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HIV-1 Tat continues to play an important role in the development of HIV-associated neurocognitive disorders (HAND), which persist in 15–55% of people living with HIV even with virological control. In the brain, Tat is present on neurons, where Tat exerts direct neuronal damaging effects by, at least in part, disrupting endolysosome functions, a pathological feature present in HAND. In this study, we determined the protective effects of 17α-estradiol (17αE2), the predominant form of estrogen in the brain, against Tat-induced endolysosome dysfunction and dendritic impairment in primary cultured hippocampal neurons. We demonstrated that pre-treatment with 17αE2 protected against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Estrogen receptor alpha (ERα) knockdown impairs the ability of 17αE2 to protect against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Furthermore, over-expressing an ERα mutant that fails to localize on endolysosomes impairs 17αE2′s protective effects against Tat-induced endolysosome dysfunction and reduction in dendritic spine density. Our findings demonstrate that 17αE2 protects against Tat-induced neuronal injury via a novel ERα-mediated and endolysosome-dependent pathway, and such a finding might lead to the development of novel adjunct therapeutics against HAND.

This paper presents an efficient fragility analysis approach for offshore structures subjected to random wave load considering parameter uncertainty. Fragility analysis of structures under random dynamic load is generally accomplished by the direct Monte Carlo simulation (MCS), which requires extensive computational time. Hence, in the present study, a new cumulative distribution function matching-based fragility analysis approach is proposed in the framework of dual response surface method to reduce this computational burden. The proposed approach does not require MCS during failure probability computations once the two response surfaces of mean and standard deviation of response are obtained. Thereby, a substantial amount of computational time is saved. The proposed approach is generic in nature and can be applied to any offshore structure, with different probability distributions of system parameters and higher uncertainty levels, as well. The record-to-record variation of random wave force time-histories (that occurs even for the same set up of hazard parameters) can be easily considered by the proposed approach. The accuracy of the proposed approach is maintained by applying the moving least-squares method, in place of the conventional least-squares method, which is often reported to be a source of error. Two illustrative examples (one simple benchmark problem and another practical steel offshore platform structure) are presented to demonstrate the efficiency of the proposed fragility analysis approach. The results indicate that the present moving least-squares method yields more accurate solutions than the conventional least-squares method. Also, the computational efficiency by the proposed approach over the usual MCS-based approach can be clearly envisaged from the numerical studies.

The blast hazards are associated with extreme randomness. Thus, structures under blast should be properly designed for such highly uncertain phenomena. However, codal stipulations and existing literature do not consider important uncertainty-related design issues, such as record-to-record variation of blast load, high uncertainty levels associated with blast parameters, and robustness of structural behaviour under extreme blast events. In the present study, a computationally efficient robust optimisation (RO) approach is proposed for blast-excited structures by addressing these issues. In doing so, a new CDF-based constraint formulation is developed in the dual response surface-based metamodeling framework to avoid the use of direct Monte Carlo simulation (MCS) during RO iterations. Thereby, the proposed approach becomes computationally much efficient than the conventional direct MCS-based RO. Since the proposed approach resorts to matching the CDF of the actual response, instead of the conventional RO formulations based on the first two statistical moments only, the approach is more realistic. The present approach adopts the moving least-squares method instead of the usual least-squares method to retain accuracy in response approximation. The blast load time-history is generated by a stochastic field model with proper validation through real blast data. Three example problems have been investigated to establish the effectiveness of the proposed RO procedure. The RO results depict that the present method yields economic design solutions, which are also insensitive to high uncertainty levels associated with the extreme blast load. The COV of performance function is substantially low even when input parameter uncertainty is high.

The ATP-dependent ClpQY system is a prokaryotic proteasome-like machinery in the mitochondrion of malaria parasite, Plasmodium. This protease system is identified as a validated target for malaria intervention. Here we have identified drug-like small chemical compounds, pyrimidotriazine derivatives, as specific inhibitors of P. falciparum ClpQ (PfClpQ) having effective parasiticidal efficacies on asexual and liver stages. Screening of the ‘Malaria Box’ compound library, using a robust in vitro activity assay, identified a hit compound targeting the PfClpQ protease with IC50 at ~750nM. Further, Structure-Activity Relationship (SAR) analysis, using a small library of derivatives of the hit compound, identified three potential lead compounds, ICGEB-L1, ICGEB-L2 and ICGEB-L3, which inhibited enzyme activity with IC50 in 100-200nM ranges. The three lead compounds effectively inhibited in vitro asexual stage parasite growth in similar concentration ranges with ~100-fold selectivity over mammalian cell line; in addition, these compounds effectively inhibited in vitro growth and maturation of hepatocytic stage parasites with EC50 in low nano-molar ranges. These compounds were found to effectively block the development of trophozoite stages to schizont stage, concurrently arresting development and segregation of parasite mitochondria, in asexual as well as hepatocytic stages of the parasite. These morphological and developmental effects of the drugs on parasites mimic the data from genetic ablation of PfClpQ. Importantly, all three compounds showed significant in vivo anti-parasitic efficacies on blood stages of P. berghei mouse malaria model. Overall these results suggest that these PfClpQ targeting compounds are promising candidates to develop new anti-malarials.

