Exceptional long-term sperm storage by a female vertebrate.

Females of many vertebrate species have the capacity to store sperm within their reproductive tracts for prolonged periods of time. Termed long-term sperm storage, this phenomenon has many important physiological, ecological, and evolutionary implications, particularly to the study of mating systems, including male reproductive success and post-copulatory sexual selection. Reptiles appear particularly predisposed to long-term sperm storage, with records in most major lineages, with a strong emphasis on turtles and squamates (lizards, snakes, but not the amphisbaenians). Because facultative parthenogenesis is a competing hypothesis to explain the production of offspring after prolonged separation from males, the identification of paternal alleles through genetic analysis is essential. However, few studies in snakes have undertaken this. Here, we report on a wild-collected female Western Diamond-backed Rattlesnake, Crotalus atrox, maintained in isolation from the time of capture in September 1999, that produced two healthy litters approximately one and six years post capture. Genetic analysis of the 2005 litter, identified paternal contribution in all offspring, thus rejecting facultative parthenogenesis. We conclude that the duration of long-term sperm storage was approximately 6 years (71 months), making this the longest period over which a female vertebrate has been shown to store sperm that resulted in the production of healthy offspring.

Multi-omics characterization and validation of MSI-related molecular features across multiple malignancies.

HEADINGS AIMS: To establish a microsatellite instability (MSI) predictive model in pan-cancer and compare the multi-omics characterization of MSI-related molecular features. MATERIALS AND METHODS: We established a 15-gene signature for predicting MSI status and performed a systematic assessment of MSI-related molecular features including gene and miRNA expression, DNA methylation, and somatic mutation, in approximately 10,000 patients across 30 cancer types from The Cancer Genome Atlas, Gene Expression Omnibus database, and our institution. Then we identified common MSI-associated dysregulated molecular features across six cancers and explored their mutual interfering relationships and the drug sensitivity. KEY FINDINGS: we demonstrated the model's high prediction performance and found the samples with high-MSI were mainly distributed in six cancers: BRCA, COAD, LUAD, LIHC, STAD, and UCEC. We found RPL22L1 was up-regulated in the high-MSI group of 5/6 cancer types. CYP27A1 and RAI2 were down-regulated in 4/6 cancer types. More than 20 miRNAs and 39 DMGs were found up-regulated in MSI-H at least three cancers. We discovered some drugs, including OSI-027 and AZD8055 had a higher sensitivity in the high MSI-score group. Functional enrichment analysis revealed the correlation between MSI score and APM score, HLA score, or glycolysis score. The complicated regulatory mechanism of tumor MSI status in multiple dimensions was explored by an integrated analysis of the correlations among MSI-related genes, miRNAs, methylation, and drug response data. SIGNIFICANCE: Our pan-cancer study provides a valuable predictive model and a comprehensive atlas of tumor MSI, which may guide more precise and personalized therapeutic strategies for tumor patients.

The Clinical Significance of Microsatellite Instability in Precision Treatment.

The recent years have seen the high heterogeneity of colorectal cancer (CRC) receiving increasing attention and being revealed step by step. Microsatellite instability (MSI), characterized by the dysfunction of mismatch repair gene, plays an important role in the heterogeneity of colorectal cancer. MSI status can be identified by immunohistochemistry for MMR protein such as MLH1, MSH2, PMS2, and MSH6 or PCR-based array for MMR gene. Recent studies have revealed MSI status is the only biomarker that can be used to select patients with high-risk stage II colon cancer for adjuvant chemotherapy. Furthermore, it always indicated better stage-adjusted survival when compared with microsatellite stable (MSS) tumors. For immunotherapy, patients with MSI tumors exhibited significant response to anti-PD-1 inhibitors after the failure to conventional therapy. In this chapter, we discuss the detection methods of MSI, the prognostic value of MSI, and its clinical guiding value in the management of precision therapy.

Molecular Mechanisms and Future Therapeutics for Spinocerebellar Ataxia Type 31 (SCA31).

