Construction and Preliminary Characterization of a Series of Mouse and Rat Testis cDNA Libraries

We have constructed a series of 23 cDNA libraries from mouse and rat testicular cells. These include libraries made from whole, intact adult testes; seminiferous tubule cells from adult testes; combined populations of primary spermatocytes from 18‐day‐old mouse testes; and isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene plus zygotene spermatocytes, juvenile pachytene spermatocytes, adult pachytene spermatocytes, round spermatids, Sertoli cells from 6‐, 8‐, 17‐, and 18–20‐day‐old mice, and peritubular cells from 18–20 day old mice, all recovered from outbred white Swiss (CD‐1) mice. We also constructed libraries from whole adult testes from five other lines of mice: C57 BI6/J, C3 HEB, BDF‐1, Balb/c, and 129 Sv. Finally, there are two libraries made from populations of Sertoli cells and peritubular cells isolated from testes of 20‐day‐old Sprague‐Dawley rats. Enzymatic dissociation, followed by gradient separation or plating/lysing techniques, was used to prepare populations of specific cell types in purities of 85–98%. cDNAs were synthesized from poly A+ mRNA primed with oligo dT and unidirectionally cloned into the lambda Uni‐Zap XR expression vector from Stratagene. Primary titers ranged from 2.1 ± 105 to 2.9 × 108 plaque‐forming units, and insert sizes averaged 1.0–1.2 kb. These libraries have been amplified once and submitted to the American Type Culture Collection (ATCC) for distribution to interested investigators. ATCC accession numbers are provided

High-Precision, In Vitro Validation of the Sequestration Mechanism for Generating Ultrasensitive Dose-Response Curves in Regulatory Networks

Our ability to recreate complex biochemical mechanisms in designed, artificial systems provides a stringent test of our understanding of these mechanisms and opens the door to their exploitation in artificial biotechnologies. Motivated by this philosophy, here we have recapitulated in vitro the “target sequestration” mechanism used by nature to improve the sensitivity (the steepness of the input/output curve) of many regulatory cascades. Specifically, we have employed molecular beacons, a commonly employed optical DNA sensor, to recreate the sequestration mechanism and performed an exhaustive, quantitative study of its key determinants (e.g., the relative concentrations and affinities of probe and depletant). We show that, using sequestration, we can narrow the pseudo-linear range of a traditional molecular beacon from 81-fold (i.e., the transition from 10% to 90% target occupancy spans an 81-fold change in target concentration) to just 1.5-fold. This narrowing of the dynamic range improves the sensitivity of molecular beacons to that equivalent of an oligomeric, allosteric receptor with a Hill coefficient greater than 9. Following this we have adapted the sequestration mechanism to steepen the binding-site occupancy curve of a common transcription factor by an order of magnitude over the sensitivity observed in the absence of sequestration. Given the success with which the sequestration mechanism has been employed by nature, we believe that this strategy could dramatically improve the performance of synthetic biological systems and artificial biosensors

Tsx Produces a Long Noncoding RNA and Has General Functions in the Germline, Stem Cells, and Brain

The Tsx gene resides at the X-inactivation center and is thought to encode a protein expressed in testis, but its function has remained mysterious. Given its proximity to noncoding genes that regulate X-inactivation, here we characterize Tsx and determine its function in mice. We find that Tsx is actually noncoding and the long transcript is expressed robustly in meiotic germ cells, embryonic stem cells, and brain. Targeted deletion of Tsx generates viable offspring and X-inactivation is only mildly affected in embryonic stem cells. However, mutant embryonic stem cells are severely growth-retarded, differentiate poorly, and show elevated cell death. Furthermore, male mice have smaller testes resulting from pachytene-specific apoptosis and a maternal-specific effect results in slightly smaller litters. Intriguingly, male mice lacking Tsx are less fearful and have measurably enhanced hippocampal short-term memory. Combined, our study indicates that Tsx performs general functions in multiple cell types and links the noncoding locus to stem and germ cell development, learning, and behavior in mammals

Preserved cognitive functions with age are determined by domain-dependent shifts in network responsivity

