Thiol reductive stress activates the hypoxia response pathway.

Owing to their capability to disrupt the oxidative protein folding environment in the endoplasmic reticulum (ER), thiol antioxidants, such as dithiothreitol (DTT), are used as ER-specific stressors. We recently showed that thiol antioxidants modulate the methionine-homocysteine cycle by upregulating an S-adenosylmethionine-dependent methyltransferase, rips-1, in Caenorhabditis elegans. However, the changes in cellular physiology induced by thiol stress that modulate the methionine-homocysteine cycle remain uncharacterized. Here, using forward genetic screens in C. elegans, we discover that thiol stress enhances rips-1 expression via the hypoxia response pathway. We demonstrate that thiol stress activates the hypoxia response pathway. The activation of the hypoxia response pathway by thiol stress is conserved in human cells. The hypoxia response pathway enhances thiol toxicity via rips-1 expression and confers protection against thiol toxicity via rips-1-independent mechanisms. Finally, we show that DTT might activate the hypoxia response pathway by producing hydrogen sulfide. Our studies reveal an intriguing interaction between thiol-mediated reductive stress and the hypoxia response pathway and challenge the current model that thiol antioxidant DTT disrupts only the ER milieu in the cell.

Phase Separation of RNA Helicase Triggers Stress-Responsive Translational Switch.

A swift response to stress requires global translational suppression, excepting stress proteins. Recently, Iserman et al. uncovered that stress-induced phase separation of the RNA helicase Ded1p results in translational suppression of housekeeping transcripts that contain complex 5' untranslated regions (UTRs). Stress-response transcripts with simpler 5' UTRs escape this global translational suppression.

Harnessing the power of genetics: fast forward genetics in Caenorhabditis elegans.

Forward genetics is a powerful tool to unravel molecular mechanisms of diverse biological processes. The success of genetic screens primarily relies on the ease of genetic manipulation of an organism and the availability of a plethora of genetic tools. The roundworm Caenorhabditis elegans has been one of the favorite models for genetic studies due to its hermaphroditic lifestyle, ease of maintenance, and availability of various genetic manipulation tools. The strength of C. elegans genetics is highlighted by the leading role of this organism in the discovery of several conserved biological processes. In this review, the principles and strategies for forward genetics in C. elegans are discussed. Further, the recent advancements that have drastically accelerated the otherwise time-consuming process of mutation identification, making forward genetic screens a method of choice for understanding biological functions, are discussed. The emphasis of the review has been on providing practical and conceptual pointers for designing genetic screens that will identify mutations, specifically disrupting the biological processes of interest.

Pharmacological reactivation of inactive X-linked Mecp2 in cerebral cortical neurons of living mice.

Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG-binding protein 2 (MECP2). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), ∼50% of cells express mutant MECP2 and the other ∼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked MECP2 in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called "XCI factors" or "XCIFs"). Whether XCIF inhibitors in living animals can reactivate Xi-linked MECP2 in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked MECP2 in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked MECP2 corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked Mecp2 in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked Mecp2 in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked MECP2 as a therapeutic approach for RTT.

Modulation of the extent of structural heterogeneity in α-synuclein fibrils by the small molecule thioflavin T.

The transition of intrinsically disordered, monomeric α-synuclein into ß-sheet-rich oligomers and fibrils is associated with multiple neurodegenerative diseases. Fibrillar aggregates possessing distinct structures that differ in toxicity have been observed in different pathological phenotypes. Understanding the mechanism of the formation of various fibril polymorphs with differing cytotoxic effects is essential for determining how the aggregation reaction could be modulated to favor nontoxic fibrils over toxic fibrils. In this study, two morphologically different α-synuclein fibrils, one helical and the other ribbon-like, are shown to form together. Surprisingly, a widely used small molecule for probing aggregation reactions, thioflavin T (ThT), was found to tune the structural heterogeneity found in the fibrils. The ribbon-like fibrils formed in the presence of ThT were found to have a longer structural core than the helical fibrils formed in the absence of ThT. The ribbon-like fibrils are also more toxic to cells. By facilitating the formation of ribbon-like fibrils over helical fibrils, ThT reduced the extent of fibril polymorphism. This study highlights the role of a small molecule such as ThT in selectively favoring the formation of a specific type of fibril by binding to aggregates formed early on one of multiple pathways, thereby altering the structural core and external morphology of the fibrils formed.

