Linking function to global and local dynamics in an elevator-type transporter

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency—yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter Glt_Ph. We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

Transporters are integral membrane proteins that move solutes across lipid bilayers. They undergo concerted conformational changes, allowing alternate exposure of their substrate-binding sites to external and internal solutions (1). In each of these so-called outward- and inward-facing states (OFS and IFS, respectively), further isomerizations accompany substrate binding and release. Transport efficiency depends on the rates of these rearrangements, but linking function and structural dynamics has presented methodological challenges. Single-molecule Forster resonance energy transfer (smFRET)-based total internal reflection fluorescence (TIRF) microscopy (2–4) has been used to monitor the dynamics of the OFS to IFS transitions (5–8) and single-transporter activity (9) in the elevator-type transporter Glt_Ph and other transporters (10–18). Hydrogen–deuterium exchange followed by mass spectrometry (HDX-MS) has been used to pinpoint local changes in structural dynamics in diverse biological systems (19–21). Here, we combine these approaches to link changes in local protein dynamics to the larger-scale conformational transitions and substrate transport in wild-type (WT) and gain-of-function mutants of Glt_Ph.Glt_Ph is an extensively studied archaeal aspartate transporter that is homologous to human excitatory amino acid transporters (EAATs). Structures of Glt_Ph (7, 22–30), and archaeal and mammalian homologs (31–37), show that the transporters assemble into homotrimers via scaffold domains. Each protomer features a mobile transport domain that binds l-Aspartate (l-Asp) and three Na⁺ ions (22, 23, 28, 31, 38) and symports the solutes by an elevator mechanism, moving ∼15 Å across the membrane from an OFS to an IFS (6, 8, 23, 24, 39). During the elevator transitions, two structurally symmetric helical hairpins (HPs) 1 and 2 form the cores of the domain interfaces in the OFS and IFS, respectively (SI Appendix, Fig. S1A) (23, 24, 40). Despite symmetry, they do not have the same function. HP1 is mostly rigid, while HP2 is a conformationally plastic “master regulator” of the transporter, gating substrate in the OFS and IFS and contributing to setting the elevator transition rates (5, 23, 24, 27, 29, 36, 41–47).In this study, we use three previously characterized mutants of Glt_Ph to pinpoint the rate-limiting steps of the transport cycle and probe the protein dynamic properties that correlate with increased transport rates. A K290A mutation at the base of HP1 disrupts a salt bridge with the scaffold domain in the OFS and dramatically increases the elevator dynamics (5, 6). A triple-mutant Y204L/A345V/V366A displays a more modest increase in elevator dynamics and substantially diminished l-Asp affinity (5). Finally, a Y204L/K290A/A345V/V366A mutant combines these substitutions and their effects (5). We compared our previously obtained smFRET data on the elevator dynamics of the WT transporter and the mutants (5) to single-transporter uptake measurements. For WT Glt_Ph, these dynamics and transport measurements established transporter subpopulations that move (5, 6) and work (9) with rates differing by orders of magnitude, with slow transporters dominating the ensemble. We now show that only mutations that both reduce the population of the slow-moving transporters and weaken substrate affinity, such as Y204L/A345V/V366A, reduce the population of the slow-working transporters and confer overall gain-of-function properties. The slow-working population comprises transporters with rare elevator transitions or slow substrate release. We then used HDX-MS to explore how the Y204L/A345V/V366A mutant differed from the WT protein. We found that the mutations decreased the stability of the secondary structure around the substrate-binding site, suggesting that the increased local dynamics underlie reduced kinetic heterogeneity within the mutant transporter ensemble.     

QT interval dispersion in the patients with central serous chorioretinopathy

AIM: To evaluate QT dispersion (QTD) in patients with central serous chorioretinopathy (CSC). METHODS: This clinical, comperative, case-control study included 30 patients with CSC at acute phase (Group 1) and 30 age- and sex-matched healthy subjects (Group 2, the control group). From all subjects, a 12-lead surface electrocardiography was obtained. The heart rate (HR), QT maximum (QTmax), QT minimum (QTmin), QT corrected (QTc), QTD and Tmean were manually measured and analyzed. Student’s t-test and Pearson’s method of correlation were used for statistical analysis. RESULTS: The patient and control groups were matched for age, smoking status (rate and duration) and gender. There were no significant differences with regard to these among the groups (P>0.05). The participants included 19 men (63.3%) and 11 women (36.7%) in Group 1, 20 men (66.7%) and 10 women (33.3%) in Group 2. QTmax, QTD and QTc were significantly higher than those of healthy controls (P<0.001 for QTmax, P=0.01 for QTD and P=0.001 for QTc). QTmin, Tmean and HR did not differ significantly between the study groups (P=0.28 for QTmin, P=0.56 for Tmean and P>0.05 for HR). No significant correlation was found between duration of the disorder and QTD values (r=0.13, P>0.05). CONCLUSION: These findings suggest that CSC may be associated with an increase in QTD and that the patients might be at risk for ventricular arrhythmia.     

