Molecular Targets for the Testing of COVID‐19

The pandemic outbreaks of coronavirus disease 2019 (COVID‐19), caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), spread all over the world in a short period of time. Efficient identification of the infection by SARS‐CoV‐2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with the infectious disease. In Taiwan, a COVID‐19 Open Science Platform adheres to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid‐February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID‐19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS‐CoV‐2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID‐19 virus.     

Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit

Ethanol often causes critical health problems by altering the neuro-nal activities of the central and peripheral nerve systems. One of the cellular targets of ethanol is the plasma membrane proteins including ion channels and receptors. Recently, we reported that ethanol elevates membrane excitability in sympathetic neurons by inhibiting Kv7.2/7.3 channels in a cell type-specific manner. Even though our studies revealed that the inhibitory effects of ethanol on the Kv7.2/7.3 channel was diminished by the increase of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI (4,5)P₂), the molecular mechanism of ethanol on Kv7.2/7.3 channel inhibition remains unclear. By investigating the kinetics of Kv7.2/7.3 current in high K⁺ solution, we found that ethanol inhibited Kv7.2/7.3 channels through a mechanism distinct from that of tetraethylammonium (TEA) which enters into the pore and blocks the gate of the channels. Using a non-stationary noise analysis (NSNA), we demonstrated that the inhibitory effect of ethanol is the result of reduction of open probability (P_O) of the Kv7.2/7.3 channel, but not of a single channel current (i) or channel number (N). Finally, ethanol selectively facilitated the kinetics of Kv7.2 current suppression by voltage-sensing phosphatase (VSP)-induced PI(4,5)P₂ depletion, while it slowed down Kv7.2 current recovery from the VSP-induced inhibition. Together our results suggest that ethanol regulates neuronal activity through the reduction of open probability and PI(4,5)P₂ sensitivity of Kv7.2/7.3 channels.     

Intestinal helminths as predictors of some malaria clinical outcomes and IL-1β levels in outpatients attending two public hospitals in Bamenda,North West Cameroon

This study aimed at determining the impact of intestinal helminths on malaria parasitaemia, anaemia and pyrexia considering the levels of IL-1β among outpatients in Bamenda. A cohort of 358 consented participants aged three (3) years and above, both males and females on malaria consultation were recruited in the study. At enrolment, patients’ axillary body temperatures were measured and recorded. Venous blood was collected for haemoglobin concentration and malaria parasitaemia determination. Blood plasma was used to measure human IL-1β levels using Human ELISA Kit. The Kato-Katz technique was used to process stool samples. Five species of intestinal helminths Ascaris lumbricoides (6.4%), Enterobius vermicularis (5.0%), Taenia species (4.2%), Trichuris trichiura (1.1%) and hookworms (0.8%) were identified. The overall prevalence of Plasmodium falciparum and intestinal helminths was 30.4% (109/358) and 17.6% (63/358) respectively. The prevalence of intestinal helminths in malaria patients was 17.4% (19/109). Higher Geometric mean parasite density (GMPD ±SD) (malaria parasitaemia) was significantly observed in patients co-infected with Enterobius vermicularis (5548 ± 2829/μL, p = 0.041) and with Taenia species (6799 ± 4584/μL, p = 0.020) than in Plasmodium falciparum infected patients alone (651 ± 6076/ μL). Higher parasitaemia of (1393 ± 3031/μL) and (3464 ± 2828/μL) were recorded in patients co-infected with Ascaris lumbricoides and with hookworms respectively but the differences were not significant (p > 0.05). Anaemia and pyrexia prevalence was 27.1% (97/358) and 33.5% (120/358) respectively. Malaria patients co-infected with Enterobius vermicularis and Ascaris lumbricoides had increased risk of anaemia (OR = 13.712, p = 0.002 and OR = 16.969, p = 0.014) respectively and pyrexia (OR = 18.07, p = 0.001 and OR = 22.560, p = 0.007) respectively than their counterparts. Increased levels of IL-1β were significantly observed in anaemic (148.884 ± 36.073 pg/mL, t = 7.411, p = 0.000) and pyretic (127.737 ± 50.322 pg/mL, t = 5.028, p = 0.000) patients than in non-anaemic (64.335 ± 38.995pg/mL) and apyretic patients (58.479 ± 36.194pg/mL). Malaria patients co-infected with each species of intestinal helminths recorded higher IL-1β levels (IL-1β > 121.68 ± 58.86 pg/mL) and the overall mean (139.63 ± 38.33pg/mL) was higher compared with levels in malaria (121.68 ± 58.86 pg/mL) and helminth (61.78 ± 31.69pg/mL) infected patients alone. Intestinal helminths exacerbated the clinical outcomes of malaria in the patients and increased levels of IL-1β were observed in co-infected patients with anaemia, pyrexia and higher parasitaemia.     

Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc₇-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc₇-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc₇-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc₇-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.Adipocytes, skeletal muscle cells, and some neurons respond to insulin stimulation by translocating intracellular glucose transporter 4 (Glut4) to the plasma membrane. In all these cells, the insulin-responsive pool of Glut4 is localized in small membrane vesicles, the insulin-responsive vesicles (IRVs; Kandror and Pilch, 2011 ; Bogan, 2012 ). The protein composition of these vesicles has been largely characterized (Kandror and Pilch, 2011 ; Bogan, 2012 ). The IRVs consist predominantly of Glut4, insulin-responsive aminopeptidase (IRAP), sortilin, low-density-lipoprotein receptor–related protein 1 (LRP1), SCAMPs, and VAMP2. Glut4, IRAP, and sortilin physically interact with each other, which might be important for the biogenesis of the IRVs (Shi and Kandror, 2007 ; Shi et al., 2008 ). In addition, the IRVs compartmentalize recycling receptors, such as the transferrin receptor and the IGF2/mannose 6-phosphate receptor, although it is not clear whether these receptors represent obligatory vesicular components or their presence in the IRVs is explained by mass action (Pilch, 2008 ), inefficient sorting, or other reasons.Deciphering of the protein composition of the IRVs is important because it is likely to explain their unique functional property: translocation to the plasma membrane in response to insulin stimulation. Even if we presume that IRV trafficking is controlled by loosely associated peripheral membrane proteins, the latter should still somehow recognize the core vesicular components that create the “biochemical individuality” of this compartment. In spite of our knowledge of the IRV protein composition, however, the identity of the protein(s) that confer insulin sensitivity to these vesicles is unknown.Insulin responsiveness of the IRVs was associated with either IRAP or Glut4. Thus it was shown that Glut4 interacted with the intracellular anchor TUG (Bogan et al., 2003 , 2012 ), whereas IRAP associated with other proteins implemented in the regulation of Glut4 translocation, such as AS160 (Larance et al., 2005 ; Peck et al., 2006 ), p115 (Hosaka et al., 2005 ), tankyrase (Yeh et al., 2007 ), and several others (reviewed in Bogan, 2012 ). Results of these studies, or at least their interpretations, are not necessarily consistent with each other, as the existence of multiple independent anchors for the IRVs is, although possible, unlikely.Ablation of the individual IRV proteins has also led to controversial data. Thus knockout of IRAP decreases total protein levels of Glut4 but does not affect its translocation in the mouse model (Keller et al., 2002 ). On the contrary, knockdown of IRAP in 3T3-L1 adipocytes has a strong inhibitory effect on translocation of Glut4 (Yeh et al., 2007 ). In yet another study, knockdown of IRAP in 3T3-L1 adipocytes did not affect insulin-stimulated translocation of Glut4 but increased its plasma membrane content under basal conditions (Jordens et al., 2010 ). By the same token, total or partial ablation of Glut4 had various effects on expression levels, intracellular localization, and translocation of IRAP (Jiang et al., 2001 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Gross et al., 2004 ; Yeh et al., 2007 ). Knockdown of either sortilin or LRP1 decreased protein levels of Glut4 (Shi and Kandror, 2005 ; Jedrychowski et al., 2010 ).One model that might explain these complicated and somewhat inconsistent results is that depletion of either major integral protein of the IRVs disrupts the network of interactions between vesicular proteins and thus decreases the efficiency of protein sorting into the IRVs (Kandror and Pilch, 2011 ). Correspondingly, the remaining IRV components that cannot be faithfully compartmentalized in the vesicles are either degraded (Jiang et al., 2001 ; Keller et al., 2002 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Shi and Kandror, 2005 ; Yeh et al., 2007 ; Jedrychowski et al., 2010 ) or mistargeted (Jiang et al., 2001 ; Jordens et al., 2010 ), depending on experimental conditions and types of cells used in these studies. In other words, knockdown of any major IRV component may decrease vesicle formation along with insulin responsiveness. Thus, in spite of a large body of literature, the identity of protein(s) that confer insulin responsiveness to the IRVs is unknown.Here we used a gain-of-function approach to address this question. Specifically, we attempted to “build” functional IRVs in undifferentiated 3T3-L1 preadipocytes by forced expression of the relevant proteins. Undifferentiated preadipocytes do not express Glut4 or sortilin and lack IRVs (ElJack et al., 1999 ; Shi and Kandror, 2005 ; Shi et al., 2008 ). Correspondingly, IRAP, which is expressed in these cells, shows low insulin response (Ross et al., 1998 ; Shi et al., 2008 ). We found that ectopic expression of increasing amounts of Glut4 in undifferentiated preadipocytes does not lead to its marked translocation to the plasma membrane upon insulin stimulation. On the contrary, sortilin expressed in undifferentiated preadipocytes was localized in the IRVs and was translocated to the plasma membrane in response to insulin stimulation. Moreover, upon coexpression with Glut4, sortilin dramatically increased its insulin responsiveness to the levels observed in fully differentiated adipocytes. Thus sortilin may represent the key component of the IRVs, which is responsible not only for the formation of vesicles (Shi and Kandror, 2005 ; Ariga et al., 2008 ; Hatakeyama and Kanzaki, 2011 ), but also for their insulin responsiveness. It is worth noting that sortilin levels are significantly decreased in obese and diabetic humans and mice (Kaddai et al., 2009 ). We thus suggest that sortilin may be a novel and important target in the fight against insulin resistance and diabetes.Our experiments also demonstrate that undifferentiated preadipocytes lack a mechanism for the full intracellular retention of Glut4 that can be achieved by ectopic expression of AS160/TBC1D4.     

