The proton electrochemical gradient induces a kinetic asymmetry in the symport cycle of LacY.

LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H+ transport (i.e., symport). While alternating access of sugar- and H+-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H+ gradient ([Formula: see text]) functions kinetically by decreasing the Km for influx 50- to 100-fold with no change in Kd The affinity of protonated LacY for sugar has an apparent pK (pKapp) of ∼10.5, due specifically to the pKa of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H+ efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that [Formula: see text] induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pKapp of ∼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 - Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the Km for efflux is almost insignificant relative to the 50- to 100-fold decrease in the Km for influx driven by ΔΨ (interior negative)

BIOCHEMISTRY OF HEMIN UPTAKE IN LISTERIA MONOCYTOGENES AND THE FUCTION OF TONB IN ESCHERICHIA COLI

Iron is an essential metal element for the metabolism pathway of most microorganisms. For pathogens, iron is also an important factor for virulence. But in the host body, the availability of free iron is very low. Iron mainly associates with heme in hemoglobin or is tightly bound to iron-associated proteins like transferrin, lactoferrin and ferritin. Both Gram-negative and Gram-positive pathogens have iron transporters in their cell envelopes to acquire iron from the environment, and one of the target iron compound for these transport systems is heme in the red blood cells.Listeria monocytogenes is a Gram-positive pathogen, which can infect human and animals and causes listeriosis. L. monocytogenes can utilize multiple iron sources including siderophores, transferrin, lactoferrin, heme and hemoglobin. In the genome of L. monocytogenes EGD-e, the hup operon encodes an ABC transporter for the uptake of heme. Site-directed deletion of hupC and hupG impaired the uptake of heme and hemoglobin in L. monocytogenes EGD-e. The function of these two genes can be complemented via a Listeria phage integration vector.[59Fe]-heme was used to measure the thermodynamic and kinetic parameters of heme transport by L. monocytohenes. The [59Fe]-heme binding experiments showed that EGD-e wild type bound heme with an affinity about 4.7nM. The deletion of genes in hup operon did not change the binding affinity very much, and also the deletions did not completely eliminate the uptake of heme in the [59Fe]-heme transport experiments. This data indicates there are other transporters available in L. monocytogenes to transport heme other than the Hup transporter.Binding experiments revealed that the sortase B-dependent surface protein (formerly called "SvpA") encoded by lmo2185, has a role in heme binding. Deletion of these proteins resulted in the increase of KD and decrease of capacity. Deletion of lmo2185 also reduced the uptake of hemoglobin in nutrition tests.The second part of this study is about the function of TonB in E. coli. TonB is an inner-membrane-associated protein in Gram-negative species. In the outer membrane of Gram-negative bacteria, there are ligand-gated receptors for the active transport of nutrients, such as siderophores and vitamins. These receptors are TonB-dependent, probably because TonB facilities the transport by transducing the proton motive force energy from the inner membrane to the outer membrane.The C-terminus of TonB contains LysM motifs, whose main function is binding to bacterial peptidoglycan. The TonB C-terminus has affinity for peptidoglycan. Site-directed mutagenesis and fluorescence quenching methods were used in this study to investigate the binding. Peptidoglycan quenched the fluorescence of FM labeled cysteine mutants of a MalE-TonB69C fusion protein. After changing two residues, E205 and D189, which may be part of the binding site, to alanine, the affinity of the C-terminus of TonB for peptidoglycan still remains

Investigation and analysis of psychological stress and professional identity of nursing students during COVID-19 pandemic

426-432Certain diseases or infections affect large number of people in short span of time. A local endemic disease can outbreak into an epidemic affecting the whole population or region which at times extend to other countries and continents and become pandemic. Pandemics results in loss of life as well as economy. Pooled efforts and resources, effective sharing of data, parallel multiple approaches as well as physical and mental state of front line staff influence management of pandemics. The coronavirus disease COVID-19 caused by SARS-CoV-2 started in December 2019 from Wuhan in China, is now a worldwide public health emergency affecting millions of people. It affects many frontline healthcare workers too. Here, we studied psychological stress and professional identity of nursing students for possible correlations, if any, and analyze influencing factors. We used purposive sampling technique with 415 nursing students in Nanjing, China through a general information questionnaire, perceived stress scale and nursing professional identity questionnaire. Students' origin, monthly living expenses and their knowledge on epidemic prevention and treatment have shown a significant impact on their psychological stress (P <0.01). Similarly, students' gender, origin, clinical practices and knowledge of prevention and treatment, and whether they actively learn such knowledge impact significantly on their professional identity (P <0.01). The overall score of psychological stress was (24.47±7.35) and professional identity had 72.47±8.07. The stress condition exhibited negative correlation with the degree of professional identity (P <0.01，r = -0.457). Increased psychological stress, had lower sense of professional identity. Overall, analysis of data on perceived stress and professional identity pandemic suggests that stress levels are inversely proportional to knowledge in effective ways of handling the pandemic. Students with clinical practice fared better in terms of professional identity. The study suggests nursing students to stay focused on studies, clinical practice and counselling, if required