This paper deals with an efficient reliability-based robust design optimization (RBRDO) of structures under extreme wind excitation. To solve such problems in the direct Monte Carlo simulation (MCS) framework, extensive computational time is required. To circumvent this, a new CDF-based RBRDO approach is proposed using the dual response surface method (RSM) framework. In the proposed approach, once the response surfaces are obtained by the dual RSM, the direct MCS can be fully avoided during optimization. Thereby, the approach evades several repetitive executions of finite element analyses code inside the optimization loop. Generally, RSM uses least squares method (LSM) which is reported to be a major source of error in few recent studies. Thus, the proposed approach utilizes the adaptive nature of moving least squares method (MLSM) for construction of response surfaces. The RBRDO is performed by using a two-criterion equivalent optimization problem, where the mean value of the cost of a structure and its standard deviation are optimized—ensuring constraint feasibility under parameter uncertainty. The dynamic response of structures necessary to define constraint functions is obtained using a random wind field model, which considers the effect of coherence and wind directionality. Two illustrative examples are presented to demonstrate the efficiency of the proposed RBRDO approach. When benchmarked through direct MCS results, the proposed MLSM-based RBRDO approach yields more accurate and robust solutions compared with that of the conventional LSM-based approach.

This paper presents robust design optimisation (RDO) of offshore jacket platforms under random wave force. It is well-established that the direct Monte Carlo Simulation (MCS) is the most accurate w...

HtrA family of serine protease is known to play role in mitochondrial homeostasis as well as in the programmed cell death. Mitochondrial homeostasis and metabolism are crucial for survival and propagation of malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin like protease activity that can be inhibited by its specific inhibitor, ucf-101. Transgenic parasite line was generated, using HA-glmS C-terminal tagging approach, for localization as well as for inducible knockdown of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, ucf-101 treatment had no effect on the parasite growth, suggesting non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase like protease as well as parasite cell death, suggesting involvement of protease activity of PfHtrA2 in apoptosis like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show specific role of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating the stress induced cell death.

Members of the HtrA family of serine proteases are known to play roles in mitochondrial homeostasis as well as in programmed cell death. Mitochondrial homeostasis and metabolism are crucial for the survival and propagation of the malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin-like protease activity that can be inhibited by its specific inhibitor, ucf-101. A transgenic parasite line was generated, using the HA-glmS C-terminal tagging approach, for localization as well as for inducible knock-down of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during the asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, the ucf-101 treatment did not affect the parasite growth, suggesting the non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase-like protease as well as parasite cell death, suggesting the involvement of protease activity of PfHtrA2 in apoptosis-like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show the specific roles of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating stress-induced cell death.

Neurotoxic HIV-1 viral proteins contribute to the development of HIV-associated neurocognitive disorder (HAND), the prevalence of which remains high (30–50%) with no effective treatment available. Estrogen is a known neuroprotective agent; however, the diverse mechanisms of estrogen action on the different types of estrogen receptors is not completely understood. In this study, we determined the extent to which and mechanisms by which 17α-estradiol (17αE2), a natural less-feminizing estrogen, offers neuroprotection against HIV-1 gp120-induced neuronal injury. Endolysosomes are important for neuronal function, and endolysosomal dysfunction contributes to HAND and other neurodegenerative disorders. In hippocampal neurons, estrogen receptor α (ERα) is localized to endolysosomes and 17αE2 acidifies endolysosomes. ERα knockdown or overexpressing an ERα mutant that is deficient in endolysosome localization prevents 17αE2-induced endolysosome acidification. Furthermore, 17αE2-induced increases in dendritic spine density depend on endolysosome localization of ERα. Pretreatment with 17αE2 protected against HIV-1 gp120-induced endolysosome deacidification and reductions in dendritic spines; such protective effects depended on endolysosome localization of ERα. In male HIV-1 transgenic rats, we show that 17αE2 treatment prevents the development of enlarged endolysosomes and reduction in dendritic spines. Our findings demonstrate a novel endolysosome-dependent pathway that governs the ERα-mediated neuroprotective actions of 17αE2, findings that might lead to the development of novel therapeutic strategies against HAND.SIGNIFICANCE STATEMENTExtranuclear presence of membrane-bound estrogen receptors (ERs) underlie the enhancing effect of estrogen on cognition and synaptic function. The estrogen receptor subtype ERα is present on endolysosomes and plays a critical role in the enhancing effects of 17αE2 on endolysosomes and dendritic spines. These findings provide novel insight into the neuroprotective actions of estrogen. Furthermore, 17αE2 protected against HIV-1 gp120-induced endolysosome dysfunction and reductions in dendritic spines, and these protective effects of 17αE2 were mediated via endolysosome localization of ERα. Such findings provide a rationale for developing 17αE2 as a therapeutic strategy against HIV-associated neurocognitive disorders.