Decreased mitochondrial OGG1 expression is linked to mitochondrial defects and delayed hepatoma cell growth

Many solid tumor cells exhibit mitochondrial respiratory impairment; however, the mechanisms of such impairment in cancer development remain unclear. Here, we demonstrate that SNU human hepatoma cells with declined mitochondrial respiratory activity showed decreased expression of mitochondrial 8-oxoguanine DNA glycosylase/lyase (mtOGG1), a mitochondrial DNA repair enzyme; similar results were obtained with human hepatocellular carcinoma tissues. Among several OGG1-2 variants with a mitochondrial-targeting sequence (OGG1-2a, -2b, -2c, -2d, and -2e), OGG1-2a was the major mitochondrial isoform in all examined hepatoma cells. Interestingly, hepatoma cells with low mtOGG1 levels showed delayed cell growth and increased intracellular reactive oxygen species (ROS) levels. Knockdown of OGG1-2 isoforms in Chang-L cells, which have active mitochondrial respiration with high mtOGG1 levels, significantly decreased cellular respiration and cell growth, and increased intracellular ROS. Overexpression of OGG1-2a in SNU423 cells, which have low mtOGG1 levels, effectively recovered cellular respiration and cell growth activities, and decreased intracellular ROS. Taken together, our results suggest that mtOGG1 plays an important role in maintaining mitochondrial respiration, thereby contributing to cell growth of hepatoma cells.     

