SARS-CoV-2 virulence factor ORF3a blocks lysosome function by modulating TBC1D5-dependent Rab7 GTPase cycle.

SARS-CoV-2, the causative agent of COVID-19, uses the host endolysosomal system for entry, replication, and egress. Previous studies have shown that the SARS-CoV-2 virulence factor ORF3a interacts with the lysosomal tethering factor HOPS complex and blocks HOPS-mediated late endosome and autophagosome fusion with lysosomes. Here, we report that SARS-CoV-2 infection leads to hyperactivation of the late endosomal and lysosomal small GTP-binding protein Rab7, which is dependent on ORF3a expression. We also observed Rab7 hyperactivation in naturally occurring ORF3a variants encoded by distinct SARS-CoV-2 variants. We found that ORF3a, in complex with Vps39, sequesters the Rab7 GAP TBC1D5 and displaces Rab7 from this complex. Thus, ORF3a disrupts the GTP hydrolysis cycle of Rab7, which is beneficial for viral production, whereas the Rab7 GDP-locked mutant strongly reduces viral replication. Hyperactivation of Rab7 in ORF3a-expressing cells impaired CI-M6PR retrieval from late endosomes to the trans-Golgi network, disrupting the biosynthetic transport of newly synthesized hydrolases to lysosomes. Furthermore, the tethering of the Rab7- and Arl8b-positive compartments was strikingly reduced upon ORF3a expression. As SARS-CoV-2 egress requires Arl8b, these findings suggest that ORF3a-mediated hyperactivation of Rab7 serves a multitude of functions, including blocking endolysosome formation, interrupting the transport of lysosomal hydrolases, and promoting viral egress.

ABCG2 transporter reduces protein aggregation in cigarette smoke condensate-exposed A549 lung cancer cells.

Cigarette smoke-induced protein aggregation damages the lung cells in emphysema and COPD; however, lung cancer cells continue to thrive, evolving to persist in the toxic environment. Here, we showed that upon the cigarette smoke condensate exposure, A549 lung cancer cells exhibit better survival and reduced level of protein aggregation when compared to non-cancerous Beas-2B and H-6053 cells. Our data suggests that upregulation of efflux pumps in cancer cells assists in reducing smoke toxicity. Specifically, we demonstrated that inhibition of the ABCG2 transporter in A549 by febuxostat or its downregulation by shRNA-mediated RNA interference resulted in a significant increase in protein aggregation due to smoke exposure.

The evolutionary loss of the Eh1 motif in FoxE1 in the lineage of placental mammals.

Forkhead box E1 (FoxE1) protein is a transcriptional regulator known to play a major role in the development of the thyroid gland. By performing sequence alignments, we detected a deletion in FoxE1, which occurred in the evolution of mammals, near the point of divergence of placental mammals. This deletion led to the loss of the majority of the Eh1 motif, which was important for interactions with transcriptional corepressors. To investigate a potential mechanism for this deletion, we analyzed replication through the deletion area in mammalian cells with two-dimensional gel electrophoresis, and in vitro, using a primer extension reaction. We demonstrated that the area of the deletion presented an obstacle for replication in both assays. The exact position of polymerization arrest in primer extension indicated that it was most likely caused by a quadruplex DNA structure. The quadruplex structure hypothesis is also consistent with the exact borders of the deletion. The exact roles of these evolutionary changes in FoxE1 family proteins are still to be determined.

Learnings from Industry 4.0 for transitioning towards Industry 4.0+: challenges and solutions for Indian pharmaceutical sector.

Industry 4.0 (I4.0) is helping production units to become smarter using cyber-physical systems and cognitive intelligence. The advanced diagnostics with I4.0 technologies (I4.0t) help in making the process highly flexible, resilient and autonomous. Still, the adoption of I4.0t especially in emerging economies like India is at a very slow pace. The present research has used an integrated approach i.e., Analytical Hierarchy Process-Combinative Distance-Based Assessment-Decision-Making Trial and Evaluation Laboratory to propose a barrier solution framework using data from pharmaceutical manufacturing sector. The findings reveal that "Costly venture" is found to be the most critical deterrent while "Customer awareness and satisfaction" is one of the potential solutions for I4.0t adoption. Further, lack of standardisation and fair benchmarking policies especially in the context of developing economies needs immediate attention. This article concludes by proposing a framework which will help to move from I4.0 towards Industry 4.0 + (I4.0+) which emphasises on role of collaboration between man and machine. And leads to sustainable supply chain management.