The HIV-1 coat protein gp120 continues to be implicated in the pathogenesis of HIV-1 associated neurocognitive disorder (HAND); a condition known to affect ~50% of people living with HIV-1 (PLWH). Autopsy brain tissues of HAND individuals display morphological changes to mitochondria and endolysosomes, and HIV-1 gp120 causes mitochondrial dysfunction including increased levels of reactive oxygen species (ROS) and de-acidification of endolysosomes. Ferrous iron is linked directly to ROS production, ferrous iron is contained in and released from endolysosomes, and PLWH have elevated iron and ROS levels. Based on those findings, we tested the hypothesis that HIV-1 gp120-induced endolysosome de-acidification and subsequent iron efflux from endolysosomes is responsible for increased levels of ROS. In U87MG glioblastoma cells, HIV-1 gp120 de-acidified endolysosomes, reduced endolysosome iron levels, increased levels of cytosolic and mitochondrial iron, and increased levels of cytosolic and mitochondrial ROS. These effects were all attenuated significantly by the endolysosome-specific iron chelator deferoxamine, by inhibitors of endolysosome-resident two-pore channels and divalent metal transporter-1 (DMT-1), and by inhibitors of mitochondria-resident DMT-1 and mitochondrial permeability transition pores. These results suggest that oxidative stress commonly observed with HIV-1 gp120 is downstream of its ability to de-acidify endolysosomes, to increase the release of iron from endolysosomes, and to increase the uptake of iron into mitochondria. Thus, endolysosomes might represent early and upstream targets for therapeutic strategies against HAND.

The present study deals with robust design optimization (RDO) of an underground bunker under stochastic blast-induced ground motion. Since the direct Monte Carlo Simulation (MCS) requires extensive computational time to solve an RDO problem, the polynomial response surface method (RSM) is highly appreciated as an alternative to reduce the computational burden. Thus, the present study attempts to explore the advantage of the moving least-squares method (MLSM) based dual RSM to take into account the record-to-record variation in the blast load in place of the conventional least-squares method (LSM). The blast-induced ground shock has been artificially generated by considering the Tajimi-Kanai power spectral density function (PSDF) and incorporating uncertainty in the blast parameters, viz. explosion distance and explosive charge weight. The application of the proposed dual RSM in RDO not only evades several finite element analyses runs in the simulation loop during the optimization process, but also improve the computational efficiency significantly. The RDO is formulated by simultaneously optimizing the expected value and variance of the performance function by using the weighted sum approach. The results show that the present MLSM-based RDO strategy yields more accurate and robust solutions than the conventional LSM-based approach when compared with the direct MCS results.

Artemisinin-based combination therapies (ACTs) have been able to reduce the clinical and pathological malaria cases in endemic areas around the globe. However, recent reports have shown a progressive decline in malaria parasite clearance in South-east Asia after ACT treatment, thus envisaging a need for new artemisinin (ART) derivatives and combinations. To address the emergence of drug resistance to current antimalarials, here we report the synthesis of artemisinin-peptidyl vinyl phosphonate hybrid molecules that show superior efficacy than artemisinin alone against chloroquine-resistant as well as multidrug-resistant Plasmodium falciparum strains with EC50 in pico-molar ranges. Further, the compounds effectively inhibited the survival of ring-stage parasite for laboratory-adapted artemisinin-resistant parasite lines as compared to artemisinin. These hybrid molecules showed complete parasite clearance in vivo using P. berghei mouse malaria model in comparison to artemisinin alone. Studies on the mode of action of hybrid molecules suggested that these artemisinin-peptidyl vinyl phosphonate hybrid molecules possessed dual activities: inhibited falcipain-2 (FP-2) activity, a P. falciparum cysteine protease involved in hemoglobin degradation, and also blocked the hemozoin formation in the food-vacuole, a step earlier shown to be blocked by artemisinin. Since these hybrid molecules blocked multiple steps of a pathway and showed synergistic efficacies, we believe that these lead compounds can be developed as effective antimalarials to prevent the spread of resistance to current antimalarials.

