Shades of white: new insights into tissue-resident leukocyte heterogeneity.

Populations of white blood cells (leukocytes) have been found in tissues and organs across the body, in states of both health and disease. The role leukocytes play within these tissues is often highly contested. For many leukocytes, there are studies outlining pro-inflammatory destructive functions, while other studies provide clear evidence of anti-inflammatory homeostatic activities of leukocytes within the same tissue. We discuss how this functional dissonance can be explained by leukocyte heterogeneity. Although cell morphology and surface receptor profiles are excellent methods to segregate cell types, the true degree of leukocyte heterogeneity that exists can only be appreciated by studying the variable and dynamic gene expression profile. Unbiased single-cell RNA sequencing profiling of tissue-resident leukocytes is transforming the way we understand leukocytes across health and disease. Recent investigations into adipose tissue-resident leukocytes have revealed unprecedented levels of heterogeneity among populations of macrophages. We use this example to pose emerging questions regarding tissue-resident leukocytes and review what is currently known (and unknown) about the diversity of tissue-resident leukocytes within different organs.

DIANA-miTED: a microRNA tissue expression database.

microRNAs (miRNAs) are short (∼23nt) single-stranded non-coding RNAs that act as potent post-transcriptional gene expression regulators. Information about miRNA expression and distribution across cell types and tissues is crucial to the understanding of their function and for their translational use as biomarkers or therapeutic targets. DIANA-miTED is the most comprehensive and systematic collection of miRNA expression values derived from the analysis of 15 183 raw human small RNA-Seq (sRNA-Seq) datasets from the Sequence Read Archive (SRA) and The Cancer Genome Atlas (TCGA). Metadata quality maximizes the utility of expression atlases, therefore we manually curated SRA and TCGA-derived information to deliver a comprehensive and standardized set, incorporating in total 199 tissues, 82 anatomical sublocations, 267 cell lines and 261 diseases. miTED offers rich instant visualizations of the expression and sample distributions of requested data across variables, as well as study-wide diagrams and graphs enabling efficient content exploration. Queries also generate links towards state-of-the-art miRNA functional resources, deeming miTED an ideal starting point for expression retrieval, exploration, comparison, and downstream analysis, without requiring bioinformatics support or expertise. DIANA-miTED is freely available at http://www.microrna.gr/mited.

SomaMutDB: a database of somatic mutations in normal human tissues.

De novo mutations, a consequence of errors in DNA repair or replication, have been reported to accumulate with age in normal tissues of humans and model organisms. This accumulation during development and aging has been implicated as a causal factor in aging and age-related pathology, including but not limited to cancer. Due to their generally very low abundance mutations have been difficult to detect in normal tissues. Only with recent advances in DNA sequencing of single-cells, clonal lineages or ultra-high-depth sequencing of small tissue biopsies, somatic mutation frequencies and spectra have been unveiled in several tissue types. The rapid accumulation of such data prompted us to develop a platform called SomaMutDB (https://vijglab.einsteinmed.org/SomaMutDB) to catalog the 2.42 million single nucleotide variations (SNVs) and 0.12 million small insertions and deletions (INDELs) thus far identified using these advanced methods in nineteen human tissues or cell types as a function of age or environmental stress conditions. SomaMutDB employs a user-friendly interface to display and query somatic mutations with their functional annotations. Moreover, the database provides six powerful tools for analyzing mutational signatures associated with the data. We believe such an integrated resource will prove valuable for understanding somatic mutations and their possible role in human aging and age-related diseases.

The Laccase Gene Family Mediate Multi-Perspective Trade-Offs during Tea Plant (Camellia sinensis) Development and Defense Processes.

