The Escherichia coli SLC26 homologue YchM (DauA) is a C4‐dicarboxylic acid transporter

The SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of secondary anion transporters. Prior studies have focused almost exclusively on eukaryotic members and bacterial members are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. In this study we have examined the function and physiological role of the Escherichia coli Slc26 homologue, YchM. We show that there is a clear YchM‐dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH. We demonstrate that YchM can also transport other C₄‐dicarboxylic acids and that its substrate specificity differs from the well‐characterized succinate transporter, DctA. Accordingly ychM was re‐designated dauA (dicarboxylic acid uptake system A). Finally, our data suggest that DauA is a protein with transport and regulation activities. This is the first report that a SLC26/SulP protein acts as a C₄‐dicarboxylic acid transporter and an unexpected new function for a prokaryotic member of this transporter family.     

Chroman-like cyclic prenylflavonoids promote neuronal differentiation and neurite outgrowth and are neuroprotective

Flavonoids target a variety of pathophysiological mechanisms and are therefore increasingly considered as compounds encompassed with therapeutic potentials in diseases such as cancer, diabetes, arteriosclerosis, and neurodegenerative diseases and mood disorders. Hops (Humulus lupulus L.) is rich in flavonoids such as the flavanone 8-prenylnaringenin, which is the most potent phytoestrogen identified so far, and the prenylchalcone xanthohumol, which has potent tumor-preventive, anti-inflammatory and antiviral activities. In the present study, we questioned whether hops-derived prenylflavonoids and synthetic derivatives thereof act on neuronal precursor cells and neuronal cell lines to induce neuronal differentiation, neurite outgrowth and neuroprotection. Therefore, mouse embryonic forebrain-derived neural precursors and Neuro2a neuroblastoma-derived cells were stimulated with the prenylflavonoids of interest, and their potential to activate the promoter of the neuronal fate-specific doublecortin gene and to stimulate neuronal differentiation and neurite outgrowth was analyzed. In this screening, we identified highly “neuroactive” compounds, which we termed “enhancement of neuronal differentiation factors” (ENDFs). The most potent molecule, ENDF1, was demonstrated to promote neuronal differentiation of neural stem cells and neurite outgrowth of cultured dorsal root ganglion neurons and protected neuronal PC12 cells from cobalt chloride-induced as well as cholinergic neurons of the nucleus basalis of Meynert from deafferentation-induced cell death. The results indicate that hops-derived prenylflavonoids such as ENDFs might be powerful molecules to promote neurogenesis, neuroregeneration and neuroprotection in cases of chronic neurodegenerative diseases, acute brain and spinal cord lesion and age-associated cognitive impairments.     

Enhancement of Tight Junctional Barrier Function by Micronutrients: Compound-Specific Effects on Permeability and Claudin Composition

Amid an increasing number of reports in the literature concerning epithelial barrier enhancement by various nutrient compounds, there has never been a study performing side-by-side comparisons of these agents in a single epithelial model. We compare five nutrient compounds (previously reported in various epithelial models to enhance barrier function) regarding their ability to increase transepithelial electrical resistance (R_t) and decrease transepithelial mannitol permeability (J_m) across LLC-PK₁ renal epithelial cell layers. The effects of these nutrients on the abundance of various tight junctional proteins are also compared. In the overall group of nutrients tested - zinc, indole, quercetin, butyrate and nicotine - only nicotine failed to improve barrier function by either parameter. Nicotine also was without effect on tight junctional proteins. Quercetin simultaneously increased R_t and decreased J_m. Zinc, butyrate and indole only exhibited statistically significant enhancement of R_t. Each of these four effective nutrient compounds had unique patterns of effects on the panel of tight junctional proteins studied. No two compounds produced the same pattern of effects. This unique pattern of effects on tight junctional complex composition by each compound establishes the chance for additive or even synergistic improvement of barrier function by combinations of compounds. A synergistic effect of the combination of quercetin and zinc on R_t is shown.     

Effect of temperature and light on seed germination of Erysimum naxense and Erysimum krendlii

Seed germination of two local Greek endemics was studied (Erysimum naxense, Erysimum krendlii). Seed viability was determined by using the tetrazolium method and germination was studied in synchronized cycles of five and four alternating temperatures [10/5 (for E. naxense only) and 15/10, 20/15, 25/20, and 30/25°C for both species, in cycles of 16 h day/8 h night], and in five light regimes (red, blue, green, white, and dark). Germination of E. naxense and E. krendlii seeds was determined daily for six and five weeks, respectively, with the data analyzed as viability adjusted accumulative seed germination at the end of each week. E. naxense’s seed viability was higher (90%) than that of E. krendlii (64%); seed germination (%) of both increased at low alternating temperatures (10/5°C, 15/10°C, 20/15°C). Germination of E. naxense seeds at low temperatures was light-independent, whereas at high temperatures it was increased with red light. Germination of E. krendlii seeds was inconsistently affected by light at the temperatures studied. Percentages of seed germination of both species were higher in experimental conditions similar to the ones of their natural habitats during autumn and/or spring (facilitated with Geographic Information Systems). These conclusions provide guidelines for species-specific propagation protocols and ex situ conservation.     