A robust design optimization (RDO) of rail bridge hollow pier (RBHP) under uncertainty is explored in the present paper. Uncertainty information about such structural system is often observed to be insufficient to define involved uncertain parameters probabilistically. Rather, information about bounds of the variability of these uncertain parameters is only available. In such case, parameters are modeled as uncertain but bounded (UBB) type. The conventional reliability-based design optimization cannot be applied with UBB type uncertainty. Keeping this in view, an RDO, which is a relatively newer approach, is explored for RBHP under UBB type uncertainty in the present study. The RDO minimizes cost and deviation of cost simultaneously ensuring constraint feasibility under uncertainty. Literature addressing RDO on RBHP with UBB parameters considering seismic effects is observed to be scarce. As the simulation-based approach would require a tremendous computational time, a moving least squares method (MLSM)-based metamodeling approach has been adopted here to alleviate the computational burden. The RDO results show that guarantee in safe performance and substantially reduced deviation of structural performance can be achieved by the RDO. The behavior of the structure is quite satisfactorily captured by the MLSM predictions than the conventional least squares method-based metamodeling. The computational time is observed to be drastically reduced in the present case in comparison to that by the direct Monte Carlo Simulation.

Hospices for people with AIDS and neuropsychiatric complications present themselves as unique case studies of the institutional behavior of patients and caregivers, as well as the effective implementation of public health policy. Using a mix of visual ethnography and first-hand narratives, I explore the workings of a hospice, Philip AIDS Centre, in rural Gujarat, India, and the daily life of its residents. I also consider the ethics and challenges of photographing people with mental illnesses, drawing my examples from the popular media and photography, while mentioning their role in palliative care.

Phospholipid metabolism is crucial for membrane dynamics in malaria parasites during entire cycle in the host cell. Plasmodium falciparum harbours several members of phospholipase family, which play key role in phospholipid metabolism. Here we have functionally characterized a parasite lysophospholipase (PfLPL1) with a view to understand its role in lipid homeostasis. We used a regulated fluorescence affinity tagging, which allowed endogenous localization and transient knock-down of the protein. PffLPL1localizes to dynamic vesicular structures that traffic from parasite periphery, through the cytosol to get associated as a multi-vesicular neutral lipid rich body next to the food-vacuole during blood stages. Down-regulation of the PfLPL1 disrupted parasite lipid-homeostasis leading to significant reduction of neutral lipids in lipid-bodies. This hindered conversion of heme to hemozoin, leading to food-vacuole abnormalities, which in turn disrupted parasite development cycle and significantly inhibited parasite growth. Detailed lipidomic analyses of inducible knock-down parasites confirmed role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Our study thus suggests a specific role of PfLPL1 to generate neutral lipids in the parasite, which are essential for parasite survival.ImportancePresent study was undertaken with a view to understand the functional role of a unique lipase (lysophopholipase, PfLPL1) of the human malaria parasite. We utilized genetic approaches for GFP tagging as well as to knock-down the target protein in the parasite. Our studies showed that PfLPL1 associates closely with the lysosome like organelle in the parasite, the food-vacuole. During the blood stage parasite cycle, the food-vacuole is involved in degradation of host haemoglobin and conversion of heme to hemozoin. Genetic knock-down approaches and detailed lipidomic studies confirmed that PfLPL1 protein plays key role in generation of neutral lipid stores in the parasite; neutral lipids are essentially required for hemozoin formation in the parasite, a vital function of the food-vacuole. Overall, this study identified specific role of PfLPL1 in the parasite which is essential for parasite survival.

This paper presents robust design optimization (RDO) of a reinforced concrete (RC) frame subjected to stochastic blast-induced ground motion (BIGM). As a full simulation approach requires extensive computational time to solve such problem, the polynomial response surface method (RSM) has been applied in the present study to alleviate the associated computational burden. The least squares method (LSM) generally adopted in the conventional RSM is often reported to be a source of error in optimization. Hence, a moving least squares method (MLSM)-based adaptive RSM is explored in the RDO. Random blast load is modelled by spectral representation method. The record-to-record variation of BIGM time history is captured by applying dual RSM. The optimization problem has been developed as a cost-minimization problem subjected to the displacement constraints. The RDO is formulated by simultaneously optimizing the expected value and the variation of the performance function by using weighted sum method. The robustness in the constraint is ensured by limiting the probability of failure of limit state function. The results show that the proposed MLSM-based RDO strategy yields more accurate solutions than the conventional LSM-based RSM taking full simulation approach as reference. At the same time, the present procedure yields Pareto-front in significantly lesser computational time than the full-simulation-based RDO approach.