Laccase (LAC) plays important roles in different plant development and defense processes. In this study, we identified laccase genes (CsLACs) in Camellia sinensis cv 'Longjing43' cultivars, which were classified into six subclades. The expression patterns of CsLACs displayed significant spatiotemporal variations across different tissues and developmental stages. Most members in subclades II, IV and subclade I exhibited contrasting expression patterns during leaf development, consistent with a trade-off model for preferential expression in the early and late developmental stages. The extensive transcriptional changes of CsLACs under different phytohormone and herbivore treatment were observed and compared, with the expression of most genes in subclades I, II and III being downregulated but genes in subclades IV, V and VI being upregulated, suggesting a growth and defense trade-off model between these subclades. Taken together, our research reveal that CsLACs mediate multi-perspective trade-offs during tea plant development and defense processes and are involved in herbivore resistance in tea plants. More in-depth research of CsLACs upstream regulation and downstream targets mediating herbivore defense should be conducted in the future.

Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing.

Apoptosis is associated with numerous phenotypical characteristics, and is thus studied with many tools. In this study, we compared two broadly used apoptotic assays: TUNEL and staining with an antibody targeting the activated form of an effector caspase. To compare them, we developed a protocol based on commonly used tools such as image filtering, z-projection, and thresholding. Even though it is commonly used in image-processing protocols, thresholding remains a recurring problem. Here, we analyzed the impact of processing parameters and readout choice on the accuracy of apoptotic signal quantification. Our results show that TUNEL is quite robust, even if image processing parameters may not always allow to detect subtle differences of the apoptotic rate. On the contrary, images from anti-cleaved caspase staining are more sensitive to handle and necessitate being processed more carefully. We then developed an open-source Fiji macro automatizing most steps of the image processing and quantification protocol. It is noteworthy that the field of application of this macro is wider than apoptosis and it can be used to treat and quantify other kind of images.

Tissue distribution of stem cell factor in adults.

Stem cell factor (SCF) is an essential cytokine during development and is necessary for gametogenesis, hematopoiesis, mast cell development, stem cell function, and melanogenesis. Here, we measure SCF concentration and distribution in adult humans and mice using gene expression analysis, tissue staining, and organ protein lysates. We demonstrate continued SCF expression in many cell types and tissues into adulthood. Tissues with high expression in adult humans included stomach, spleen, kidney, lung, and pancreas. In mice, we found high SCF expression in the esophagus, ovary, uterus, kidney, and small intestine. Future studies may correlate our findings of increased, organ-specific SCF concentrations within adult tissues with increased risk of SCF/CD117-related disease.

Effects of dietary active soybean trypsin inhibitors on pancreatic weights, histology and expression of STAT3 and receptors for androgen and estrogen in different tissues of rats.

Our previous study showed that soy milks could contain high levels of active soybean trypsin inhibitors (SBTI) if they were not properly processed. This study investigated the effects of consuming active SBTI on pancreatic weights, histology, trypsinogen production and expression of STAT3, receptors for androgen (AR) and estrogen (ER) in pancreas, liver and uterus of rats. Weanling Sprague-Dawley rats were randomly divided into 3 groups (8 females and 8 males/group) and fed diets containing either 20% casein protein (Casein) or 20% soy protein (SP) in the presence of high (1.42 BAEE unit/µg, SP + SBTI) or low (0.2 BAEE unit/µg, SP-SBTI) levels of active SBTI for 8 weeks. Ingestion of SP + SBTI diet markedly increased pancreatic weights and trypsinogen content (p < 0.01), and caused acinar cell hypertrophy, and reduced pancreatic STAT3, p-STAT3, AR and ERß content, and increased uterine ERα and ERß compared to the Casein or SP-SBTI diets (p < 0.05). The two SP-containing diets lowered hepatic STAT3, p-STAT3, and pancreatic ERα, and increased hepatic ERα and ERß content in the female rats compared to the Casein diet (p < 0.05). This study demonstrated for the first time that consumption of high level of active SBTI not only increased pancreatic weights and acinar cell secretions, but also attenuated the expression of pancreatic STAT3, p-STAT3, AR, and ERß proteins in both sexes and increased uterine ERα and ERß content, and that dietary soy protein affected hepatic STAT3, p-STAT3, ERα and ERß in a gender-dependent manner.

