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Background. Acute appendicitis (AA) might be amenable to conservative antibiotic treatment, whereas a perforated appendix (PA) necessitates surgery. We investigated the value of clinical–laboratory markers in distinguishing AA from a PA. Methods. Retrospectively obtained preoperative parameters for 306 consecutive patients ( n = 237 ) vs. PA ( n = 69 )), treated at our institution between January 2014 and December 2017. Results. A PA was associated with male preponderance, younger age, decreased sodium level and increased white blood cell count, Tzanakis score, C-reactive protein (CRP) level, and CRP-to-lymphocyte ratio (CLR). Upon discrimination analysis, CLR and CRP displayed the highest accuracy in differentiating a PA from AA. Regression analysis identified levels of CRP, sodium, and the Tzanakis score as independent predictors for a PA. Conclusion. Levels of CLR, CRP, sodium, and Tzanakis score might support decision-making regarding treatment options for pediatric appendicitis.

Lynch Syndrome (LS) is the most common dominantly inherited colorectal cancer (CRC) predisposition and is caused by a heterozygous germline defect in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. High microsatellite instability (MSI-H) and loss of MMR protein expression in tumours reflecting a defective MMR are indicators for LS, as well as a positive family history of early onset CRC. MSH2 and MSH6 form a major functional heterodimer, and MSH3 is an alternative binding partner for MSH2. So far, the role of germline MSH3 variants remains unclear, as to our knowledge heterozygous truncating variants are not regarded causative for LS, but were detected in patients with CRC, and recently biallelic MSH3 defects have been identified in two patients with adenomatous polyposis. By gene screening we investigated the role of MSH3 in 11 LS patients with truncating MSH6 germline variants and an unexplained MSH2 protein loss in their corresponding MSI-H tumours. We report the first two LS patients harbouring heterozygous germline variants c.1035del and c.2732T>G in MSH3 coincidentally with truncating variants in MSH6. In the patient with truncating germline variants in MSH3 and MSH6, two additional somatic second hits in both genes abrogate all binding partners for the MSH2 protein which might subsequently be degraded. The clinical relevance of MSH3 germline variants is currently under re-evaluation, and heterozygous MSH3 defects alone do not seem to induce a LS phenotype, but might aggravate the MSH6 phenotype in affected family members.

Background In kindreds carrying path_BRCA1/2 variants, some women in these families will develop cancer despite testing negative for the family’s pathogenic variant. These families may have additional genetic variants, which not only may increase the susceptibility of the families’ path_BRCA1/2, but also be capable of causing cancer in the absence of the path_BRCA1/2 variants. We aimed to identify novel genetic variants in prospectively detected breast cancer (BC) or gynecological cancer cases tested negative for their families’ pathogenic BRCA1/2 variant (path_BRCA1 or path_BRCA2). Methods Women with BC or gynecological cancer who had tested negative for path_BRCA1 or path_BRCA2 variants were included. Forty-four cancer susceptibility genes were screened for genetic variation through a targeted amplicon-based sequencing assay. Protein- and RNA splicing-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as the ones most likely affecting pre-mRNA splicing were experimentally analyzed in a minigene assay. Results We identified 48 women who were tested negative for their family’s path_BRCA1 (n = 13) or path_BRCA2 (n = 35) variants. Pathogenic variants in the ATM, BRCA2, MSH6 and MUTYH genes were found in 10% (5/48) of the cases, of whom 15% (2/13) were from path_BRCA1 and 9% (3/35) from path_BRCA2 families. Out of the 26 unique VUS, 3 (12%) were predicted to affect RNA splicing (APC c.721G > A, MAP3K1 c.764A > G and MSH2 c.815C > T). However, by using a minigene, assay we here show that APC c.721G > A does not cause a splicing defect, similarly to what has been recently reported for the MAP3K1 c.764A > G. The MSH2 c.815C > T was previously described as causing partial exon skipping and it was identified in this work together with the path_BRCA2 c.9382C > T (p.R3128X). Conclusion All women in breast or breast/ovarian cancer kindreds would benefit from being offered genetic testing irrespective of which causative genetic variants have been demonstrated in their relatives.

