Tim50's presequence receptor domain is essential for signal driven transport across the TIM23 complex

N-terminal targeting signals (presequences) direct proteins across the TOM complex in the outer mitochondrial membrane and the TIM23 complex in the inner mitochondrial membrane. Presequences provide directionality to the transport process and regulate the transport machineries during translocation. However, surprisingly little is known about how presequence receptors interact with the signals and what role these interactions play during preprotein transport. Here, we identify signal-binding sites of presequence receptors through photo-affinity labeling. Using engineered presequence probes, photo cross-linking sites on mitochondrial proteins were mapped mass spectrometrically, thereby defining a presequence-binding domain of Tim50, a core subunit of the TIM23 complex that is essential for mitochondrial protein import. Our results establish Tim50 as the primary presequence receptor at the inner membrane and show that targeting signals and Tim50 regulate the Tim23 channel in an antagonistic manner.     

Glucose-induced production of recombinant proteins in Hansenula polymorpha mutants deficient in catabolite repression

The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system.     

Changes in ischemic stroke presentations,management and outcomes during the first year of the COVID-19 pandemic in Alberta: a population study

Background:Pandemics may promote hospital avoidance, and added precautions may exacerbate treatment delays for medical emergencies such as stroke. We sought to evaluate ischemic stroke presentations, management and outcomes during the first year of the COVID-19 pandemic.Methods:We conducted a population-based study, using linked administrative and stroke registry data from Alberta to identify all patients presenting with stroke before the pandemic (Jan. 1, 2016 to Feb. 27, 2020) and in 5 periods over the first pandemic year (Feb. 28, 2020 to Mar. 31, 2021), reflecting changes in case numbers and restrictions. We evaluated changes in hospital admissions, emergency department presentations, thrombolysis, endovascular therapy, workflow times and outcomes.Results:The study included 19 531 patients in the prepandemic period and 4900 patients across the 5 pandemic periods. Presentations for ischemic stroke dropped in the first pandemic wave (weekly adjusted incidence rate ratio [IRR] 0.54, 95% confidence interval [CI] 0.50 to 0.59). Population-level incidence of thrombolysis (adjusted IRR 0.50, 95% CI 0.41 to 0.62) and endovascular therapy (adjusted IRR 0.63, 95% CI 0.47 to 0.84) also decreased during the first wave, but proportions of patients presenting with stroke who received acute therapies did not decline. Rates of patients presenting with stroke did not return to prepandemic levels, even during a lull in COVID-19 cases between the first 2 waves of the pandemic, and fell further in subsequent waves. In-hospital delays in thrombolysis or endovascular therapy occurred in several pandemic periods. The likelihood of in-hospital death increased in Wave 2 (adjusted odds ratio [OR] 1.48, 95% CI 1.25 to 1.74) and Wave 3 (adjusted OR 1.46, 95% CI 1.07 to 2.00). Out-of-hospital deaths, as a proportion of stroke-related deaths, rose during 4 of 5 pandemic periods.Interpretation:The first year of the COVID-19 pandemic saw persistently reduced rates of patients presenting with ischemic stroke, recurrent treatment delays and higher risk of in-hospital death in later waves. These findings support public health messaging that encourages care-seeking for medical emergencies during pandemic periods, and stroke systems should re-evaluate protocols to mitigate inefficiencies.