Exosomal miR-125b-5p deriving from mesenchymal stem cells promotes tubular repair by suppression of p53 in ischemic acute kidney injury.

Mesenchymal stem cells-derived exosomes (MSC-exos) have attracted great interest as a cell-free therapy for acute kidney injury (AKI). However, the in vivo biodistribution of MSC-exos in ischemic AKI has not been established. The potential of MSC-exos in promoting tubular repair and the underlying mechanisms remain largely unknown. Methods: Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of human umbilical cord mesenchymal stem cells (hucMSCs) derived exosomes. The biodistribution of MSC-exos in murine ischemia/reperfusion (I/R) induced AKI was imaged by the IVIS spectrum imaging system. The therapeutic efficacy of MSC-exos was investigated in renal I/R injury. The cell cycle arrest, proliferation and apoptosis of tubular epithelial cells (TECs) were evaluated in vivo and in HK-2 cells. The exosomal miRNAs of MSC-exos were profiled by high-throughput miRNA sequencing. One of the most enriched miRNA in MSC-exos was knockdown by transfecting miRNA inhibitor to hucMSCs. Then we investigated whether this candidate miRNA was involved in MSC-exos-mediated tubular repair. Results:Ex vivo imaging showed that MSC-exos was efficiently homing to the ischemic kidney and predominantly accumulated in proximal tubules by virtue of the VLA-4 and LFA-1 on MSC-exos surface. MSC-exos alleviated murine ischemic AKI and decreased the renal tubules injury in a dose-dependent manner. Furthermore, MSC-exos significantly attenuated the cell cycle arrest and apoptosis of TECs both in vivo and in vitro. Mechanistically, miR-125b-5p, which was highly enriched in MSC-exos, repressed the protein expression of p53 in TECs, leading to not only the up-regulation of CDK1 and Cyclin B1 to rescue G2/M arrest, but also the modulation of Bcl-2 and Bax to inhibit TEC apoptosis. Finally, inhibiting miR-125b-5p could mitigate the protective effects of MSC-exos in I/R mice. Conclusion: MSC-exos exhibit preferential tropism to injured kidney and localize to proximal tubules in ischemic AKI. We demonstrate that MSC-exos ameliorate ischemic AKI and promote tubular repair by targeting the cell cycle arrest and apoptosis of TECs through miR-125b-5p/p53 pathway. This study provides a novel insight into the role of MSC-exos in renal tubule repair and highlights the potential of MSC-exos as a promising therapeutic strategy for AKI.

Cyclin-dependent Kinase 1 and Aurora Kinase choreograph mitotic storage and redistribution of a growth factor receptor.

Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis.

The BMP signaling gradient is interpreted through concentration thresholds in dorsal-ventral axial patterning.

Bone Morphogenetic Protein (BMP) patterns the dorsal-ventral (DV) embryonic axis in all vertebrates, but it is unknown how cells along the DV axis interpret and translate the gradient of BMP signaling into differential gene activation that will give rise to distinct cell fates. To determine the mechanism of BMP morphogen interpretation in the zebrafish gastrula, we identified 57 genes that are directly activated by BMP signaling. By using Seurat analysis of single-cell RNA sequencing (scRNA-seq) data, we found that these genes are expressed in at least 3 distinct DV domains of the embryo. We distinguished between 3 models of BMP signal interpretation in which cells activate distinct gene expression through interpretation of thresholds of (1) the BMP signaling gradient slope; (2) the BMP signal duration; or (3) the level of BMP signal activation. We tested these 3 models using quantitative measurements of phosphorylated Smad5 (pSmad5) and by examining the spatial relationship between BMP signaling and activation of different target genes at single-cell resolution across the embryo. We found that BMP signaling gradient slope or BMP exposure duration did not account for the differential target gene expression domains. Instead, we show that cells respond to 3 distinct levels of BMP signaling activity to activate and position target gene expression. Together, we demonstrate that distinct pSmad5 threshold levels activate spatially distinct target genes to pattern the DV axis.

