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Lysosome-related organelles (LROs) are a group of functionally diverse, cell type-specific compartments. LROs include melanosomes, alpha and dense granules, lytic granules, lamellar bodies and other compartments with distinct morphologies and functions allowing specialised and unique functions of their host cells. The formation, maturation and secretion of specific LROs are compromised in a number of hereditary rare multisystem disorders, including Hermansky-Pudlak syndromes, Griscelli syndrome and the Arthrogryposis, Renal dysfunction and Cholestasis syndrome. Each of these disorders impacts the function of several LROs, resulting in a variety of clinical features affecting systems such as immunity, neurophysiology and pigmentation. This has demonstrated the close relationship between LROs and led to the identification of conserved components required for LRO biogenesis and function. Here, we discuss aspects of this conserved machinery among LROs in relation to the heritable multisystem disorders they associate with, and present our current understanding of how dysfunctions in the proteins affected in the disease impact the formation, motility and ultimate secretion of LROs. Moreover, we have analysed the expression of the members of the CHEVI complex affected in Arthrogryposis, Renal dysfunction and Cholestasis syndrome, in different cell types, by collecting single cell RNA expression data from the human protein atlas. We propose a hypothesis describing how transcriptional regulation could constitute a mechanism that regulates the pleiotropic functions of proteins and their interacting partners in different LROs.

Controversy surrounds the use of parenteral nutrition in critical illness. Previous overviews used composite scales to identify high-quality trials, which may mask important differences in true methodological quality. Using a component-based approach this meta-analysis investigated the effect of trial quality on overall conclusions reached when standard enteral nutrition is compared to standard parenteral nutrition in critically ill patients. An extensive literature search was undertaken to identify all eligible trials. We retrieved 465 publications, and 11 qualified for inclusion. Nine trials presented complete follow-up, allowing the conduct of an intention to treat analysis. Aggregation revealed a mortality benefit in favour of parenteral nutrition, with no heterogeneity. A priori specified subgroup analysis demonstrated the presence of a potentially important treatment-subgroup interaction between studies of parenteral vs. early enteral nutrition compared to parenteral vs. late enteral. Six trials with complete follow-up reported infectious complications. Infectious complications were increased with parenteral use. The I(2) measure of heterogeneity was 37.7%. Intention to treat trials demonstrated reduced mortality associated with parenteral nutrition use. A priori subgroup analysis attributed this reduction to trials comparing parenteral to delayed enteral nutrition. Despite an association with increased infectious complications, a grade B+ evidence-based recommendation (level II trials, no heterogeneity) can be generated for parenteral nutrition use in patients in whom enteral nutrition cannot be initiated within 24 h of ICU admission or injury.

Importance Acute kidney injury (AKI) is characterized by severe loss of glomerular filtration rate (GFR) and is associated with a prolonged intensive care unit (ICU) stay and increased risk of death. No interventions have yet been shown to prevent AKI or preserve GFR in critically ill patients. Evidence from mammalian physiology and small clinical trials suggests higher amino acid intake may protect the kidney from ischemic insults and thus may preserve GFR during critical illness. Objective To determine whether amino acid therapy, achieved through daily intravenous (IV) supplementation with standard amino acids, preserves kidney function in critically ill patients. Design, setting, and participants Multicenter, phase II, randomized clinical trial conducted between December 2010 and February 2013 in the ICUs of 16 community and tertiary hospitals in Australia and New Zealand. Participants were adult critically ill patients expected to remain in the study ICU for longer than 2 days. Interventions Random allocation to receive a daily supplement of up to 100 g of IV amino acids or standard care. Main outcomes and measures Duration of renal dysfunction (primary outcome); estimated GFR (eGFR) derived from creatinine; eGFR derived from cystatin C; urinary output; renal replacement therapy (RRT) use; fluid balance and other measures of renal function. Results 474 patients were enrolled and randomized (235 to standard care, 239 to IV amino acid therapy). At time of enrollment, patients allocated to receive amino acid therapy had higher APACHE II scores (20.2 ± 6.8 vs. 21.7 ± 7.6, P  = 0.02) and more patients had pre-existing renal dysfunction (29/235 vs. 44/239, P  = 0.07). Duration of renal dysfunction after enrollment did not differ between groups (mean difference 0.21 AKI days per 10 patient ICU days, 95 % CI −0.27 to 1.04, P  = 0.45). Amino acid therapy significantly improved eGFR (treatment group × time interaction, P  = 0.004), with an early peak difference of 7.7 mL/min/1.73 m 2 (95 % CI 1.0–14.5 mL/min/1.73 m 2 , P  = 0.02) on study day 4. Daily urine output was also significantly increased (+300 mL/day, 95 % CI 145–455 mL, P  = 0.0002). There was a trend towards increased RRT use in patients receiving amino acid therapy (13/235 vs. 25/239, P  = 0.062); however, this trend was not present after controlling for baseline imbalance ( P  = 0.21). Conclusion and relevance Treatment with a daily IV supplement of standard amino acids did not alter our primary outcome, duration of renal dysfunction. Trial registration anzctr.org.au Identifier: ACTRN12609001015235.

