Cysteine Accessibility in ClC-0 Supports Conservation of the ClC Intracellular Vestibule

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity

Botryosphaeriaceae on native and exotic Myrtaceae trees in southern and eastern Africa

Preface Members of the Botryosphaeriaceae represent a diverse family of fungi with over 180 species. They have been reported from a wide range of hosts occurring as endophytes, saprophytes or pathogens. These fungi are commonly referred to as latent opportunistic pathogens as they cause disease when the host is under abiotic or biotic stress. Symptoms associated with infection include tip die-back, stem and branch cankers, fruit rots, leaf spots and in severe cases mortality of trees. However, they are best known as canker and die-back pathogens on woody plants including species such as Eucalyptus (Myrtaceae). This dissertation focusses on the Botryosphaeriaceae on native and introduced Myrtaceae in southern and eastern Africa and aimed to increase current scientific knowledge of these fungi in the region. The literature review of this dissertation provides background to the research chapter by discussing the historic taxonomic changes in the Botryosphaeriaceae, and the current methods used for identification and classification of the species in this family. Furthermore, it summarizes the knowledge pertaining to Botryosphaeriaceae species on Eucalyptus and native Myrtaceae trees, particularly in eastern and southern Africa. Importantly, it considers fungal host jumps/shifts and their future impact in relation to Eucalyptus plantation forestry in Africa. Consideration is also given to the biology, ecology and possible future impacts of the Botryosphaeriaceae. Eucalyptus is the largest genus in the family Myrtaceae. Most species in this genus originates from Australia and were introduced to other countries as non-natives. In most parts of the world, Eucalyptus plantations are established in close proximity to native trees making it possible for pathogens to move from the native to the non-native trees and vice versa. This movement could result in considerable damage to the Eucalyptus plantations and the native trees. Chapter two of this dissertation focuses on identifying the Botryosphaeriaceae species occurring on both native and the non-native Myrtaceae trees in the Limpopo Province of South Africa and selected countries in southern and eastern Africa. Furthermore, it aimed at understanding the implications of the host jumps/shifts between the native and the non-native trees and their importance in relation to plantation forestry in eastern and southern Africa. This was achieved by sampling trees from South Africa, Malawi, Mozambique, Tanzania and Zimbabwe. Species resembling Botryosphaeriaceae were identified to species level using multi-gene DNA sequence data. To study the implications of the host jumps/shifts on Eucalyptus plantations, isolates obtained from native Myrtaceae trees were tested for pathogenicity under glasshouse conditions. The work presented in this dissertation provides a foundation of knowledge regarding the occurance and distribution of Botryosphaeriaceae species on several native Myrtaceae and Eucalyptus trees in eastern and southern Africa. This information is especially important in light of the fact that the areas from which isolates were obtained had not been considered previously. Furthermore, it suggests future directions to better understand the biology and taxonomy, movement, role and influence of Botryosphaeriaceae species on both native and non-native Myrtaceae trees.Dissertation (MSc)--University of Pretoria, 2017.Microbiology and Plant PathologyMScUnrestricte

The Mechanism of Fast-Gate Opening in ClC-0

ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108:237–250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chen and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ec1. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent

The Role of a Conserved Lysine in Chloride- and Voltage-dependent ClC-0 Fast Gating

ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account for the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening

Botryosphaeriaceae species associated with branch dieback and decline of macadamia trees in South Africa

DATA AVAILABILITY : All sequence data generated for this study have been submitted to GenBank (accession numbers are listed in Online Resource Table 1) and any additional data are available on request.Botryosphaeriaceae species are important latent pathogens causing diseases on trees utilized in forestry and agriculture. In recent years, there has been an increase in the incidence and severity of branch dieback and decline on macadamia trees in South Africa, and species of Botryosphaeriaceae have been considered as a possible cause. Although botryosphaeria dieback has been well-studied in Australia, there is little information regarding these fungi on Macadamia in South Africa. The aims of this study were consequently to (i) identify species of Botryosphaeriaceae from Macadamia branches from main production regions in South Africa, (ii) compare the diversity of species between symptomatic and asymptomatic branches, as well as between different growing regions, (iii) and to consider their relative importance in causing dieback. Eight species and three putative hybrids of the Botryosphaeriaceae were identified based on a phylogenetic comparison of sequence data from the ITS rDNA, tub2, tef-1α and rpb2 loci. These included an unidentified Diplodia sp., and Lasiodiplodia sp., as well as L. gilanensis, L. theobromae, L. pseudotheobromae, Neofusicoccum kwambonambiense, N. luteum, N. parvum and three hybrid species. The unidentified species of Diplodia., Lasiodiplodia sp., L. gilanensis, and N. kwambonambiense are reported for the first time on Macadamia in South Africa. All species showed a potential to cause branch dieback symptoms, with species of Neofusicoccum identified as the most aggressive species. This study revealed a high level of diversity of Botryosphaeriaceae species and illustrates their potential as causal agents of dieback on Macadamia in South Africa.Macadamias South Africa NPC (SAMAC), the University of Pretoria, and the Forestry and Agricultural Biotechnology Institute (FABI). Open access funding provided by University of Pretoria.http://link.springer.com/journal/13313hj2024Biochemistry, Genetics and Microbiology (BGM)Forestry and Agricultural Biotechnology Institute (FABI)SDG-15:Life on lan

