Cells Deploy a Two-Pronged Strategy to Rectify Misfolded Proinsulin Aggregates.

Insulin gene coding sequence mutations are known to cause mutant INS-gene-induced diabetes of youth (MIDY), yet the cellular pathways needed to prevent misfolded proinsulin accumulation remain incompletely understood. Here, we report that Akita mutant proinsulin forms detergent-insoluble aggregates that entrap wild-type (WT) proinsulin in the endoplasmic reticulum (ER), thereby blocking insulin production. Two distinct quality-control mechanisms operate together to combat this insult: the ER luminal chaperone Grp170 prevents proinsulin aggregation, while the ER membrane morphogenic protein reticulon-3 (RTN3) disposes of aggregates via ER-coupled autophagy (ER-phagy). We show that enhanced RTN-dependent clearance of aggregated Akita proinsulin helps to restore ER export of WT proinsulin, which can promote WT insulin production, potentially alleviating MIDY. We also find that RTN3 participates in the clearance of other mutant prohormone aggregates. Together, these results identify a series of substrates of RTN3-mediated ER-phagy, highlighting RTN3 in the disposal of pathogenic prohormone aggregates.

20,000 picometers under the OMM: diving into the vastness of mitochondrial metabolite transport.

The metabolic compartmentalization enabled by mitochondria is key feature of many cellular processes such as energy conversion to ATP production, redox balance, and the biosynthesis of heme, urea, nucleotides, lipids, and others. For a majority of these functions, metabolites need to be transported across the impermeable inner mitochondrial membrane by dedicated carrier proteins. Here, we examine the substrates, structural features, and human health implications of four mitochondrial metabolite carrier families: the SLC25A family, the mitochondrial ABCB transporters, the mitochondrial pyruvate carrier (MPC), and the sideroflexin proteins.

The science behind wet wipes for infant skin: Ingredient review, safety, and efficacy.

In the diapered area, the continuous exposure to excess moisture and irritants from urine and feces weakens the stratum corneum, making the skin more susceptible to irritation. The use of wet wipes for infants (baby wipes) is a common practice to clean skin after urine or a bowel movement, and this practice even extends to cleaning the hands and face, resulting in repeated daily use. Therefore, ensuring that baby wipes contain ingredients that are safe and mild on skin is important to help minimize skin irritation and discomfort. While disposable baby wipes have been shown to be effective and gentle at cleaning infant skin, even the skin of premature infants, there is growing public concern regarding their safety and tolerability. Not all products are made the same, as differences exist in manufacturing processes, ingredients, materials, safety, and quality testing. Therefore, it is important that healthcare professionals have accessible evidenced-based information on the safety and tolerability of common ingredients found in baby wipes to optimally educate their patients and families. Herein, we provide a review on best practices for ingredient selection, safety, and efficacy of baby wipes.

International consensus statement: methods for recording and reporting of epidemiological data on injuries and illnesses in golf.

Epidemiological studies of injury in elite and recreational golfers have lacked consistency in methods and definitions employed and this limits comparison of results across studies. In their sports-generic statement, the Consensus Group recruited by the IOC (2020) called for sport-specific consensus statements. On invitation by International Golf Federation, a group of international experts in sport and exercise medicine, golf research and sports injury/illness epidemiology was selected to prepare a golf-specific consensus statement. Methodological stages included literature review and initial drafting, online feedback from the consensus group, revision and second draft, virtual consensus meetings and completion of final version. This consensus statement provides golf-specific recommendations for data collection and research reporting including: (i) injury and illness definitions, and characteristics with golf-specific examples, (ii) definitions of golf-specific exposure measurements and recommendations for the calculation of prevalence and incidence, (iii) injury, illness and exposure report forms for medical staff and for golfers, and (iv) a baseline questionnaire. Implementation of the consensus methodology will enable comparison among golf studies and with other sports. It facilitates analysis of causative factors for injuries and illness in golf, and can also be used to evaluate the effects of prevention programmes to support the health of golfers.

Human TREX2 components PCID2 and centrin 2, but not ENY2, have distinct functions in protein export and co-localize to the centrosome.