Human immunodeficiency virus type 1 (HIV-1) associated neuropathy is the most common neurological complication of HIV-1, with debilitating pain affecting the quality of life. HIV-1 gp120 plays an important role in the pathogenesis of HIV neuropathy via direct neurotoxic effects or indirect pro-inflammatory responses. Studies have shown that gp120-induced release of mediators from Schwann cells induce CCR5-dependent DRG neurotoxicity, however, CCR5 antagonists failed to improve pain in HIV- infected individuals. Thus, there is an urgent need for a better understanding of neuropathic pain pathogenesis and developing effective therapeutic strategies. Because lysosomal exocytosis in Schwann cells is an indispensable process for regulating myelination and demyelination, we determined the extent to which gp120 affected lysosomal exocytosis in human Schwann cells. We demonstrated that gp120 promoted the movement of lysosomes toward plasma membranes, induced lysosomal exocytosis, and increased the release of ATP into the extracellular media. Mechanistically, we demonstrated lysosome de-acidification, and activation of P2X4 and VNUT to underlie gp120-induced lysosome exocytosis. Functionally, we demonstrated that gp120-induced lysosome exocytosis and release of ATP from Schwann cells leads to increases in intracellular calcium and generation of cytosolic reactive oxygen species in DRG neurons. Our results suggest that gp120-induced lysosome exocytosis and release of ATP from Schwann cells and DRG neurons contribute to the pathogenesis of HIV-1 associated neuropathy.

Background Glioblastoma multiforme (GBM) is a very aggressive form of brain cancer that carries with it a tragically poor prognosis. As with many other forms of cancer, the extracellular environment near GBM tumors is acidified and is relevant to the pathogenesis of GBM because decreased pH promotes tumor cell invasion, increases angiogenesis, decreases immune surveillance, and increases resistance to possible treatments. Recently, vacuolar ATPase (v-ATPase), a proton pump that helps maintain the acidic environment in endosomes and lysosomes (hereafter referred to endolysosomes) as well as proton gradients across the plasma membrane, was identified as a novel therapeutic target for GBM. However, information is lacking about cancer cell and tissue pH of endolysosomes, cytosol and extracellular fluid. Aim Here, we measured endolysosome, cytosolic, and extracellular pH in U87MG cells in the absence and presence of the v-ATPase inhibitor bafilomycin A1. Methods In vitro measurements of U87MG cells were conducted using LysoSensor dye and a Lysosome-RFP dye for lysosome pH, BCECF-AM for cytosolic pH, and a pH-sensitive microprobe for extracellular pH. Results Bafilomycin A1 increased endolysosome pH from 5.28 to 5.57, decreased cytosolic pH from 7.01 to 6.46, and increased extracellular pH from 7.18 to 7.40. Conclusions Here, we report the ability to make pH measurements in U87MG glioblastoma cells and discuss these results in the context of GBM pathogenesis and possible treatment. This might be of some importance in understanding the pathogenesis of GBM because the highly regulated stores of hydrogen (H+) ions in endolysosomes can influence cytosolic and extracellular pH as well as the distribution, numbers, and sizes of endolysosomes.

BACKGROUND: Glioblastoma multiforme (GBM) is a Grade IV astrocytoma with an aggressive disease course and a uniformly poor prognosis. Pathologically, GBM is characterized by rapid development of primary tumors, diffuse infiltration into the brain parenchyma, and robust angiogenesis. The treatment options that are limited and largely ineffective include a combination of surgical resection, radiotherapy, and chemotherapy with the alkylating agent temozolomide. RECENT FINDINGS: Similar to many other forms of cancer, the extracellular environment near GBM tumors is acidified. Extracellular acidosis is particularly relevant to tumorgenesis and the concept of tumor cell dormancy because of findings that decreased pH reduces proliferation, increases resistance to apoptosis and autophagy, promotes tumor cell invasion, increases angiogenesis, obscures immune surveillance, and promotes resistance to drug and radio-treatment. Factors known to participate in the acidification process are nutrient starvation, oxidative stress, hypoxia and high levels of anaerobic glycolysis that lead to increases in lactate. Also involved are endosomes and lysosomes (hereafter termed endolysosomes), acidic organelles with highly regulated stores of hydrogen (H(+)) ions. Endolysosomes contain more than 60 hydrolases as well as about 50 proteins that are known to affect the number, sizes and distribution patterns of these organelles within cells. Recently, vacuolar ATPase (v-ATPase), the main proton pump that is responsible for maintaining the acidic environment in endolysosomes, was identified as a novel therapeutic target for glioblastoma. CONCLUSIONS: Thus, a greater understanding of the role of endolysosomes in regulating cellular and extracellular acidity could result in a better elucidation of GBM pathogenesis and new therapeutic strategies.

