Evolution of Spliceosomal snRNA Genes in Metazoan Animals

While studies of the evolutionary histories of protein families are commonplace, little is known on noncoding RNAs beyond microRNAs and some snoRNAs. Here we investigate in detail the evolutionary history of the nine spliceosomal snRNA families (U1, U2, U4, U5, U6, U11, U12, U4atac, and U6atac) across the completely or partially sequenced genomes of metazoan animals. Representatives of the five major spliceosomal snRNAs were found in all genomes. None of the minor splicesomal snRNAs were detected in nematodes or in the shotgun traces of Oikopleura dioica, while in all other animal genomes at most one of them is missing. Although snRNAs are present in multiple copies in most genomes, distinguishable paralogue groups are not stable over long evolutionary times, although they appear independently in several clades. In general, animal snRNA secondary structures are highly conserved, albeit, in particular, U11 and U12 in insects exhibit dramatic variations. An analysis of genomic context of snRNAs reveals that they behave like mobile elements, exhibiting very little syntenic conservation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Efficient algorithms to explore conformation spaces of flexible protein loops

Several applications in biology - e.g., incorporation of protein flexibility in ligand docking algorithms, interpretation of fuzzy X-ray crystallographic data, and homology modeling - require computing the internal parameters of a flexible fragment (usually, a loop) of a protein in order to connect its termini to the rest of the protein without causing any steric clash. One must often sample many such conformations in order to explore and adequately represent the conformational range of the studied loop. While sampling must be fast, it is made difficult by the fact that two conflicting constraints - kinematic closure and clash avoidance - must be satisfied concurrently. This paper describes two efficient and complementary sampling algorithms to explore the space of closed clash-free conformations of a flexible protein loop. The "seed sampling" algorithm samples broadly from this space, while the "deformation sampling" algorithm uses seed conformations as starting points to explore the conformation space around them at a finer grain. Computational results are presented for various loops ranging from 5 to 25 residues. More specific results also show that the combination of the sampling algorithms with a functional site prediction software (FEATURE) makes it possible to compute and recognize calcium-binding loop conformations. The sampling algorithms are implemented in a toolkit (LoopTK), which is available at https://simtk.org/home/looptk.     

DC-SIGN and CD150 have distinct roles in transmission of measles virus from dendritic cells to T-lymphocytes

Measles virus (MV) is among the most infectious viruses that affect humans and is transmitted via the respiratory route. In macaques, MV primarily infects lymphocytes and dendritic cells (DCs). Little is known about the initial target cell for MV infection. Since DCs bridge the peripheral mucosal tissues with lymphoid tissues, we hypothesize that DCs are the initial target cells that capture MV in the respiratory tract and transport the virus to the lymphoid tissues where MV is transmitted to lymphocytes. Recently, we have demonstrated that the C-type lectin DC-SIGN interacts with MV and enhances infection of DCs in cis. Using immunofluorescence microscopy, we demonstrate that DC-SIGN+ DCs are abundantly present just below the epithelia of the respiratory tract. DC-SIGN+ DCs efficiently present MV-derived antigens to CD4+ T-lymphocytes after antigen uptake via either CD150 or DC-SIGN in vitro. However, DC-SIGN+ DCs also mediate transmission of MV to CD4+ and CD8+ T-lymphocytes. We distinguished two different transmission routes that were either dependent or independent on direct DC infection. DC-SIGN and CD150 are both involved in direct DC infection and subsequent transmission of de novo synthesized virus. However, DC-SIGN, but not CD150, mediates trans-infection of MV to T-lymphocytes independent of DC infection. Together these data suggest a prominent role for DCs during the initiation, dissemination, and clearance of MV infection.     

A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the gamma subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the gamma subunit is not required for enzymatic activity, the gamma subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and gamma subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the gamma subunit. Therefore, we suggest that the gamma subunit contributes to adaptation of P. pastoris to energy resources.     

Does treatment of subsyndromal depression improve depression-related and diabetes-related outcomes? A randomised controlled comparison of psychoeducation,physical exercise and enhanced treatment as usual

Background

Elevated depressive symptoms that do not reach criteria for a clinical diagnosis of depression are highly prevalent in persons with diabetes. This study was aimed at determining the efficacy of psychoeducation and physical exercise compared with enhanced treatment as usual on 1-year changes in depressive symptoms, diabetes distress and self-management, and quality of life and metabolic control in type 2 diabetes patients with subsyndromal depression.

Methods

Adult type 2 diabetes patients who screened positively for depression and expressed a need for professional help with mood-related issues were eligible. Exclusion criteria were clinical depression, current psychiatric treatment and advanced diabetes complications. Out of 365 eligible patients 209 consented to either 6 weekly sessions of psychoeducation (A) and physical exercise (B), or to enhanced treatment as usual (C). Computer-generated sequences for block randomisation stratified by gender were used. Depressive symptoms (primary outcome) and diabetes distress, diabetes self-care, metabolic control and health-related quality of life (secondary outcomes) were analysed at 6-month and 12-month follow-up using repeated-measures ANOVAs.

