Thioflavin T indicates mitochondrial membrane potential in mammalian cells.

The fluorescent benzothiazole dye thioflavin T (ThT) is widely used as a marker for protein aggregates, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilization of ThT in research and diagnosis. Here, we show that ThT localizes into the mitochondria of HeLa cells in a membrane-potential-dependent manner. Specifically, ThT colocalized in cells with the mitochondrial membrane potential indicator tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ = 405 nm), but neither factor alone, caused depolarization of mitochondrial membrane potential. This additive effect of the concentration and blue light was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its effect on mitochondrial membrane potential through photosensitization. These results show that ThT can act as a mitochondrial membrane potential indicator in mammalian cells, when used at low concentrations and with low blue light exposure. However, it causes dissipation of the mitochondrial membrane potential depending additively on its concentrations and blue light exposure. This conclusion motivates a re-evaluation of ThT's use at micromolar range in live-cell analyses and indicates that this dye can enable future studies on the potential connections between mitochondrial membrane potential dynamics and protein aggregation.

A novel COVID-19 program, delivering vaccines throughout rural and remote Australia.

Background: The Royal Flying Doctor Service of Australia (RFDS) established a unique SARS-CoV-2 vaccination program for vaccinating Australians that live in rural and remote areas. This paper describes the preparation and response phases of the RFDS response. Methods: This study includes vaccinations conducted by the RFDS from 01 January 2021 until 31 December 2021 when vaccines were mandatory for work and social activities. Prior to each clinic, we conducted community consultation to determine site requirements, patient characteristics, expected vaccination numbers, and community transmission rates. Findings: Ninety-five organizations requested support. The majority (n = 60; 63.2%) came from Aboriginal Community Controlled Health Organizations. Following consultation, 360 communities were approved for support. Actual vaccinations exceeded expectations (n = 70,827 vs. 49,407), with a concordance correlation coefficient of 0.88 (95% CI, 0.83, 0.93). Areas that reported healthcare workforce shortages during the preparation phase had the highest population proportion difference between expected and actual vaccinations. Areas that reported high vaccine hesitancy during the preparation phase had fewer than expected vaccines. There was a noticeable increase in vaccination rates in line with community outbreaks and positive polymerase chain reaction cases [r (41) = 0.35, p = 0.021]. Engagement with community leaders prior to clinic deployment was essential to provide a tailored response based on community expectations.

Particular genomic and virulence traits associated with preterm infant-derived toxigenic Clostridium perfringens strains.

Clostridium perfringens is an anaerobic toxin-producing bacterium associated with intestinal diseases, particularly in neonatal humans and animals. Infant gut microbiome studies have recently indicated a link between C. perfringens and the preterm infant disease necrotizing enterocolitis (NEC), with specific NEC cases associated with overabundant C. perfringens termed C. perfringens-associated NEC (CPA-NEC). In the present study, we carried out whole-genome sequencing of 272 C. perfringens isolates from 70 infants across 5 hospitals in the United Kingdom. In this retrospective analysis, we performed in-depth genomic analyses (virulence profiling, strain tracking and plasmid analysis) and experimentally characterized pathogenic traits of 31 strains, including 4 from CPA-NEC patients. We found that the gene encoding toxin perfringolysin O, pfoA, was largely deficient in a human-derived hypovirulent lineage, as well as certain colonization factors, in contrast to typical pfoA-encoding virulent lineages. We determined that infant-associated pfoA+ strains caused significantly more cellular damage than pfoA- strains in vitro, and further confirmed this virulence trait in vivo using an oral-challenge C57BL/6 murine model. These findings suggest both the importance of pfoA+ C. perfringens as a gut pathogen in preterm infants and areas for further investigation, including potential intervention and therapeutic strategies.

Aeromedical retrieval services characteristics globally: a scoping review.