The present paper deals with the Robust Design Optimization (RDO) of an offshore steel structure under wave loading considering parameter uncertainty. A novel Moving Least Squares Method (MLSM)-based metamodelling strategy has been adopted in the framework of MCS to evade extensive computational time requirement. The optimization problem is posed as weight minimization problem under displacement constraint. The proposed MLSM-based RDO strategy yields more accurate solutions than the conventional Least Squares Method (LSM)-based metamodelling when compared with the direct MCS results as benchmark. The proposed approach requires less computational time than the direct MCS. The results show that by compromising a small increment in structural weight, one can achieve robust and reliable design solution within affordable computational time by the proposed RDO approach.

The adaptability of micro-tunneling of underground pipeline is getting importance day by day because of minimum disturbance requirement to the busy city life. In the present paper, a Robust Design Optimization (RDO) approach under uncertainty of such buried concrete pipeline is presented incorporating seismic effect. Often sufficient reliable data is unavailable for such problems to treat the involved parameters probabilistically. Hence, these parameters have been treated as Uncertain but Bounded (UBB) type. The limit state functions considered are the bending moment, torsional moment, crack control, shear, and minimum and maximum steel requirements as per ASCE and BS guideline. The results depict that the RDO yields solutions which are insensitive to the variation of input uncertainty. The parametric study shows that by accommodating a small increment in the optimal cost of the pipe, a more robust and reliable design solution can be achieved by the RDO approach in comparison to the deterministic design.

Background Apolipoprotein E (ApoE) is the major carrier protein that mediates the transport and delivery of cholesterol and other lipids in the brain. Three isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans, and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1-associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE uptake in the brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation. Methods Using U87MG glioblastoma cells expressing LTR-driven luciferase, we determined the extent to which LRP1 as well as ApoE2, ApoE3, and ApoE4 affected Tat-mediated HIV-1 LTR transactivation. Results A specific LRP1 antagonist and siRNA knockdown of LRP1 both restricted significantly Tat-mediated LTR transactivation. Of the three ApoEs, ApoE4 was the least potent and effective at preventing HIV-1 Tat internalization and at decreasing Tat-mediated HIV-1 LTR transactivation. Further, Tat-mediated LTR transactivation was attenuated by an ApoE mimetic peptide, and ApoE4-induced restriction of Tat-mediated LTR transactivation was potentiated by an ApoE4 structure modulator that changes ApoE4 into an ApoE3-like phenotype. Conclusions These findings help explain observed differential effects of ApoEs on HIV-1 infectivity and the prevalence of HAND in people living with HIV-1 infection and suggest that ApoE mimetic peptides and ApoE4 structure modulator might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders. Electronic supplementary material The online version of this article (10.1186/s12974-018-1129-1) contains supplementary material, which is available to authorized users.

The human malaria parasite Plasmodium falciparum possesses sophisticated systems of protein secretion to modulate host cell invasion and remodeling. In the present study, we provide insights into the function of the AP-1 complex in P. falciparum. We utilized GFP fusion constructs for live cell imaging, as well as fixed parasites in immunofluorescence analysis, to study adaptor protein mu1 (Pfμ1) mediated protein trafficking in P. falciparum. In trophozoites Pfμ1 showed similar dynamic localization to that of several Golgi/ER markers, indicating Golgi/ER localization. Treatment of transgenic parasites with Brefeldin A altered the localization of Golgi-associated Pfμ1, supporting the localization studies. Co-localization studies showed considerable overlap of Pfμ1 with the resident rhoptry proteins, rhoptry associated protein 1 (RAP1) and Cytoadherence linked asexual gene 3.1 (Clag3.1) in schizont stage. Immunoprecipitation experiments with Pfμ1 and PfRAP1 revealed an interaction, which may be mediated through an intermediate transmembrane cargo receptor. A specific role for Pfμ1 in trafficking was suggested by treatment with AlF4, which resulted in a shift to a predominantly ER-associated compartment and consequent decrease in co-localization with the Golgi marker GRASP. Together, these results suggest a role for the AP-1 complex in rhoptry protein trafficking in P. falciparum.