Spinocerebellar ataxia type 31 (SCA31) is one of the autosomal-dominant neurodegenerative disorders that shows progressive cerebellar ataxia as a cardinal symptom. This disease is caused by a 2.5- to 3.8-kb-long complex pentanucleotide repeat containing (TGGAA)n, (TAGAA)n, (TAAAA)n, and (TAAAATAGAA)n in an intron of the gene called BEAN1 (brain expressed, associated with Nedd4). By comparing various pentanucleotide repeats in this particular locus among control Japanese and Caucasian populations, it was found that (TGGAA)n was the only sequence segregating with SCA31, strongly suggesting the pathogenicity of (TGGAA)n. The complex repeat also lies in an intron of another gene, TK2 (thymidine kinase 2), which is transcribed in the opposite direction, indicating that the complex repeat is bi-directionally transcribed as noncoding repeats. In SCA31 human brains, (UGGAA)n, the BEAN1 transcript of SCA31 mutation was found to form abnormal RNA structures called RNA foci in cerebellar Purkinje cell nuclei. Subsequent RNA pulldown analysis disclosed that (UGGAA)n binds to RNA-binding proteins TDP-43, FUS, and hnRNP A2/B1. In fact, TDP-43 was found to co-localize with RNA foci in human SCA31 Purkinje cells. To dissect the pathogenesis of (UGGAA)n in SCA31, we generated transgenic fly models of SCA31 by overexpressing SCA31 complex pentanucleotide repeats in Drosophila. We found that the toxicity of (UGGAA)n is length- and expression level-dependent, and it was dampened by co-expressing TDP-43, FUS, and hnRNP A2/B1. Further investigation revealed that TDP-43 ameliorates (UGGAA)n toxicity by directly fixing the abnormal structure of (UGGAA)n. This led us to propose that TDP-43 acts as an RNA chaperone against toxic (UGGAA)n. Further research on the role of RNA-binding proteins as RNA chaperones may provide a novel therapeutic strategy for SCA31.

A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival.

Functions of many long noncoding RNAs (lncRNAs) depend on their ability to interact with multiple copies of specific RNA-binding proteins (RBPs). Here, we devised a workflow combining bioinformatics and experimental validation steps to systematically identify RNAs capable of multivalent RBP recruitment. This uncovered a number of previously unknown transcripts encoding high-density RBP recognition arrays within genetically normal short tandem repeats. We show that a top-scoring hit in this screen, lncRNA PNCTR, contains hundreds of pyrimidine tract-binding protein (PTBP1)-specific motifs allowing it to sequester a substantial fraction of PTBP1 in a nuclear body called perinucleolar compartment. Importantly, PNCTR is markedly overexpressed in a variety of cancer cells and its downregulation is sufficient to induce programmed cell death at least in part by stimulating PTBP1 splicing regulation activity. This work expands our understanding of the repeat-containing fraction of the human genome and illuminates a novel mechanism driving malignant transformation of cancer cells.

The proline-arginine repeat protein linked to C9-ALS/FTD causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat (DPR) proteins. Although it has been reported that DPR proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we have first confirmed that proline-arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity and found that the poly-PR-induced neurotoxicity is repressed by the acceleration of ribosomal RNA synthesis. These results suggest that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. We have further identified DEAD-box RNA helicases as poly-PR-binding proteins, the functions of which are inhibited by poly-PR. The enforced reduction in the expression of DEAD-box RNA helicases causes impairment of ribosome biogenesis and neuronal cell death. These results together suggest that poly-PR causes neurotoxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

Disease-Associated Short Tandem Repeats Co-localize with Chromatin Domain Boundaries.

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.

Toward deciphering the mechanistic role of variations in the Rep1 repeat site in the transcription regulation of SNCA gene.

Short structural variants-variants other than single nucleotide polymorphisms-are hypothesized to contribute to many complex diseases, possibly by modulating gene expression. However, the molecular mechanisms by which noncoding short structural variants exert their effects on gene regulation have not been discovered. Here, we study simple sequence repeats (SSRs), a common class of short structural variants. Previously, we showed that repetitive sequences can directly influence the binding of transcription factors to their proximate recognition sites, a mechanism we termed non-consensus binding. In this study, we focus on the SSR termed Rep1, which was associated with Parkinson's disease (PD) and has been implicated in the cis-regulation of the PD-risk SNCA gene. We show that Rep1 acts via the non-consensus binding mechanism to affect the binding of transcription factors from the GATA and ELK families to their specific sites located right next to the Rep1 repeat. Next, we performed an expression analysis to further our understanding regarding the GATA and ELK family members that are potentially relevant for SNCA transcriptional regulation in health and disease. Our analysis indicates a potential role for GATA2, consistent with previous reports. Our study proposes non-consensus transcription factor binding as a potential mechanism through which noncoding repeat variants could exert their pathogenic effects by regulating gene expression.