Screening and functional analysis of mutations in NEUROD1 gene in pedigrees of maturity-onset diabetes of the young

Objective·To screen the mutations of NEUROD1 gene in families of maturity-onset diabetes of the young (MODY), and investigate the correlation between the mutation and MODY6 and its potential pathogenesis in Chinese.Methods·PCR-direct sequencing was used for screening NEUROD1 mutations from 96 MODY probands who were negative for mutations in the GCK/MODY2, HNF1A/MODY3 and HNF1B/MODY5 genes, and the genotypic frequency of NEUROD1 variations were compared between the 96 MODY probands and 100 non-diabetic control subjects. A de novo modeling method was used to predict the three-dimensional (3D) structures of wild type (WT) and mutated NEUROD1 proteins. Transcriptional activities of both WT and mutant of NEUROD1 on insulin gene were detected by using dual luciferase reporter gene system.Results·Glu59Gln (NM_002500.5, c.175G>C), a heterozygous missense mutation in the NEUROD1 gene, was identified in a MODY pedigree. 3D structural analysis showed that the mutation transformed the negatively charged Glu59 of WT into uncharged mutation Gln59, leading to the loss of Glu59-Arg54 and Glu59-Lys88, two salt bridge bonds, and the formation of Gln59-Arg54, one new hydrogen bond. Transcriptional activity of Glu59Gln mutant for insulin gene was reduced by 36.3% when compared with that of WT (P<0.05). A common variation Ala45Thr (G-A) was identified, and AA+GA genotypic frequency of the variation was significantly elevated in the 96 MODY probands in comparison to non-diabetic control subjects (P=0.002).Conclusion·Glu59Gln mutation alters the N-terminal molecular conformation of NEUROD1 protein, resulting in decreased transcriptional activity of insulin gene, which is the cause of the defective insulin secretion in mutation carriers of the MODY6 pedigree. The Ala45Thr variation is associated with earlier age of onset of diabetes in MODY6 probands

Thermodynamics of Nanobody Binding to Lactose Permease

Camelid nanobodies (Nbs) raised against the outward-facing conformer of a double-Trp mutant of the lactose permease of Escherichia coli (LacY) stabilize the permease in outward-facing conformations. Isothermal titration calorimetry is applied herein to dissect the binding thermodynamics of two Nbs, one that markedly improves access to the sugar-binding site and another that dramatically increases the affinity for galactoside. The findings presented here show that both enthalpy and entropy contribute favorably to binding of the Nbs to wild-type (WT) LacY and that binding of Nb to double-Trp mutant G46W/G262W is driven by a greater enthalpy at an entropic penalty. Thermodynamic analyses support the interpretation that WT LacY is stabilized in outward-facing conformations like the double-Trp mutant with closure of the water-filled cytoplasmic cavity through conformational selection. The LacY conformational transition required for ligand binding is reflected by a favorable entropy increase. Molecular dynamics simulations further suggest that the entropy increase likely stems from release of immobilized water molecules primarily from the cytoplasmic cavity upon closure

No Consistent Simulated Trends in the Atlantic Meridional Overturning Circulation for the Past 6,000 Years

The Atlantic Meridional Overturning Circulation (AMOC) is a key feature of the North Atlantic with global ocean impacts. The AMOC's response to past changes in forcings during the Holocene provides important context for the coming centuries. Here, we investigate AMOC trends using an emerging set of transient simulations using multiple global climate models for the past 6,000 years. Although some models show changes, no consistent trend in overall AMOC strength during the mid-to-late Holocene emerges from the ensemble. We interpret this result to suggest no overall change in AMOC, which fits with our assessment of available proxy reconstructions. The decadal variability of the AMOC does not change in ensemble during the mid- and late-Holocene. There are interesting AMOC changes seen in the early Holocene, but their nature depends a lot on which inputs are used to drive the experiment

