Novel testis germ cell-specific transcript of the CREB gene contains an alternatively spliced exon with multiple in-frame stop codons.

The gene encoding the cAMP-responsive transcription factor CREB consists of multiple small exons some of which undergo alternative RNA splicing. We describe the finding of a novel transcript of the CREB gene expressed at high levels in the germ cells of the rat testis. The transcript contains an alternatively spliced exon inserted within the sequence encoding the transcriptional transactivation domain of CREB and this exon contains multiple in-frame stop codons. Furthermore, the exon is conserved in both rat and human genes (75% nucleotide identity). Although the function(s) of this RNA or the truncated CREB protein predicted to result from the translation of this unusual transcript is unknown, the high level of expression in the testicular germ cells and remarkable conservation of sequences in rat and human suggests that it may have a unique biological function in these cells.

Systemic mastocytosis following a malignant ovarian germ cell tumour.

Cases of mediastinal germ cell tumours associated with haematological disorders (two cases of systemic mastocytosis included) have been reported previously. This combination is more frequent than would be expected by chance alone. We report the case of a 30-year-old woman, who presented with a systemic mastocytosis following a malignant ovarian germ cell tumour which was treated by chemo- and radiotherapy. The patient predominantly complained of skeletal pains, which led to an erroneous radiological diagnosis of fibrous dysplasia for years. An aggressive variant of systemic mastocytosis was diagnosed on bone marrow examination. Systemic mastocytosis was confirmed by splenectomy, liver biopsy and finally autopsy. The present case is unique because of the ovarian location of the germ cell tumour. We suggest our observation could be related to the broad group of haematological malignancies associated with germ cell tumours.

Mixed testicular germ cell tumor presenting as metastatic pure choriocarcinoma involving the maxillary gingiva.

Gingival metastases are infrequent and invariably associated with a widespread disease and a poor prognosis. Because of their unremarkable clinical appearance, they can be difficult to distinguish from more common gingival hyperplastic or reactive lesions, such as pyogenic granuloma, peripheral giant cell granuloma, and peripheral ossifying granuloma. We are reporting here an unusual case of a 36-year-old man with a mixed testicular germ cell tumor presenting as a metastatic pure choriocarcinoma involving the maxillary gingiva, extending from the first left premolar to the left second maxillary molar, mimicking a 'benign looking' gingival mass. Gingival metastases may be the first manifestation of a widespread metastatic disease and therefore particular attention must be paid to gingival lesions associated with atypical clinical symptoms and/or signs.

Predicted effect of climate change on the invasibility and distribution of the Western corn root-worm

1 Insect pests, biological invasions and climate change are considered to representmajor threats to biodiversity, ecosystem functioning, agriculture and forestry.Deriving hypothesis of contemporary and/or future potential distributions of insectpests and invasive species is becoming an important tool for predicting the spatialstructure of potential threats.2 The western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte is apest of maize in North America that has invaded Europe in recent years, resultingin economic costs in terms of maize yields in both continents. The present studyaimed to estimate the dynamics of potential areas of invasion by the WCR under aclimate change scenario in the Northern Hemisphere. The areas at risk under thisscenario were assessed by comparing, using complementary approaches, the spatialprojections of current and future areas of climatic favourability of the WCR. Spatialhypothesis were generated with respect to the presence records in the native rangeof the WCR and physiological thresholds from previous empirical studies.3 We used a previously developed protocol specifically designed to estimatethe climatic favourability of the WCR. We selected the most biologicallyrelevant climatic predictors and then used multidimensional envelope (MDE) andMahalanobis distances (MD) approaches to derive potential distributions for currentand future climatic conditions.4 The results obtained showed a northward advancement of the upper physiologicallimit as a result of climate change, which might increase the strength of outbreaksat higher latitudes. In addition, both MDE and MD outputs predict the stability ofclimatic favourability for the WCR in the core of the already invaded area in Europe,which suggests that this zone would continue to experience damage from this pestin Europe.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Non-Risk-Adapted Surveillance in Clinical Stage I Nonseminomatous Germ Cell Tumors: The Princess Margaret Hospital's Experience.