Background Retinitis pigmentosa in combination with hearing loss can be a feature of different Mendelian disorders. We describe a novel syndrome caused by biallelic mutations in the ‘exosome component 2’ ( EXOSC2 ) gene. Methods Clinical ascertainment of three similar affected patients followed by whole exome sequencing. Results Three individuals from two unrelated German families presented with a novel Mendelian disorder encompassing childhood myopia, early onset retinitis pigmentosa, progressive sensorineural hearing loss, hypothyroidism, short stature, brachydactyly, recognisable facial gestalt, premature ageing and mild intellectual disability. Whole exome sequencing revealed homozygous or compound heterozygous missense variants in the EXOSC2 gene in all three patients. EXOSC2 encodes the ‘ribosomal RNA-processing protein 4’ (RRP4)—one of the core components of the RNA exosome. The RNA exosome is a multiprotein complex that plays key roles in RNA processing and degradation. Intriguingly, the EXOSC2 -associated phenotype shows only minimal overlap with the previously reported diseases associated with mutations in the RNA exosome core component genes EXOSC3 and EXOSC8 . Conclusion We report a novel condition that is probably caused by altered RNA exosome function and expands the spectrum of clinical consequences of impaired RNA metabolism.

Background The genetic mechanisms for families who meet the clinical criteria for Lynch syndrome (LS) but do not carry pathogenic variants in the mismatch repair (MMR) genes are still undetermined. We aimed to study the potential contribution of genes other than MMR genes to the biological and clinical characteristics of Norwegian families fulfilling Amsterdam (AMS) criteria or revised Bethesda guidelines. Methods The Hereditary Cancer Biobank of the Norwegian Radium Hospital was interrogated to identify individuals with a high risk of developing colorectal cancer (CRC) for whom no pathogenic variants in MMR genes had been found in routine diagnostic DNA sequencing. Forty-four cancer susceptibility genes were selected and analyzed by using our in-house designed TruSeq amplicon-based assay for targeted sequencing. RNA splicing- and protein-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as likely to affect splicing were experimentally analyzed by resorting to minigene assays. Results We identified a patient who met the revised Bethesda guidelines and carried a likely pathogenic variant in CHEK2 (c.470 T > C, p.I157T). In addition, 25 unique VUS were identified in 18 individuals, of which 2 exonic variants (MAP3K1 c.764A > G and NOTCH3 c.5854G >A) were analyzed in the minigene splicing assay and found not to have an effect on RNA splicing. Conclusions Among high-risk CRC patients that fulfill the AMS criteria or revised Bethesda guidelines, targeted gene sequencing identified likely pathogenic variant and VUS in other genes than the MMR genes (CHEK2, NOTCH3 and MAP3K1). Our study suggests that the analysis of genes currently excluded from routine molecular diagnostic screens may confer cancer susceptibility.

To study the potential contribution of genes other than BRCA1/2, PTEN, and TP53 to the biological and clinical characteristics of multiple early-onset cancers in Norwegian families, including early-onset breast cancer, Cowden-like and Li-Fraumeni-like syndromes (BC, CSL and LFL, respectively). The Hereditary Cancer Biobank from the Norwegian Radium Hospital was used to identify early-onset BC, CSL or LFL for whom no pathogenic variants in BRCA1/2, PTEN, or TP53 had been found in routine diagnostic DNA sequencing. Forty-four cancer susceptibility genes were selected and analyzed by our in-house designed TruSeq amplicon-based assay for targeted sequencing. Protein- and RNA splicing-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as the more likely to affect splicing were experimentally analyzed by minigene assay. We identified a CSL individual carrying a variant in CHEK2 (c.319+2T>A, IVS2), here considered as likely pathogenic. Out of the five VUS (BRCA2, CDH1, CHEK2, MAP3K1, NOTCH3) tested in the minigene splicing assay, only NOTCH3 c.14090C>T (p.Ser497Leu) showed a significant effect on RNA splicing, notably by inducing partial skipping of exon 9. Among 13 early-onset BC, CSL and LFL patients, gene panel sequencing identified a potentially pathogenic variant in CHEK2 that affects a canonical RNA splicing signal. Our study provides new information on genetic loci that may affect the risk of developing cancer in these patients and their families, demonstrating that genes presently not routinely tested in molecular diagnostic settings may be important for capturing cancer predisposition in these families.