Green, resilient, agile, and sustainable fresh food supply chain enablers: evidence from India.

The existing research on fresh food supply chains (FFSC) sustainability consisting of fur fundamental pillars, namely green (G), resilient (R), agile (A), and sustainability (S) (hereafter GRAS), is explored sparsely and needs thorough investigation. Further, conceptualization and mutual interactions among GRAS enablers that can help perpetuate sustainable supply chains (SSC) still need to be addressed. This study proposes a methodological framework to evaluate the SCS from the perspective of GRAS enablers with an application for the Indian FFSC. A mixed-method sequential approach was used with interviews followed by integrated fuzzy interpretive structural modelling-decision-making trial and evaluation laboratory (FISM-DEMATEL) techniques. The study recognizes twenty supply chain sustainability (SCS) enablers through an extensive literature review and discussions with the expert group. The research discloses that the firms' 'organization culture' acts as the most powerful driver in achieving sustainability in FFSC, followed by the firms' 'environmental certification program' and 'financial strength.' This investigation helps the managers/policymakers of the Indian FFSC to ascertain and comprehend the most significant SCS enablers to achieve sustainability in the supply chain (SC). The causation of SCS enablers supports the managers in systematically focusing on the most significant enablers and working towards their successful implementation. According to our knowledge, this is the first scholarly work that establishes hierarchies and interrelationships among GRAS enablers, thereby providing a holistic picture to decision-makers while adapting such practices. Supplementary Information: The online version contains supplementary material available at 10.1007/s10479-023-05176-x.

RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes.

Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.

RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning.

The bidirectional movement of lysosomes on microtubule tracks regulates their whole-cell spatial arrangement. Arl8b, a small GTP-binding (G) protein, promotes lysosome anterograde trafficking mediated by kinesin-1. Herein, we report an Arl8b effector, RUFY3, which regulates the retrograde transport of lysosomes. We show that RUFY3 interacts with the JIP4-dynein-dynactin complex and facilitates Arl8b association with the retrograde motor complex. Accordingly, RUFY3 knockdown disrupts the positioning of Arl8b-positive endosomes and reduces Arl8b colocalization with Rab7-marked late endosomal compartments. Moreover, we find that RUFY3 regulates nutrient-dependent lysosome distribution, although autophagosome-lysosome fusion and autophagic cargo degradation are not impaired upon RUFY3 depletion. Interestingly, lysosome size is significantly reduced in RUFY3 depleted cells, which could be rescued by inhibition of the lysosome reformation regulatory factor PIKFYVE. These findings suggest a model in which the perinuclear cloud arrangement of lysosomes regulates both the positioning and size of these proteolytic compartments.

Supply chain resilience during the COVID-19 pandemic.

The COVID-19 pandemic has challenged supply chains more seriously challenged than ever before. During this prolonged global health crisis, supply chain managers were forced to rely primarily on solutions developed for limited and foreseeable crises. This study aimed to understand how well existing solutions facilitated supply chain resilience in the UK perishable goods market. Consistent with this aim, we developed a research model based on the supply chain resilience literature and tested it with covariance-based structural equation modelling. Data were collected from 282 retail employees. Supply chain velocity was the preferred measure of resilience. The findings demonstrate that pandemic-related disruptions have affected resilience-building activities. While both proactive and reactive approaches have promoted resilience building during the pandemic, they have not been sufficient to ameliorate all the pandemic's negative effects. Innovation featured as the most effective factor, followed by robustness, empowerment, and risk management via reduced risk. The effect of firm size was significant only on supply chain risk management, with larger companies more efficiently applying risk management practices. The results emphasise the importance of innovation for supply chain resilience. Regardless of firm size, innovation works for every company. Empowerment is another costless and effective tool. Therefore, it is safe to conclude that innovation and empowerment can help organisations to manage their supply chains effectively during crises. Companies can strengthen their supply chain resilience by developing strong relationships with their supplier and employees.

A CRACker of an adaptor connects dynein-mediated transport to calcium signaling.

Many different adaptor proteins activate the processivity of dynein-dynactin complexes and determine the specific cargo for retrograde transport by binding cargo receptors such as Rab GTP-binding (G) proteins. In this issue, Wang et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201806097) identify two GTPases that can function directly as dynein adaptors during endocytosis and are regulated by calcium.

