Mitochondrial physiology

Internal and external respiration (mt) Mitochondrial catabolic respiration JkO2 is the O2 consumption in the oxidation of fuel substrates (electron donors) and reduction of O2 catalysed by the electron transfer system ETS, which drives the protonmotive force pmF. JkO2 excludes mitochondrial residual oxygen consumption, mt-Rox (). (ce) Cell respiration JrO2 is internal cellular O2 consumption, taking into account all chemical reactions r that consume O2 in the cells. Catabolic cell respiration is the O2 consumption associated with catabolic pathways in the cell, including mitochondrial (mt) catabolism, and: mt-Rox (); non-mt O2 consumption by catabolic reactions, particularly peroxisomal oxidases and microsomal cytochrome P450 systems (); non-mt Rox by reactions unrelated to catabolism (). (ext) External respiration balances internal respiration at steady state, including extracellular Rox () and aerobic respiration by the microbiome (). External O2 is transported from the environment across the respiratory cascade by circulation between tissues and diffusion across cell membranes, to the intracellular compartment. The respiratory quotient RQ is the molar CO2/O2 exchange ratio; combined with the nitrogen quotient N/O2 (mol N given off per mol O2 consumed), the RQ reflects the proportion of carbohydrate, lipid and protein utilized in cell respiration during aerobically balanced steady states. Bicarbonate and CO2 are transported in reverse to the extracellular milieu and the organismic environment. Hemoglobin provides the molecular paradigm for the combined CO2/O2 exchange, as do lungs and gills on the morphological level, but CO2/O2 exchange across the skin and other surfaces is less interdependent, and highly independent in cell respiration

Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling

Bone morphogenetic proteins (BMPs) control multiple cellular processes in embryos and adult tissues. BMPs signal through the activation of type I BMP receptor kinases, which then phosphorylate SMADs 1/5/8. In the canonical pathway, this triggers the association of these SMADs with SMAD4 and their translocation to the nucleus, where they regulate gene expression. BMPs can also signal independently of SMAD4, but this pathway is poorly understood. Here, we report the discovery and characterization of PAWS1/FAM83G as a novel SMAD1 interactor. PAWS1 forms a complex with SMAD1 in a SMAD4-independent manner, and BMP signalling induces the phosphorylation of PAWS1 through BMPR1A. The phosphorylation of PAWS1 in response to BMP is essential for activation of the SMAD4-independent BMP target genes NEDD9 and ASNS. Our findings identify PAWS1 as the first non-SMAD substrate for type I BMP receptor kinases and as a novel player in the BMP pathway. We also demonstrate that PAWS1 regulates the expression of several non-BMP target genes, suggesting roles for PAWS1 beyond the BMP pathway

Passive UHF RFID Voice Prosthesis Mounted Sensor for Microbial Growth Detection

Capacitive loading due to human tissue can lead to low efficiency for implantable Passive Radio Frequency Identification (RFID) antennas. The presented passive UHF antenna sensor provides read distances above 0.5 meters (within a body phantom) by utilizing a convoluted half-wave dipole design. It is able to detect simulated early to mature Candida albicans biofilm growth when mounted upon a voice prosthesis (up to a 30 μm biofilm thickness). Depending on the propagation frequency of interest, as early 4-hour growth (5 to 10 μm biofilm thickness) equivalent could be detected and before any device failure could occur due to the colonization. This was accomplished by utilising thin layers of polyurethane to decouple the saliva from the presented UHF sensor (biofilm growth is known to increase layer hydrophobicity). This presented sensor has better functionality within the US UHF frequency band as it detects changes above 5 μm. If there is a need for implantation within additional tissues with variable dielectric properties, a shunt capacitance of 2.6 pF could allow the system functionality within the permittivity range of 21 to 58. Allowing for immediate medical intervention before medical prosthesis failur

High Resolution Respirometry in Candida albicans

Many Candida species, such as the opportunistic human pathogen Candida albicans, are Crabtree-Negative yeasts and are therefore highly dependent on the energy generated through oxidative phosphorylation. Respiration control is linked to a range of aspects of C. albicans cell physiology that appear to be important for virulence, most notably its ability to switch from yeast to hyphal forms and the maintenance of the cell wall. The following protocol allows for the measurement and characterisation of respiration in C. albicans using high resolution respirometry. We outline how addition of respiration inhibitors can be used to assay the “mode” of respiration, mitochondrial health and the level of electron transport that is coupled to ATP synthase activity in living cell cultures. These data provide useful insight into the effects of external factors, such as exposure to anti-fungal compounds, or internal changes such as genetic alterations on respiratory performance

The potential of respiration inhibition as a new approach to combat human fungal pathogens

The respiratory chain has been proposed as an attractive target for the development of new therapies to tackle human fungal pathogens. This arises from the presence of fungal-specific electron transport chain components and links between respiration and the control of virulence traits in several pathogenic species. However, as the physiological roles of mitochondria remain largely undetermined with respect to pathogenesis, its value as a potential new drug target remains to be determined. The use of respiration inhibitors as fungicides is well developed but has been hampered by the emergence of rapid resistance to current inhibitors. In addition, recent data suggest that adaptation of the human fungal pathogen, Candida albicans, to respiration inhibitors can enhance virulence traits such as yeast-to-hypha transition and cell wall organisation. We conclude that although respiration holds promise as a target for the development of new therapies to treat human fungal infections, we require a more detailed understanding of the role that mitochondria play in stress adaption and virulence