TREX-2 is a five protein complex, conserved from yeast to humans, involved in linking mRNA transcription and export. The centrin 2 subunit of TREX-2 is also a component of the centrosome and is additionally involved in a distinctly different process of nuclear protein export. While centrin 2 is a known multifunctional protein, the roles of other human TREX-2 complex proteins other than mRNA export are not known. In this study, we found that human TREX-2 member PCID2 but not ENY2 is involved in some of the same cellular processes as those of centrin 2 apart from the classical TREX-2 function. PCID2 is present at the centrosome in a subset of HeLa cells and this localization is centrin 2 dependent. Furthermore, the presence of PCID2 at the centrosome is prevalent throughout the cell cycle as determined by co-staining with cyclins E, A and B. PCID2 but not ENY2 is also involved in protein export. Surprisingly, siRNA knockdown of PCID2 delayed the rate of nuclear protein export, a mechanism distinct from the effects of centrin 2, which when knocked down inhibits export. Finally we showed that co-depletion of centrin 2 and PCID2 leads to blocking rather than delaying nuclear protein export, indicating the dominance of the centrin 2 phenotype. Together these results represent the first discovery of specific novel functions for PCID2 other than mRNA export and suggest that components of the TREX-2 complex serve alternative shared roles in the regulation of nuclear transport and cell cycle progression.

Australian Institute of Sport and the Australian Paralympic Committee position statement: urinary tract infection in spinal cord injured athletes.

Patients with spinal cord injuries are at increased risk of developing symptomatic urinary tract infections. Current evidence-based knowledge regarding prevention and treatment of urinary tract infection in the spinal cord injured population is limited. There are currently no urinary tract infection prevention and management guidelines specifically targeted towards elite spinal cord injured athletes. This position statement represents a set of recommendations intended to provide clinical guidelines for sport and exercise medicine physicians and other healthcare providers for the prevention and treatment of urinary tract infection in spinal cord injured athletes. It has been endorsed by the Australian Institute of Sport (AIS) and the Australian Paralympic Committee (APC).

Mesenchymal stem cell-secretome laden photopolymerizable hydrogels for wound healing.

Mesenchymal stem cell-derived secretome represents an emerging acellular therapeutic which possess significant opportunity for clinical applications due to its anti-inflammatory, immunomodulatory, and wound healing properties. However, maintaining therapeutic efficacy and ensuring stability of cell-based products is challenging, requiring a robust delivery method. Therefore, we designed a hydrogel-based scaffold loaded with CK Cell Technologies' proprietary Mesenchymal stem cell-secretome for controlled release treatment of acute and chronic wounds. We incorporated both conditioned media (CM) and extracellular vesicles (EVs) into gelatin methacryloyl (GelMA) hydrogels and demonstrated how we can tune the diffusive release of the EVs from them. To demonstrate viability of the approach, we developed a wound healing scratch assay where we see in situ release of CM and EVs promote enhanced migration of human dermal fibroblasts (hDFs). We see the colocalization of these EVs in the fibroblasts using fluorescent microscopy. Finally, as a surrogate for in vivo neovascularization, we conducted an in vitro tube formation assay for the MSC-secretome using matrigel-embedded human microvascular endothelial cells. By adding CM and EVs, we observe an increase in tubulogenesis. Collectively, our data demonstrates by tuning the GelMA properties, we can influence the controlled release of the MSC-secretome for a wound dressing and bandage application for chronic and acute wounds.

Phosphate starvation signaling increases mitochondrial membrane potential through respiration-independent mechanisms.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Isolation and Characterisation of an Adipose-derived human mesenchymal stem cell line - 'CKC-Endeavour-1'.

Mesenchymal stem cells derived from adipose tissue (ADMSCs) are being increasingly considered in regenerative medicine-based clinical applications. Apart from possessing therapeutic applications themselves, ADMSCs also secrete a myriad of soluble factors which are promising candidates for treating several degenerative diseases such as osteoarthritis and neurodegenerative diseases, wound repair as well as for cosmeceutical purposes. In our research study, we successfully isolated ADMSCs in-house, now called CKC-Endeavour-1 from the lipoaspirate sample of a patient who underwent liposuction. The subsequent expansion of cells was performed in xeno-free and serum-free conditions and their characterisation was performed using tri-lineage differentiation studies. The levels of differentiation were assessed by staining and gene expression which was observed to be comparable between the in-house developed ADMSC cell line and the commercially purchased ADMSCs. Following characterisation, the secretory components from these MSCs, namely, conditioned media (ADMSC-CM) and exosomes (ADMSC-EXO) were harvested from CKC-Endeavour-1 under xeno-free, serum-free, and supplement-free conditions followed by lyophilisation in order to attempt to prolong its shelf-life. The comprehensive analysis of the secretome profile of ADMSC-CM using carried out using cytokine array and demonstrated the presence of 105 cytokines and growth factors. Also, clinical grade Izon columns were used to isolate the exosomes from ADMSC-CM obtaining exosomes in the size range of <200nm, analysed using nanoparticle tracking analysis. Overall, our study developed an ADMSC cell line, CKC-Endeavour-1, along with their CM and exosome (EXO) products under clinically safe conditions. Additionally, we have obtained a comprehensive understanding of the secreted factors present in the ADMSC-CM which could be further explored in detail to tap the best therapeutic benefits from them.

