DNA double-strand breaks & poptosis in the testis

During spermatogenesis, DNA damage is a naturally occurring event. At a certain stage, during the first meiotic prophase, DNA breaks are endogenously induced and even required for meiotic recombination. We studied these DNA breaks but also used ionizing radiation (IR) to induce DNA double-strand breaks (DSBs). Spermatogenesis is a multi step process that begins with mitotically dividing diploid stem cells and ends with highly differentiated haploid elongated spermatids. Every phase of spermatogenesis is tightly regulated, and during the process the different spermatogenic cells completely change in chromatin structure and response to DNA damage. A proper response to DNA damage is essential for germ cell development and loss-of-function mutations of many DNA damage response proteins lead to abrogation of spermatogenesis. We studied the testicular response to IR and DNA double-strand breaks (DSBs) mainly at two levels: at the spermatogonial level, during which the germ cells undergo mitotic divisions, and at the first meiotic prophase during which DSBs required for meiotic recombination are induced. Firstly, we investigated whether spermatogonia, being connected by cytoplasmic intercellular bridges, can individually undergo apoptosis in response IR or whether they, like during germ cell density, degenerate in interconnected clusters. In response to IR the germ cells were found to individually undergo apoptosis. Secondly, we investigated whether the c-Abl-p73 pathway, and possibly p63, can be an alternative apoptotic pathway as described for p53 deficient mice. Therefore we studied the testicular expression patterns and interactions of these proteins before and after irradiation. c-Abl was found to activate p73, but not p63, in the testis in response to IR and the c-Abl-p73 pathway can be an additional apoptotic pathway for damaged male germ cells. Furthermore, we studied the expression pattern of ?-H2AX, which is commonly used as a marker for DSBs, in the testis before and after irradiation. We also investigated whether ?-H2AX signaling is connected with p53 induced spermatogonial apoptosis and whether DNA-PK is required for p53 signaling and ?-H2AX foci formation and signaling during spermatogenesis. In the testis, ?-H2AX appeared not only to be present at DSBs but also just before the onset of meiosis and at the X&Y chromosomes, spermatogonial ?-H2AX appeared to be connected to p53 and totally independent of DNA-PK. We also studied non-homologues end joining (NHEJ) and the protein complex DNA-PK in the testis. We localized the three subunits of DNA-PK; Ku70, Ku86 and DNA-PKcs, in different cell types during spermatogenesis and found that the Ku heterodimer is absent during meiotic recombination, thereby preventing NHEJ to happen instead of meiotic recombination. Additionally, using DNA-PKcs deficient scid mice, we described a function for DNA-PKcs in the spermatogonial DNA damage response and during meiosis. Finally, we studied expression and activation of ATM, which lies at the basis of most responses to DSBs, during spermatogenesis. Using an antibody specific for phosphorylated ATM we were able to compare ATM activation in response to IR with the presence of endogenously active ATM during the meiosis. ATM phosphorylation in the testis was found to be coherent with p53 induction, however, during meiosis ATM seems to be phosphorylated at different unknown site

Tumors Widely Express Hundreds of Embryonic Germline Genes.

We have recently described a class of 756 genes that are widely expressed in cancers, but are normally restricted to adult germ cells, referred to as germ cell cancer genes (GC genes). We hypothesized that carcinogenesis involves the reactivation of biomolecular processes and regulatory mechanisms that, under normal circumstances, are restricted to germline development. This would imply that cancer cells share gene expression profiles with primordial germ cells (PGCs). We therefore compared the transcriptomes of human PGCs (hPGCs) and PGC-like cells (PGCLCs) with 17,382 samples from 54 healthy somatic tissues (GTEx) and 11,003 samples from 33 tumor types (TCGA), and identified 672 GC genes, expanding the known GC gene pool by 387 genes (51%). We found that GC genes are expressed in clusters that are often expressed in multiple tumor types. Moreover, the amount of GC gene expression correlates with poor survival in patients with lung adenocarcinoma. As GC genes specific to the embryonic germline are not expressed in any adult tissue, targeting these in cancer treatment may result in fewer side effects than targeting conventional cancer/testis (CT) or GC genes and may preserve fertility. We anticipate that our extended GC dataset enables improved understanding of tumor development and may provide multiple novel targets for cancer treatment development

Dysbiosis of bifidobacteria and Clostridium cluster XIVa in the cystic fibrosis fecal microbiota