BACKGROUND: Aeromedical emergency retrieval services play an important role in supporting patients with critical and often life-threatening clinical conditions. Aeromedical retrieval services help to provide fast access to definitive care for critically ill patients in under-served regions. Typically, fixed-wing aeromedical retrieval becomes the most viable transport option compared with rotary-wing aircraft when distances away from centres of definitive care extend beyond 200 kms. To our knowledge, there are no studies that have investigated fixed-wing aeromedical services in the member countries of the organisation for economic cooperation and development (OECD). A description of the global characteristics of aeromedical services will inform international collaboration to optimise clinical outcomes for patients. AIM: In this scoping review, we aimed to describe the features of government- and not-for-profit organisation-owned fixed-wing aeromedical retrieval services in some of the member countries of the OECD. METHODS: We followed scoping review methodology based on the grey literature search strategy identified in earlier studies. This mostly involved internet-based searches of the websites of fixed-wing aeromedical emergency retrieval services affiliated with the OECD member countries. RESULTS: We identified 460 potentially relevant records after searching Google Scholar (n = 24) and Google search engines (n = 436). After removing ineligible and duplicate information, this scoping review identified 86 government-and not-for-profit-operated fixed-wing aeromedical retrieval services as existing in 17 OECD countries. Concentrations of the services were greatest in the USA followed by Australia, Canada, and the UK. The most prevalent business models used across the identified OECD member countries comprised the government, not-for-profit, and hybrid models. Three-quarters of the not-for-profit and two-fifths of the hybrid business models were in the USA compared to other countries studied. The government or state-funded business model was most common in Australia (11/24, 46%), Canada (4/24, 17%), and the UK (4/24, 17%). The frequently used service delivery models adopted for patients of all ages included primary/secondary retrievals, secondary retrievals only, and service specialisation models. Of these service models, primary/secondary retrieval involving the transportation of adults and children from community clinics and primary health care facilities to centres of definitive care comprised the core tasks performed by most of the aeromedical retrieval services studied. The service specialisation model provided an extra layer of specialist health care dedicated to the transportation of neonates and paediatrics. At least eight aeromedical retrieval services catered solely for children from birth to 16 years of age. One aeromedical service, the royal flying doctor service in Australia also provided primary health care and telehealth services in addition to primary retrieval and interhospital transfer of patients. The doctor and registered nurse/paramedic (Franco-German model) and the nurse and/or paramedic (Anglo-American model) configurations were the most common staffing models used across the aeromedical services studied. CONCLUSIONS: The development and composition of fixed-wing aeromedical emergency retrieval services operated by not-for-profit organisations and governments in the OECD countries showed diversity in terms of governance arrangements, services provided, and staffing models used. We do not fully understand the impact of these differences on the quality of service provision, including equitable service access, highlighting a need for further research.

Maternal gut microbiota Bifidobacterium promotes placental morphogenesis, nutrient transport and fetal growth in mice.

The gut microbiota plays a central role in regulating host metabolism. While substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Notably, in pregnant women, Bifidobacterium represents a key beneficial microbiota genus, with levels observed to increase across pregnancy. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. Maternal and placental metabolome were affected by maternal gut microbiota (i.e. acetate, formate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. Our work emphasizes the importance of the maternal gut microbiota on feto-placental development and sets a foundation for future research towards the use of probiotics during pregnancy.

The role of valve stiffness in the insurgence of deep vein thrombosis.

Deep vein thrombosis is a life-threatening development of blood clots in deep veins. Immobility and blood flow stagnancy are typical risk factors indicating that fluid dynamics play an important role in the initiation of venous clots. However, the roles of physical parameters of the valves and flow conditions in deep vein thrombosis initiation have not been fully understood. Here, we describe a microfluidics in vitro method that enabled us to explore the role of valve elasticity using in situ fabrication and characterisation. In our experimental model the stiffness of each valve leaflet can be controlled independently, and various flow conditions were tested. The resulting complex flow patterns were detected using ghost particle velocimetry and linked to localised thrombus formation using whole blood and an aqueous suspension of polystyrene particles. In particular, valves with leaflets of similar stiffness had clot formation on the valve tips whereas valves with leaflets of different stiffness had clot formation in the valve pocket.

Acetate protects against intestinal ischemia-reperfusion injury independent of its cognate free fatty acid 2 receptor.

Free fatty acid 2 receptor (FFA2) is highly expressed on neutrophils and, when activated by its cognate ligand acetate, generates potent anti-inflammatory activities. The roles of FFA2 and acetate have not been explored in ischemia-reperfusion injury (IRI). We therefore examined the function of FFA2 and the therapeutic potential of acetate to reduce tissue injury in an acute model of intestinal IRI. The superior mesenteric artery of wild-type (WT) and FFA2-/- mice was briefly occluded then reperfused following treatment with acetate or vehicle. The absence of FFA2 resulted in intestinal injury similar to that observed in WT mice, indicating a minimal causal role for FFA2 in this model. Acetate treatment to WT mice prior to ischemia profoundly protected the intestine from IRI-induced damage. Amelioration of IRI was also observed, although to a lesser extent, when acetate was administered to FFA2-/- mice demonstrating that certain protective effects of acetate were FFA2-independent. Remarkably, despite the lack of tissue damage following IRI, acetate-treated mice had markedly increased neutrophil infiltration to the reperfused intestine which was dependent on FFA2. These studies reveal a minimal causal role for FFA2 in intestinal IRI but highlight the novel therapeutic potential for acetate in the amelioration of ischemia-mediated tissue damage.