Mitochondrial pyruvate supports lymphoma proliferation by fueling a glutamate pyruvate transaminase 2-dependent glutaminolysis pathway.

The fate of pyruvate is a defining feature in many cell types. One major fate is mitochondrial entry via the mitochondrial pyruvate carrier (MPC). We found that diffuse large B cell lymphomas (DLBCLs) consume mitochondrial pyruvate via glutamate-pyruvate transaminase 2 to enable α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid cycle in DLBCLs. As a result, MPC inhibition led to decreased glutaminolysis in DLBCLs, opposite to previous observations in other cell types. We also found that MPC inhibition or genetic depletion decreased DLBCL proliferation in an extracellular matrix (ECM)-like environment and xenografts, but not in a suspension environment. Moreover, the metabolic profile of DLBCL cells in ECM is markedly different from cells in a suspension environment. Thus, we conclude that the synergistic consumption and assimilation of glutamine and pyruvate enables DLBCL proliferation in an extracellular environment-dependent manner.

Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction.

The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion.

Cancer cells have often lost genetic sequences that control when and how cell division takes place. Deleting these genes, however, is not an exact art, and neighboring sequences regularly get removed in the process. For example, the loss of the tumor suppressor gene PTEN, the second most deleted gene in cancer, frequently involves the removal of the nearby ATAD1 gene. While hundreds of thousands of human tumors completely lack ATAD1, individuals born without a functional version of this gene do not survive past early childhood. How can tumor cells cope without ATAD1 ­ and could these coping strategies become the target for new therapies? Winter et al. aimed to answer these questions by examining a variety of cancer cells lacking ATAD1 in the laboratory. Under normal circumstances, the enzyme that this gene codes for sits at the surface of mitochondria, the cellular compartments essential for energy production. There, it extracts any faulty, defective proteins that may otherwise cause havoc and endanger mitochondrial health. Experiments revealed that without ATAD1, cancer cells started to rely more heavily on an alternative mechanism to remove harmful proteins: the process centers on MARCH5, an enzyme which tags molecules that require removal so the cell can recycle them. Drugs that block the pathway involving MARCH5 already exist, but they have so far been employed to treat other types of tumors. Winter et al. showed that using these compounds led to the death of cancerous ATAD1-deficient cells, including in human tumors grown in mice. Overall, this work demonstrates that cancer cells which have lost ATAD1 become more vulnerable to disruptions in the protein removal pathway mediated by MARCH5, including via already existing drugs. If confirmed by further translational work, these findings could have important clinical impact given how frequently PTEN and ATAD1 are lost together in cancer.

Do rugby league players under-report concussion symptoms? A cross-sectional study of elite teams based in Australia.

OBJECTIVE: To determine the rate of under-reporting of concussion and its symptoms in elite rugby league players in Australia. METHODS: The study was conducted in the preseason of the 2020 National Rugby League (NRL) competition.A total of 151 male, NRL club contracted rugby league players across three professional clubs participated.The participants completed a voluntary, anonymous survey exploring player demographics, concussion data, under-reporting instances and reasons for under-reporting over the 2018 and 2019 rugby league seasons. RESULTS: 17.2% of surveyed players reported sustaining a likely concussion over the past 2 years and not reporting to medical staff. 22% of NRL first grade players admitted to not reporting at least one concussion during the 2018 and 2019 seasons. The most common reason not to report was the player 'not wanting to be ruled out of the game or training session' (57.7%), followed by 'not wanting to let down the coaches or teammates' (23.1%). 85.4% of surveyed players reported having concussion education by their club in the previous two seasons. CONCLUSIONS: 17.2 % of elite rugby league players in Australia chose not to report likely concussive episodes and concussion-related symptoms during the 2018 and 2019 seasons. Clinicians need to be aware of under-reporting in athletes when assessing players following head injuries. The findings highlight the need for development of validated, objective testing for concussion following sports-associated head injury.

The pyruvate-lactate axis modulates cardiac hypertrophy and heart failure.