BACKGROUND: Recurrent antimicrobial interventions and disease-related intestinal dysfunction are suspected to contribute to the dysbiosis of the gastrointestinal microbial ecosystem in patients with cystic fibrosis (CF). The present study set out to detect and identify microbial discriminants in the gut microbiota composition that are associated with CF-related intestinal dysbiosis. METHODS: An in-depth description of CF-associated gut dysbiosis was obtained by screening denaturing gradient gel electrophoresis (DGGE) fingerprints for potentially discriminating bacterial species, and quantification by means of real-time PCR analyses using group-specific primers. RESULTS: A total of 8 DGGE band-classes assigned to the genus Bifidobacterium (n=3), and members of Clostridium clusters XIVa (n=3) and IV (n=2), were significantly (p<0.05) underrepresented in samples of patients with CF. Real-time PCR analyses confirmed a significantly lower abundance and temporal stability of bifidobacteria and Clostridium cluster XIVa in the faecal microbiota of patients with CF. CONCLUSION: This study is the first to report specific microbial determinants of dysbiosis in patients with CF

Preantral follicular atresia occurs mainly through autophagy, while antral follicles degenerate mostly through apoptosis

There is a general agreement that granulosa cell apoptosis is the cause of antral follicle attrition. Less clear is whether this pathway is also activated in case of preantral follicle degeneration, as several reports mention that the incidence of granulosa cell apoptosis in preantral follicles is negligible. Our objective is therefore to determine which cell-death pathways are involved in preantral and antral follicular degeneration.Atretic preantal and antral follicles were investigated using immunohistochemistry and laser-capture microdissection followed by quantitative real-time reverse transcription polymerase chain reaction. Microtubule-associated light-chain protein 3 (LC3), sequestosome 1 (SQSTM1/P62), Beclin1, autophagy-related protein 7 (ATG7), and cleaved caspase 3 (cCASP3) were used as markers for autophagy and apoptosis, respectively. P62 immunostaining was far less intense in granulosa cells of atretic compared to healthy preantral follicles, while no difference in LC3 and BECLIN1 immunostaining intensity was observed. This difference in P62 immunostaining was not observed in atretic antral follicles. mRNA levels of LC3 and P62 were not different between healthy and atretic (pre)antral follicles. ATG7 immunostaining was observed in granulosa cells of preantral atretic follicles, not in granulosa cells of degenerating antral follicles. The number of cCASP3-positive cells was negligible in preantral atretic follicles, while numerous in atretic antral follicles. Taken together, we conclude that preantral and antral follicular atresia is the result of activation of different cell-death pathways as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy.</p

Imaging practice in low-grade gliomas among European specialized centers and proposal for a minimum core of imaging

Objective: Imaging studies in diffuse low-grade gliomas (DLGG) vary across centers. In order to establish a minimal core of imaging necessary for further investigations and clinical trials in the field of DLGG, we aimed to establish the status quo within specialized European centers. Methods: An online survey composed of 46 items was sent out to members of the European Low-Grade Glioma Network, the European Association of Neurosurgical Societies, the German Society of Neurosurgery and the Austrian Society of Neurosurgery. Results: A total of 128 fully completed surveys were received and analyzed. Most centers (n=96, 75%) were academic and half of the centers (n=64, 50%) adhered to a dedicated treatment program for DLGG. There were national differences regarding the sequences enclosed in MRI imaging and use of PET, however most included T1 (without and with contrast, 100%), T2 (100%) and TIRM or FLAIR (20, 98%). DWI is performed by 80% of centers and 61% of centers regularly performed PWI.ConclusionA minimal core of imaging composed of T1 (w/wo contrast), T2, TIRM/FLAIR, PWI and DWI could be identified. All morphologic images should be obtained in a slice thickness of 3mm. No common standard could be obtained regarding advanced MRI protocols and PET. Importance of the study: We believe that our study makes a significant contribution to the literature because we were able to determine similarities in numerous aspects of LGG imaging. Using the proposed minimal core of imaging in clinical routine will facilitate future cooperative studies

Imaging practice in low-grade gliomas among European specialized centers and proposal for a minimum core of imaging.

OBJECTIVE: Imaging studies in diffuse low-grade gliomas (DLGG) vary across centers. In order to establish a minimal core of imaging necessary for further investigations and clinical trials in the field of DLGG, we aimed to establish the status quo within specialized European centers. METHODS: An online survey composed of 46 items was sent out to members of the European Low-Grade Glioma Network, the European Association of Neurosurgical Societies, the German Society of Neurosurgery and the Austrian Society of Neurosurgery. RESULTS: A total of 128 fully completed surveys were received and analyzed. Most centers (n = 96, 75%) were academic and half of the centers (n = 64, 50%) adhered to a dedicated treatment program for DLGG. There were national differences regarding the sequences enclosed in MRI imaging and use of PET, however most included T1 (without and with contrast, 100%), T2 (100%) and TIRM or FLAIR (20, 98%). DWI is performed by 80% of centers and 61% of centers regularly performed PWI. CONCLUSION: A minimal core of imaging composed of T1 (w/wo contrast), T2, TIRM/FLAIR, PWI and DWI could be identified. All morphologic images should be obtained in a slice thickness of ≤ 3 mm. No common standard could be obtained regarding advanced MRI protocols and PET. IMPORTANCE OF THE STUDY: We believe that our study makes a significant contribution to the literature because we were able to determine similarities in numerous aspects of LGG imaging. Using the proposed "minimal core of imaging" in clinical routine will facilitate future cooperative studies