The Pathogenic A116V Mutation Enhances Ion-Selective Channel Formation by Prion Protein in Membranes.

Prion diseases are a group of fatal neurodegenerative disorders that afflict mammals. Misfolded and aggregated forms of the prion protein (PrP(Sc)) have been associated with many prion diseases. A transmembrane form of PrP favored by the pathogenic mutation A116V is associated with Gerstmann-Sträussler-Scheinker syndrome, but no accumulation of PrP(Sc) is detected. However, the role of the transmembrane form of PrP in pathological processes leading to neuronal death remains unclear. This study reports that the full-length mouse PrP (moPrP) significantly increases the permeability of living cells to K(+), and forms K(+)- and Ca(2+)-selective channels in lipid membranes. Importantly, the pathogenic mutation A116V greatly increases the channel-forming capability of moPrP. The channels thus formed are impermeable to sodium and chloride ions, and are blocked by blockers of voltage-gated ion channels. Hydrogen-deuterium exchange studies coupled with mass spectrometry (HDX-MS) show that upon interaction with lipid, the central hydrophobic region (109-132) of the protein is protected against exchange, making it a good candidate for inserting into the membrane and lining the channel. HDX-MS also shows a dramatic increase in the protein-lipid stoichiometry for A116V moPrP, providing a rationale for its increased channel-forming capability. The results suggest that ion channel formation may be a possible mechanism of PrP-mediated neurodegeneration by the transmembrane forms of PrP.

The Pathogenic Mutation T182A Converts the Prion Protein into a Molten Globule-like Conformation Whose Misfolding to Oligomers but Not to Fibrils Is Drastically Accelerated.

Delineation of the effects of pathogenic mutations linked with familial prion diseases on the structure and misfolding of prion protein (PrP) will be useful in understanding the molecular mechanism of PrP misfolding. Here, it has been shown that the pathogenic mutation T182A causes a drastic reduction in the apparent cooperativity and enthalpy of unfolding of the mouse prion protein (moPrP) under misfolding-prone conditions by converting the protein into a molten globule (MG)-like conformation. Hydrogen-deuterium exchange studies in conjunction with mass spectrometry indicate that the T182A mutation disrupts the core of the protein, thereby increasing overall structural dynamics. T182A moPrP is shown to misfold to oligomers very much faster than does wild-type (wt) moPrP but to misfold to fibrils at a rate similar to that of wt moPrP. This observation suggests that oligomers are unlikely to play a productive role in the direct pathway of aggregation from monomer to fibrils. The observation that fully folded T182A moPrP has a MG-like structure, and that it misfolds to oligomers much faster than does wt moPrP, suggests that a MG-like intermediate, whose structure resembles that of fully folded T182A moPrP, might be populated early on the pathway of misfolding of wt moPrP to oligomers.

Molecular Mechanism of the Misfolding and Oligomerization of the Prion Protein: Current Understanding and Its Implications.

Prion diseases, also known as transmissible spongiform encephalopathies, make up a group of fatal neurodegenerative disorders linked with the misfolding and aggregation of the prion protein (PrP). Although it is not yet understood how the misfolding of PrP induces neurodegeneration, it is widely accepted that the formation of misfolded prion protein (termed PrP(Sc)) is both the triggering event in the disease and the main component of the infectious agent responsible for disease transmission. Despite the clear involvement of PrP(Sc) in prion diseases, the exact composition of PrP(Sc) is not yet well-known. Recent studies show that misfolded oligomers of PrP could, however, be responsible for neurotoxicity and/or infectivity in the prion diseases. Hence, understanding the molecular mechanism of formation of the misfolded oligomers of PrP is critical for developing an understanding about the prion diseases and for developing anti-prion therapeutics. This review discusses recent advances in understanding the molecular mechanism of misfolded oligomer formation by PrP and its implications for the development of anti-prion therapeutics.

Structural effects of multiple pathogenic mutations suggest a model for the initiation of misfolding of the prion protein.

A molecular understanding of the prion diseases requires delineation of the origin of misfolding of the prion protein (PrP). An understanding of how different disease-linked mutations affect the structure and dynamics of native monomeric PrP can provide a clue about how misfolding commences. In this study, hydrogen-deuterium exchange mass spectrometry was used to show that several disease-linked mutant variants, which are thermodynamically destabilized, share a common structural perturbation in their native states: helix 1 is destabilized to an extent that correlates well with the destabilization of the native protein. The mutant variants misfold and form oligomers faster than does the wild-type protein, at rates that increase exponentially with the extent to which helix 1 is destabilized in the native protein. It appears, therefore, that the loss of helix 1 structure marks the beginning of PrP misfolding and oligomerization.

Rational stabilization of helix 2 of the prion protein prevents its misfolding and oligomerization.

Designed stabilization of helix 2 of the mouse prion protein is shown to lead to an increase in global stability of the protein. Studies of hydrogen exchange coupled to mass spectrometry confirm that the increase in stability is confined primarily to helix 2, and that it accounts for the global stabilization of the protein. Importantly, such localized stabilization of the protein can completely inhibit its ability to form oligomers and slows down amyloid fibril formation.

Process intensification for sustainable extraction of metals from e-waste: challenges and opportunities.

The electrical and electronic waste is expected to increase up to 74.7 million metric tons by 2030 due to the unparalleled replacement rate of electronic devices, depleting the conventional sources of valuable metals such as rare earth elements, platinum group metals, Co, Sb, Mo, Li, Ni, Cu, Ag, Sn, Au, and Cr. Most of the current techniques for recycling, recovering, and disposing of e-waste are inappropriate and therefore contaminate the land, air, and water due to the release of hazardous compounds into the environment. Hydrometallurgy and pyrometallurgy are two such conventional methods used extensively for metal recovery from waste electrical and electronic equipment (WEEE). However, environmental repercussions and higher energy requirements are the key drawbacks that prevent their widespread application. Thus, to ensure the environment and elemental sustainability, novel processes and technologies must be developed for e-waste management with enhanced recovery and reuse of the valued elements. Therefore, the goal of the current work is to examine the batch and continuous processes of metal extraction from e-waste. In addition to the conventional devices, microfluidic devices have been also analyzed for microflow metal extraction. In microfluidic devices, it has been observed that the large specific surface area and short diffusion distance of microfluidic devices are advantageous for the efficient extraction of metals. Additionally, cutting-edge technologies have been proposed to enhance the recovery, reusability, and recycling of e-waste. The current study may support decision-making by researchers in deciding the direction of future research and moving toward sustainable development.

Polyoxometalate-HKUST-1 composite derived nanostructured Na-Cu-Mo2C catalyst for efficient reverse water gas shift reaction.

Transforming CO2 to CO via reverse water-gas shift (RWGS) reaction is widely regarded as a promising technique for improving the efficiency and economics of CO2 utilization processes. Moreover, it is also considered as a pathway towards e-fuels. Cu-oxide catalysts are widely explored for low-temperature RWGS reactions; nevertheless, they tend to deactivate significantly under applied reaction conditions due to the agglomeration of copper particles at elevated temperatures. Herein, we have synthesized homogeneously distributed Cu metallic nanoparticles supported on Mo2C for the RWGS reaction by a unique approach of in situ carburization of metal-organic frameworks (MOFs) using a Cu-based MOF i.e. HKUST-1 encapsulating molybdenum-based polyoxometalates. The newly derived Na-Cu-Mo2C nanocomposite catalyst system exhibits excellent catalytic performance with a CO production rate of 3230.0 mmol gcat-1 h-1 with 100% CO selectivity. Even after 250 h of a stability test, the catalyst remained active with more than 80% of its initial activity.