Transcriptome profiles of sunflower reveal the potential role of microsatellites in gene expression divergence.

The mechanisms by which natural populations generate adaptive genetic variation are not well understood. Some studies propose that microsatellites can function as drivers of adaptive variation. Here, we tested a potentially adaptive role for transcribed microsatellites with natural populations of the common sunflower (Helianthus annuus L.) by assessing the enrichment of microsatellites in genes that show expression divergence across latitudes. Seeds collected from six populations at two distinct latitudes in Kansas and Oklahoma were planted and grown in a common garden. Morphological measurements from the common garden demonstrated that phenotypic variation among populations is largely explained by underlying genetic variation. An RNA-Seq experiment was conducted with 96 of the individuals grown in the common garden and differentially expressed (DE) transcripts between the two latitudes were identified. A total number of 825 DE transcripts were identified. DE transcripts and nondifferentially expressed (NDE) transcripts were then scanned for microsatellites. The abundance of different motif lengths and types in both groups were estimated. Our results indicate that DE transcripts are significantly enriched with mononucleotide repeats and significantly depauperate in trinucleotide repeats. Further, the standardized mononucleotide repeat motif A and dinucleotide repeat motif AG were significantly enriched within DE transcripts while motif types, C, AT, ACC and AAC in DE transcripts, are significantly differentiated in microsatellite tract length between the two latitudes. The tract length differentiation at specific microsatellite motif types across latitudes and their enrichment within DE transcripts indicate a potential functional role for transcribed microsatellites in gene expression divergence in sunflower.

Association mapping of seed and disease resistance traits in Theobroma cacao L.

MAIN CONCLUSION: Microsatellite and single nucleotide polymorphism markers that could be used in marker assisted breeding of cacao were identified for number of filled seeds, black pod resistance and witches' broom disease resistance. An association mapping approach was employed to identify markers for seed number and resistance to black pod and witches' broom disease (WBD) in cacao (Theobroma cacao L.). Ninety-five microsatellites (SSRs) and 775 single nucleotide polymorphisms (SNPs) were assessed on 483 unique trees in the International Cocoa Genebank Trinidad (ICGT). Linkage disequilibrium (LD) and association mapping studies were conducted to identify markers to tag the phenotypic traits. Decay of LD occurred over an average 9.3 cM for chromosomes 1-9 and 2.5 cM for chromosome 10. Marker/trait associations were generally identified based on general linear models (GLMs) that incorporated principal components from molecular information on relatedness factor. Seven markers (mTcCIR 8, 66, 126, 212; TcSNP368, 697, 1370) on chromosomes 1 and 9 were identified for number of filled seeds (NSEED). A single marker was found for black pod resistance (mTcCIR280) on chromosome 3, whereas six markers on chromosomes 4, 5, 6, 8, and 10 were detected for WBD (mTcCIR91, 183; TcSNP375, 720, 1230 and 1374). It is expected that this association mapping study in cacao would contribute to the knowledge of the genetic determinism of cocoa traits and that the markers identified herein would prove useful in marker assisted breeding of cacao.

A single center's approach to discriminating donor versus host origin of renal neoplasia in the allograft kidney.

Renal cell carcinoma (RCC) in the allograft of kidney transplant recipient (KTR) patients is rare and may represent a de novo process arising from the transplanted organ or metastasis from a clinically undetectable host primary. Determination of host versus donor origin is important for staging and management. We report our experience utilizing Penta-C (PC) and Penta-D (PD) short-tandem repeat (STR) microsatellite analysis to discriminate between host and donor origin of RCC identified in renal allografts. We identified 5 KTR patients with RCC in the allograft kidney. The PC and PD microsatellite analysis was applied to tumor, host, and donor formalin-fixed, paraffin-embedded tissue sections and/or fresh blood leukocytes to identify the origin of the neoplastic cells. The PC and PD microsatellite alleles were robustly amplified in all samples. Each case showed one or more informative alleles indicating that the neoplastic cells originate from donor tissue. Allele frequency data indicate that by using both PC and PD markers, we will be able to discriminate between host and donor cell of origin in over 99% of cases. The PC and PD microsatellite analysis is a convenient, robust, and efficient strategy to determine donor versus host origin or RCC in transplant kidney specimens.

