Crystal Structure of Miner1: The Redox-active 2Fe-2S Protein Causative in Wolfram Syndrome 2

The endoplasmic reticulum protein Miner1 is essential for health and longevity. Mis-splicing of CISD2, which codes for Miner1, is causative in Wolfram Syndrome 2 (WFS2) resulting in early onset optic atrophy, diabetes mellitus, deafness and decreased lifespan. In knock-out studies, disruption of CISD2 leads to accelerated aging, blindness and muscle atrophy. In this work, we characterized the soluble region of human Miner1 and solved its crystal structure to a resolution of 2.1 Å (R-factor = 17%). Although originally annotated as a zinc finger, we show that Miner1 is a homodimer harboring two redox-active 2Fe-2S clusters, indicating for the first time an association of a redox-active FeS protein with WFS2. Each 2Fe-2S cluster is bound by a rare Cys₃-His motif within a 17 amino acid segment. Miner1 is the first functionally different protein that shares the NEET fold with its recently identified paralog mitoNEET, an outer mitochondrial membrane protein. We report the first measurement of the redox potentials (E_m) of Miner1 and mitoNEET, showing that they are proton-coupled with E_m ∼ 0 mV at pH 7.5. Changes in the pH sensitivity of their cluster stabilities are attributed to significant differences in the electrostatic distribution and surfaces between the two proteins. The structural and biophysical results are discussed in relation to possible roles of Miner1 in cellular Fe-S management and redox reactions.     

Identification of novel large genomic rearrangements at the BRCA1 locus in Malaysian women with breast cancer

Background: The incidence of breast cancer has been on the rise in Malaysia. It is suggested that a subset of breast cancer cases were associated with germline mutation in breast cancer susceptibility (BRCA) genes. Most of the BRCA mutations reported in Malaysia were point mutations, small deletions and insertions. Here we report the first study of BRCA large genomic rearrangements (LGRs) in Malaysia. We aimed to detect the presence of LGRs in the BRCA genes of Malaysian patients with breast cancer. Methods: Multiplex ligation-dependent probe amplification (MLPA) for BRCA LGRs was carried out on 100 patients (60 were high-risk breast cancer patients previously tested negative/positive for BRCA1 and BRCA2 mutations, and 40 were sporadic breast cancer patients), recruited from three major referral centres, Universiti Kebangsaan Malaysia Medical Centre (UKMMC), Hospital Kuala Lumpur (HKL) and Hospital Putrajaya (HPJ). Results: Two novel BRCA1 rearrangements were detected in patients with sporadic breast cancer; both results were confirmed by quantitative PCR. No LGRs were found in patients with high-risk breast cancer. The two large genomic rearrangements detected were genomic amplifications of exon 3 and exon 10. No BRCA2 genomic rearrangement was found in both high-risk and sporadic breast cancer patients. Conclusion: These results will be helpful to understand the mutation spectrum of BRCA1 and BRCA2 genes in Malaysian patients with breast cancer. Further studies involving larger samples are required to establish a genetic screening strategy for both high-risk and sporadic breast cancer patients.     

Epistasis for founder-specific inbreeding depression in rabbits

Inbreeding depression is a topic of main interest in experimental and domestic species, although previous studies simplified this genetically complex effect to the linear (or quadratic) regression coefficient linked to the inbreeding coefficient of each individual or, in more recent studies, to founder-specific inbreeding coefficients. Going beyond generalizing to these traditional scenarios, our research focused on the analysis of gene-by-gene interactions leading to epistasis for inbreeding depression effects. Under a Bayesian context, inbreeding depression effects were evaluated for weaning weight (WW) in a commercial rabbit population founded from 4 bucks and 1 doe (MARET population). Founder-specific inbreeding depression effects for the 4 bucks ranged between -81.1 and 38.3 g for each 1% inbreeding. More interestingly, 2 epistatic interactions between the partial inbreeding coefficient of 2 bucks were also significant and negative, showing a -1.9 and -1.0 g reduction on WW. These results provide the first evidence of epistatic inbreeding depression phenomena in domestic species, emphasizing the complexity of the genetic architecture in mammals.     

Long range temporal correlations in EEG oscillations of subclinically depressed individuals: their association with brooding and suppression

Long-range temporal correlations (LRTC) in brain oscillations have been found to be associated with depression severity in clinically depressed patients. Less is known, however, about the relationships between LRTC and proneness to engage in depression-related cognitive emotion regulation (ER) strategies which characterize both clinically and subclinically depressed (SBD) people. In this study we applied detrended fluctuation analysis to the amplitude envelope of broad band, theta band, and alpha band spontaneous EEG oscillations of a group of SBD individuals and a group of non-depressed individuals (both groups from a sample of healthy adults, N = 120), to whom brooding and thought suppression questionnaires were administered. Between-groups differences were not found for any band scaling exponents at any brain location, but linear correlations pointed out several associations between exponents at frontal, central, parietal, temporal, and occipital sites and maladaptive ER strategies. These results suggest that alterations in brain dynamics are related with the proneness that depressive individuals show to engage in brooding and thought suppression in order to cognitively regulate their emotions.     