Learning from Lockdown: Listening to students' voices about the challenges and benefits in the post-COVID-19 digital practices

The sudden move to remote teaching and learning in Spring 2020 brought together staff and students in order to learn from each other how to best address the challenges of the new ways of teaching and learning. In this synchronous presentation the seven authors will take turns: *to introduce the two institutionally-supported studies conducted last year, one surveying the UCL undergraduates, and the other one surveying the postgraduate taught students in the CPA department of UCL IoE; *to share the framework that guided the analysis of students’ views of their experiences and priorities surrounding online learning, gathered via online surveys and focussed group discussions; *to propose alternative conceptions for what could be a meaningful and rich online educative experience; *to discuss our findings and their implications for theory, research, policy and practice in a post-pandemic context; and last and most important, *to describe how staff and students collaborated on carrying out these projects, and later worked together to write an academic paper about to the two projects

Engagement discourses, relationality and the student voice: connectedness, questioning and inclusion in post-Covid digital practices

The Covid-19 crisis has led to a rapid pivot to online teaching and student engagement across higher education internationally, due to public health ‘lockdown’ measures. In March 2020 in the UK this move was sudden, and universities were forced to move their provision to digital formats with little preparatory time, and in many cases, inadequate training and experience. In the subsequent period, higher education institutions have prioritised the enhancement of digital education, with a range of strategic initiatives and training programmes for teaching staff. This paper, written by a staff-student partnership of authors, reports on two institutionally-supported studies conducted at a large, research-focused university in England, in which student views were sought on their experiences and priorities surrounding online engagement during the Covid-19 crisis. In our discussion of the findings, we argue that the student accounts challenge some of the mainstream assumptions about constructs such as student ‘inclusivity’, academic ‘community’ online, and teaching which encourages ‘questioning’, requiring us to think more deeply about what constitutes a meaningful and rich online educative experience. In the spirit of ‘lessons learned’ from the Covid-19 pandemic, the paper proposes alternative conceptions of these values, emphasising relationality, communities, difference, and the importance of an ethos of care. We conclude with a discussion of findings, implications for theory, research, policy and practice in a post-pandemic context, proposing that an ethos of care be recognised as central to the development of digital education and the practices and ethics of student engagement

Alternative Oxidase – Aid or obstacle to combat the rise of fungal pathogens?

Fungal pathogens present a growing threat to both humans and global health security alike. Increasing evidence of antifungal resistance in fungal populations that infect both humans and plant species has increased reliance on combination therapies and shown the need for new antifungal therapeutic targets to be investigated. Here, we review the roles of mitochondria and fungal respiration in pathogenesis and discuss the role of the Alternative Oxidase enzyme (Aox) in both human fungal pathogens and phytopathogens. Increasing evidence exists for Aox within mechanisms that underpin fungal virulence. Aox also plays important roles in adaptability that may prove useful within dual targeted fungal-specific therapeutic approaches. As improved fungal specific mitochondrial and Aox inhibitors are under development we may see this as an emerging target for future approaches to tackling the growing challenge of fungal infection

The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+-Cl- co-transporters

This is the final version of the article. Available from Portland Press via the DOI in this record.There is another ORE record for this publication: http://hdl.handle.net/10871/32310Precise homoeostasis of the intracellular concentration of Cl- is achieved via the co-ordinated activities of the Cl- influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+-K+ ion co-transporters), also promote inhibition of the KCCs (K+-Cl- co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl- influx, we propose that the targeting of WNK-SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl- extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.This work was supported by the Medical Research Council and the Wellcome Trust [grant number 091415] as well as the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KgaA, Janssen Pharmaceutica and Pfizer). K.T.K. is supported by the Manton Center for Orphan Diseases at Children's Hospital Boston at Harvard Medical School, and the Harvard/MIT Joint Research Grants Program in Basic Neuroscience

Elevated Levels of Mislocalised, Constitutive Ras Signalling Can Drive Quiescence by Uncoupling Cell-Cycle Regulation from Metabolic Homeostasis

The small GTPase Ras plays an important role in connecting external and internal signalling cues to cell fate in eukaryotic cells. As such, the loss of RAS regulation, localisation, or expression level can drive changes in cell behaviour and fate. Post-translational modifications and expression levels are crucial to ensure Ras localisation, regulation, function, and cell fate, exemplified by RAS mutations and gene duplications that are common in many cancers. Here, we reveal that excessive production of yeast Ras2, in which the phosphorylation-regulated serine at position 225 is replaced with alanine or glutamate, leads to its mislocalisation and constitutive activation. Rather than inducing cell death, as has been widely reported to be a consequence of constitutive Ras2 signalling in yeast, the overexpression of RAS2S225A or RAS2S225E alleles leads to slow growth, a loss of respiration, reduced stress response, and a state of quiescence. These effects are mediated via cAMP/PKA signalling and transcriptional changes, suggesting that quiescence is promoted by an uncoupling of cell-cycle regulation from metabolic homeostasis. The quiescent cell fate induced by the overexpression of RAS2S225A or RAS2S225E could be rescued by the deletion of CUP9, a suppressor of the dipeptide transporter Ptr2, or the addition of peptone, implying that a loss of metabolic control, or a failure to pass a metabolic checkpoint, is central to this altered cell fate. Our data suggest that the combination of an increased RAS2 copy number and a dominant active mutation that leads to its mislocalisation can result in growth arrest and add weight to the possibility that approaches to retarget RAS signalling could be employed to develop new therapies