The relationships of preventive behaviors and psychological resilience with depression, anxiety, and stress among university students during the COVID-19 pandemic: A two-wave longitudinal study in Shandong Province, China

IntroductionStudies have shown that the psychological impact of the COVID-19 pandemic may lead to long-term health problems; therefore, more attention should be paid to the mental health of university students. This study aimed to explore the longitudinal effects of preventive behaviors and psychological resilience on the mental health of Chinese college students during COVID-19.MethodsWe recruited 2,948 university students from five universities in Shandong Province. We used a generalized estimating equation (GEE) model to estimate the impact of preventive behaviors and psychological resilience on mental health.ResultsIn the follow-up survey, the prevalence of anxiety (44.8% at T1 vs 41.2% at T2) and stress (23.0% at T1 vs 19.6% at T2) decreased over time, whereas the prevalence of depression (35.2% at T1 vs 36.9% at T2) increased significantly (P &lt; 0.001). Senior students were more likely to report depression (OR = 1.710, P &lt; 0.001), anxiety (OR = 0.815, P = 0.019), and stress (OR = 1.385, P = 0.011). Among all majors, medical students were most likely to report depression (OR = 1.373, P = 0.021), anxiety (OR = 1.310, P = 0.040), and stress (OR = 1.775, P &lt; 0.001). Students who wore a mask outside were less likely to report depression (OR = 0.761, P = 0.027) and anxiety (OR = 0.686, P = 0.002) compared to those who did not wear masks. Students who complied with the standard hand-washing technique were less likely to report depression (OR = 0.628, P &lt; 0.001), anxiety (OR = 0.701, P &lt; 0.001), and stress (OR = 0.638, P &lt; 0.001). Students who maintained a distance of one meter in queues were less likely to report depression (OR = 0.668, P &lt; 0.001), anxiety (OR = 0.634, P &lt; 0.001), and stress (OR = 0.638, P &lt; 0.001). Psychological resilience was a protective factor against depression (OR = 0.973, P &lt; 0.001), anxiety (OR = 0.980, P &lt; 0.001), and stress (OR = 0.976, P &lt; 0.001).DiscussionThe prevalence of depression among university students increased at follow-up, while the prevalence of anxiety and stress decreased. Senior students and medical students are vulnerable groups. University students should continue to follow relevant preventive behaviors to protect their mental health. Improving psychological resilience may help maintain and promote university students' mental health

Development and verification of a combined immune- and cancer-associated fibroblast related prognostic signature for colon adenocarcinoma

IntroductionTo better understand the role of immune escape and cancer-associated fibroblasts (CAFs) in colon adenocarcinoma (COAD), an integrative analysis of the tumor microenvironment was performed using a set of 12 immune- and CAF-related genes (ICRGs).MethodsUnivariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses were used to establish a prognostic signature based on the expression of these 12 genes (S1PR5, AEN, IL20RB, FGF9, OSBPL1A, HSF4, PCAT6, FABP4, KIF15, ZNF792, CD1B and GLP2R). This signature was validated in both internal and external cohorts and was found to have a higher C-index than previous COAD signatures, confirming its robustness and reliability. To make use of this signature in clinical settings, a nomogram incorporating ICRG signatures and key clinical parameters, such as age and T stage, was developed. Finally, the role of S1PR5 in the immune response of COAD was validated through in vitro cytotoxicity experiments.ResultsThe developed nomogram exhibited slightly improved predictive accuracy compared to the ICRG signature alone, as indicated by the areas under the receiver operating characteristic curves (AUC, nomogram:0.838; ICRGs:0.807). The study also evaluated the relationships between risk scores (RS) based on the expression of the ICRGs and other key immunotherapy variables, including immune checkpoint expression, immunophenoscore (IPS), and microsatellite instability (MSI). Integration of these variables led to more precise prediction of treatment efficacy, enabling personalized immunotherapy for COAD patients. Knocking down S1PR5 can enhance the efficacy of PD-1 monoclonal antibody, promoting the cytotoxicity of T cells against HCT116 cells ((p&lt;0.05).DiscussionThese findings indicate that the ICRG signature may be a valuable tool for predicting prognostic risk, evaluating the efficacy of immunotherapy, and tailoring personalized treatment options for patients with COAD