The complete mitochondrial genome of the Byasa alcinous (Lepidoptera: Papilionidae: Papilioninae).

The complete mitochondrial genome (mitogenome) of Byasa alcinous (Lepidoptera: Papilionidae: Papilioninae) is a circular molecule of 15,266 bp in length, containing 37 typical insect mitochondrial genes: 13 protein coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and a non-coding AT-rich region. Its gene order and arrangement are identical to all other available butterfly mitogenomes. All PCGs start with a typical ATN initiation codon, except for COI, which is initiated by the CGA codon as observed in other butterfly species. Ten PCGs terminate in the complete stop codon TAA or TAG, whereas the COI, COII and ND4 genes end with single T. Ten intergenic spacers (73 bp in total), and 12 overlapping regions (28 bp in total) are dispersed throughout the whole genome. The non-coding AT-rich region is 405 bp long and contains some conserved structures similar to those found in other butterfly mitogenomes, such as the motif ATAGA followed by a 12-bp poly-T stretch and a microsatellite-like (AT)14 element preceded by the ATTTA motif. Additionally, a 11-bp poly-T sequences and a microsatellite-like (AT)7 repeated elements are detected in this region.

Genetic biodiversity of Italian olives (Olea europaea) germplasm analyzed by SSR markers.

The olive is an important fruit species cultivated for oil and table olives in Italy and the Mediterranean basin. The conservation of cultivated plants in ex situ collections is essential for the optimal management and use of their genetic resources. The largest ex situ olive germplasm collection consists of approximately 500 Italian olive varieties and corresponding to 85% of the total Italian olive germplasm is maintained at the Consiglio per la Ricerca e sperimentazione per l'Agricoltura, Centro di Ricerca per l'Olivicoltura e l'Industria Olearia (CRA-OLI), in Italy. In this work, eleven preselected nuclear microsatellite markers were used to assess genetic diversity, population structure, and gene flows with the aim of assembling a core collection. The dendrogram obtained utilizing the unweighted pair group method highlights the presence of homonymy and synonymy in olive tree datasets analyzed in this study. 439 different unique genotype profiles were obtained with this combination of 11 loci nSSR, representing 89.8% of the varieties analyzed. The remaining 10.2% comprises different variety pairs in which both accessions are genetically indistinguishable. Clustering analysis performed using BAPS software detected seven groups in Italian olive germplasm and gene flows were determined among identified clusters. We proposed an Italian core collection of 23 olive varieties capturing all detected alleles at microsatellites. The information collected in this study regarding the CRA-OLI ex situ collection can be used for breeding programs, for germplasm conservation, and for optimizing a strategy for the management of olive gene pools.

PAAR-repeat proteins sharpen and diversify the type VI secretion system spike.

The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.

Populus euphratica: the transcriptomic response to drought stress.

Populus euphratica Olivier is widely established in arid and semiarid regions but lags in the availability of transcriptomic resources in response to water deficiency. To investigate the mechanisms that allow P. euphratica to maintain growth in arid regions, the responses of the plant to soil water deficit were analyzed at a systems level using physiological and pyrosequencing approaches. We generated 218,601 and 287,120 reads from non-stressed control and drought-stressed P. euphratica leaves respectively, totaling over 200 million base pairs. After assembly, 24,013 transcripts were yielded with an average length of 1,128 bp. We determined 2,279 simple sequence repeats, which may have possible information for understanding drought adaption of woody plants. Stomatal closure was inhibited under moderate drought to maintain a relatively high rate of CO2 assimilation and water transportation, which was supposed to be important for P. euphratica to maintain normal growth and develop vigorous root systems in an adverse environment. This was accompanied by strong transcriptional remodeling of stress-perception, signaling and transcription regulation, photoprotective system, oxidative stress detoxification, and other stress responsive genes. In addition, genes involved in stomatal closure inhibition, ascorbate-glutathione pathway and ubiquitin-proteasome system that may specially modulate the drought stress responses of P. euphratica are highlighted. Our analysis provides a comprehensive picture of how P. euphratica responds to drought stress at physiological and transcriptome levels which may help to understand molecular mechanisms associated with drought response and could be useful for genetic engineering of woody plants.