Importance of Ethnicity,CYP2B6 and ABCB1 Genotype for Efavirenz Pharmacokinetics and Treatment Outcomes: A Parallel-Group Prospective Cohort Study in Two Sub-Saharan Africa Populations

Objectives

We evaluated the importance of ethnicity and pharmacogenetic variations in determining efavirenz pharmacokinetics, auto-induction and immunological outcomes in two African populations.

Methods

ART naïve HIV patients from Ethiopia (n = 285) and Tanzania (n = 209) were prospectively enrolled in parallel to start efavirenz based HAART. CD4+ cell counts were determined at baseline, 12, 24 and 48 weeks. Plasma and intracellular efavirenz and 8-hydroxyefvairenz concentrations were determined at week 4 and 16. Genotyping for common functional CYP2B6, CYP3A5, ABCB1, UGT2B7 and SLCO1B1 variant alleles were done.

Result

Patient country, CYP2B6*6 and ABCB1 c.4036A>G (rs3842A>G) genotype were significant predictors of plasma and intracellular efavirenz concentration. CYP2B6*6 and ABCB1 c.4036A>G (rs3842) genotype were significantly associated with higher plasma efavirenz concentration and their allele frequencies were significantly higher in Tanzanians than Ethiopians. Tanzanians displayed significantly higher efavirenz plasma concentration at week 4 (p<0.0002) and week 16 (p = 0.006) compared to Ethiopians. Efavirenz plasma concentrations remained significantly higher in Tanzanians even after controlling for the effect of CYP2B6*6 and ABCB1 c.4036A>G genotype. Within country analyses indicated a significant decrease in the mean plasma efavirenz concentration by week 16 compared to week 4 in Tanzanians (p = 0.006), whereas no significant differences in plasma concentration over time was observed in Ethiopians (p = 0.84). Intracellular efavirenz concentration and patient country were significant predictors of CD4 gain during HAART.

Conclusion

We report substantial differences in efavirenz pharmacokinetics, extent of auto-induction and immunologic recovery between Ethiopian and Tanzanian HIV patients, partly but not solely, due to pharmacogenetic variations. The observed inter-ethnic variations in efavirenz plasma exposure may possibly result in varying clinical treatment outcome or adverse event profiles between populations.     

Ixabepilone Administered Weekly or Every Three Weeks in HER2-Negative Metastatic Breast Cancer Patients; A Randomized Non-Comparative Phase II Trial

To explore the activity and safety of two schedules of ixabepilone, as first line chemotherapy, in patients with metastatic breast cancer previously treated with adjuvant chemotherapy, a randomized non-comparative phase II study was conducted. From November 2008 until December 2010, 64 patients were treated with either ixabepilone 40 mg/m² every 3 weeks (Group A, 32 patients) or ixabepilone 20 mg/m² on days 1, 8 and 15 every 4 weeks (Group B, 32 patients). Overall response rate (the primary end point) was 47% in Group A and 50% in Group B. The most frequent severe adverse events were neutropenia (32% vs. 23%), metabolic disturbances (29% vs. 27%) and sensory neuropathy (12% vs. 27%). Two patients in Group A and 3 in Group B developed febrile neutropenia. After a median follow-up of 22.7 months, median progression-free survival (PFS) was 9 months in Group A and 12 months in Group B. Median survival was 26 months in Group A, whereas it was not reached in Group B. Multiple genetic and molecular markers were examined in tumor and peripheral blood DNA, but none of them was associated with ORR or drug toxicity. Favorable prognostic markers included: the T-variants of ABCB1 SNPs c.2677G/A/T, c.1236C/T and c.3435C/T, as well as high MAPT mRNA and Tau protein expression, which were all associated with the ER/PgR-positive phenotype; absence of TopoIIa; and, an interaction between low TUBB3 mRNA expression and Group B. Upon multivariate analysis, tumor ER-positivity was a favorable (p = 0.0092) and TopoIIa an unfavorable (p = 0.002) prognostic factor for PFS; PgR-positivity was favorable (p = 0.028) for survival. In conclusion, ixabepilone had a manageable safety profile in both the 3-weekly and weekly schedules. A number of markers identified in the present trial appear to deserve further evaluation for their prognostic and/or predictive value in larger multi-arm studies.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT 00790894","term_id":"NCT00790894"}}NCT 00790894     

Human CD4+ T cells displaying viral epitopes elicit a functional virus-specific memory CD8+ T cell response

