PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid‐encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β‐barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion‐conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco‐harboring pathogens.     

Impact of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex lineages as a determinant of disease phenotypes from an immigrant rich moderate tuberculosis burden country

Background

Growing evidences suggested that the Mycobacterium tuberculosis complex (MTBC) lineages can determine the clinical outcome of pulmonary and extra-pulmonary tuberculosis. However, limited data only available revealing such association of bacterial genotypes and clinical phenotypes from immigrant rich countries.

Methods

A multicenter study has been carried out on a collection of 2092 (1003 extrapulmonary and 1089 pulmonary) MTBC isolates. Genotyping of all the isolates were carried out by spoligotyping and 24 loci based MIRU-VNTR typing.

Results

Demographically domination of young Saudi nationals (61.4%) and men (61.2%) were found in this cohort. Lymph nodes (62.4%) and gastrointestinal sites (16.7%) were the most common anatomical sites of infection. The predominant lineages were Delhi/CAS (26.9%), EAI (14.2%) and Ghana (9.9%). Mycobacterium africanum type I and II were reported for the first time in the country among extrapulmonary cases. ‘Ancestral’ lineages M.bovis (OR-5.22; 95% CI-2.23-8.22, p-?<?0.001) and Delhi/CAS (OR-0.57; 95% CI-0.411-0.734, p-?<?0.001) were directly associated with lymph node tuberculosis and gastrointestinal tuberculosis (M. bovis-OR-0.33; 95% CI-0.085-0.567, p-0.001 and Delhi/CAS-OR-1.87; 95% CI-1.22-2.53, p-?<?0.001) respectively. Among the ‘Modern’ lineages, EAI showed significant association to central nervous system tuberculosis (OR-1.98; 95% CI-0.76-3.19, p-0.04) and Uganda-I to gastrointestinal tuberculosis (OR-2.41; 95% CI-0.77-4.06, p-0.02).

Conclusions

The findings substantially contribute to the emerging evidences that MTBC lineages influence disease phenotypes and epidemiological consequences.

     

The ERK-1/2 Signaling Pathway Is Involved in the Stimulation of Branching Morphogenesis of Fetal Mouse Submandibular Glands by EGF

We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149–161) and that the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651–1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG.     

Changes at panicle emergence in the water relations of a wetland and a dryland Japonica rice cultivar under wetland conditions

The diurnal and seasonal changes in plant water relations of two Japonica rice ( Oryza sativa L.) cultivars, Nipponbare and Tachiminori, were studied under flooded conditions at Kyoto University. The dryland cv. Tachiminori maintained higher predawn and midday leaf osmotic potentials relative to the wetland cv. Nipponbare during the vegetative stage, but the ranking was reversed after flowering. The relationship between leaf water potential and leaf osmotic potential showed that prior to panicle emergence Nipponbare was able to adjust osmotically to maintain turgor, whereas after heading there was little turgor maintenance. Tachiminori showed little difference in osmotic adjustment before and after panicle emergence. Fertilizer treatment during panicle development also helped to maintain the degree of osmotic adjustment in both cultivars.     

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF’s role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.     

Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates

Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity.     

Suppression of mitochondria-dependent neutrophil apoptosis with thermal injury

Neutrophil apoptosis is delayed under trauma and/or sepsis conditions. The mechanism for the delay has remained unclear. We hypothesize that modulation of the mitochondrial pathway of apoptosis contributes to the delay in neutrophil apoptosis with burn injury. Rats were subjected to burn injury (30% of total body surface area, 98°C for 10 s) and euthanatized 24 h postinjury. Blood neutrophils from sham and burn-injured rats were isolated by Ficoll gradient centrifugation and cultured for 2 or 8 h. Neutrophil apoptosis was determined by annexin V and propidium iodide (PI) labeling and flow cytometry. Neutrophil mitochondrial morphology was assessed via histochemical staining (MitoTracker GreenFM) and confocal microscopy. Neutrophils from rats with burn injury showed a decreased level of apoptosis compared with sham rat neutrophils at both 2 and 8 h of incubation. In incubated sham rat neutrophils, mitochondria showed a change from normal "tubular" to an "aggregated" morphology. In contrast, cultured neutrophils from burn rats did not exhibit this mitochondrial morphological transition until 8 h of incubation. Compared with sham rat neutrophils, neutrophils from burn rats showed decreased levels of active caspase-9 and -3. Whereas an upregulation of Bcl-xL and a downregulation of Bax seemed to contribute to decreased apoptosis in burn rat neutrophils at 2 h of incubation, the decreased apoptosis at 8 h appeared to be associated with a decrease in Bax and increased phosphorylated Bad. These data suggest that suppression of the mitochondrial pathway plays an essential role in the delay of polymorphonuclear neutrophil apoptosis with burn injury. burn; rat; polymorphonuclear leukocytes; caspase-3; caspase-9; cytochrome c; Bcl-xL; Bax; Bad; MitoTracker GreenFM; confocal microscopy     