Colocalization of sterol isomerase and sigma(1) receptor at endoplasmic reticulum and nuclear envelope level.

SR31747A is a sigma ligand previously described as having original immunosuppressive properties. Two SR31747A targets were recently identified and termed sigma(1) or SR-BP-1 (SR31747A-binding protein-1) and hSI (human sterol isomerase). In order to characterize these proteins further, we examined their expression and localization at the subcellular level. Based on the amino acid sequence deduced from the cloned hSI, anti-hSI polyclonal antibody was raised against the N-terminal fragment of the protein. Using this antibody, we performed Western-blot experiments to demonstrate the presence of hSI in various B and T cell lines, and hSI expression was quantified in these cell lines by flow cytometry and estimated at 15 000-30 000 sites per cell. Subcellular localization studies by both confocal and electron microscopy, performed on THP1 cells with anti-hSI antibody and with the previously described anti-(SR-BP-1) monoclonal antibody, demonstrated that: (a) hSI was colocalized with SR-BP-1; (b) hSI and SR-BP-1 were associated with the endoplasmic reticulum and with the outer and inner membranes of the nuclear envelope; (c) both proteins were delocalized during the cell cycle at the mitosis step when the nuclear membranes disappeared. Taken together our results suggest that both SR31747A-binding proteins not only play a role in sterol metabolism but indirectly affect lipoprotein functions.     

The molecular interactions of buspirone analogues with the serotonin transporter

A major problem with the selective serotonin reuptake inhibitors (SSRIs) is the delayed onset of action. A reason for that may be that the initial SSRI-induced increase in serotonin levels activates somatodendritic 5-HT(1A) autoreceptors, causing a decrease in serotonin release in major forebrain areas. It has been suggested that compounds combining inhibition of the serotonin transport protein with antagonistic effects on the 5-HT(1A) receptor will shorten the onset time. The anxiolytic drug buspirone is known as 5-HT(1A) partial agonist. In the present work, we are studying the inhibition of the serotonin transporter protein by a series of buspirone analogues by molecular modelling and by experimental affinity measurements. Models of the transporter protein were constructed using the crystal structure of the Escherichia coli major facilitator family transporter-LacY and the X-ray structure of the neurotransmitter symporter family (NSS) transporter-LeuT(Aa) as templates. The buspirone analogues were docked into both SERT models and the interactions with amino acids within the protein were analyzed. Two putative binding sites were identified on the LeuT(Aa) based model, one suggested to be a high-affinity site, and the other suggested to be a low-affinity binding site. Molecular dynamic simulations of the LacY based model in complex with ligands did not induce a helical architecture of the LacY based model into an arrangement more similar to that of the LeuT(Aa) based model.     

Short-Term Effect of Antibiotics on Human Gut Microbiota

From birth onwards, the human gut microbiota rapidly increases in diversity and reaches an adult-like stage at three years of age. After this age, the composition may fluctuate in response to external factors such as antibiotics. Previous studies have shown that resilience is not complete months after cessation of the antibiotic intake. However, little is known about the short-term effects of antibiotic intake on the gut microbial community. Here we examined the load and composition of the fecal microbiota immediately after treatment in 21 patients, who received broad-spectrum antibiotics such as fluoroquinolones and β-lactams. A fecal sample was collected from all participants before treatment and one week after for microbial load and community composition analyses by quantitative PCR and pyrosequencing of the 16S rRNA gene, respectively. Fluoroquinolones and β-lactams significantly decreased microbial diversity by 25% and reduced the core phylogenetic microbiota from 29 to 12 taxa. However, at the phylum level, these antibiotics increased the Bacteroidetes/Firmicutes ratio (p = 0.0007, FDR = 0.002). At the species level, our findings unexpectedly revealed that both antibiotic types increased the proportion of several unknown taxa belonging to the Bacteroides genus, a Gram-negative group of bacteria (p = 0.0003, FDR<0.016). Furthermore, the average microbial load was affected by the treatment. Indeed, the β-lactams increased it significantly by two-fold (p = 0.04). The maintenance of or possible increase detected in microbial load and the selection of Gram-negative over Gram-positive bacteria breaks the idea generally held about the effect of broad-spectrum antibiotics on gut microbiota.     

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes.