The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules
receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for ...disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown.
To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach.
Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology.
Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.
Mutations in PTEN inducible kinase-1 (PINK1) induce mitochondrial dysfunction in dopaminergic neurons resulting in an inherited form of Parkinson's disease. Although PINK1 is present in the heart its ...exact role there is unclear. We hypothesized that PINK1 protects the heart against acute ischemia reperfusion injury (IRI) by preventing mitochondrial dysfunction.
Over-expressing PINK1 in HL-1 cardiac cells reduced cell death following simulated IRI (29.2±5.2% PINK1 versus 49.0±2.4% control; N = 320 cells/group P<0.05), and delayed the onset of mitochondrial permeability transition pore (MPTP) opening (by 1.3 fold; P<0.05). Hearts excised from PINK1+/+, PINK1+/- and PINK1-/- mice were subjected to 35 minutes regional ischemia followed by 30 minutes reperfusion. Interestingly, myocardial infarct size was increased in PINK1-/- hearts compared to PINK1+/+ hearts with an intermediate infarct size in PINK1+/- hearts (25.1±2.0% PINK1+/+, 38.9±3.4% PINK1+/- versus 51.5±4.3% PINK1-/- hearts; N>5 animals/group; P<0.05). Cardiomyocytes isolated from PINK1-/- hearts had a lower resting mitochondrial membrane potential, had inhibited mitochondrial respiration, generated more oxidative stress during simulated IRI, and underwent rigor contracture more rapidly in response to an uncoupler when compared to PINK1+/+ cells suggesting mitochondrial dysfunction in hearts deficient in PINK1.
We show that the loss of PINK1 increases the heart's vulnerability to ischemia-reperfusion injury. This may be due, in part, to increased mitochondrial dysfunction. These findings implicate PINK1 as a novel target for cardioprotection.
To determine the duration of protection from hepatitis B vaccine given in infancy and early childhood and asses risk factors for HBV infection and chronic infection.
In 1984 infant HBV vaccination ...was started in two Gambian villages. Cross sectional serological surveys have been undertaken every 4 years to determine vaccine efficacy. In the current survey 84.6% of 1508 eligible participants aged 1-28 years were tested. A spouse study was conducted in females (aged 14 years and above) and their male partners.
Vaccine efficacy against chronic infection with hepatitis B virus was 95.1% (95% confidence interval 91.5% to 97.1%), which did not vary significantly between age groups or village. Efficacy against infection was 85.4% (82.7% to 87.7%), falling significantly with age. Concentrations of hepatitis B antibody fell exponentially with age varying according to peak response: 20 years after vaccination only 17.8% (95% CI 10.1-25.6) of persons with a low peak response (10-99 mIU/ml) had detectable HBs antibody compared to 27% (21.9% to 32.2%) of those with a high peak response (>999 mIU/ml). Time since vaccination and a low peak response were the strongest risk factors for HBV infections; males were more susceptible, marriage was not a significant risk for females. Hepatitis B DNA was not detected after infection, which tested soley core antibody positive. An undetectable peak antibody response of <10 mIU/ml and a mother who was hepatitis B e antigen positive were powerful risk factors for chronic infection.
Adolescents and young adults vaccinated in infancy are at increased risk of hepatitis B infection, but not chronic infection. Married women were not at increased risk. There is no compelling evidence for the use of a booster dose of HBV vaccine in The Gambia.
The COVID-19 pandemic has disrupted healthcare systems around the world, impacting how we deliver medical education. The normal day-to-day routines have been altered for a number of reasons, ...including changes to scheduled training rotations, physical distancing requirements, trainee redeployment, and heightened level of concern. Medical educators will likely need to adapt their programs to maximize learning, maintain effective care delivery, and ensure competent graduates. Along with a continued focus on learner/faculty wellness, medical educators will have to optimize existing training experiences, adapt those that are no longer viable, employ new technologies, and be flexible when assessing competencies. These practical tips offer guidance on how to adapt medical education programs within the constraints of the pandemic landscape, stressing the need for communication, innovation, collaboration, flexibility, and planning within the era of competency-based medical education.
The mechanism through which the protein kinase Akt (also called PKB), protects the heart against acute ischaemia-reperfusion injury (IRI) is not clear. Here, we investigate whether Akt mediates its ...cardioprotective effect by modulating mitochondrial morphology. Transfection of HL-1 cardiac cells with constitutively active Akt (caAkt) changed mitochondrial morphology as evidenced by an increase in the proportion of cells displaying predominantly elongated mitochondria (73 ± 5.0 % caAkt vs 49 ± 5.8 % control: N=80 cells/group; p< 0.05). This effect was associated with delayed time taken to induce mitochondrial permeability transition pore (MPTP) opening (by 2.4 ± 0.5 fold; N=80 cells/group: p< 0.05); and reduced cell death following simulated IRI (32.8 ± 1.2 % caAkt vs 63.8 ± 5.6 % control: N=320 cells/group: p< 0.05). Similar effects on mitochondrial morphology, MPTP opening, and cell survival post-IRI, were demonstrated with pharmacological activation of Akt using the known cardioprotective cytokine, erythropoietin (EPO). The effect of Akt on inducing mitochondrial elongation was found to be dependent on the mitochondrial fusion protein, Mitofusin-1 (Mfn1), as ablation of Mfn1 in mouse embryonic fibroblasts (MEFs) abrogated Akt-mediated mitochondrial elongation. Finally, in vivo pre-treatment with EPO reduced myocardial infarct size (as a % of the area at risk) in adult mice subjected to IRI (26.2 ± 2.6 % with EPO vs 46.1 ± 6.5 % in control; N=7/group: p< 0.05), and reduced the proportion of cells displaying myofibrillar disarray and mitochondrial fragmentation observed by electron microscopy in adult murine hearts subjected to ischaemia from 5.8 ± 1.0 % to 2.2 ± 1.0 % (N=5 hearts/group; p< 0.05). In conclusion, we found that either genetic or pharmacological activation of Akt protected the heart against acute ischaemia-reperfusion injury by modulating mitochondrial morphology.