Results

Out of the 74 patients randomised into group A, 66 into B and 69 into group C, 203 completed the interventions, and 179 patients with all 3 assessments were analysed. Depressive symptoms in participants from the psychoeducational, physical exercise and the enhanced treatment as usual groups improved equally from baseline to 12-month follow-up (time versus time x group effect; F = 12.51, p < 0.001, η² = 0.07 and F = 0.609, p = 0.656, η² = 0.007 respectively), as did diabetes distress and quality of life (all p < 0.001), diabetes self-care (p < 0.001 to < 0.05), triglycerides, and total cholesterol and LDL-cholesterol (p < 0.001).

Conclusions

The employed interventions had comparable positive effects on 12-month psychological and diabetes-related outcomes suggesting that even minimal intervention addressing patients’ diabetes-related problems and concerns had favourable clinical implications and might be sufficient to treat subsyndromal depression. Further investigation is warranted to clarify possible mechanisms of improvement.

Trial registration

Current Controlled Trials ISRCTN05673017The message on assigning the above mentioned ISRCTN was received on 11 August 2010

     

Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.     

Tissue distribution of a human Ca v 1.2 alpha1 subunit splice variant with a 75 bp insertion

Mesenchymal stem cells from human bone marrow (MSC) express mRNA encoding the L-type Ca2+ channel Ca v 1.2 alpha1 subunit (alpha(1)1.2). We now describe a splice variant including an alternative exon of 75 bp in the region between exons 9 and 10, which we identified in MSC by semi-quantitative RT-PCR. With primers specific for variants including (+9*) or excluding the 75 bp insertion (-9*), we found comparable mRNA expression patterns in MSC and in primary cultures of related connective tissue cells (chondrocytes, osteoblasts and fibroblasts). Since culture conditions might have altered variant expression, we investigated mRNA levels in various native human tissue samples (cartilage, bone, fat, liver, kidney, aorta, bladder, cardiac ventricle and atrium, CNS). We found highest levels of the +9* variant in aorta, containing smooth muscle and connective tissue cells, but the variant was expressed in all tissues. We therefore hypothesized that broad expression of +9* might be linked to the presence of vasculature and/or connective tissue structures, rather than to tissue-specific parenchymal cells (e.g. cardiomyocytes). To test this hypothesis we separated human atrium into a cardiomyocyte-enriched fraction and a cardiomyocyte-depleted fraction. RT-PCR demonstrated significantly larger levels of the +9* variant in the non-cardiomyocyte fraction. The result was even more clear in single cell RT-PCR experiments, where the +9* variant was undetectable in cardiomyocytes but present in non-cardiomyocytes. We conclude that the +9* variant is present in all human tissues investigated so far, and suggest that expression in human atrium is associated with vascular smooth muscle and/or connective tissue cells.     

Cellulolytic activity of four Fusarium spp.

Fusarium lini, F. lycopersici, F. pallidoroseum and F. semitectum grown in shake flasks produced, respectively, 0.19, 0.33, 0.13 and 0.09 units filter-paper cellulase/ml. Trichoderma reesei, in comparison, produced 0.8 U/ml.The authors are with Defence Food Research Laboratory, Mysore-570 011, India.     

Age-Related and Heteroplasmy-Related Variation in Human mtDNA Copy Number

The mitochondrial (mt) genome is present in many copies in human cells, and intra-individual variation in mtDNA sequences is known as heteroplasmy. Recent studies found that heteroplasmies are highly tissue-specific, site-specific, and allele-specific, however the functional implications have not been explored. This study investigates variation in mtDNA copy numbers (mtCN) in 12 different tissues obtained at autopsy from 152 individuals (ranging in age from 3 days to 96 years). Three different methods to estimate mtCN were compared: shotgun sequencing (in 4 tissues), capture-enriched sequencing (in 12 tissues) and droplet digital PCR (ddPCR, in 2 tissues). The highest precision in mtCN estimation was achieved using shotgun sequencing data. However, capture-enrichment data provide reliable estimates of relative (albeit not absolute) mtCNs. Comparisons of mtCN from different tissues of the same individual revealed that mtCNs in different tissues are, with few exceptions, uncorrelated. Hence, each tissue of an individual seems to regulate mtCN in a tissue-related rather than an individual-dependent manner. Skeletal muscle (SM) samples showed an age-related decrease in mtCN that was especially pronounced in males, while there was an age-related increase in mtCN for liver (LIV) samples. MtCN in SM samples was significantly negatively correlated with both the total number of heteroplasmic sites and with minor allele frequency (MAF) at two heteroplasmic sites, 408 and 16327. Heteroplasmies at both sites are highly specific for SM, accumulate with aging and are part of functional elements that regulate mtDNA replication. These data support the hypothesis that selection acting on these heteroplasmic sites is reducing mtCN in SM of older individuals.