ERASing endoplasmic reticulum stress: the faster, the better.

The endoplasmic reticulum (ER) has evolved multiple mechanisms to maintain homeostasis under stress conditions. A recent study by Efstathiou et al. identified a novel mechanism of silencing ER-associated RNAs by the exogenous RNA interference pathway. This adaptive response reduces protein flux in the ER under stressful conditions.

α-Terpineol synergizes with gentamicin to rescue Caenorhabditis elegans from Pseudomonas aeruginosa infection by attenuating quorum sensing-regulated virulence.

AIMS: This study scrutinized α-Terpineol (α-T) for its anti-virulence and anti-fouling potential against P. aeruginosa PAO1 in conjunction with gentamicin (GeN) using in-vitro, in-silico, and in-vivo approaches. MAIN METHODS: The quorum quenching (QQ) potential of the drug combination was studied using a quorum sensing (QS) biosensor strain and tested for synergy using chequerboard and time-kill kinetics assays. The effect of α-T and GeN on bacterial motility, QS-regulated virulence factor production, and biofilm formation was assessed in P. aeruginosa PAO1 along with molecular docking analysis. The protective effects of α-T-GeN combination were also examined in a Caenorhabditis elegans infection model through slow-killing (SK) assays. KEY FINDINGS: The drug combination displayed synergy, enhanced QQ activity, and suppressed AHL production in PAO1. At sub-inhibitory concentrations, the drug combination suppressed the expression of genes regulating QS and pseudomonal virulence, thereby inhibiting the production of virulence factors in PAO1. The drug combination compromised all forms of pseudomonal motility, strongly inhibited biofilm formation, and successfully eradicated preformed biofilms. Based on these findings, it is concluded that GeN (alone) does not harbor any QQ properties, but enhances the QQ potential of α-T. Moreover, combinational treatment protected C. elegans from pseudomonal infection and improved survival rates by 73 % at 96 h. SIGNIFICANCE: For the first time, the molecular mechanism responsible for the anti-QS activity of α-T was unraveled through a comprehensive investigation, thereby asserting its potential as an anti-virulent drug against P. aeruginosa.

Optimization of 'on farm' hydropriming conditions in wheat: Soaking time and water volume have interactive effects on seed performance.

Seed priming is a simple and cost effective method to obtain a better plant stand under diverse environmental conditions. The current study was designed to determine the optimal priming duration and water volume for wheat seed. For this experiment, three wheat genotypes with distinct genetic and adaptive backgrounds were chosen. Seeds of each genotype were hydroprimed for 7 durations, i.e. 1, 2, 4, 8, 12, 16, and 20 hours, in three different water volumes, i.e. half, equal, and double volume with respect to seed weight and then surface dried for 1 hour. The control was unprimed (dry) seed. The germination characteristics and seedling vigour potential of hydroprimed seeds were evaluated in the lab by recording several parameters such as germination percentage and speed, seedling growth, and vigour indices at two different temperature levels. The results showed that optimal duration for hydropriming of wheat seed is 12 hours with an equal volume with respect to original seed weight, closely followed by 8 hours with double volume. Reduction in seed performance was observed at 16 and 20 hours priming particularly at double volume treatment. Effect of temperature on seed germination showed improvement in seedling vigour at 25°C when compared to 20°C, although effect on germination percentage was non-significant. Volume of water and priming duration showed significant interactive effects demonstrating that a higher volume can give equivalent results at a shorter duration and vice versa. Another experiment was also conducted to compare the on-farm priming (surface dried seed) with conventional priming (seed re-dried to original moisture) taking 3 potential durations i.e. 8, 12 and 16 hours. Results revealed that both priming methods were statistically at par in terms of germination percentage, while, surface drying resulted in better seedling vigour and speed of germination.

Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer.

Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.

Dithiothreitol causes toxicity in C. elegans by modulating the methionine-homocysteine cycle.