Overexpression of the Dual-specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) gene contributes to the retardation, craniofacial anomalies, cognitive impairment, and learning and memory deficits associated with Down Syndrome (DS). DCAF7/HAN11/WDR68 (hereafter WDR68) binds DYRK1A and is required for craniofacial development. Accumulating evidence suggests DYRK1A-WDR68 complexes enable proper growth and patterning of multiple organ systems and suppress inappropriate cell growth/transformation by regulating the balance between proliferation and differentiation in multiple cellular contexts. Here we report, using engineered mouse C2C12 and human HeLa cell lines, that WDR68 is required for normal levels of DYRK1A. However, Wdr68 does not significantly regulate Dyrk1a mRNA expression levels and proteasome inhibition did not restore DYRK1A in cells lacking Wdr68 (Δwdr68 cells). Overexpression of WDR68 increased DYRK1A levels while overexpression of DYRK1A had no effect on WDR68 levels. We further report that WDR68 is similarly required for normal levels of the closely related DYRK1B kinase and that both DYRK1A and DYRK1B are essential for the transition from proliferation to differentiation in C2C12 cells. These findings reveal an additional role of WDR68 in DYRK1A-WDR68 and DYRK1B-WDR68 complexes.

Intrauterine growth restriction (IUGR) occurs naturally in pigs and leads to low birth weight of piglets due to undernutrition caused by placental insufficiency. For 2 main reasons, low birth weight causes economic loss. First, low birth weight pigs have a greater mortality and increasing the litter size causes more low birth weight piglets within litters. Second, surviving low birth weight piglets have reduced performance (i.e., ADG, feed conversion rate, and percentage meat). To develop dietary strategies for preventing IUGR, knowledge of the biological basis of IUGR is required. Muscle fiber number, formed during myogenesis, is correlated positively with performance traits and has been shown in several studies to be reduced in low birth weight pigs. Postnatal muscle hypertrophy is due to satellite cell number per fiber at birth and their rate of proliferation as well as protein deposition (i.e., protein synthesis and degradation). Previous studies and some recent ones indicate that low birth weight littermates in mice are born with fewer satellite cells and studies on pigs show that the rate of satellite cell proliferation may vary within litters. Proteomics studies show that protein synthesis and degradation is downregulated in IUGR pigs and low birth weight pigs also produce meat with less tenderness. Alternative maternal feeding strategies to prevent IUGR have been examined. Increasing maternal global nutrition had no beneficial effect on performance and muscle growth traits in several studies. Feeding excess maternal dietary protein also did not influence muscle growth traits whereas moderately decreased maternal dietary protein may decrease muscle fiber number and performance. On the other hand, addition of L-carnitine to the maternal gestation or lactation diet may increase birth and weaning weights or the muscle fiber number, respectively, in low birth weight pig offspring. Finally, promising data have been obtained on reproductive traits in pigs after addition of functional AA, such as arginine and glutamine, to the gestational diet. Although much is known about the biological basis of IUGR, we still need to learn much more about the mode of action before maternal dietary strategies can be developed to prevent IUGR.

The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription). In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC) predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense), all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of “bonafide” breast cancer susceptibility genes., Author Summary Approximately 5–10% of breast/ovarian cancer (BC/OC) cases have inherited an increased risk of developing this malignancy. However, mutations in the two major breast cancer susceptibility genes BRCA1 and BRCA2 explain only 15–20% of all familial BC/OC cases. With the emergence of the high throughput NGS-technology, the number of proposed novel candidate genes for breast cancer predisposition continuously increases. However, a “bonafide” proof of cancer susceptibility requires a detailed characterization of candidate mutations, which we addressed in the current study. Using the DNA repair gene ERCC2 as an example, we performed a comprehensive multi-center approach, analyzing ERCC2 mutations in 1000+ patients with hereditary BC/OC. We identified 25 potential candidate mutations for cancer breast cancer susceptibility, some of them affecting ERCC2 functional activity in appropriate cell-culture based assays. However, a more dissected analysis showed no convincing co-segregation with BC/OC and there was no longer a significant overrepresentation in BC/OC when compared to regionally matched controls instead of the global ExAc variant data base, pointing to the relevance of founder-mutations. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we highlight the complexity of correct interpretation for the discovery of “bonafide” breast cancer susceptibility genes.