Assessment of morphological and molecular similarity of Hungarian white grape varieties.

The aim of this paper was to find possible link between molecular and morphological similarities of 38 Hungarian white grape varieties. Three aspects of morphological and molecular similarity were assessed in the study: comparison of the ordered variety pairs, assessment of molecular and morphological mean similarity differences and separation of varieties into similar groups by divisive cluster analysis to define (DIANA). Molecular similarity was calculated from binary data based on allele sizes obtained in DNA analysis. DNA fingerprints were determined at 9 SSR loci recommended by the European GrapeGen06 project. Morphological similarity was calculated on the basis of quantitative morphological descriptors. Morphological and molecular similarity values were ordered and categorized after pairwise comparison. Overall correlation was found to be weak but case by case assessment of the variety pairs confirmed some coincidence of molecular and morphological similarity. General similarity position of each variety was characterized by Mean Similarity Index (MSI). It was calculated as the mean of n-1 pair similarity values of the variety concerned. Varieties were ordered and compared by the difference of the index. Five varieties had low morphological and high molecular MSI meaning that they share several SSR marker alleles with the others but seems relatively distinct according to the expression of their morphological traits. Divisive cluster analysis was carried out to find similar groups. Eight and twelve cluster solutions proved to be sufficient to distinct varieties. Morphological and molecular similarity groups partly coincided according to the results. Several clusters reflected parent offspring relations but molecular clustering gave more realistic results concerning pedigree.

Evaluation of a new panel of six mononucleotide repeat markers for the detection of DNA mismatch repair-deficient tumours.

BACKGROUND: Microsatellite instability (MSI) is a molecular phenotype due to defective DNA mismatch repair (MMR) system. It is used to predict outcome of colorectal tumours and to screen tumours for Lynch syndrome (LS). A pentaplex panel composed of five mononucleotide markers has been largely recommended for determination of the MSI status. However, its sensitivity may be taken in default in occasional situations. The aim of the study was to optimise this panel for the detection of MSI. METHODS: We developed an assay allowing co-amplification of six mononucleotide repeat markers (BAT25, BAT26, BAT40, NR21, NR22, NR27) and one polymorphic dinucleotide marker (D3S1260) in a single reaction. Performances of the new panel were evaluated on a cohort of patients suspected of LS. RESULTS: We demonstrate that our assay is technically as easy to use as the pentaplex assay. The hexaplex panel shows similar performances for the identification of colorectal and non-MSH6-deficient tumours. On the other hand, the hexaplex panel has higher sensitivity for the identification of MSH6-deficient tumours (94.7% vs 84.2%) and MMR-deficient tumours other than colorectal cancer (92.9% vs 85.7%). CONCLUSION: The hexaplex panel could thus be an attractive alternative to the pentaplex panel for the identification of patients with LS.

Outcome and status of microsatellite stability in Japanese atomic bomb survivors with early gastric carcinoma.

BACKGROUND: In the decade after the 1945 atomic bombing of Hiroshima, a high incidence of leukemia was observed among atomic bomb survivors. However, the incidence of other cancers gradually increased, while that of leukemia decreased after this period. We evaluated the clinical outcome of early gastric cancer and microsatellite stability over a long-term period in atomic bomb survivors. METHODS: The results of surgical treatment for early gastric cancer were reviewed for 117 atomic bomb survivors and 394 control patients between 1995 and 2006. In addition, immunohistochemical staining for hMSH2 and hMLH1 expression was performed to evaluate the status of microsatellite stability in 57 atomic bomb survivors and 82 control patients. RESULTS: The long-term survival rate for early gastric cancer in atomic bomb survivors was significantly lower than that in control patients (p < 0.01). Multivariable analysis revealed that age and sex were significant and independent prognostic factors for early gastric cancer. Defective hMSH2 and/or hMLH1 expression was also significantly higher in survivors than in control patients (p < 0.001). Logistic regression analysis revealed that atomic bomb survivorship was related to defective hMSH2 and/or hMLH1 expression. CONCLUSIONS: The prognosis of early gastric cancer in atomic bomb survivors was poor and was related to age and sex, rather than to being an atomic bomb survivor. Furthermore, a higher rate of defective hMSH2 and/or hMLH1 expression was observed in the survivors.