Ectoderm to mesoderm transition by down-regulation of actomyosin contractility.

Collective migration of cohesive tissues is a fundamental process in morphogenesis and is particularly well illustrated during gastrulation by the rapid and massive internalization of the mesoderm, which contrasts with the much more modest movements of the ectoderm. In the Xenopus embryo, the differences in morphogenetic capabilities of ectoderm and mesoderm can be connected to the intrinsic motility of individual cells, very low for ectoderm, high for mesoderm. Surprisingly, we find that these seemingly deep differences can be accounted for simply by differences in Rho-kinases (Rock)-dependent actomyosin contractility. We show that Rock inhibition is sufficient to rapidly unleash motility in the ectoderm and confer it with mesoderm-like properties. In the mesoderm, this motility is dependent on two negative regulators of RhoA, the small GTPase Rnd1 and the RhoGAP Shirin/Dlc2/ArhGAP37. Both are absolutely essential for gastrulation. At the cellular and tissue level, the two regulators show overlapping yet distinct functions. They both contribute to decrease cortical tension and confer motility, but Shirin tends to increase tissue fluidity and stimulate dispersion, while Rnd1 tends to favor more compact collective migration. Thus, each is able to contribute to a specific property of the migratory behavior of the mesoderm. We propose that the "ectoderm to mesoderm transition" is a prototypic case of collective migration driven by a down-regulation of cellular tension, without the need for the complex changes traditionally associated with the epithelial-to-mesenchymal transition.

Functional characterization, tissue distribution and nutritional regulation of the Elovl4 gene in golden pompano, Trachinotus ovatus (Linnaeus, 1758).

The elongases of very long-chain fatty acids (Elovls) are involved in the rate-limiting of the carbon chain elongation reaction in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovls family, Elovl4, has been regarded as a critical enzyme involved in the biosynthesis pathway of polyunsaturated fatty acids (PUFAs). To explore the role of Elovl4 in PUFA synthesis in Trachinotus ovatus, the cDNA of the Elovl4b gene is cloned from T. ovatus (ToElovl4b). The ORF of ToElovl4b was 918 bp and encoded 305 amino acid (aa) protein sequences. Sequence alignment showed that the deduced amino acids contained significant structural features of the Elovl4 family, such as a histidine box motif (HXXHH), multiple transmembrane domains and an endoplasmic reticulum (ER) retention signal. Moreover, phylogenetic analysis revealed that ToElovl4b was highly conserved with that of Rachycentron canadum Elovl4b. Moreover, heterologous expression in yeast demonstrated that ToElovl4b could efficiently elongate 18:2n-6, 18:3n-6 and 20:5n-3 FAs up to 20:2n-6, 20:3n-6 and 22:5n-3, respectively. Furthermore, the tissue expression profile indicated that mRNA expression of ToElovl4b was higher in the gonads and brain than in other tissues. Additionally, nutritional regulation suggested the highest mRNA levels of ToElovl4b in liver and brain were under feeding with 1:1 FO-SO (fish oil, FO; soybean oil, SO) and 1:1 FO-CO (corn oil, CO)), respectively. These new insights were useful for understanding the molecular basis and regulation of LC-PUFA biosynthesis in fish.

Planar cell polarity: moving from single cells to tissue-scale biology.