HIV-associated neurocognitive disorders (HAND) remain prevalent and are aggravated by µ-opioid use. We have previously shown that morphine and other µ-opioids may contribute to HAND by inhibiting the homeostatic and neuroprotective chemokine receptor CXCR4 in cortical neurons, and this novel mechanism depends on upregulation of the protein ferritin heavy chain (FHC). Here, we examined the cellular events and potential mechanisms involved in morphine-mediated FHC upregulation using rat cortical neurons of either sex in vitro and in vivo. Morphine dose dependently increased FHC protein levels in primary neurons through µ-opioid receptor (µOR) and Gαi-protein signaling. Cytoplasmic FHC levels were significantly elevated, but nuclear FHC levels and FHC gene expression were unchanged. Morphine-treated rats also displayed increased FHC levels in layer 2/3 neurons of the prefrontal cortex. Importantly, bothin vitroandin vivoFHC upregulation was accompanied by loss of mature dendritic spines, which was also dependent on µOR and Gαi-protein signaling. Moreover, morphine upregulated ferritin light chain (FLC), a component of the ferritin iron storage complex, suggesting that morphine altered neuronal iron metabolism. Indeed, prior to FHC upregulation, morphine increased cytoplasmic labile iron levels as a function of decreased endolysosomal iron. In line with this, chelation of endolysosomal iron (but not extracellular iron) blocked morphine-induced FHC upregulation and dendritic spine reduction, whereas iron overloading mimicked the effect of morphine on FHC and dendritic spines. Overall, these data demonstrate that iron mediates morphine-induced FHC upregulation and consequent dendritic spine deficits and implicate endolysosomal iron efflux to the cytoplasm in these effects.

Background Malaria remains the world’s most important devastating parasitic disease. Of the five species of Plasmodium known to infect and cause human malaria, Plasmodium falciparum is the most virulent and responsible for majority of the deaths caused by this disease. Mainstream drug therapy targets the asexual blood stage of the malaria parasite, as the disease symptoms are mainly associated with this stage. The prevalence of malaria parasite strains resistance to existing anti-malarial drugs has made the control of malaria even more challenging and hence the development of a new class of drugs is inevitable. Methods Screening against different drug resistant and sensitive strains of P. falciparum was performed for few bicyclic lactam-based motifs, exhibiting a broad spectrum of activity with low toxicity generated via a focussed library obtained from diversity oriented synthesis (DOS). The synthesis and screening was followed by an in vitro assessment of the possible cytotoxic effect of this class of compounds on malaria parasite. Results The central scaffold a chiral bicyclic lactam (A) and (A’) which were synthesized from (R)-phenylalaninol, levulinic acid and 3-(2-nitrophenyl) levulinic acid respectively. The DOS library was generated from A and from A’, by either direct substitution with o-nitrobenzylbromide at the carbon α- to the amide functionality or by conversion to fused pyrroloquinolines. Upon screening this diverse library for their anti-malarial activity, a dinitro/diamine substituted bicyclic lactam was found to demonstrate exceptional activity of >85% inhibition at 50 μM concentration across different strains of P. falciparum with no toxicity against mammalian cells. Also, loss of mitochondrial membrane potential, mitochondrial functionality and apoptosis was observed in parasite treated with diamine-substituted bicyclic lactams. Conclusions This study unveils a DOS-mediated exploration of small molecules with novel structural motifs that culminates in identifying a potential lead molecule against malaria. In vitro investigations further reveal their cytocidal effect on malaria parasite growth. It is not the first time that DOS has been used as a strategy to identify therapeutic leads against malaria, but this study establishes the direct implications of DOS in scouting novel motifs with anti-malarial activity. Electronic supplementary material The online version of this article (doi:10.1186/1475-2875-13-467) contains supplementary material, which is available to authorized users.