he Mediterranean storm track constitutes a well-defined branch of the North Hemisphere storm track and is characterised by small but intense features and frequent cyclogenesis. The goal of this study is to assess the level of consensus among cyclone detection and tracking methods (CDTMs), to identify robust features and to explore sources of disagreement. A set of 14 CDTMs has been applied for computing the climatology of cyclones crossing the Mediterranean region using the ERA-Interim dataset for the period 1979–2008 as common testbed. Results show large differences in actual cyclone numbers identified by different methods, but a good level of consensus on the interpretation of results regarding location, annual cycle and trends of cyclone tracks. Cyclogenesis areas such as the north-western Mediterranean, North Africa, north shore of the Levantine basin, as well as the seasonality of their maxima are robust features on which methods show a substantial agreement. Differences among methods are greatly reduced if cyclone numbers are transformed to a dimensionless index, which, in spite of disagreement on mean values and interannual variances of cyclone numbers, reveals a consensus on variability, sign and significance of trends. Further, excluding ‘weak’ and ‘slow’ cyclones from the computation of cyclone statistics improves the agreement among CDTMs. Results show significant negative trends of cyclone frequency in spring and positive trends in summer, whose contrasting effects compensate each other at annual scale, so that there is no significant long-term trend in total cyclone numbers in the Mediterranean basin in the 1979–2008 period. Keywords: Mediterranean region, cyclones, automatic tracking methods, tracks, cyclogenesis (Published: 20 May 2016) Citation: Tellus A 2016, 68, 29391, http://dx.doi.org/10.3402/tellusa.v68.29391

Selective breeding is an effective tool to improve livestock. Several selection experiments have been conducted to study direct selection responses as well as correlated responses in traits of skeletal muscle growth and function. Moreover, comparisons of domestic with wild-type species and of extreme breeds provide information on the genetic background of the skeletal muscle phenotype. Structural muscular components that differed with increasing distance in lean growth or meat quality in mammals were found to be myofibre number, myofibre size, proportions of fibre types as well as the numbers and proportions of secondary and primary fibres. Furthermore, markers of satellite cell proliferation, metabolic enzyme activities, glycogen and fat contents, the expression of myosin heavy chain isoforms, of activated AMPKα and other proteins in skeletal muscle tissue and circulating IGF1 and IGF-binding proteins have been identified to be involved in selection responses observed in pigs, cattle and/or chicken. The use of molecular methods for selective breeding of fish has only recently been adopted in aquaculture and studies of the genetic basis of growth and flesh quality traits are scarce. Some of the molecular markers of muscle structure/metabolism in livestock have also been identified in fish, but so far no studies have linked them with selection response. Genome scans have been applied to identify genomic regions exhibiting quantitative trait loci that control traits of interest, for example, muscle structure and meat quality in pigs and growth rate in chicken. As another approach, polymorphisms in candidate genes reveal the relationship between genetic variation and target traits. Thus, in large-scale studies with pigs' associations of polymorphisms in the HMGA2, CA3, EPOR, NME1 and TTN genes with traits of carcass and meat quality were detected. Other studies revealed the significance of mutations in the IGF2 and RYR1 genes for carcass lean and muscle fibre traits in pigs. Mutations in the myostatin (MSTN) gene in fish were also examined. Advances in research of the genetic and environmental control of traits related to meat quality and growth have been made by the application of holistic 'omics' techniques that studied the whole muscle-specific genome, transcriptome and proteome in relation to muscle and meat traits, the development of new methods for muscle fibre typing and the adaptation of biophysical measures to develop parameters of muscle fibre traits as well as the application of in vitro studies. Finally, future research priorities in the field are defined. Copyright © 2010 The Animal Consortium.