Geomagnetic storms generate heightened magnetovariational activity, which induces electric fields that drive hazardous currents known as geomagnetically induced currents (GICs) through man-made technological conductors including power transmission lines, railway networks and gas pipelines. We multiply magnetotelluric (MT) impedances from 23 sites in Scotland and northern England with measured geomagnetic field spectra from the Halloween 2003 and September 2017 storms to estimate maximum peak-to-peak, electric field magnitudes and directions for these storms, which we present as hazard maps. By sampling these electric fields in the direction of the longest (>50 km), high-voltage (275 and 400 kV) Scottish power transmission lines and integrating along their lengths, we estimate their associated transmission-line voltages. Lateral electrical conductivity variations in the Earth generate horizontal magnetic field gradients. We investigate the effect of these gradients on electric field estimates obtained using remote magnetic fields by applying a correction to the impedance tensor derived from the magnetic perturbation tensor between the local MT site and the remote magnetic field site. For the September 2017 storm, we also compare our estimated electric fields with a unique dataset comprising measured storm-time electric fields from 7 MT sites. We find that peak-to-peak, electric field magnitudes may have reached 13 V/km during the Halloween storm in some areas of the Scottish Highlands, with line-averaged electric fields >5 V/km sustained along a number of long-distance, high-voltage power transmission lines; line-averaged electric fields for the September 2017 storm are 1 V/km or less. Our surface electric fields show significant site-to-site variability that arises due to Earth’s internal 3D electrical conductivity structure, as characterised by the MT impedance tensors.

Although the randomized controlled trial is the most important tool currently available to objectively assess the impact of new treatments, the act of randomization itself is often poorly conducted and incompletely reported. The primary purpose of randomizing patients into treatment arms is to prevent researchers, clinicians, and patients from predicting, and thus influencing, which patients will receive which treatments. This important source of bias can be eliminated by concealing the upcoming allocation sequence from researchers and participants. Although there are many approaches to randomization that are known to effectively conceal the randomization sequence, the use of sequentially numbered, opaque sealed envelopes (SNOSE) is both cheap and effective. The purpose of this tutorial is to describe a step-by-step process for the preparation of SNOSE. We will outline how to prepare SNOSE to preserve allocation concealment in a trial that ( a) uses unrestricted (simple) randomization, ( b) stratifies randomization on one factor, ( c) uses permuted blocks and, and ( d) is conducted at more than 1 study site.

Drug resistance continues to be a major barrier to the delivery of curative therapies in cancer. Historically, drug resistance has been associated with over‐expression of drug transporters, changes in drug kinetics or amplification of drug targets. However, the emergence of resistance in patients treated with new‐targeted therapies has provided new insight into the complexities underlying cancer drug resistance. Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance. Single cell sequencing studies that identified multiple genetically distinct variants within human tumours clearly demonstrate the heterogeneous nature of human tumours. The major contributors to intratumoural heterogeneity are (i) genetic variation, (ii) stochastic processes, (iii) the microenvironment and (iv) cell and tissue plasticity. Each of these factors impacts on drug sensitivity. To deliver curative therapies to patients, modification of current therapeutic strategies to include methods that estimate intratumoural heterogeneity and plasticity will be essential.

Globally, lung cancer is the leading cause of cancer-related death. The majority of non-small cell lung cancer (NSCLC) tumours express epidermal growth factor receptor (EGFR), which allows for precise and targeted therapy in these patients. The dysregulation of EGFR in solid epithelial cancers has two distinct mechanisms: either a kinase-activating mutation in EGFR (EGFR-mutant) and/or an overexpression of wild-type EGFR (wt-EGFR). The underlying mechanism of EGFR dysregulation influences the efficacy of anti-EGFR therapy as well as the nature of resistance patterns and secondary mutations. This review will critically analyse the mechanisms of EGFR expression in NSCLC, its relevance to currently approved targeted treatment options, and the complex nature of secondary mutations and intrinsic and acquired resistance patterns in NSCLC.