In response to the COVID-19 pandemic, affected countries implemented various public health measures to decrease viral transmission. An unintended consequence of these measures could be hospital avoidance by patients with medical emergencies, as observed during other outbreaks in the 2000s.^1,2 Some public health messaging specifically warned groups at high cardiovascular risk, such as older people or those with heart disease, that they were at elevated risk of severe COVID-19.³ Physical distancing may also result in loss of services and support networks, impairing patients’ ability to seek medical assistance.⁴ Furthermore, pandemics generate new challenges of managing personal protective equipment and cleaning protocols,⁵ and additional information bottlenecks, which could result in workflow delays for emergencies like stroke.⁶Previous studies have reported declines in patients presenting to hospital with stroke or acute coronary syndrome during the pandemic.^7,8 A World Stroke Organization survey of members in several countries indicated a sharp reduction in stroke admissions by 50%–80% in the first weeks of the pandemic.⁹ A cross-sectional study reported a global decline in hospital admissions for stroke.¹⁰ Patients who present to hospital seem to be doing so later than usual, perhaps waiting until their condition becomes more severe.^11–14 However, studies have not been at a population level, consequently suffering from selection bias, and have generally focused only on the first wave of the pandemic. As the associations between the pandemic and the incidence, treatment, workflow and outcomes of stroke are likely to be modified by several events — including changing COVID-19 case counts, public health restrictions and health system strains — it is important to explore population data from pandemic periods beyond the first wave to better understand these phenomena.Verifying and quantifying the pandemic’s effect on stroke presentations and workflow can help tailor public health messaging to continue emphasizing the time-critical nature of emergencies like stroke. Such data may also help optimize pandemic stroke workflow protocols. We sought to explore patterns of hospital admissions, treatment rates, workflow delays and outcomes for ischemic stroke during the first year of the COVID-19 pandemic in Alberta, Canada.     

The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo‐ATP synthase

Mitochondrial F₁F_o‐ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear‐ and mitochondria‐encoded subunits. Whereas chaperones for formation of the matrix‐exposed hexameric F₁‐ATPase core domain have been identified, insight into how the nuclear‐encoded F₁‐domain assembles with the membrane‐embedded F_o‐region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F₁‐module and peripheral stalk, but not with the assembled F₁F_o‐ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F₁‐module to the membrane embedded F_o‐domain. We conclude that INAC represents a matrix‐exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F₁F_o‐ATP synthase.     

Combinational spinal GAD65 gene delivery and systemic GABA-mimetic treatment for modulation of spasticity

Background

Loss of GABA-mediated pre-synaptic inhibition after spinal injury plays a key role in the progressive increase in spinal reflexes and the appearance of spasticity. Clinical studies show that the use of baclofen (GABA_B receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments.

Methods/Principal Findings

Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene.

Conclusions/Significance

These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel and highly effective anti-spasticity treatment which is associated with minimal side effects and is restricted to GAD65-gene over-expressing spinal segments.     

A general mathematical framework for the analysis of spatiotemporal point processes

Spatial and stochastic models are often straightforward to simulate but difficult to analyze mathematically. Most of the mathematical methods available for nonlinear stochastic and spatial models are based on heuristic rather than mathematically justified assumptions, so that, e.g., the choice of the moment closure can be considered more of an art than a science. In this paper, we build on recent developments in specific branch of probability theory, Markov evolutions in the space of locally finite configurations, to develop a mathematically rigorous and practical framework that we expect to be widely applicable for theoretical ecology. In particular, we show how spatial moment equations of all orders can be systematically derived from the underlying individual-based assumptions. Further, as a new mathematical development, we go beyond mean-field theory by discussing how spatial moment equations can be perturbatively expanded around the mean-field model. While we have suggested such a perturbation expansion in our previous research, the present paper gives a rigorous mathematical justification. In addition to bringing mathematical rigor, the application of the mathematically well-established framework of Markov evolutions allows one to derive perturbation expansions in a transparent and systematic manner, which we hope will facilitate the application of the methods in theoretical ecology.     

The ferroxidase activity of yeast frataxin

Frataxin is required for maintenance of normal mitochondrial iron levels and respiration. The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (alpha(48)) that accumulates iron in a water-soluble form. Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (alpha --> alpha(3)). A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O(2) is detected at Fe(II)/mYfh1p ratios of < or = 0.5. Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of >0.5. Gel filtration analysis indicates that an oligomer of mYfh1p, alpha(3), is responsible for both reactions. The observed 2 Fe(II)/O(2) stoichiometry implies production of H(2)O(2) during the ferroxidase reaction. However, only a fraction of the expected total H(2)O(2) is detected in solution. Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H(2)O(2) reacts with the protein. Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H(2)O(2) results in significant attenuation of Fenton chemistry. Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O(2). This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer.     