The metabolic rewiring of cardiomyocytes is a widely accepted hallmark of heart failure (HF). These metabolic changes include a decrease in mitochondrial pyruvate oxidation and an increased export of lactate. We identify the mitochondrial pyruvate carrier (MPC) and the cellular lactate exporter monocarboxylate transporter 4 (MCT4) as pivotal nodes in this metabolic axis. We observed that cardiac assist device-induced myocardial recovery in chronic HF patients was coincident with increased myocardial expression of the MPC. Moreover, the genetic ablation of the MPC in cultured cardiomyocytes and in adult murine hearts was sufficient to induce hypertrophy and HF. Conversely, MPC overexpression attenuated drug-induced hypertrophy in a cell-autonomous manner. We also introduced a novel, highly potent MCT4 inhibitor that mitigated hypertrophy in cultured cardiomyocytes and in mice. Together, we find that alteration of the pyruvate-lactate axis is a fundamental and early feature of cardiac hypertrophy and failure.

Benefits of Using an Appropriately Formulated Wipe to Clean Diapered Skin of Preterm Infants.

The skin of premature infants is underdeveloped rendering it more prone to break down and irritation. Therefore, special care is needed to protect premature skin and ensure it is not adversely affected. Many health care professionals advise using just water and cloth to clean diapered skin after a bowel movement despite evidence that shows improved infant skin health with the use of modern appropriately formulated baby wipes. This article describes the unique physiology of premature infant skin, reviews clinical evidence comparing use of baby wipes to water and cloth, and describes attributes of appropriately formulated baby wipes.

Misfolded proinsulin in the endoplasmic reticulum during development of beta cell failure in diabetes.

The endoplasmic reticulum (ER) is broadly distributed throughout the cytoplasm of pancreatic beta cells, and this is where all proinsulin is initially made. Healthy beta cells can synthesize 6000 proinsulin molecules per second. Ordinarily, nascent proinsulin entering the ER rapidly folds via the formation of three evolutionarily conserved disulfide bonds (B7-A7, B19-A20, and A6-A11). A modest amount of proinsulin misfolding, including both intramolecular disulfide mispairing and intermolecular disulfide-linked protein complexes, is a natural by-product of proinsulin biosynthesis, as is the case for many proteins. The steady-state level of misfolded proinsulin-a potential ER stressor-is linked to (1) production rate, (2) ER environment, (3) presence or absence of naturally occurring (mutational) defects in proinsulin, and (4) clearance of misfolded proinsulin molecules. Accumulation of misfolded proinsulin beyond a certain threshold begins to interfere with the normal intracellular transport of bystander proinsulin, leading to diminished insulin production and hyperglycemia, as well as exacerbating ER stress. This is most obvious in mutant INS gene-induced Diabetes of Youth (MIDY; an autosomal dominant disease) but also likely to occur in type 2 diabetes owing to dysregulation in proinsulin synthesis, ER folding environment, or clearance.

Chaperone-Driven Degradation of a Misfolded Proinsulin Mutant in Parallel With Restoration of Wild-Type Insulin Secretion.

In heterozygous patients with a diabetic syndrome called mutant INS gene-induced diabetes of youth (MIDY), there is decreased insulin secretion when mutant proinsulin expression prevents wild-type (WT) proinsulin from exiting the endoplasmic reticulum (ER), which is essential for insulin production. Our previous results revealed that mutant Akita proinsulin is triaged by ER-associated degradation (ERAD). We now find that the ER chaperone Grp170 participates in the degradation process by shifting Akita proinsulin from high-molecular weight (MW) complexes toward smaller oligomeric species that are competent to undergo ERAD. Strikingly, overexpressing Grp170 also liberates WT proinsulin, which is no longer trapped in these high-MW complexes, enhancing ERAD of Akita proinsulin and restoring WT insulin secretion. Our data reveal that Grp170 participates in preparing mutant proinsulin for degradation while enabling WT proinsulin escape from the ER. In principle, selective destruction of mutant proinsulin offers a rational approach to rectify the insulin secretion problem in MIDY.

Opportunistic intruders: how viruses orchestrate ER functions to infect cells.

Viruses subvert the functions of their host cells to replicate and form new viral progeny. The endoplasmic reticulum (ER) has been identified as a central organelle that governs the intracellular interplay between viruses and hosts. In this Review, we analyse how viruses from vastly different families converge on this unique intracellular organelle during infection, co-opting some of the endogenous functions of the ER to promote distinct steps of the viral life cycle from entry and replication to assembly and egress. The ER can act as the common denominator during infection for diverse virus families, thereby providing a shared principle that underlies the apparent complexity of relationships between viruses and host cells. As a plethora of information illuminating the molecular and cellular basis of virus-ER interactions has become available, these insights may lead to the development of crucial therapeutic agents.

PDI reductase acts on Akita mutant proinsulin to initiate retrotranslocation along the Hrd1/Sel1L-p97 axis.

In mutant INS gene-induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.