The diagnostic,predictive, and prognostic role of serum epithelial cell adhesion molecule (EpCAM) and vascular cell adhesion molecule-1 (VCAM-1) levels in breast cancer

The purpose of this study was to determine the clinical significance of vascular cell adhesion molecule-1 (VCAM-1) and epithelial cell adhesion molecule (EpCAM) in breast cancer (BC) patients. Ninety-six BC patients and 30 age- and sex-matched healthy controls were enrolled into this study. Pretreatment serum markers were determined by the solid-phase sandwich (enzyme-linked immunosorbent assay (ELISA)). The median age at diagnosis was 48 years (range 29–80 years). Majority of the patients (71 %) had luminal subtype, and 38.5 % had metastatic disease. Twenty-nine (30 %) patients showed tumor progression, and 20 (21 %) patients died during follow-up. Median progression-free survival (PFS) and overall survival (OS) were 8.6?±?1.7 and 35.5?±?1.5 months, respectively. The baseline serum EpCAM levels of the patients were significantly higher than those of the controls (p?VCAM-1 between the patients and controls (p?=?0.47). No significant correlation was detected between the levels of the serum markers and other clinical parameters (p?>?0.05). Patients with HER-2-positive and triple-negative tumors had significantly poorer PFS (p?=?0.04 and p?=?0.001, respectively), while metastatic disease and chemotherapy unresponsiveness had significantly adverse effect on OS analysis (p?p?VCAM-1 levels nor serum EpCAM levels were identified to have a prognostic role on either PFS or OS (VCAM-1 p?=?0.76 and p?=?0.32; EpCAM p?=?0.16 and p?=?0.69, respectively). Even though any predictive or prognostic role could not be determined for both markers, serum levels of EpCAM were found to have diagnostic value in BC patients.     

High plasma D-dimer level is associated with decreased survival in patients with lung cancer

AimsAn elevated plasma d-dimer level indicates the activation of coagulation and fibrinolysis. In the present study, we investigated the association of pre-treatment haemostatic parameters (d-dimer, fibrinogen and prothrombin fragment 1+2) with clinicopathological parameters and outcome in patients with lung cancer.Materials and methodsPlasma levels of d-dimer and other parameters were measured in 78 evaluable patients with lung cancer (60 non-small cell lung cancers, 18 small cell lung cancers). At diagnosis, 35 patients (44.9%) were locally advanced stage (IIIA/B) and 43 patients (55.1%) had metastatic disease (IV). Multivariate statistical analysis was carried out using Cox's proportional hazards model. The receiver operating characteristic curve was used to determine the cut-off values for d-dimer, fibrinogen and prothrombin fragment 1+2.ResultsThe median survival for all patients was 264 days (95% confidence interval 200–328 days). A significant association between the plasma levels of d-dimer and the response to chemotherapy was observed (P = 0.03). With the univariate analysis, tumour stage, pre-treatment plasma levels of d-dimer, fibrinogen, platelet count, lactate dehydrogenase concentration and Karnofsky performance status were predictive for survival. With the multivariate analysis (P ≤ 0.1), the plasma level of d-dimer (P < 0.001), tumour stage (P = 0.01) and Karnofsky performance status (P = 0.02) were identified as independent predictive factors. The median survival times were 405 days (95% confidence interval 165–644 days) and 207 days (95% confidence interval 146–267 days, P < 0.001), respectively, for patients with a low d-dimer level (≤0.65 μg/ml) and a high d-dimer level (>0.65 μg/ml).ConclusionsElevated plasma levels of d-dimer in patients with lung cancer are associated with decreased survival and a poor response to treatment. Pre-treatment for the d-dimer level may be useful in the prediction of survival and the response to treatment.     

Incorporation of resident macrophages in engineered tissues: Multiple cell type response to microenvironment controlled macrophage‐laden gelatine hydrogels

The success of tissue engineering strategy is strongly related to the inflammatory response, mainly through the activity of macrophages that are key cells in initial immune response to implants. For engineered tissues, the presence of resident macrophages can be beneficial for maintenance of homeostasis and healing. Thus, incorporation of macrophages in engineered tissues can facilitate the integration upon implantation. In this study, an in‐vitro model of interaction was developed between encapsulated naive monocytes, macrophages induced with M1/M2 stimulation and incoming cells for immune assisted tissue engineering applications. To mimic the wound healing cascade, naive THP‐1 monocytes, endothelial cells and fibroblasts were seeded on the gels as incoming cells. The interaction was first monitored in the absence of the gels. To mimic resident macrophages, THP‐1 cells were encapsulated in the presence or absence of IL‐4 to control their phenotype and then these hydrogels were seeded with incoming cells. Without encapsulation, activated macrophages induce apoptosis in endothelial cells. Once encapsulated no adverse effects were seen. Macrophage‐laden hydrogels attracted more endothelial cells and fibroblasts compared to monocytes‐laden hydrogels. The induction (M2 stimulation) of encapsulated macrophages did not change the overall number of attracted cells; but significantly affected their morphology. M1 stimulation by a defined media resulted in more secretion of both pro‐ and anti‐inflammatory cytokines compared to M2 stimulation. It was demonstrated that there is a distinct effect of encapsulated macrophages on the behaviour of the incoming cells; this effect can be harnessed to establish a microenvironment more prone to regeneration upon implantation.     