Planar cell polarity (PCP) reflects cellular orientation within the plane of an epithelium. PCP is crucial during many biological patterning processes and for organ function. It is omnipresent, from convergent-extension mechanisms during early development through to terminal organogenesis, and it regulates many aspects of cell positioning and orientation during tissue morphogenesis, organ development and homeostasis. Suzanne Eaton used the power of Drosophila as a model system to study PCP, but her vision of, and impact on, PCP studies in flies translates to all animal models. As I highlight here, Suzanne's incorporation of quantitative biophysical studies of whole tissues, integrated with the detailed cell biology of PCP phenomena, completely changed how the field studies this intriguing feature. Moreover, Suzanne's impact on ongoing and future PCP studies is fundamental, long-lasting and transformative.

Protein fractional synthesis rates within tissues of high- and low-active mice.

With the rise in physical inactivity and its related diseases, it is necessary to understand the mechanisms involved in physical activity regulation. Biological factors regulating physical activity are studied to establish a possible target for improving the physical activity level. However, little is known about the role metabolism plays in physical activity regulation. Therefore, we studied protein fractional synthesis rate (FSR) of multiple organ tissues of 12-week-old male mice that were previously established as inherently low-active (n = 15, C3H/HeJ strain) and high-active (n = 15, C57L/J strain). Total body water of each mouse was enriched to 5% deuterium oxide (D2O) via intraperitoneal injection and maintained with D2O enriched drinking water for about 24 h. Blood samples from the jugular vein and tissues (kidney, heart, lung, muscle, fat, jejunum, ileum, liver, brain, skin, and bone) were collected for enrichment analysis of alanine by LC-MS/MS. Protein FSR was calculated as -ln(1-enrichment). Data are mean±SE as fraction/day (unpaired t-test). Kidney protein FSR in the low-active mice was 7.82% higher than in high-active mice (low-active: 0.1863±0.0018, high-active: 0.1754±0.0028, p = 0.0030). No differences were found in any of the other measured organ tissues. However, all tissues resulted in a generally higher protein FSR in the low-activity mice compared to the high-activity mice (e.g. lung LA: 0.0711±0.0015, HA: 0.0643±0.0020, heart LA: 0.0649± 0.0013 HA: 0.0712±0.0073). Our observations suggest that high-active mice in most organ tissues are no more inherently equipped for metabolic adaptation than low-active mice, but there may be a connection between protein metabolism of kidney tissue and physical activity level. In addition, low-active mice have higher organ-specific baseline protein FSR possibly contributing to the inability to achieve higher physical activity levels.

First echinoderm alpha-amylase from a tropical sea cucumber (Holothuria leucospilota): Molecular cloning, tissue distribution, cellular localization and functional production in a heterogenous E.coli system with codon optimization.

Holothuria leucospilota (Echinodermata: Holothuroidea) is a widespread tropical sea cucumber with strong value for the ecological restoration of coral reefs. Therefore, some studies regarding the artificial breeding and cultivation of H. leucospilota have been undertaken recently. However, the biological functions of the digestive system of this species have not been elucidated. In this study, a cDNA coding for α-amylase, an indicator of digestive maturity in animals, was identified from H. leucospilota and designated Hl-Amy. The full-length cDNA of the Hl-Amy gene, which is 1734 bp in length with an open reading frame (ORF) of 1578 bp, encodes a 525 amino acid (a.a.) protein with a deduced molecular weight of 59.34 kDa. According to the CaZy database annotation, Hl-Amy belongs to the class of GH-H with the official nomenclature of α-amylase (EC 3.2.1.1) or 4-α-D-glucan glucanohydrolase. The Hl-Amy protein contains a signal peptide at the N-terminal followed by a functional amylase domain, which includes the catalytic activity site. The mRNA expression of Hl-Amy was abundantly exhibited in the intestine, followed by the transverse vessel with a low level, but was hardly detected in other selected tissues. During embryonic and larval development, Hl-Amy was constitutively expressed in all stages, and the highest expression level was observed in the blastula. By in situ hybridization (ISH), positive Hl-Amy signals were observed in different parts of the three different intestinal segments (foregut, midgut and hindgut). The Hl-Amy recombinant protein was generated in an E. coli system with codon optimization, which is necessary for Hl-Amy successfully expressed in this heterogenous system. The Hl-Amy recombinant protein was purified by immobilized metal ion affinity chromatography (IMAC), and its activity of starch hydrolysis was further detected. The optimal temperatures and pH for Hl-Amy recombinant protein were 55°C and 6.0, respectively, with an activity of 62.2 U/mg. In summary, this current study has filled a knowledge gap on the biological function and expression profiles of an essential digestive enzyme in sea cucumber, which may encourage future investigation toward rationalized diets for H. leucospilota in artificial cultivation, and optimized heterogenous prokaryotic systems for producing recombinant enzymes of marine origins.