Skeletal muscle development in vertebrates - also termed myogenesis - is a highly integrated process. Evidence to date indicates that the processes are very similar across mammals, poultry and fish, although the timings of the various steps differ considerably. Myogenesis is regulated by the myogenic regulatory factors and consists of two to three distinct phases when different fibre populations appear. The critical times when myogenesis is prone to hormonal or environmental influences depend largely on the developmental stage. One of the main mechanisms for both genetic and environmental effects on muscle fibre development is via the direct action of the growth hormone-insulin-like growth factor (GH-IGF) axis. In mammals and poultry, postnatal growth and function of muscles relate mainly to the hypertrophy of the fibres formed during myogenesis and to their fibre-type composition in terms of metabolic and contractile properties, whereas in fish hyperplasia still plays a major role. Candidate genes that are important in skeletal muscle development, for instance, encode for IGFs and IGF-binding proteins, myosin heavy chain isoforms, troponin T, myosin light chain and others have been identified. In mammals, nutritional supply in utero affects myogenesis and the GH-IGF axis may have an indirect action through the partitioning of nutrients towards the gravid uterus. Impaired myogenesis resulting in low skeletal myofibre numbers is considered one of the main reasons for negative long-term consequences of intrauterine growth retardation. Severe undernutrition in utero due to natural variation in litter or twin-bearing species or insufficient maternal nutrient supply may impair myogenesis and adversely affect carcass quality later in terms of reduced lean and increased fat deposition in the progeny. On the other hand, increases in maternal feed intake above standard requirement seem to have no beneficial effects on the growth of the progeny with myogenesis not or only slightly affected. Initial studies on low and high maternal protein feeding are published. Although there are only a few studies, first results also reveal an influence of nutrition on skeletal muscle development in fish and poultry. Finally, environmental temperature has been identified as a critical factor for growth and development of skeletal muscle in both fish and poultry. Copyright © 2010 The Animal Consortium.

Intrauterine growth restriction (IUGR), resulting in low birth body weight (LBW) occurs naturally in pigs. However, IUGR may also cause persistent changes in physiology and metabolism resulting in poorer performance, organogenesis and meat quality. As IUGR pigs have a lower daily gain from birth to slaughter they may differ in utilization of nutrients and requirements for dietary protein compared with their larger littermates. Thus, the objective in this study was to examine the interaction between birth body weight (BW) and the postnatal dietary protein level, in relation to postnatal performance, organogenesis, muscle metabolism and meat quality. The experiment was carried out with offspring from 16 purebred Danish Landrace gilts mated to Danish Landrace boars. The female and entire male pigs with LBW that survived at weaning were compared with the female and male pigs with the highest/high birth body weight (HBW) within each litter. The offspring were reared individually from weaning and were fed ad libitum a diet containing either a normal level of protein (NP) for optimal growth or an isocaloric diet containing a 30% lower protein content (LP) from 3 weeks to 150 days of age. At slaughter, we found no interactions between birth weight group and dietary protein level for any of the measured traits. The relative crown–rump length (cm/kg) at birth indicates that LBW pigs were thinner than HBW pigs. Daily gain and feed intake were reduced by 14% and 10%, respectively, while the kg feed/kg gain was slightly increased by 3% in LBW pigs compared with HBW pigs. The LP diet reduced daily gain by 27% due to reduced feed intake and increased kg feed/kg gain by 12% and 21%, respectively compared with the NP diet. LBW male pigs produced meat with a higher shear force than male HBW pigs and also LP pigs produced meat with higher shear force than NP pigs. The activity of lactate dehydrogenase in the Longissimus dorsi muscle (LD) was reduced in pigs fed the LP diet. Calpastatin was increased in LD of LBW pigs and decreased in pigs fed the NP diet. In conclusion, these results suggest a rejection of our hypothesis that low birth weight littermates have a lower requirement for dietary protein compared with heavy weight littermates. Furthermore, LBW male pigs and LP fed pigs of both genders produced less tender meat than HBW pigs or NP fed pigs, respectively.

The objective of this study was to examine the effect of increased maternal nutrition in early to mid gestation on changes in serum growth factors of the sow and foetuses. Furthermore, the effect of the foetal sera on in vitro proliferation and differentiation of porcine primary myoblasts was examined. Pregnant sows were either given food either in accordance to requirements (2 kg/day; C) until day 50 or 70 of gestation or given food in accordance to requirements until day 25 and then ad libitum (A) until day 50 or 70. Sows were slaughtered at the Institute's veterinary controlled slaughterhouse at day 50 or 70, respectively. Serum from sows and pools of cord-blood serum from each litter were analysed for glucose, lactate, insulin, insulin-like growth factor (IGF; IGF-1 and -2) and IGF binding proteins (IGFBPs). IGF-1 (P < 0·001) was higher in A compared with C sows, and a 28-kDa IGFBP (P < 0·05) and a 24-kDa IGFBP (P < 0·05) was higher in serum from day 70 compared with day 50 sows. There was no significant effect of food intake on growth factor concentrations in foetal serum or on serum-induced proliferation and differentiation of myoblasts. A 28 kDa IGFBP, IGFBP-2 and -3 were all higher (P < 0·06) and serum-induced proliferation (P < 0·001) and differentiation (P < 0·1) lower at day 70 than day 50. Maternal food intake did not influence the DNA and RNA concentrations and the CPK activity in the foetal longissimus dorsi muscle. The glucose concentration in the liver was higher in C than A foetuses at day 70 of gestation, but not at day 50.In conclusion, no significant effects of maternal nutrition were found on serum growth factor concentrations in the foetuses or on serum-induced proliferation and differentiation of primary myoblasts.