The Ag-specific cellular recall response to herpes virus infections is characterized by a swift recruitment of virus-specific memory T cells. Rapid activation is achieved through formation of the immunological synapse and supramolecular clustering of signal molecules at the site of contact. During the formation of the immunological synapse, epitope-loaded MHC molecules are transferred via trogocytosis from APCs to T cells, enabling the latter to function as Ag-presenting T cells (T-APCs). The contribution of viral epitope expressing T-APCs in the regulation of the herpes virus-specific CD8+ T cell memory response remains unclear. Comparison of CD4+ T-APCs with professional APCs such as Ag-presenting CD40L-activated B cells (CD40B-APCs) demonstrated reduced levels of costimulatory ligands. Despite the observed differences, CD4+ T-APCs are as potent as CD40B-APCs in stimulating herpes virus-specific CD8+ T cells resulting in a greater than 35-fold expansion of CD8+ T cells specific for dominant and subdominant viral epitopes. Virus-specific CD8+ T cells generated by CD4+ T-APCs or CD40B-APCs showed both comparable effector function such as specific lysis of targets and cytokine production and also did not differ in their phenotype after expansion. These results indicate that viral epitope presentation by Ag-specific CD4+ T cells may contribute to the rapid recruitment of virus-specific memory CD8+ T cells during a viral recall response.     

Recognition of LD motifs by the focal adhesion targeting domains of focal adhesion kinase and proline‐rich tyrosine kinase 2‐beta: Insights from molecular dynamics simulations

The focal adhesion kinase (FAK) and the proline‐rich tyrosine kinase 2‐beta (PYK2) are implicated in cancer progression and metastasis and represent promising biomarkers and targets for cancer therapy. FAK and PYK2 are recruited to focal adhesions (FAs) via interactions between their FA targeting (FAT) domains and conserved segments (LD motifs) on the proteins Paxillin, Leupaxin, and Hic‐5. A promising new approach for the inhibition of FAK and PYK2 targets interactions of the FAK domains with proteins that promote localization at FAs. Advances toward this goal include the development of surface plasmon resonance, heteronuclear single quantum coherence nuclear magnetic resonance (HSQC‐NMR) and fluorescence polarization assays for the identification of fragments or compounds interfering with the FAK‐Paxillin interaction. We have recently validated this strategy, showing that Paxillin mimicking polypeptides with 2 to 3 LD motifs displace FAK from FAs and block kinase‐dependent and independent functions of FAK, including downstream integrin signaling and FA localization of the protein p130Cas. In the present work we study by all‐atom molecular dynamics simulations the recognition of peptides with the Paxillin and Leupaxin LD motifs by the FAK‐FAT and PYK2‐FAT domains. Our simulations and free‐energy analysis interpret experimental data on binding of Paxillin and Leupaxin LD motifs at FAK‐FAT and PYK2‐FAT binding sites, and assess the roles of consensus LD regions and flanking residues. Our results can assist in the design of effective inhibitory peptides of the FAK‐FAT: Paxillin and PYK2‐FAT:Leupaxin complexes and the construction of pharmacophore models for the discovery of potential small‐molecule inhibitors of the FAK‐FAT and PYK2‐FAT focal adhesion based functions.     

The <Emphasis Type="Italic">VKORC1</Emphasis> Asp36Tyr variant and <Emphasis Type="Italic">VKORC1</Emphasis> haplotype diversity in Ashkenazi and Ethiopian populations

The vitamin K epoxide reductase (VKORC1) is a key enzyme in the vitamin K cycle impacting various biological processes. VKORC1 genetic variability has been extensively studied in the context of warfarin pharmacogenetics revealing different distributions of VKORC1 haplotypes in various populations. We previously identified the VKORC1 Asp36Tyr mutation that was associated with warfarin resistance and with distinctive ethnic distribution. In this study, we performed haplotype analysis using Asp36Tyr and seven other VKORC1 markers in Ashkenazi and Ethiopian-Jewish and non-Jewish individuals. The VKORC1 variability was represented by nine haplotypes (V1-V9) that could be grouped into two distinct clusters (V1-V3 and V4-V9) with intra-cluster difference limited to two nucleotide changes. Phylogeny analysis suggested that these haplotypes could have developed from an ancestral variant, the common V8 haplotype (40 % in all population samples), after ten single mutation events. Asp36Tyr was exclusive to the V5 haplotype of the second cluster. Two haplotypes V5 and V4, distinguished only by Asp36Tyr, were prevalent in both Ethiopian population samples. The V2 haplotype, belonging to the first cluster, was the second most prevalent haplotype in the Ashkenazi population sample (15.8 %) but relatively uncommon in the Ethiopian origin (4.5-4.7 %). We discuss the genetic diversity among studied populations and its potential impact on warfarin-dose management in certain populations of African and European origin.     

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.