Hypoxia divergently regulates production of reactive oxygen species in human pulmonary and coronary artery smooth muscle cells

Acute hypoxia causes pulmonary vasoconstriction and coronary vasodilation. The divergent effects of hypoxia on pulmonary and coronary vascular smooth muscle cells suggest that the mechanisms involved in oxygen sensing and downstream effectors are different in these two types of cells. Since production of reactive oxygen species (ROS) is regulated by oxygen tension, ROS have been hypothesized to be a signaling mechanism in hypoxia-induced pulmonary vasoconstriction and vascular remodeling. Furthermore, an increased ROS production is also implicated in arteriosclerosis. In this study, we determined and compared the effects of hypoxia on ROS levels in human pulmonary arterial smooth muscle cells (PASMC) and coronary arterial smooth muscle cells (CASMC). Our results indicated that acute exposure to hypoxia (Po(2) = 25-30 mmHg for 5-10 min) significantly and rapidly decreased ROS levels in both PASMC and CASMC. However, chronic exposure to hypoxia (Po(2) = 30 mmHg for 48 h) markedly increased ROS levels in PASMC, but decreased ROS production in CASMC. Furthermore, chronic treatment with endothelin-1, a potent vasoconstrictor and mitogen, caused a significant increase in ROS production in both PASMC and CASMC. The inhibitory effect of acute hypoxia on ROS production in PASMC was also accelerated in cells chronically treated with endothelin-1. While the decreased ROS in PASMC and CASMC after acute exposure to hypoxia may reflect the lower level of oxygen substrate available for ROS production, the increased ROS production in PASMC during chronic hypoxia may reflect a pathophysiological response unique to the pulmonary vasculature that contributes to the development of pulmonary vascular remodeling in patients with hypoxia-associated pulmonary hypertension.     

Overexpression of TRPC1 enhances pulmonary vasoconstriction induced by capacitative Ca2+ entry

Transient receptor potential (TRP) cation channels are a critical pathway for Ca2+ entry during pulmonary artery (PA) smooth muscle contraction. However, whether canonical TRP (TRPC) subunits and which TRP channel isoforms are involved in store depletion-induced pulmonary vasoconstriction in vivo remain unclear. This study was designed to test whether overexpression of the human TRPC1 gene (hTRPC1) in rat PA enhances pulmonary vasoconstriction due to store depletion-mediated Ca2+ influx. The hTRPC1 was infected into rat PA rings with an adenoviral vector. RT-PCR and Western blot analyses confirmed the mRNA and protein expression of hTRPC1 in the arterial rings. The amplitude of active tension induced by 40 mM K+ (40K) in PA rings infected with an empty adenoviral vector (647 +/- 88 mg/mg) was similar to that in PA rings infected with hTRPC1 (703 +/- 123 mg/mg, P = 0.3). However, the active tension due to capacitative Ca2+ entry (CCE) induced by cyclopiazonic acid was significantly enhanced in PA rings overexpressing hTRPC1 (91 +/- 13% of 40K-induced contraction) compared with rings infected with an empty adenoviral vector (61 +/- 14%, P < 0.001). Endothelial expression of hTRPC1 was not involved since the CCE-induced vasoconstriction was also enhanced in endothelium-denuded PA rings infected with the adenoviral vector carrying hTRPC1. These observations demonstrate that hTRPC1 is an important Ca(2+)-permeable channel that mediates pulmonary vasoconstriction when PA smooth muscle cell intracellular Ca2+ stores are depleted.     

A new record of Gobius jarosi (Teleostei,Gobiidae) from the Early Miocene of Poland with inference to paleogeography and palaeoecology of the Carpathian Basin

Abstract

Gobies (family Gobiidae) were in the past and now important components of marine ecosystems as an essential part of the food chain. However, the early fossil record of this group is relatively meager, with only scarce skeletal remains. The oldest known representative of the genus Gobius has been recently described from the Early Miocene of Czech Republic as Gobius jarosi P?ikryl & Reichenbacher, 2018. Here we present a detailed study of a well-preserved goby skeleton of the same age from the Harta locality (Poland). This specimen is assumed as belonging to Gobius jarosi based on its almost complete morphological and meristic identity with the type material from the Vá?any nad Litavou locality. Some aspects of the paleogeography and paleoecology of the Early Miocene fish assemblage from Harta with special reference to the Carpathian Basin are also discussed in this paper.