstruction, following that bladder outlet obstruction cascades detrusor hypertrophy, bladder neck hypertrophy/obstructions, and ureterovesical junction obstruction/reflux, causing gradual damage to the bladder and upper tract and deterioration of renal function as a consequence.