Detection and localization of early- and late-stage cancers using platelet RNA

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I–IV cancer patients and in half of 352 stage I–III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening

DNA double-strand breaks & poptosis in the testis

During spermatogenesis, DNA damage is a naturally occurring event. At a certain stage, during the first meiotic prophase, DNA breaks are endogenously induced and even required for meiotic recombination. We studied these DNA breaks but also used ionizing radiation (IR) to induce DNA double-strand breaks (DSBs). Spermatogenesis is a multi step process that begins with mitotically dividing diploid stem cells and ends with highly differentiated haploid elongated spermatids. Every phase of spermatogenesis is tightly regulated, and during the process the different spermatogenic cells completely change in chromatin structure and response to DNA damage. A proper response to DNA damage is essential for germ cell development and loss-of-function mutations of many DNA damage response proteins lead to abrogation of spermatogenesis. We studied the testicular response to IR and DNA double-strand breaks (DSBs) mainly at two levels: at the spermatogonial level, during which the germ cells undergo mitotic divisions, and at the first meiotic prophase during which DSBs required for meiotic recombination are induced. Firstly, we investigated whether spermatogonia, being connected by cytoplasmic intercellular bridges, can individually undergo apoptosis in response IR or whether they, like during germ cell density, degenerate in interconnected clusters. In response to IR the germ cells were found to individually undergo apoptosis. Secondly, we investigated whether the c-Abl-p73 pathway, and possibly p63, can be an alternative apoptotic pathway as described for p53 deficient mice. Therefore we studied the testicular expression patterns and interactions of these proteins before and after irradiation. c-Abl was found to activate p73, but not p63, in the testis in response to IR and the c-Abl-p73 pathway can be an additional apoptotic pathway for damaged male germ cells. Furthermore, we studied the expression pattern of ?-H2AX, which is commonly used as a marker for DSBs, in the testis before and after irradiation. We also investigated whether ?-H2AX signaling is connected with p53 induced spermatogonial apoptosis and whether DNA-PK is required for p53 signaling and ?-H2AX foci formation and signaling during spermatogenesis. In the testis, ?-H2AX appeared not only to be present at DSBs but also just before the onset of meiosis and at the X&Y chromosomes, spermatogonial ?-H2AX appeared to be connected to p53 and totally independent of DNA-PK. We also studied non-homologues end joining (NHEJ) and the protein complex DNA-PK in the testis. We localized the three subunits of DNA-PK; Ku70, Ku86 and DNA-PKcs, in different cell types during spermatogenesis and found that the Ku heterodimer is absent during meiotic recombination, thereby preventing NHEJ to happen instead of meiotic recombination. Additionally, using DNA-PKcs deficient scid mice, we described a function for DNA-PKcs in the spermatogonial DNA damage response and during meiosis. Finally, we studied expression and activation of ATM, which lies at the basis of most responses to DSBs, during spermatogenesis. Using an antibody specific for phosphorylated ATM we were able to compare ATM activation in response to IR with the presence of endogenously active ATM during the meiosis. ATM phosphorylation in the testis was found to be coherent with p53 induction, however, during meiosis ATM seems to be phosphorylated at different unknown site

Mutations causing specific arrests in the development of mouse primordial germ cells and gonocytes

This review focuses on those mouse mutations that cause an effect on the morphology, viability, and/or behavior of primordial germ cells (PGCs) and gonocytes at specific steps of their fetal development up to the start of spermatogenesis, a few days after birth. To restrict the area covered, mice with mutations that cause abnormal hormone levels or mutations of genes not expressed in germ cells that secondarily cause spermatogenic problems are not discussed. To make our literature search as comprehensive as possible, Pubmed was searched for "(primordial germ cells OR prospermatogonia OR prespermatogonia OR gonocytes OR spermatogonia ormeiosis or spermiogenesis or spermatogenesis) AND mouse AND (knockout or mutant or transgenic)." This search started at 2003 as mutants created earlier were already retrieved for a previous review. The resulting citations were then further selected for complete or partial arrests at the level of PGCs and/or gonocytes. Fifty-nine protein coding genes and two miRNA coding genes were found that arrest the development of PGCs and gonocytes at specific steps providing a better insight into the regulation of the development of these cells. As to be expected, often problems in fetal germ cell development have an effect on the fertility of the mice at adulthood. Summary Sentence: Many gene mutations cause an arrest at specific developmental steps of mouse primordial germ cells and gonocytes, also called prospermatogonia providing valuable clues for the regulation of their development