The objective of this study was to examine how increased feed intake of the sow during early to mid-gestation affects sow performance and the muscle fiber number, performance, and technological meat quality of the offspring. Thirty-nine pregnant sows (Landrace x Large White sows mated to Landrace or Large White boars) in their fourth parity were assigned to one of three treatments: 1) the sows were either fed restrictively (control = 15 MJ of NE/d from d 1 to 90, then 24 MJ of NE/d from d 91 to 112, and again 15 MJ of NE/d from d 113 to 115 of gestation); 2) fed ad libitum from d 25 to 50 (A25-50); or 3) ad libitum from d 25 to 70 (A25-70) and as control in the remaining periods. The offspring were weaned at 4 wk of age and had free access to feed from 2 wk of age until slaughter. They were slaughtered litterwise at an average body weight of 104 +/- 14 kg. Estimates for total, primary (P-), and secondary (S-) muscle fiber number; muscle fiber area; and DNA and RNA content were analyzed in semitendinosus muscle (ST) samples from the heaviest, middle, and lightest weight (LW) pigs of each sex within litter selected at slaughter. Technological meat quality traits (pH at 24 h postmortem, drip loss, Minolta color, and pigment) were analyzed in longissimus dorsi muscle. Fiber number, fiber area, and concentrations and content of DNA and RNA of the offspring were not significantly affected by increased maternal nutrition. The ST muscle weight was lower in offspring from A25-50 than control sows (P = 0.019). Average daily gain, carcass weight, and the muscle deposition rate also were numerically lower for A25-50 than control and A25-70 pigs. An interaction between treatment and pig weight was found for muscle deposition rate (P = 0.006), in that LW pigs from treatment A25-50 had a lower deposition rate than LW pigs from control. We found no effect of treatment on the meat quality traits in the offspring. Also, barrows had a higher (P < 0.05) number of P-fibers, higher daily gain, and carcass weight than female pigs. No differences were found on any meat quality traits between sexes. Thus, ad libitum feeding of pregnant sows from d 25 to 50 or d 25 to 70 of gestation did not have any beneficial effect on muscle fiber number and area in the offspring. It seems that maternal ad libitum feeding from d 25 to 50 in gestation had a negative effect on postnatal muscle growth, with especially the LW pigs being affected.

To rapidly identify genes required for early vertebrate development, we are carrying out a large-scale, insertional mutagenesis screen in zebrafish, using mouse retroviral vectors as the mutagen. We will obtain mutations in 450 to 500 different genes--roughly 20% of the genes that can be mutated to produce a visible embryonic phenotype in this species--and will clone the majority of the mutated alleles. So far, we have isolated more than 500 insertional mutants. Here we describe the first 75 insertional mutants for which the disrupted genes have been identified. In agreement with chemical mutagenesis screens, approximately one-third of the mutants have developmental defects that affect primarily one or a small number of organs, body shape or swimming behavior; the rest of the mutants show more widespread or pleiotropic abnormalities. Many of the genes we identified have not been previously assigned a biological role in vivo. Roughly 20% of the mutants result from lesions in genes for which the biochemical and cellular function of the proteins they encode cannot be deduced with confidence, if at all, from their predicted amino-acid sequences. All of the genes have either orthologs or clearly related genes in human. These results provide an unbiased view of the genetic construction kit for a vertebrate embryo, reveal the diversity of genes required for vertebrate development and suggest that hundreds of genes of unknown biochemical function essential for vertebrate development have yet to be identified.