The aims were to assess the utility of: 1) virtual reality-mediated simulation, and 2) a multi-modality 'Bootcamp' in the delivery of total knee arthroplasty (TKA) teaching to orthopaedic surgical ...trainees.
Surgical training opportunities are diminished as a result of the COVID-19 pandemic which may result in delays to training completion and gaps in the permanent workforce. Modern and technology-enhanced learning methods have been identified as having the potential to support high-quality and sustainable education.
This mixed-methods study assessed the educational benefit of two activities designed to teach TKA to junior (ST1-3) orthopaedic trainees. A multi-modality training Bootcamp was delivered that included: virtual reality (VR) and saw-bone simulation; tutorials, and case-based symposia. The VR component was delivered to different participants (surgical trainees, scrub nurses, and consultants) on a further two separate occasions. Qualitative and quantitative data were collected pertaining to utility and performance.
Trainees reported that the Bootcamp improved comprehension of arthroplasty principles including component alignment, knee balancing, and intraoperative strategies. Case-based discussions helped develop diagnostic and decision-making skills. The VR activity improved understanding of the surgical process map, increased ability to anticipate steps, and consider the procedure strategically. All staff groups found the VR activity beneficial and would recommend it as a useful addition to a surgical department.
VR-mediated simulation could augment the education of surgical trainees and scrub team staff by improving comprehension of the surgical process map. Integrated multi-modality 'Bootcamp-style' training activities constructed around trainees' needs may provide a sustainable solution to bridge the experience gap related to reduced exposure to elective orthopaedic practice.
Glycolytic activity is increased in proliferating cells, leading to the concept that glycolysis could be a therapeutic target in cystic diseases and kidney cancer. Preclinical studies using the ...glucose analog 2-deoxy-d-glucose have shown promise; however, inhibiting glycolysis in humans is unlikely to be without risks. While proximal tubules are predominantly aerobic, later segments are more glycolytic. Understanding exactly where and why glycolysis is important in the physiology of the distal nephron is thus crucial in predicting potential adverse effects of glycolysis inhibitors. Live imaging techniques could play an important role in the process of characterizing cellular metabolism in the functioning kidney. The goal of this review is to briefly summarize recent findings on targeting glycolysis in proliferative kidney diseases and to highlight the necessity for future research focusing on glycolysis in the healthy kidney.
Mitochondrial superoxide (O2⋅−) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O2⋅−, but are of limited applicability in vivo, while in cells their usefulness is ...constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O2⋅− probe, MitoNeoD, which can assess O2⋅− changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O2⋅−-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O2⋅− over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O2⋅− from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O2⋅− production in health and disease.
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•MitoNeoD is a mitochondria-targeted O2⋅− probe that can be used in vivo•Neopentyl groups prevent DNA intercalation by MitoNeoD and its derivatives•Incorporation of a carbon-deuterium bond enhances O2⋅− selectivity by MitoNeoD•MitoNeoD extends methods available to assess mitochondrial O2⋅−
Current methods to assess mitochondrial O2⋅− cannot be applied in vivo and are artifact prone. Here Shchepinova et al. introduce MitoNeoD, which can be used to assess changes in mitochondrial O2⋅− by fluorescence and by mass spectrometry.
ABSTRACT
Some philosophers think that fostering children's autonomy – in the sense of critical, rational reflection on our beliefs and goals – is an appropriate aim of public educational policy. ...Critics say that this amounts to championing an Enlightenment outlook over ways of life rooted in faith and tradition. The most common response to the critics is to assert that children have an interest in autonomy, not because autonomy is intrinsically worthwhile, but because it is instrumentally valuable in discovering how to lead a good life. Finding fault with this Instrumental Argument, I argue that the best case for autonomy is that critical, rational reflection is something we all already rely on in everyday life to figure out which beliefs are worthy of our assent and which goals are worthy of our pursuit. The question is not whether children will reason, but whether they will reason well or poorly. Teaching children how to think critically is, thus, best understood as helping them meet their own emerging standards of rationality. And, given that we have a duty to respect others as reasoning beings, we in turn have a collective duty to foster young people's capacity to distinguish good reasoning from bad.
Tenofovir (TFV) is a widely used and effective treatment for HIV infection. Numerous studies have shown that TFV exposure is associated with small but significant declines in estimated glomerular ...filtration rate (eGFR). However, TFV toxicity is targeted mainly at the proximal tubule (PT), and in severe cases can cause the renal Fanconi syndrome or acute kidney injury. Severe toxicity occurs in a minority of patients, but milder PT dysfunction is more common; the long-term significance of this on kidney and bone health is uncertain. Recent work suggests that changes in eGFR on TFV therapy might be explained by inhibition of PT creatinine secretion rather than actual alterations in glomerular function. Risk factors for nephrotoxicity include pre-existing kidney disease, increased age, and low body mass. Mitochondria in the PT are the targets of TFV toxicity, but the exact mechanisms remain unclear. Substantial improvement of renal function occurs in many patients with TFV toxicity upon stopping therapy, but function does not always return to baseline. In recent years, TFV usage has been extended to new clinical spheres, including pediatrics, resource-poor settings and treatment of Hepatitis B infection; theoretical reasons exist as to why some of these patients might be at higher or lower risk of TFV toxicity. Finally, strategies have been proposed to prevent TFV toxicity or enhance recovery.
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