Bioelectrical understanding and engineering of cell biology.

The last five decades of molecular and systems biology research have provided unprecedented insights into the molecular and genetic basis of many cellular processes. Despite these insights, however, it is arguable that there is still only limited predictive understanding of cell behaviours. In particular, the basis of heterogeneity in single-cell behaviour and the initiation of many different metabolic, transcriptional or mechanical responses to environmental stimuli remain largely unexplained. To go beyond the status quo, the understanding of cell behaviours emerging from molecular genetics must be complemented with physical and physiological ones, focusing on the intracellular and extracellular conditions within and around cells. Here, we argue that such a combination of genetics, physics and physiology can be grounded on a bioelectrical conceptualization of cells. We motivate the reasoning behind such a proposal and describe examples where a bioelectrical view has been shown to, or can, provide predictive biological understanding. In addition, we discuss how this view opens up novel ways to control cell behaviours by electrical and electrochemical means, setting the stage for the emergence of bioelectrical engineering.

The early life microbiota protects neonatal mice from pathological small intestinal epithelial cell shedding.

The early life gut microbiota plays a crucial role in regulating and maintaining the intestinal barrier, with disturbances in these communities linked to dysregulated renewal and replenishment of intestinal epithelial cells. Here we sought to determine pathological cell shedding outcomes throughout the postnatal developmental period, and which host and microbial factors mediate these responses. Surprisingly, neonatal mice (Day 14 and 21) were highly refractory to induction of cell shedding after intraperitoneal administration of liposaccharide (LPS), with Day 29 mice showing strong pathological responses, more similar to those observed in adult mice. These differential responses were not linked to defects in the cellular mechanisms and pathways known to regulate cell shedding responses. When we profiled microbiota and metabolites, we observed significant alterations. Neonatal mice had high relative abundances of Streptococcus, Escherichia, and Enterococcus and increased primary bile acids. In contrast, older mice were dominated by Candidatus Arthromitus, Alistipes, and Lachnoclostridium, and had increased concentrations of SCFAs and methyamines. Antibiotic treatment of neonates restored LPS-induced small intestinal cell shedding, whereas adult fecal microbiota transplant alone had no effect. Our findings further support the importance of the early life window for microbiota-epithelial interactions in the presence of inflammatory stimuli and highlights areas for further investigation.

Characterisation of small molecule ligands 4CMTB and 2CTAP as modulators of human FFA2 receptor signalling.

Short chain fatty acids (SCFAs) are protective against inflammatory diseases. Free fatty acid receptor 2 (FFA2), is a target of SCFAs however, their selectivity for FFA2 over other FFA receptors is limited. This study aimed to functionally characterise 2-(4-chlorophenyl)-3-methyl-N-(thiazole-2-yl)butanamide (4CMTB) and 4-((4-(2-chlorophenyl)thiazole-2-yl)amino)-4oxo-3-phenylbutanoic acid (2CTAP), and their enantiomers, in modulating FFA2 activity. The racemic mixture (R/S) and its constituents (R-) and (S-) 4CMTB or 2CTAP were used to stimulate human (h)FFA2 in the absence or presence of acetate. Calcium ions (Ca2+), phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) and cyclic adenosine monophosphate (cAMP) were measured. R/S-4CMTB is a functionally selective ago-allosteric ligand that enhances Ca2+ response to acetate. Both R/S-4CMTB and S-4CMTB are more potent activators of pERK1/2 and inhibitors of forskolin-induced cAMP than acetate. S-4CMTB increased neutrophil infiltration in intestinal ischemia reperfusion injury (IRI). 2CTAP inhibited constitutive Ca2+ levels, antagonised acetate-induced pERK1/2 and prevented damage following IRI. This study characterises enantiomers of functionally selective ligands for FFA2 in cells stably expressing hFFA2. It highlights the novel roles of selective FFA2 enantiomers 4CMTB and 2CTAP on Ca2+, pERK1/2 and cAMP and their roles as allosteric modulators which, may assist in efforts to design novel therapeutic agents for FFA2-driven inflammatory diseases.

