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.

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.

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.

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.

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.

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.

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.

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.

A Feasible Alternative Strategy Targeting Furin Disrupts SARS-CoV-2 Infection Cycle.

The COVID-19 causing coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human lung cells via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). Notably, the cleavage of the spike by the host cell protease furin in virus-producing cells is critical for subsequent spike-driven entry into lung cells. Thus, effective targeted therapies blocking the spike cleavage and activation in viral producing cells may provide an alternate strategy to break the viral transmission cycle and to overcome disease pathology. Here we engineered and described an antibody-based targeted strategy, which directly competes with the furin mediated proteolytic activation of the spike in virus-producing cells. The described approach involves engineering competitive furin substrate residues in the IgG1 Fc-extended flexible linker domain of SARS-CoV-2 spike targeting antibodies. Considering the site of spike furin cleavage and SARS-CoV-2 egress remains uncertain, the experimental strategy pursued here revealed novel mechanistic insights into proteolytic processing of the spike protein, which suggest that processing does not occur in the constitutive secretory pathway. Furthermore, our results show blockade of furin-mediated cleavage of the spike protein for membrane fusion activation and virus host-cell entry function. These findings provide an alternate insight of targeting applicability to SARS-CoV-2 and the future coronaviridae family members, exploiting the host protease system to gain cellular entry and subsequent chain of infections. IMPORTANCE Since its emergence in December 2019, COVID-19 has remained a global economic and health threat. Although RNA and DNA vector-based vaccines induced antibody response and immunological memory have proven highly effective against hospitalization and mortality, their long-term efficacy remains unknown against continuously evolving SARS-CoV-2 variants. As host cell-enriched furin-mediated cleavage of SARS-CoV-2 spike protein is critical for viral entry and chain of the infection cycle, the solution described here of an antibody Fc-conjugated furin competing peptide is significant. In a scenario where spike mutational drifts do not interfere with the Fc-conjugated antibody's epitope, the proposed furin competing strategy confers a broad-spectrum targeting design to impede the production of efficiently transmissible SARS-CoV-2 viral particles. In addition, the proposed approach is plug-and-play against other potentially deadly viruses that exploit secretory pathway independent host protease machinery to gain cellular entry and subsequent transmissions to host cells.

Bispecific antibodies for the treatment of breast cancer.

INTRODUCTION: There are more than two dozen bispecific antibodies (BsAbs) in development with a variety of designs which are relevant to breast cancer. The field of BsAbs for breast cancer includes agents that co-direct immune recognition of the cancer cell, target unique cancer antigens, and target the microenvironment. BsAbs are being developed for use as antibody-drug conjugates and as homing signals for engineered T-cells. AREAS COVERED: This review covers potential targets for bispecific antibodies, agents in pre-clinical development, agents in clinical trials, combinatorial therapies, and future directions. EXPERT OPINION: There is no BsAb approval expected for breast cancer in the near term, but late-stage trials are underway. Future BsAb roles in breast cancer are possible given unmet needs in estrogen receptor+ disease, residual disease, and de-escalating chemotherapy use. The HER2+ space shows hints of success for BsAbs, but is already crowded. Areas of unmet need still exist.

A patch of positively charged residues regulates the efficacy of clinical DR5 antibodies in solid tumors.

Receptor clustering is the first and critical step to activate apoptosis by death receptor-5 (DR5). The recent discovery of the autoinhibitory DR5 ectodomain has challenged the long-standing view of its mechanistic activation by the natural ligand Apo2L. Because the autoinhibitory residues have remained unknown, here we characterize a crucial patch of positively charged residues (PPCR) in the highly variable domain of DR5. The PPCR electrostatically separates DR5 receptors to autoinhibit their clustering in the absence of ligand and antibody binding. Mutational substitution and antibody-mediated PPCR interference resulted in increased apoptotic cytotoxic function. A dually specific antibody that enables sustained tampering with PPCR function exceptionally enhanced DR5 clustering and apoptotic activation and distinctively improved the survival of animals bearing aggressive metastatic and recurrent tumors, whereas clinically tested DR5 antibodies without PPCR blockade function were largely ineffective. Our study provides mechanistic insights into DR5 activation and a therapeutic analytical design for potential clinical success.

Arming "old guards" with "new dual-targeting weapons".

The long-held paradigm that tumor suppressors are un-targetable in cancer therapy is challenged by a study published in Science. This recent work elegantly describes and characterizes a p53 mutant peptide-selective TCR-mimic antibody and its co-targeting T cell-activating bispecific antibody to eliminate neoantigen-expressing tumors.

Unexpected PD-L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies.

Lack of effective immune infiltration represents a significant barrier to immunotherapy in solid tumors. Thus, solid tumor-enriched death receptor-5 (DR5) activating antibodies, which generates tumor debulking by extrinsic apoptotic cytotoxicity, remains a crucial alternate therapeutic strategy. Over past few decades, many DR5 antibodies moved to clinical trials after successfully controlling tumors in immunodeficient tumor xenografts. However, DR5 antibodies failed to significantly improve survival in phase-II trials, leading in efforts to generate second generation of DR5 agonists to supersize apoptotic cytotoxicity in tumors. Here we have discovered that clinical DR5 antibodies activate an unexpected immunosuppressive PD-L1 stabilization pathway, which potentially had contributed to their limited success in clinics. The DR5 agonist stimulated caspase-8 signaling not only activates ROCK1 but also undermines proteasome function, both of which contributes to increased PD-L1 stability on tumor cell surface. Targeting DR5-ROCK1-PD-L1 axis markedly increases immune effector T-cell function, promotes tumor regression, and improves overall survival in animal models. These insights have identified a potential clinically viable combinatorial strategy to revive solid cancer immunotherapy using death receptor agonism.

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.

Extracellular Proteostasis: Laying Siege to Pathogens.

How protein homeostasis is maintained in the extracellular space remains poorly studied. A recent study employed a Caenorhabditis elegans model to carry out a systematic analysis of the extracellular proteostasis network and uncovered its role in combating a pathogenic attack.

Oncogenic TRIM37 Links Chemoresistance and Metastatic Fate in Triple-Negative Breast Cancer.

The majority of clinical deaths in patients with triple-negative breast cancer (TNBC) are due to chemoresistance and aggressive metastases, with high prevalence in younger women of African ethnicity. Although tumorigenic drivers are numerous and varied, the drivers of metastatic transition remain largely unknown. Here, we uncovered a molecular dependence of TNBC tumors on the TRIM37 network, which enables tumor cells to resist chemotherapeutic as well as metastatic stress. TRIM37-directed histone H2A monoubiquitination enforces changes in DNA repair that rendered TP53-mutant TNBC cells resistant to chemotherapy. Chemotherapeutic drugs triggered a positive feedback loop via ATM/E2F1/STAT signaling, amplifying the TRIM37 network in chemoresistant cancer cells. High expression of TRIM37 induced transcriptomic changes characteristic of a metastatic phenotype, and inhibition of TRIM37 substantially reduced the in vivo propensity of TNBC cells. Selective delivery of TRIM37-specific antisense oligonucleotides using antifolate receptor 1-conjugated nanoparticles in combination with chemotherapy suppressed lung metastasis in spontaneous metastatic murine models. Collectively, these findings establish TRIM37 as a clinically relevant target with opportunities for therapeutic intervention. SIGNIFICANCE: TRIM37 drives aggressive TNBC biology by promoting resistance to chemotherapy and inducing a prometastatic transcriptional program; inhibition of TRIM37 increases chemotherapy efficacy and reduces metastasis risk in patients with TNBC.

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.

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.