Summary The ATP-dependent ClpQY system is a prokaryotic proteasome-like multi-subunit machinery localized in the mitochondrion of malaria parasite. The ClpQY machinery consists of ClpQ threonine pro- tease and ClpY ATPase. In the present study, we have assessed cellular effects of transient interference of PfClpQ protease activity in Plas- modium falciparum using a trans-dominant nega- tive approach combined with FKBP degradation domain system. A proteolytically inactive mutant PfClpQ protein (PfClpQ(mut)) fused with FKBP degradation domain was expressed in parasites, which gets stabilized by Shield1 drug treatment. We show that the inactive PfClpQ(mut) interacts with wild-type PfClpQ and associates within multi- subunit complex in the parasite. Stabilization of the PfClpQ(mut) and its association in the protease machinery caused dominant negative effect in the transgenic parasites, which disrupted the growth cycle of asexual blood stage parasites. The mito- chondria in these parasites showed abnormal mor- phology, these mitochondria were not able to grow and divide in the parasite. We further show that the dominant negative effect of PfClpQ(mut) disrupted transcription of mitochondrial genome encoded genes, which in turn blocked normal development and functioning of the mitochondria.

Malaria remains a major parasitic disease in the tropical and sub-tropical countries mainly due to dramatic increase in parasite lines resistant to commonly used anti-malarials. Characterization of novel metabolic pathways in the parasites and understanding their functional role is a prerequisite to design new anti-malarial strategies. Parasite proteases play key role in growth and differentiation of all the developmental stages across the parasite life cycle and present the most promising targets to develop new drugs against malaria. In Plasmodium falciparum genome database a total of 123 proteases are identified; these proteases belong to five different clans: Cysteine, Aspartic, Serine, Metallo-, and Threonine. Some of the most studied parasite proteases are those that are functional in the asexual blood stage cycle. Starting with the processing of key parasite ligand in merozoite, the invasive form of blood stage parasite, degradation of host hemoglobin in food-vacuole, regulation of levels of key metabolic pathways in cytosol and cellular organelles, degradation of misfolded and unused proteins, and rupture of host membrane for egress of daughter merozoites is mediated by these proteases. Here we discuss roles of some of the parasite proteases involved in various steps of the parasite intra-erythrocytic cycle.

The ATP-dependent ClpQY system is a prokaryotic proteasome-like multi-subunit machinery localized in the mitochondrion of malaria parasite. The ClpQY machinery consists of ClpQ threonine protease and ClpY ATPase. In the present study, we have assessed cellular effects of transient interference of PfClpQ protease activity in Plasmodium falciparum using a trans-dominant negative approach combined with FKBP degradation domain system. A proteolytically inactive mutant PfClpQ protein [PfClpQ(mut)] fused with FKBP degradation domain was expressed in parasites, which gets stabilized by Shield1 drug treatment. We show that the inactive PfClpQ(mut) interacts with wild-type PfClpQ and associates within multi-subunit complex in the parasite. Stabilization of the PfClpQ(mut) and its association in the protease machinery caused dominant negative effect in the transgenic parasites, which disrupted the growth cycle of asexual blood stage parasites. The mitochondria in these parasites showed abnormal morphology, these mitochondria were not able to grow and divide in the parasite. We further show that the dominant negative effect of PfClpQ(mut) disrupted transcription of mitochondrial genome encoded genes, which in turn blocked normal development and functioning of the mitochondria.

A regulated protein turnover machinery in the cell is essential for effective cellular homeostasis; any interference with this system induces cellular stress and alters the normal functioning of proteins important for cell survival. In this study, we show that persistent cellular stress and organelle dysfunction because of disruption of cellular homeostasis in human malaria parasite Plasmodium falciparum, leads to apoptosis-like cell death. Quantitative global proteomic analysis of the stressed parasites before onset of cell death, showed upregulation of a number of proteins involved in cellular homeostasis; protein network analyses identified upregulated metabolic pathways that may be associated with stress tolerance and pro-survival mechanism. However, persistent stress on parasites cause structural abnormalities in endoplasmic reticulum and mitochondria, subsequently a cascade of reactions are initiated in parasites including rise in cytosolic calcium levels, loss of mitochondrial membrane potential and activation of VAD-FMK-binding proteases. We further show that activation of VAD-FMK-binding proteases in the parasites leads to degradation of phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), a known target of metacaspases, as well as degradation of other components of spliceosomal complex. Loss of spliceosomal machinery impairs the mRNA splicing, leading to accumulation of unprocessed RNAs in the parasite and thus dysregulate vital cellular functions, which in turn leads to execution of apoptosis-like cell death. Our results establish one of the possible mechanisms of instigation of cell death by organelle stress in Plasmodium.

Figure S1. Chloroquine enhances Tat-mediated LTR transactivation in U87MG cells. Figure S2. Endolysosome de-acidifying reagents enhance HIV-1 Tat-mediated LTR transactivation in U87MG cells. Figure S3. Endotoxin (LPS) does not affect Tat-mediated HIV-1 Tat transactivation in U87MG cells. (DOCX 1981kb)