Healthy ageing has disparate effects on different cognitive domains. The neural basis of these differences, however, is largely unknown. We investigated this question by using Independent Components Analysis to obtain functional brain components from 98 healthy participants aged 23-87 years from the population-based Cam-CAN cohort. Participants performed two cognitive tasks that show age-related decrease (fluid intelligence and object naming) and a syntactic comprehension task that shows age-related preservation. We report that activation of task-positive neural components predicts inter-individual differences in performance in each task across the adult lifespan. Furthermore, only the two tasks that show performance declines with age show age-related decreases in task-positive activation of neural components and decreasing default mode (DM) suppression. Our results suggest that distributed, multi-component brain responsivity supports cognition across the adult lifespan, and the maintenance of this, along with maintained DM deactivation, characterizes successful ageing and may explain differential ageing trajectories across cognitive domains

Identification of novel Y chromosome encoded transcripts by testis transcriptome analysis of mice with deletions of the Y chromosome long arm.

BACKGROUND: The male-specific region of the mouse Y chromosome long arm (MSYq) is comprised largely of repeated DNA, including multiple copies of the spermatid-expressed Ssty gene family. Large deletions of MSYq are associated with sperm head defects for which Ssty deficiency has been presumed to be responsible. RESULTS: In a search for further candidate genes associated with these defects we analyzed changes in the testis transcriptome resulting from MSYq deletions, using testis cDNA microarrays. This approach, aided by accumulating mouse MSYq sequence information, identified transcripts derived from two further spermatid-expressed multicopy MSYq gene families; like Ssty, each of these new MSYq gene families has multicopy relatives on the X chromosome. The Sly family encodes a protein with homology to the chromatin-associated proteins XLR and XMR that are encoded by the X chromosomal relatives. The second MSYq gene family was identified because the transcripts hybridized to a microarrayed X chromosome-encoded testis cDNA. The X loci ('Astx') encoding this cDNA had 92-94% sequence identity to over 100 putative Y loci ('Asty') across exons and introns; only low level Asty transcription was detected. More strongly transcribed recombinant loci were identified that included Asty exons 2-4 preceded by Ssty1 exons 1, 2 and part of exon 3. Transcription from the Ssty1 promotor generated spermatid-specific transcripts that, in addition to the variable inclusion of Ssty1 and Asty exons, included additional exons because of the serendipitous presence of splice sites further downstream. CONCLUSION: We identified further MSYq-encoded transcripts expressed in spermatids and deriving from multicopy Y genes, deficiency of which may underlie the defects in sperm development associated with MSYq deletions.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Multiple determinants of lifespan memory differences

Memory problems are among the most common complaints as people grow older. Using structural equation modeling of commensurate scores of anterograde memory from a large (N = 315), population-derived sample (www.cam-can.org), we provide evidence for three memory factors that are supported by distinct brain regions and show differential sensitivity to age. Associative memory and item memory are dramatically affected by age, even after adjusting for education level and fluid intelligence, whereas visual priming is not. Associative memory and item memory are differentially affected by emotional valence, and the age-related decline in associative memory is faster for negative than for positive or neutral stimuli. Gray-matter volume in the hippocampus, parahippocampus and fusiform cortex, and a white-matter index for the fornix, uncinate fasciculus and inferior longitudinal fasciculus, show differential contributions to the three memory factors. Together, these data demonstrate the extent to which differential ageing of the brain leads to differential patterns of memory loss

Differential Expression of Non-Coding RNAs and Continuous Evolution of the X Chromosome in Testicular Transcriptome of Two Mouse Species

BACKGROUND: Tight regulation of testicular gene expression is a prerequisite for male reproductive success, while differentiation of gene activity in spermatogenesis is important during speciation. Thus, comparison of testicular transcriptomes between closely related species can reveal unique regulatory patterns and shed light on evolutionary constraints separating the species. METHODOLOGY/PRINCIPAL FINDINGS: Here, we compared testicular transcriptomes of two closely related mouse species, Mus musculus and Mus spretus, which diverged more than one million years ago. We analyzed testicular expression using tiling arrays overlapping Chromosomes 2, X, Y and mitochondrial genome. An excess of differentially regulated non-coding RNAs was found on Chromosome 2 including the intronic antisense RNAs, intergenic RNAs and premature forms of Piwi-interacting RNAs (piRNAs). Moreover, striking difference was found in the expression of X-linked G6pdx gene, the parental gene of the autosomal retrogene G6pd2. CONCLUSIONS/SIGNIFICANCE: The prevalence of non-coding RNAs among differentially expressed transcripts indicates their role in species-specific regulation of spermatogenesis. The postmeiotic expression of G6pdx in Mus spretus points towards the continuous evolution of X-chromosome silencing and provides an example of expression change accompanying the out-of-the X-chromosomal retroposition