The redox reagent dithiothreitol (DTT) causes stress in the endoplasmic reticulum (ER) by disrupting its oxidative protein folding environment, which results in the accumulation and misfolding of the newly synthesized proteins. DTT may potentially impact cellular physiology by ER-independent mechanisms; however, such mechanisms remain poorly characterized. Using the nematode model Caenorhabditis elegans, here we show that DTT toxicity is modulated by the bacterial diet. Specifically, the dietary component vitamin B12 alleviates DTT toxicity in a methionine synthase-dependent manner. Using a forward genetic screen, we discover that loss-of-function of R08E5.3, an S-adenosylmethionine (SAM)-dependent methyltransferase, confers DTT resistance. DTT upregulates R08E5.3 expression and modulates the activity of the methionine-homocysteine cycle. Employing genetic and biochemical studies, we establish that DTT toxicity is a result of the depletion of SAM. Finally, we show that a functional IRE-1/XBP-1 unfolded protein response pathway is required to counteract toxicity at high, but not low, DTT concentrations.

Animal and plant cells synthesize a significant fraction of their proteins on a structure known as the endoplasmic reticulum. Researchers often use the molecule dithiothreitol to specifically target this compartment and learn more about its role. The toxin works by disturbing the complex chemical environment present in the reticulum, which is required for the proteins to assemble properly. However, it is important to clarify whether dithiothreitol could also affect other parts of the cell, as this could give rise to misleading results. To explore this possibility, Gokul G and Jogender Singh studied the effects of dithiothreitol on the millimetre-long roundworm Caenorhabditis elegans. Their experiments revealed that vitamin B12 could protect against dithiothreitol toxicity via a complex cascade of molecular events which reduced the levels of an important regulatory molecule known as S-adenosylmethionine. Crucially, the chemical reactions that dithiothreitol targeted took place outside the reticulum, suggesting that the toxin impairs processes in the wider cell. These results suggest that dithiothreitol should be reconsidered for use in endoplasmic reticulum studies. However, they also imply that this toxin could be beneficial in small doses, as a reduced concentration of S-adenosylmethionine increases lifespan and health in a variety of organisms.

Gentamicin Augments the Quorum Quenching Potential of Cinnamaldehyde In Vitro and Protects Caenorhabditis elegans From Pseudomonas aeruginosa Infection.

The quorum sensing (QS) circuitry of Pseudomonas aeruginosa represents an attractive target to attenuate bacterial virulence and antibiotic resistance. In this context, phytochemicals harboring anti-virulent properties have emerged as an alternative medicine to combat pseudomonal infections. Hence, this study was undertaken to investigate the synergistic effects and quorum quenching (QQ) potential of cinnamaldehyde (CiNN) in combination with gentamicin (GeN) against P. aeruginosa. The QQ activity of this novel combination was evaluated using a QS reporter strain and synergism was studied using chequerboard assays. Further, the genotypic and phenotypic expression of pseudomonal virulence factors was examined alongside biofilm formation. The combination of CiNN and GeN exhibited synergy and promising anti-QS activity. This drug combination was shown to suppress AHL production and downregulate the expression of critical QS genes in P. aeruginosa PAO1. Molecular docking revealed strong interactions between the QS receptors and CiNN, asserting its QQ potential. Bacterial motility was compromised along with a significant reduction in pyocyanin (72.3%), alginate (58.7%), rhamnolipid (33.6%), hemolysin (82.6%), protease (70.9%), and elastase (63.9%) production. The drug combination successfully eradicated preformed biofilms and inhibited biofilm formation by abrogating EPS production. Our findings suggest that although GeN alone could not attenuate QS, but was able to augment the anti-QS potential of CiNN. To validate our results using an infection model, we quantified the survival rates of Caenorhabditis elegans following PAO1 challenge. The combination significantly rescued C. elegans from PAO1 infection and improved its survival rate by 54% at 96 h. In summary, this study is the first to elucidate the mechanism behind the QQ prospects of CiNN (augmented in presence of GeN) by abrogating AHL production and increasing the survival rate of C. elegans, thereby highlighting its anti-virulent properties.