Regulation of Organelle Acidity

Intracellular organelles have characteristic pH ranges that are set and maintained by a balance between ion pumps, leaks, and internal ionic equilibria. Previously, a thermodynamic study by Rybak et al. (Rybak, S., F. Lanni, and R. Murphy. 1997. Biophys. J. 73:674–687) identified the key elements involved in pH regulation; however, recent experiments show that cellular compartments are not in thermodynamic equilibrium. We present here a nonequilibrium model of lumenal acidification based on the interplay of ion pumps and channels, the physical properties of the lumenal matrix, and the organelle geometry. The model successfully predicts experimentally measured steady-state and transient pH values and membrane potentials. We conclude that morphological differences among organelles are insufficient to explain the wide range of pHs present in the cell. Using sensitivity analysis, we quantified the influence of pH regulatory elements on the dynamics of acidification. We found that V-ATPase proton pump and proton leak densities are the two parameters that most strongly influence resting pH. Additionally, we modeled the pH response of the Golgi complex to varying external solutions, and our findings suggest that the membrane is permeable to more than one dominant counter ion. From this data, we determined a Golgi complex proton permeability of 8.1 × 10−6 cm/s. Furthermore, we analyzed the early-to-late transition in the endosomal pathway where Na,K-ATPases have been shown to limit acidification by an entire pH unit. Our model supports the role of the Na,K-ATPase in regulating endosomal pH by affecting the membrane potential. However, experimental data can only be reproduced by (1) positing the existence of a hypothetical voltage-gated chloride channel or (2) that newly formed vesicles have especially high potassium concentrations and small chloride conductance

Feasibility and acceptability of an HPV self-testing strategy : lessons from a research context to assess for ability to implement into primary care at a national level in Botswana

DATA AVAILABILITY STATEMENT : The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.BACKGROUND : The WHO strategy for cervical cancer elimination strives to achieve 70% coverage with high-performance cervical screening. While few low- and middle-income countries have achieved this, high-risk human papillomavirus (hrHPV) self-testing creates the possibility to rapidly upscale access to highperformance cervical screening across resource settings. However, effective hrHPV screening requires linkage to follow-up, which has been variable in prior studies. This study developed and tested an implementation strategy aimed at improving screening and linkage tofollow-up care in South EastDistrict in Botswana. METHODS : This study performed primary hrHPV self-testing; those with positive results were referred for a triage visit. Withdrawals for any reason, loss-to follow-up between hrHPV test and triage visit, and number of call attempts to give hrHPV results were also documented. Acceptability of the program to patients was measured as the proportion of patients who completed a triage visit when indicated, meeting the a priori threshold of 80%. Feasibility was defined as the proportion of participants receiving the results and attending follow-up. To assess the associations between participant characteristics and loss-to-follow-up we used log-binomial regressions to estimate risk ratios and 95% confidence intervals (CI). RESULTS : Enrollment of 3,000 women occurred from February 2021 to August 2022. In total, 10 participants withdrew and an additional 33 were determined ineligible after consent, leaving a final cohort of 2,957 participants who underwent self-swab hrHPV testing. Half (50%) of participants tested positive for hrHPV and nearly all (98%) of participants received their hrHPV results, primarily via telephone. Few calls to participants were required to communicate results: 2,397 (82%) required one call, 386 (13%) required 2 calls, and only 151 (5%) required 3–5 calls. The median time from specimen collection to participant receiving results was 44 days (IQR, 27–65). Of all hrHPV positive participants, 1,328 (90%) attended a triage visit. DISCUSSION : In a large cohort we had low loss-to-follow-up of 10%, indicating that the strategy is acceptable. Telephonic results reporting was associated with high screening completion, required few calls to participants, and supports the feasibility of hrHPV self-testing in primary care followed by interval triage.The Young Investigator Award from the Department of Obstetrics and Gynecology at Beth Israel Deaconess Medical Center, the National Cancer Institute, National Institutes of Health Award.https://www.frontiersin.org/journals/global-womens-healtham2024Obstetrics and GynaecologySDG-03:Good heatlh and well-bein

A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+