Parma consensus statement on metabolic disruptors

A multidisciplinary group of experts gathered in Parma Italy for a workshop hosted by the University of Parma, May 16–18, 2014 to address concerns about the potential relationship between environmental metabolic disrupting chemicals, obesity and related metabolic disorders. The objectives of the workshop were to: 1. Review findings related to the role of environmental chemicals, referred to as “metabolic disruptors”, in obesity and metabolic syndrome with special attention to recent discoveries from animal model and epidemiology studies; 2. Identify conclusions that could be drawn with confidence from existing animal and human data; 3. Develop predictions based on current data; and 4. Identify critical knowledge gaps and areas of uncertainty. The consensus statements are intended to aid in expanding understanding of the role of metabolic disruptors in the obesity and metabolic disease epidemics, to move the field forward by assessing the current state of the science and to identify research needs on the role of environmental chemical exposures in these diseases. We propose broadening the definition of obesogens to that of metabolic disruptors, to encompass chemicals that play a role in altered susceptibility to obesity, diabetes and related metabolic disorders including metabolic syndrome

Bupropion for the treatment of apathy in Huntington's disease:A multicenter, randomised, double-blind, placebo-controlled, prospective crossover trial

OBJECTIVE:To evaluate the efficacy and safety of bupropion in the treatment of apathy in Huntington's disease (HD). METHODS:In this phase 2b multicentre, double-blind, placebo-controlled crossover trial, individuals with HD and clinical signs of apathy according to the Structured Clinical Interview for Apathy-Dementia (SCIA-D), but not depression (n = 40) were randomized to receive either bupropion 150/300mg or placebo daily for 10 weeks. The primary outcome parameter was a significant change of the Apathy Evaluation Scale (AES) score after ten weeks of treatment as judged by an informant (AES-I) living in close proximity with the study participant. The secondary outcome parameters included changes of 1. AES scores determined by the patient (AES-S) or the clinical investigator (AES-C), 2. psychiatric symptoms (NPI, HADS-SIS, UHDRS-Behavior), 3. cognitive performance (SDMT, Stroop, VFT, MMSE), 4. motor symptoms (UHDRS-Motor), 5. activities of daily function (TFC, UHDRS-Function), and 6. caregiver distress (NPI-D). In addition, we investigated the effect of bupropion on brain structure as well as brain responses and functional connectivity during reward processing in a gambling task using magnetic resonance imaging (MRI). RESULTS:At baseline, there were no significant treatment group differences in the clinical primary and secondary outcome parameters. At endpoint, there was no statistically significant difference between treatment groups for all clinical primary and secondary outcome variables. Study participation, irrespective of the intervention, lessened symptoms of apathy according to the informant and the clinical investigator. CONCLUSION:Bupropion does not alleviate apathy in HD. However, study participation/placebo effects were observed, which document the need for carefully controlled trials when investigating therapeutic interventions for the neuropsychiatric symptoms of HD. TRIAL REGISTRATION:ClinicalTrials.gov 01914965

De Novo Origin of Human Protein-Coding Genes

The de novo origin of a new protein-coding gene from non-coding DNA is considered to be a very rare occurrence in genomes. Here we identify 60 new protein-coding genes that originated de novo on the human lineage since divergence from the chimpanzee. The functionality of these genes is supported by both transcriptional and proteomic evidence. RNA–seq data indicate that these genes have their highest expression levels in the cerebral cortex and testes, which might suggest that these genes contribute to phenotypic traits that are unique to humans, such as improved cognitive ability. Our results are inconsistent with the traditional view that the de novo origin of new genes is very rare, thus there should be greater appreciation of the importance of the de novo origination of genes