Transcriptome Profiling across Five Tissues of Giant Panda.

Gene differential expression studies can serve to explore and understand the laws and characteristics of animal life activities, and the difference in gene expression between different animal tissues has been well demonstrated and studied. However, for the world-famous rare and protected species giant panda (Ailuropoda melanoleuca), only the transcriptome of the blood and spleen has been reported separately. Here, in order to explore the transcriptome differences between the different tissues of the giant panda, transcriptome profiles of the heart, liver, spleen, lung, and kidney from five captive giant pandas were constructed with Illumina HiSeq 2500 platform. The comparative analysis of the intertissue gene expression patterns was carried out based on the generated RNA sequencing datasets. Analyses of Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network were performed according to the identified differentially expressed genes (DEGs). We generated 194.52 GB clean base data from twenty-five sequencing libraries and identified 18,701 genes, including 3492 novel genes. With corrected p value <0.05 and |log2FoldChange| >2, we finally obtained 921, 553, 574, 457, and 638 tissue-specific DEGs in the heart, liver, spleen, lung, and kidney, respectively. In addition, we identified TTN, CAV3, LDB3, TRDN, and ACTN2 in the heart; FGA, AHSG, and SERPINC1 in the liver; CD19, CD79B, and IL21R in the spleen; NKX2-4 and SFTPB in the lung; GC and HRG in the kidney as hub genes in the PPI network. The results of the analyses showed a similar gene expression pattern between the spleen and lung. This study provided for the first time the heart, liver, lung, and kidney's transcriptome resources of the giant panda, and it provided a valuable resource for further genetic research or other potential research.

siRNA based drug design, quality, delivery and clinical translation.

Gene silencing by RNA interference represents a promising therapeutic approach. The development of carriers, e.g., polymers, lipids, peptides, antibodies, aptamers, small molecules, exosome and red blood cells, is crucial for the systemic delivery of siRNA. Cell-specific targeting ligands in the nano-carriers can improve the pharmacokinetics, biodistribution, and selectivity of siRNA therapeutics. The safety, effectiveness, quality and prosperity of production and manufacturing are important considerations for selecting the appropriate siRNA carriers. Efficacy of systemic delivery of siRNA requires considerations of trafficking through the blood, off-target effects, innate immune response and endosomal escape avoiding lysosomal degradation for entering into RNAi process. Multifunctional nanocarriers with stimuli-responsive properties such as pH, magnetic and photo-sensitive segments can enhance the efficacy of siRNA delivery. The improved preclinical characterization of suitable siRNA drugs, good laboratory practice, that reduce the differences between in vitro and in vivo results may increase the success of siRNA drugs in clinical settings.

AMP-activated protein kinase in the grass carp Ctenopharyngodon idellus: Molecular characterization, tissue distribution and mRNA expression in response to overwinter starvation stress.