Incorporating Motility in the Motor: Role of the Hook Protein Family in Regulating Dynein Motility.

Cytoplasmic dynein is a retrograde microtubule-based motor transporting cellular cargo, including organelles, vesicular intermediates, RNA granules, and proteins, thus regulating their subcellular distribution and function. Mammalian dynein associates with dynactin, a multisubunit protein complex that is necessary for the processive motility of dynein along the microtubule tracks. Recent studies have shown that the interaction between dynein and dynactin is enhanced in the presence of a coiled-coil activating adaptor protein, which performs dual functions of recruiting dynein and dynactin to their cargoes and inducing the superprocessive motility of the motor complex. One such family of coiled-coil activating adaptor proteins is the Hook family of proteins that are conserved across evolution with three paralogs in the case of mammals, namely, HOOK1-HOOK3. This Perspective aims to provide an overview of the Hook protein structure and the cellular functions of Hook proteins, with an emphasis on the recent developments in understanding their role as activating dynein adaptors.

The dynein adaptor Hook2 plays essential roles in mitotic progression and cytokinesis.

Hook proteins are evolutionarily conserved dynein adaptors that promote assembly of highly processive dynein-dynactin motor complexes. Mammals express three Hook paralogs, namely Hook1, Hook2, and Hook3, that have distinct subcellular localizations and expectedly, distinct cellular functions. Here we demonstrate that Hook2 binds to and promotes dynein-dynactin assembly specifically during mitosis. During the late G2 phase, Hook2 mediates dynein-dynactin localization at the nuclear envelope (NE), which is required for centrosome anchoring to the NE. Independent of its binding to dynein, Hook2 regulates microtubule nucleation at the centrosome; accordingly, Hook2-depleted cells have reduced astral microtubules and spindle positioning defects. Besides the centrosome, Hook2 localizes to and recruits dynactin and dynein to the central spindle. Dynactin-dependent targeting of centralspindlin complex to the midzone is abrogated upon Hook2 depletion; accordingly, Hook2 depletion results in cytokinesis failure. We find that the zebrafish Hook2 homologue promotes dynein-dynactin association and was essential for zebrafish early development. Together, these results suggest that Hook2 mediates assembly of the dynein-dynactin complex and regulates mitotic progression and cytokinesis.

How to do business with lysosomes: Salmonella leads the way.

Pathogens have devised various strategies to alter the host endomembrane system towards building their replicative niche. This is aptly illustrated by Salmonella Typhimurium, whereby it remodels the host endolysosomal system to form a unique niche, also known as Salmonella-containing vacuole (SCV). Decades of research using in vitro cell-based infection studies have revealed intricate details of how Salmonella effectors target endocytic trafficking machinery of the host cell to acquire membrane and nutrients for bacterial replication. Unexpectedly, Salmonella requires host factors involved in endosome-lysosome fusion for its intravacuolar replication. Understanding how Salmonella obtains selective content from lysosomes, that is nutrients, but not active hydrolases, needs further exploration. Recent studies have described heterogeneity in the composition and pH of lysosomes, which will be highly relevant to explore, not only in the context of Salmonella infection, but also for other intracellular pathogens that interact with the endolysosomal pathway.

Rare Coexistence of Acute Monoblastic Leukemia with Chronic Lymphocytic Leukemia.

Acute monoblastic leukemia (AMoL) is a rare hematopoietic neoplasm, and simultaneous occurrence of acute monoblastic leukemia with chronic lymphocytic leukemia is very rare and only a few cases have been reported in the literature. We here report a rare case of dual hematological malignancy in an 85-year-old male. The peripheral blood and bone marrow examination revealed dual population of atypical cells, comprising large cells with opened-up chromatin having monocytic appearance and small mature-appearing lymphocytes. Flowcytometric immunophenotyping confirmed the monocytic lineage of cells, whereas small lymphocytes showed the immunophenotype consistent with chronic lymphocytic leukemia (CLL). The final diagnosis was made as acute monoblastic leukemia with associated CLL. This is a rare case scenario, and this highlights the importance of careful morphological examination and flowcytometric immunophenotyping in the exact characterization of hematopoietic malignancies.

Hsp70 and gama-Semino protein as possible prognostic marker of prostate cancer.