Aims/hypothesis. ART2.2 is a mouse T-cell surface ectoenzyme [mono (ADP-ribosyl) transferase] shed upon strong activation. We analysed temporal changes in ART2.2 expression in unmanipulated and cyclophosphamide-treated NOD/Lt mice compared with diabetes-resistant control strains. We used NAD, the ART2.2 substrate, to test whether ART-mediated ADP-ribosylation could retard diabetogenic activation of islet-reactive T cells in vitro. Methods. ART2.2 and CD38, another NAD-utilizing enzyme, were measured by flow cytometry. ADP-ribosylation from ethano-NAD was followed by flow cytometry using a reagent specific for etheno-ADP ribose. Results. Although mature NOD CD4 + and C D8 + T cells expressed ART2.2, this expression was delayed in young NOD mice when compared with control strains. This ontological delay at 3 weeks of age correlated with an early burst of CD25 expression unique to NOD splenic T cells. This pattern was reproduced in cyclophosphamide-accelerated diabetes in young NOD/Lt males, wherein a retarded repopulation of ART2.2 T cells in spleen and islets correlated with development of heavy insulitis and diabetes. NAD inhibited anti-CD3 induced activation of splenic T cells in vitro and also retarded killing of beta-cell targets by NOD islet-reactive CD8 effectors in vitro at concentrations equal to or greater than 1 μmol/l. Evidence suggested that CD38 on B lymphocytes competes with ART2.2 for substrate needed by B lymphocytes for ADP ribosylation. Conclusions. ART2.2 on T cells may not simply mark the resting state, but could also contribute to it via ADP-ribosylation. [Diabetologia (2001) 44: 848–858]

STUDY QUESTION: Do children born to fathers of advanced age have an increased risk of dying before the age of 5 years?SUMMARY ANSWER: Children born to fathers aged 40 years or more have an increased risk of dying in early childhood due to an excess risk of fatal congenital anomalies, malignancies and external causes.WHAT IS KNOWN ALREADY: Advanced paternal age has previously been associated with adverse reproductive outcomes and some long-term health problems in the offspring. This is possibly due to specific point mutations, a condition known to increase in the sperm with increasing paternal age.STUDY DESIGN, SIZE, DURATION: A Danish population-based register study, designed as a prospective cohort study, of 1 575 521 live born children born from 1978 to 2004. The age of the child (in days) was used as the underlying time and the children entered the cohort the day they were born and were followed until 31 December 2009. The children were censored on date of turning 5 years, date of death or date of emigration, whichever occurred first.PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from population-covering registers from Statistics Denmark including the Integrated Database for Labour Market Research, the Medical Birth Registry and the Registry of Causes of Death was linked using the unique civil registry number. Hazard ratios (HR) with 95% confidence intervals (CI) were used to estimate the risk of under-five mortality. The effect of paternal age was examined using restricted cubic splines and paternal age groups.MAIN RESULTS AND THE ROLE OF CHANCE: Compared with children born to fathers aged 30-34 years, a statistically significant excess risk was found for children born to fathers aged 40-44 years [HR: 1.10 (95% CI: 1.00-1.21)] and children born to fathers aged 45+ years [HR: 1.16 (95% CI: 1.02-1.32)]. When only looking at 1-5 year olds, the relative risk (HR) among children born to fathers aged 40-44 years increased to 1.24 (95% CI: 1.00-1.53) and the risk in the oldest paternal age group (45+ years) rose to 1.65 (95% CI: 1.24-2.18). The results suggest that the elevated risk for children of fathers aged 40 years or more was primarily attributed to an elevated risk of dying from congenital malformations, malignancies and external causes.LIMITATIONS, REASONS FOR CAUTION: Specific causes of death might be misclassified; however, this is not likely to be dependent on paternal age. In some cases, the biological father may differ from the father registered. This misclassification is most likely non-differential.WIDER IMPLICATIONS OF THE FINDINGS: The excess risk of mortality among children born to older fathers is in accordance with the literature. The association needs further attention as it can provide valuable knowledge of the etiology of genetic diseases. Also, the association could become of greater importance in the future if the proportion of fathers aged 40+ years keeps growing.STUDY FUNDING/COMPETING INTEREST (S): None.