Sterilization of biodegradable, collagen-based implants is challenging as irradiation sterilization methods can alter their mechanical properties. Electron beam (EB) irradiation is a terminal sterilization method that has been used for biologically-derived implants. Here, recombinant human collagen type III-phosphorylcholine (RHCIII-MPC) hydrogels were irradiated with EB doses of 17, 19, or 21 kGy and their subsequent biocompatibility and ability to promote regeneration in rabbit corneas was evaluated. Unirradiated hydrogels stored in 1% chloroform in phosphate-buffered saline (C-PBS) were the controls. There were no significant differences between irradiated and non-irradiated samples in optical or physical properties (tensile strength, modulus, elasticity), or the ability to support cell growth. However, irradiated implants were more sensitive to high levels of collagenase than unirradiated controls and the C-PBS implants had increased cell growth compared to EB and controls at 72 h. Corneal implants e-beamed at 17 kGy or e-beamed and subsequently frozen (EB-F) to increase shelf-life showed no adverse biological effects of the irradiation. EB, EB-F, and C-PBS implanted corneas all rapidly re-epithelialized but showed mild neovascularization that resolved over 6 months. The regenerated neo-corneas were transparent at 6 months post-operation. In vivo confocal microscopy confirmed normal morphology for the epithelium, stroma, sub-basal nerves and unoperated endothelium. Histology showed that all the regenerated corneas were morphologically similar to the normal. Immunohistochemistry indicated the presence of a differentiated corneal epithelium and functional tear film. In conclusion, the e-beamed corneal implants performed as well as non-irradiated control implants, resulting in fully regenerated neo-corneas with new nerves and without blood vessels or inflammation that may impede vision or corneal function. Therefore, a complete validation study to establish EB irradiation as an effective means for corneal implant sterilization prior to clinical application is necessary as a next step.

Perineural spread of tumour cells along cranial nerves is a severe complication of primary cutaneous squamous cell carcinomas of the head and neck region. While surgical excision of the tumour is the treatment of choice, removal of all the tumour is often complicated by the neural location and recurrence is frequent. Non-invasive immune treatments such as checkpoint inhibitor blockade may be useful in this set of tumours although little is understood about the immune response to perineural spread of squamous cell carcinomas. Immunohistochemistry studies suggest that perineural tumour contains a lymphocyte infiltrate but it is difficult to quantitate the different proportions of immune cell subsets and expression of checkpoint molecules such as PD-1, Tim-3 and CTLA-4. Using flow cytometry of excised perineural tumour tissue, we show that a T cell infiltrate is prominent in addition to less frequent B cell, NK cell and NKT cell infiltrates. CD8 T cells are more frequent than other T cells in the tumour tissue. Amongst CD8 T cells, the frequency of Tim-3, CTLA-4 and PD-1 expressing cells was significantly greater in the tumour relative to the blood, a pattern that was repeated for Tim-3, CTLA-4 and PD-1 amongst non-CD8 T cells. Using immunohistochemistry, PD-1 and PD-L1-expression could be detected in close proximity amongst perineural tumour tissue. The data suggest that perineural SCC contains a mixture of immune cells with a predominant T cell infiltrate containing CD8 T cells. Elevated frequencies of tumour-associated Tim-3+, CTLA-4+ and PD-1+ CD8 T cells suggests that a subset of patients may benefit from local antibody blockade of these checkpoint inhibitors.

The development of cancer vaccines has been intensively pursued over the past 50 years with modest success. However, recent advancements in the fields of genetics, molecular biology, biochemistry, and immunology have renewed interest in these immunotherapies and allowed the development of promising cancer vaccine candidates. Numerous clinical trials testing the response evoked by tumour antigens, differing in origin and nature, have shed light on the desirable target characteristics capable of inducing strong tumour-specific non-toxic responses with increased potential to bring clinical benefit to patients. Novel delivery methods, ranging from a patient’s autologous dendritic cells to liposome nanoparticles, have exponentially increased the abundance and exposure of the antigenic payloads. Furthermore, growing knowledge of the mechanisms by which tumours evade the immune response has led to new approaches to reverse these roadblocks and to re-invigorate previously suppressed anti-tumour surveillance. The use of new drugs in combination with antigen-based therapies is highly targeted and may represent the future of cancer vaccines. In this review, we address the main antigens and delivery methods used to develop cancer vaccines, their clinical outcomes, and the new directions that the vaccine immunotherapy field is taking.