Changes in human hepatic metabolism in steatosis and cirrhosis.

AIM: To understand the underlying metabolic changes in human liver disease we have applied nuclear magnetic resonance (NMR) metabolomics analysis to human liver tissue. METHODS: We have carried out pilot study using 1H-NMR to derive metabolomic signatures from human liver from patients with steatosis, nonalcoholic steatohepatitis (NASH) or alcohol-related liver damage (ARLD) to identify species that can predict outcome and discriminate between alcohol and metabolic-induced liver injuries. RESULTS: Changes in branched chain amino acid homeostasis, tricarboxylic acid cycle and purine biosynthesis intermediates along with betaine were associated with the development of cirrhosis in both ARLD and nonalcoholic fatty liver disease. Species such as propylene glycol and as yet unidentified moieties that allowed discrimination between NASH and ARLD samples were also detected using our approach. CONCLUSION: Our high throughput, non-destructive technique for multiple analyte quantification in human liver specimens has potential for identification of biomarkers with prognostic and diagnostic significance.

Exploring the role of the microbiota member Bifidobacterium in modulating immune-linked diseases.

The gut-associated microbiota is essential for multiple physiological processes, including immune development. Acquisition of our initial pioneer microbial communities, including the dominant early life genus Bifidobacterium, occurs at a critical period of immune maturation and programming. Bifidobacteria are resident microbiota members throughout our lifetime and have been shown to modulate specific immune cells and pathways. Notably, reductions in this genus have been associated with several diseases, including inflammatory bowel disease. In this review, we provide an overview of bifidobacteria profiles throughout life and how different strains of bifidobacteria have been implicated in immune modulation in disease states. The focus will be examining preclinical models and outcomes from clinical trials on immune-linked chronic conditions. Finally, we highlight some of the important unresolved questions in relation to Bifidobacterium-mediated immune modulation and implications for future directions, trials, and development of new therapies.

Discovery of functionally selective C5aR2 ligands: novel modulators of C5a signalling.

The complement cascade is comprised of a highly sophisticated network of innate immune proteins that are activated in response to invading pathogens or tissue injury. The complement activation peptide, C5a, binds two seven transmembrane receptors, namely the C5a receptor 1 (C5aR1) and C5a receptor 2 (C5aR2, or C5L2). C5aR2 is a non-G-protein-signalling receptor whose biological role remains controversial. Some of this controversy arises owing to the lack of selective ligands for C5aR2. In this study, a library of 61 peptides based on the C-terminus of C5a was assayed for the ability to selectively modulate C5aR2 function. Two ligands (P32 and P59) were identified as functionally selective C5aR2 ligands, exhibiting selective recruitment of ß-arrestin 2 via C5aR2, partial inhibition of C5a-induced ERK1/2 activation and lipopolysaccharide-stimulated interleukin-6 release from human monocyte-derived macrophages. Importantly, neither ligand could induce ERK1/2 activation or inhibit C5a-induced ERK1/2 activation via C5aR1 directly. Finally, P32 inhibited C5a-mediated neutrophil mobilisation in wild-type, but not C5aR2(-/-) mice. These functionally selective ligands for C5aR2 are novel tools that can selectively modulate C5a activity in vitro and in vivo, and thus will be valuable tools to interrogate C5aR2 function.

Neutrophils--a key component of ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) is a common occurrence following myocardial infarction, transplantation, stroke, and trauma that can lead to multiple organ failure, which remains the foremost cause of death in critically ill patients. Current therapeutic strategies for IRI are mainly palliative, and there is an urgent requirement for a therapeutic that could prevent or reverse tissue damage caused by IRI. Neutrophils are the primary responders following ischemia and reperfusion and represent important components in the protracted inflammatory response and severity associated with IRI. Experimental studies demonstrate neutrophil infiltration at the site of ischemia and show that inducing neutropenia can protect organs from IRI. In this review, we highlight the mechanisms involved in neutrophil recruitment, activation, and adherence and how this contributes to disease severity in IRI. Inhibiting neutrophil mobilization, tissue recruitment, and ultimately neutrophil-associated activation of local and systemic inflammatory responses may have therapeutic potential in the amelioration of local and remote tissue damage following IRI.