Selenium-induced alteration of lipids,lipid peroxidation,and thiol group in circadian rhythm centers of rat

The effect of sodium selenite (0.05, 0.1, and 0.2 mg/kg body weight, ip) on the contents of lipids (phospholipids, cholesterol, esterified fatty acids, gangliosides), thiobarbituric acid reactive substance (TBARS), and thiol group in circadian rhythm centers (preoptic area, brainstem, and posterior hypothalamus) of male Wistar rats was studied after 7 d of treatment. The content of phospholipids was elevated significantly with a dose of 0.1 mg/kg of selenite in the preoptic area and brainstem, but a 0.2-mg/kg dose has depleted its level significantly in these regions. The alteration of phospholipids in posterior hypothalamus was not significant with three doses of sodium selenite. The level of cholesterol in the preoptic area was inhibited significantly with a dose of 0.05 mg/kg sodium selenite, but its level was elevated significantly with a dose of 0.2 mg/kg selenite in the preoptic area and brainstem. Alteration with three doses of sodium selenite in the posterior hypothalamus was not significant. The ganglioside level in the preoptic area and brainstem was elevated significantly with a 0.1-mg dose of sodium selenite; conversely, a 0.2 mg dose of sodium selenite caused a significant depletion on its content in these areas. In the posterior hypothalamus, the ganglioside level was depleted significantly with a dose of 0.1 mg, but elevated significantly with a dose of 0.2 mg of sodium selenite. The level of esterified fatty acids was decreased significantly in the preoptic area and brainstem with a dose of 0.1 mg/kg sodium selenite, but in these regions, its level was elevated with a dose of 0.2 mg/kg sodium selenite and its elevation was significant in the preoptic area. In the posterior hypothalamus, the alteration of esterified fatty acids with three doses of sodium selenite was not significant. The effect of 0.1 and 0.2 mg/kg sodium selenite on the TBARS level and thiol group in sleep centers was significantly opposite to the wakefulness center. A sodium selenite dose of 0.1 mg/kg had depleted the content of TBARS in the preoptic area and brainstem but elevated the content of the thiol group significantly in the posterior hypothalamus. On the other hand, a 0.2-mg/kg dose of sodium selenite has significantly elevated the content of TBARS but depleted the content of the thiol group significantly in the posterior hypothalamus. No dose-dependent alteration was observed on the content of lipids, TBARS, and thiol group in the circadian rhythm centers of rats.     

Osteopontin as a biomarker for COVID-19 severity and multisystem inflammatory syndrome in children: A pilot study

This study sought to evaluate the candidacy of plasma osteopontin (OPN) as a biomarker of COVID-19 severity and multisystem inflammatory condition in children (MIS-C) in children. A retrospective analysis of 26 children (0–21 years of age) admitted to Children’s Healthcare of Atlanta with a diagnosis of COVID-19 between March 17 and May 26, 2020 was undertaken. The patients were classified into three categories based on COVID-19 severity levels: asymptomatic or minimally symptomatic (control population, admitted for other non-COVID-19 conditions), mild/moderate, and severe COVID-19. A fourth category of children met the Centers for Disease Control and Prevention''s case definition for MIS-C. Residual blood samples were analyzed for OPN, a marker of inflammation using commercial ELISA kits (R&D), and results were correlated with clinical data. This study demonstrates that OPN levels are significantly elevated in children hospitalized with moderate and severe COVID-19 and MIS-C compared to OPN levels in mild/asymptomatic children. Further, OPN differentiated among clinical levels of severity in COVID-19, while other inflammatory markers including maximum erythrocyte sedimentation rate, C-reactive protein and ferritin, minimum lymphocyte and platelet counts, soluble interleukin-2R, and interleukin-6 did not. We conclude OPN is a potential biomarker of COVID-19 severity and MIS-C in children that may have future clinical utility. The specificity and positive predictive value of this marker for COVID-19 and MIS-C are areas for future larger prospective research studies.