In the United States, Prostate Cancer (PCa) is the leading cause of cancer-related mortality in men. PCa resulted in abnormal growth and function of prostate gland such as secretion of high level of gamma-seminoprotein (gama-SM)/Prostate-Specific Antigen (PSA) which could be detected in the blood. Beside gama-SM protein, the levels of heat shock proteins (Hsp70) were also observed significantly high. Therefore, gama-SM and Hsp70 are unique proteins with high potential for PCa therapeutics and diagnostics. High level of Hsp70 suppresses apoptosis, thus allowing PCa cells to exist; however, depletion of Hsp70 induces apoptosis in PCa cells. Gama-SM is the most prominent biomarker for PCa screening; however, its accuracy is still questionable. Thus, a more suitable streamline biomarker for PCa screening is urgently needed. Hsp70 and gama-SM proteins could be used as a revolutionary biomarker for PCa, and could help to identify possible therapeutic target(s). In this review article we will discuss the relationship between the Hsp70 and gama-SM proteins with PCa, their potential as a dual biomarker, and the possibility for both proteins being used as therapeutic targets.

Lysosome-Mediated Plasma Membrane Repair Is Dependent on the Small GTPase Arl8b and Determines Cell Death Type in Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis is an extremely successful pathogen, and its success is widely attributed to its ability to manipulate the intracellular environment of macrophages. A central phenomenon of tuberculosis pathology enabling immune evasion is the capacity of virulent M. tuberculosis (H37Rv) to induce macrophage necrosis, which facilitates the escape of the mycobacteria from the macrophage and spread of infection. In contrast, avirulent M. tuberculosis (H37Ra) induces macrophage apoptosis, which permits Ag presentation and activation of adaptive immunity. Previously, we found that H37Rv induces plasma membrane microdisruptions, leading to necrosis in the absence of plasma membrane repair. In contrast, H37Ra permits plasma membrane repair, which changes the host cell death modality to apoptosis, suggesting that membrane repair is critical for sequestering the pathogen in apoptotic vesicles. However, mechanisms of plasma membrane repair induced in response to M. tuberculosis infection remain unknown. Plasma membrane repair is known to induce a Ca2+-mediated signaling, which recruits lysosomes to the area of damaged plasma membrane sites for its resealing. In this study, we found that the small GTPase Arl8b is required for plasma membrane repair by controlling the exocytosis of lysosomes in cell lines and in human primary macrophages. Importantly, we found that the Arl8b secretion pathway is crucial to control the type of cell death of the M. tuberculosis-infected macrophages. Indeed, Arl8b-depleted macrophages infected with avirulent H37Ra undergo necrotic instead of apoptotic cell death. These findings suggest that membrane repair mediated by Arl8b may be an important mechanism distinguishing avirulent from virulent M. tuberculosis-induced necrotic cell death.

Multiple Roles, Multiple Adaptors: Dynein During Cell Cycle.

Dynein is an essential protein complex present in most eukaryotes that regulate biological processes ranging from ciliary beating, intracellular transport, to cell division. Elucidating the detailed mechanism of dynein function has been a challenging task owing to its large molecular weight and high complexity of the motor. With the advent of technologies in the last two decades, studies have uncovered a wealth of information about the structural, biochemical, and cell biological roles of this motor protein. Cytoplasmic dynein associates with dynactin through adaptor proteins to mediate retrograde transport of vesicles, mRNA, proteins, and organelles on the microtubule tracts. In a mitotic cell, dynein has multiple localizations, such as at the nuclear envelope, kinetochores, mitotic spindle and spindle poles, and cell cortex. In line with this, dynein regulates multiple events during the cell cycle, such as centrosome separation, nuclear envelope breakdown, spindle assembly checkpoint inactivation, chromosome segregation, and spindle positioning. Here, we provide an overview of dynein structure and function with focus on the roles played by this motor during different stages of the cell cycle. Further, we review in detail the role of dynactin and dynein adaptors that regulate both recruitment and activity of dynein during the cell cycle.

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication.

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor-HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication.

DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes.

Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3'-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) etc., are routinely used by researchers to analyze the role of various proteins in endocytic pathway. Trafficking of DQ-BSA in cells depleted of or over-expressing the gene of interest is a useful assay for identifying the role of various proteins in endocytic trafficking pathway. The protocol describes the DQ-Red BSA trafficking assay that can be used to study endocytic trafficking in various cell types.