Adaptor Protein Complex 2–Mediated Endocytosis Is Crucial for Male Reproductive Organ Development in Arabidopsis

Fertilization in flowering plants requires the temporal and spatial coordination of many developmental processes, including pollen production, anther dehiscence, ovule production, and pollen tube elongation. However, it remains elusive as to how this coordination occurs during reproduction. Here, we present evidence that endocytosis, involving heterotetrameric adaptor protein complex 2 (AP-2), plays a crucial role in fertilization. An Arabidopsis thaliana mutant ap2m displays multiple defects in pollen production and viability, as well as elongation of staminal filaments and pollen tubes, all of which are pivotal processes needed for fertilization. Of these abnormalities, the defects in elongation of staminal filaments and pollen tubes were partially rescued by exogenous auxin. Moreover, DR5rev:GFP (for green fluorescent protein) expression was greatly reduced in filaments and anthers in ap2m mutant plants. At the cellular level, ap2m mutants displayed defects in both endocytosis of N-(3-triethylammonium-propyl)-4-(4-diethylaminophenylhexatrienyl) pyridinium dibromide, a lypophilic dye used as an endocytosis marker, and polar localization of auxin-efflux carrier PIN FORMED2 (PIN2) in the stamen filaments. Moreover, these defects were phenocopied by treatment with Tyrphostin A23, an inhibitor of endocytosis. Based on these results, we propose that AP-2–dependent endocytosis plays a crucial role in coordinating the multiple developmental aspects of male reproductive organs by modulating cellular auxin level through the regulation of the amount and polarity of PINs.     

Induction of the Differentiation of HL-60 Promyelocytic Leukemia Cells by l-Ascorbic Acid

The present study was undertaken to examine the effect of l-ascorbic acid (LAA) on the growth of HL-60 promyelocytic leukemia cells, besides induction of apoptosis. LAA (≥10^-4?M) was found to markedly inhibit the proliferation of HL-60 in liquid culture and clonogenicity in semisolid culture. Moreover, LAA-treated HL-60 showed activity to produce chemiluminescence and expressed CD 66b cell surface antigens, indicating that LAA induces the differentiation of HL-60 mainly into granulocytes. The results are supported by morphological changes of LAA-treated HL-60 into segmented neutrophils. Therefore, the inhibitory effect of LAA on the growth of HL-60 cells seems to arise from the induction of differentiation. To assess the potential role of LAA, cells were exposed to oxygen radical scavengers in the absence or presence of LAA. Catalase abolished and superoxide dismutase promoted LAA-induced differentiation of HL-60. Thus, H²O² produced as a result of LAA treatment seems to play a major role in induction of HL-60 differentiation.     

ICAM-1 expression in vaginal cells as a potential biomarker for inflammatory response

This study aimed to elucidate the mechanisms that may lead to an efficient strategy to induce a suitable host response of the vaginal mucosa upon exposure to intravaginally delivered exogenous compounds. It was hypothesized that the upregulation of intercellular adhesion molecule (ICAM)-1 gene expression may reflect the inflammatory response evoked by exogenous compounds. Major emphasis was placed on ethylenediamine tetraacetic acid (EDTA) which was added as a synergistic agent to conventional spermicidal agents or anti-HIV drugs. The levels of ICAM-1 mRNA were examined as a surrogate marker for inflammatory response in human vaginal epithelial cells upon exposure to EDTA or interleukin (IL)-1β (i.e. positive control, 25 mM). The effects of estrogen on EDTA-induced ICAM-1 expression were also evaluated for the estrogen involvement in the inflammatory process of the vaginal mucosa. ICAM-1 expression in human vaginal cells (VK2/E6E7 cells) increased as EDTA concentration added to human vaginal cell lines increased. The effects of estrogen on EDTA-induced ICAM-1 expression in human vaginal epithelial cells were estrogen-concentration dependent; estrogen at lower concentrations (～1–10 nM) did not affect ICAM-1 expression, whereas estrogen at higher concentrations (～100 nM–1 µM) attenuated ICAM-1 expression. The influence of estrogen in ICAM-1 expression suggests the beneficial effects of estrogen on the regulation of vaginal homeostasis. Identification and quantification of specific surrogate markers for the inflammatory response evoked by exogenous compounds and their regulation by estrogen will lead to an efficient strategy against sexually transmitted diseases including AIDS.