In Vitro Antifungal Activity of Ankaferd Blood Stopper Against Candida albicans

Background

Candida albicans is a memeber of the oral flora that can lead to various complications in immunosupresive patients after oral surgery processes. Ankaferd Blood Stopper® (ABS) is a medical plant extract that is safe to use in patients with dental surgery bleedings in Turkey.

Objective

The study evaluated the antifungal activity of ABS medicinal plant extract against C albicans using the agar diffusion and broth microdilution methods.

Methods

The plant extract antifungal activity was assessed in vitro either by applying the ABS extract directly and by applying different concentrations of ABS onto Candida culture. For these experiments, an agar diffusion method was used. To determine the minimum inhibitory concentration (MIC), a broth microdilution method was used.

Results

Different volumes of the active substance (10, 20, 30, and 40 μL) were applied onto Candida (0.5 McFarland solution) cultivated plate; Candida growth was inhibited in accordance with the volumes of ABS. However, when various dilutions of ABS (1:2, 1:20, 1:40, and 1:80) were added as drops containing 20 μL, no antifungal effects were found. No MIC values were identified using broth microdilution. When different dilutions of ABS containing 100 μL of 0.5 McFarland solution of C albicans were cultured depending on the time (10, 20, 30, and 40 minutes), the effect of the duration was not significant.

Conclusion

The various tests were carried out to investigate antifungal effects of ABS on Candida, but none were found.     

Heart-type, fatty-acid binding protein can be a diagnostic marker in acute coronary syndromes

OBJECTIVES: Chest pain is one of the most common complaints among patients admitted to emergency departments. Cardiac troponins, CK-MB and myoglobin, which are used routinely in the diagnosis of acute coronary syndrome (ACS), are not elevated in the initial hours of ACS--precluding their usefulness in the early diagnosis. The aim of this study is to determine the efficacy of H-FABP compared to myoglobin and CK-MB in the early diagnosis of ACS. METHODS: Sixty-seven patients with ACS were enrolled in the study. An initial blood sample was obtained for CK-MB, cTnT, myoglobin and H-FABP. At the fourth, eighth, and 12th hours, repeat ECGs and cardiac enzyme samples were obtained. H-FABP test was repeated at the fourth hour. RESULTS: H-FABP has sensitivity equal to that of CK-MB and superior to that of myoglobin (97.6%, 96.7%, 85.4%, respectively) on the first hour. This trend extends to the fourth hour of myocardial injury as well. H-FABP was more specific than CK-MB, myoglobin and troponin T at the first hour (38.5%, 34.6%, 34.6%, 23.1%, respectively), whereas its specificity at the fourth hour was equal to those of CK-MB and troponin T and exceeded that of myoglobin. CONCLUSIONS: It can be suggested that in patients with an initial diagnosis of ACS and within 20 hours from symptom onset, H-FABP levels may be measured. For this purpose, point-of-care H-FABP test may be utilized, which has the advantage of bedside testing and rapid test results.     

The beneficial effects of nerolidol and hesperidin on surgically induced endometriosis in a rat model*

Abstract

The objective of this article is to analyze the effects of nerolidol and hesperidin treatment on surgically induced endometriosis in a rat model. Endometriosis was induced in 24 healthy adult female Wistar albino rats via homologous uterine horn transplantation. Three operations were performed on each rat. After the second operation, the rats were randomized into control, nerolidol, and hesperidin treatment groups, and medications were administered for 2?weeks. The effects of the drugs on the endometriotic foci were evaluated after the third operation. Compared with the endometriosis control group, the average volume of the lesions was significantly lower in rats treated with hesperidin and nerolidol. Malondialdehyde levels were significantly reduced in the nerolidol-treated group, and glutathione levels and superoxide dismutase activity were significantly elevated in the endometriotic foci of both the hesperidin- and nerolidol-treated groups compared with the endometriosis group. Hesperidin and nerolidol treatment also improved histological parameters, such as hemorrhage, vascular congestion, necrosis, and inflammatory cell infiltration in the endometriotic foci. The results of this study demonstrated that treatment with the potent antioxidants nerolidol and hesperidin caused a significant regression of surgically induced endometriotic foci in rats.