Adenosine monophosphate-activated protein kinase (AMPK) is the main energy sensor in mammals, but limited information is available regarding its role as an energy sensor in nutrient-restricted fish particularly in period of overwinter starvation. The present study aimed to investigate the role of AMPK in the grass carp Ctenopharyngodon idellus through characterization of AMPK full-length cDNAs and the measurement of transcriptional activity in response to overwinter starvation. AMPK is a heterotrimeric serine/threonine kinase that consists of a catalytic alpha (α) subunit complexed with two regulatory subunits, beta (ß) and gamma (γ). In our study, we identified nine isoforms of the AMPK family in grass carp and obtained their complete coding sequences (CDS). In the grass carp, the α subunit is encoded by two isoforms (α1 and α2). The ß and γ subunits are encoded by three (ß1a, ß1b, ß2) and four isoforms (γ1, γ2a, γ2b, γ3), respectively. AMPK isoforms in grass carp possess structural features similar to mammalian AMPK and exhibit a high degree of homology with other fish and vertebrate AMPK sequences. The mRNA of nine grass carp AMPK isoforms were found to be expressed in a wide range of tissues in vivo, but the abundance of each AMPK mRNA demonstrated a tissue-dependent expression pattern, indicating that they might be key complexes playing the role of energy metabolism sensors during overwinter starvation conditions. Compared to expression levels in control fish (week 0), the expression of various AMPK isoforms significantly increased in the hepatopancreas of fish exposed to 1 week or more of overwinter starvation conditions as follows: week 1 (AMPK α1 and AMPK α2), week8 (AMPK ß1b and AMPK γ2b), week 12 (AMPK ß2 and AMPK γ1), and week 16 (AMPK ß1a, AMPK γ2a, and AMPK γ3). Additionally, compared to expression levels in control fish (week 0), the expression of various AMPK isoforms significantly increased in the adipose tissue of fish exposed to 1 week or more of overwinter starvation conditions as follows: week 1(AMPK ß1a and AMPK ß1b), week 4 (AMPK α1, AMPK α2, AMPK γ1, AMPK γ2b and AMPK γ3), and week 8 (AMPK ß2 and AMPK γ2a). Further in vitro analysis revealed that the mRNA levels of AMPK isoforms in hepatocytes (AMPK α1, AMPK α2, AMPK ß1a, AMPK ß1b, AMPK ß2, AMPK γ2b and AMPK γ3) and adipocytes (AMPK γ2a, AMPK γ2b and AMPK γ3) changed significantly with in the first 24 h of exposure to the overwinter starvation conditions. These findings confirm that nine AMPK subunits are present in grass carp and that all encode proteins with conserved functional domains. The nine AMPK subunits are all regulated at the transcriptional levels to manage excess energy expenditure during overwinter starvation stress.

Germline and somatic DNA repair gene alterations in prostate cancer.

BACKGROUND: Emerging evidence has suggested that DNA repair gene alterations may be important in prostate cancer pathogenesis. In the current study, the authors sought to characterize alterations in DNA repair pathway genes in both primary and metastatic prostate tumors with attention to tissue distribution as well as specific genomic alterations. METHODS: The authors studied the distribution and type of alterations in 24 genes that are considered important for DNA repair in 944 prostate cancers harvested from localized and metastatic tumors. Tumor DNA underwent hybrid capture for all coding exons of 287 or 395 cancer-related genes plus select introns from 19 or 31 genes frequently rearranged in cancer. Captured libraries were sequenced to a median exon coverage depth of >×500. Specific genomic alterations were characterized and the frequencies of mutations by tissue site (prostate vs metastases) were compared using logistic regression. RESULTS: A total of 152 patients from the cohort of 944 men (16%) harbored a germline or somatic mutation in ≥1 DNA repair genes. The most frequently mutated genes were BRCA2 (11.4%) and ATM (5.8%), followed by MSH6 (2.5%) and MSH2 (2.1%). Mutations were identified in approximately 20.1% of primary prostate tumors compared with 18.8% of bone metastases. When stratified by tissue site, the highest rates of DNA repair mutations were found in solid organ metastases, including brain and visceral metastases, compared with prostate. CONCLUSIONS: DNA repair gene mutations are more common in metastatic than localized prostate tumors. Visceral and other solid organ metastases appear enriched for these mutations compared with localized tumors or bone and lymph node metastases.