BACKGROUND: Since 1981 Princess Margaret Hospital has used initial active surveillance (AS) with delayed treatment at relapse as the preferred management for all patients with clinical stage I nonseminomatous germ cell tumors (NSGCT). OBJECTIVE: Our aim was to report our overall AS experience and compare outcomes over different periods using this non-risk-adapted approach. DESIGN, SETTING, AND PARTICIPANTS: Three hundred and seventy-one patients with stage I NSGCT were managed by AS from 1981 to 2005. For analysis by time period, patients were divided into two cohorts by diagnosis date: initial cohort, 1981-1992 (n=157), and recent cohort, 1993-2005 (n=214). INTERVENTION: Patients were followed at regular intervals, and treatment was only given for relapse. MEASUREMENTS: Recurrence rates, time to relapse, risk factors for recurrence, disease-specific survival, and overall survival were determined. RESULTS AND LIMITATIONS: With a median follow-up of 6.3 yr, 104 patients (28%) relapsed: 53 of 157 (33.8%) in the initial group and 51 of 214 (23.8%) in the recent group. Median time to relapse was 7 mo. Lymphovascular invasion (p<0.0001) and pure embryonal carcinoma (p=0.02) were independent predictors of recurrence; 125 patients (33.7%) were designated as high risk based on the presence of one or both factors. In the initial cohort, 66 of 157 patients (42.0%) were high risk and 36 of 66 patients (54.5%) relapsed versus 17 of 91 low-risk patients (18.7%) (p<0.0001). In the recent cohort, 59 of 214 patients (27.6%) were high risk and 29 of 59 had a recurrence (49.2%) versus 22 of 155 low-risk patients (14.2%) (p<0.0001). Three patients (0.8%) died from testis cancer. The estimated 5-yr disease-specific survival was 99.3% in the initial group and 98.9% in the recent one. CONCLUSIONS: Non-risk-adapted surveillance is an effective, simple strategy for the management of all stage I NSGCT.

46, XY gonadal dysgenesis: new SRY point mutation in two siblings with paternal germ line mosaicism.

Stoppa-Vaucher S, Ayabe T, Paquette J, Patey N, Francoeur D, Vuissoz J-M, Deladoëy J, Samuels ME, Ogata T, Deal CL. 46, XY gonadal dysgenesis: new SRY point mutation in two siblings with paternal germ line mosaicism. Familial recurrence risks are poorly understood in cases of de novo mutations. In the event of parental germ line mosaicism, recurrence risks can be higher than generally appreciated, with implications for genetic counseling and clinical practice. In the course of treating a female with pubertal delay and hypergonadotropic hypogonadism, we identified a new missense mutation in the SRY gene, leading to somatic feminization of this karyotypically normal XY individual. We tested a younger sister despite a normal onset of puberty, who also possessed an XY karyotype and the same SRY mutation. Imaging studies in the sister revealed an ovarian tumor, which was removed. DNA from the father's blood possessed the wild type SRY sequence, and paternity testing was consistent with the given family structure. A brother was 46, XY with a wild type SRY sequence strongly suggesting paternal Y-chromosome germline mosaicism for the mutation. In disorders of sexual development (DSDs), early diagnosis is critical for optimal psychological development of the affected patients. In this case, preventive karyotypic screening allowed early diagnosis of a gonadal tumor in the sibling prior to the age of normal puberty. Our results suggest that cytological or molecular diagnosis should be applied for siblings of an affected DSD individual.

Germ cell-specific targeting of DICER or DGCR8 reveals a novel role for endo-siRNAs in the progression of mammalian spermatogenesis and male fertility.

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.

Dicer1 depletion in male germ cells leads to infertility due to cumulative meiotic and spermiogenic defects.

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis.

Pituitary blastoma: a pathognomonic feature of germ-line DICER1 mutations.

Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5' arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 "hits" occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis.

Functional studies of a germ-line polymorphism at codon 47 within the p53 gene.

A rare germ-line polymorphism in codon 47 of the p53 gene replaces the wild-type proline (CCG) with a serine (TCG). Restriction analysis of 101 human samples revealed the frequency of the rare allele to be 0% (n = 69) in Caucasians and 4.7% (3/64, n = 32) among African-Americans. To investigate the consequence of this amino acid substitution, a cDNA construct (p53 mut47ser) containing the mutation was introduced into a lung adenocarcinoma cell line (Calu-6) that does not express p53. A growth suppression similar to that obtained after introduction of a wild-type p53 cDNA construct was observed, in contrast to the result obtained by introduction of p53 mut143ala. Furthermore, expression of neither p53 mut47ser nor wild-type p53 was tolerated by growing cells. In transient expression assays, both mut47ser and wild-type p53 activated the expression of a reporter gene linked to a p53 binding sequence (PG13-CAT) and inhibited the expression of the luciferase gene under the control of the Rous sarcoma virus promoter (RSVluc). In the same assay, mut143ala did not activate the expression of PG13-CAT and produced only a slight inhibitory effect on RSVluc. These findings indicate that the p53 variant with a serine at codon 47 should be considered as a rare germ-line polymorphism that does not alter the growth-suppression activity of p53.