Epidemiological studies in man and with experimental animal models have shown that intrauterine growth restriction (IUGR) resulting in low birth weight is associated with higher risk of programming welfare diseases in later life. In the pig, severe IUGR occurs naturally and contribute substantially to a large intralitter variation in birth weight and may therefore be a good model for man. In the present paper the natural form of IUGR in pigs was studied close to term by nuclear magnetic resonance (NMR-)based metabolomics. The NMR-based investigations revealed different metabolic profiles of plasma samples from low-birth weight (LW) and high-birth weight (HW) piglets, respectively, and differences were assigned to levels of glucose and myo-inositol. Further studies by GC-MS revealed that LW piglets had a significant higher concentration of myoinositol and D-chiro-inositol in plasma compared to larger littermates. Myo-inositol and D-chiro-inositol have been coupled with glucose intolerance and insulin resistance in adults, and the present paper therefore suggests that IUGR is related to impaired glucose metabolism during fetal development, which may cause type 2 diabetes in adulthood.

von Recklinghausen's neurofibromatosis (NF1) is a common inherited human disease. The events leading to patient symptoms from inheritance of a defective NF1 gene are unknown. Since knowledge of the distribution of the normal NF1 gene product should improve understanding of the pathogenesis of the disease, we raised antibodies against peptides coded by portions of the recently cloned human NF1 cDNA. These antibodies specifically recognize a 220 kd protein (neurofibromin) in both human and rat spinal cord. Neurofibromin is most abundant in the nervous system. Immunostaining of tissue sections indicates that neurons, oligodendrocytes, and nonmyelinating Schwann cells contain neurofibromin while astrocytes and myelinating Schwann cells do not. These results suggest a function for neurofibromin in the normal nervous system. Some NF1 disease manifestations, such as Schwann cell tumors and learning disabilities, may result from abnormalities in the cells that express neurofibromin.

Nodal-signaling is required for specification of mesoderm, endoderm, establishing left–right asymmetry, and craniofacial development. Wdr68 is a WD40-repeat domain-containing protein recently shown to be required for endothelin-1 (edn1) expression and subsequent lower jaw development. Previous reports detected the Wdr68 protein in multiprotein complexes containing mammalian members of the dual-specificity tyrosine-regulated kinase (dyrk) family. Here we describe the characterization of the zebrafish dyrk1b homolog. We report the detection of a physical interaction between Dyrk1b and Wdr68. We also found perturbations of nodal signaling in dyrk1b antisense morpholino knockdown (dyrk1b-MO) animals. Specifically, we found reduced expression of lft1 and lft2 (lft1/2) during gastrulation and a near complete loss of the later asymmetric lft1/2 expression domains. Although wdr68-MO animals did not display lft1/2 expression defects during gastrulation, they displayed a near complete loss of the later asymmetric lft1/2 expression domains. While expression of ndr1 was not substantially effected during gastrulation, ndr2 expression was moderately reduced in dyrk1b-MO animals. Analysis of additional downstream components of the nodal signaling pathway in dyrk1b-MO animals revealed modestly expanded expression of the dorsal axial mesoderm marker gsc while the pan-mesodermal marker bik was largely unaffected. The endodermal markers cas and sox17 were also moderately reduced in dyrk1b-MO animals. Notably, and similar to defects previously reported for wdr68 mutant animals, we also found reduced expression of the pharyngeal pouch marker edn1 in dyrk1b-MO animals. Taken together, these data reveal a role for dyrk1b in endoderm formation and craniofacial patterning in the zebrafish. genesis 48:20–30, 2010. © 2009 Wiley-Liss, Inc.

Pigs are a litter bearing species that give birth to an average between 9 and 13 pigs per litter depending on breed and country. The gestation length is 113–115 days. Early gestation ends at around day 40, and mid gestation at about day 80. In conventional, indoor pig production the lactation period is 3–4 weeks, thus pigs are weaned at around days 21–28 at a mean live body weight of between 6 and 8 kg. In most countries, pigs are slaughtered when they reach a live body weight between 90 and 120 kg, although production of heavier pigs is common in some counties.

Introduction: Breast and ovarian cancer (BC/OC) predisposition is associated with a number of high- and low-penetrance susceptibility genes. Despite comprehensive testing there is still a large portion of high risk cases without mutation in any of the known susceptibility loci. Therefore novel candidate genes need to be identified. Here we report on the results of testing 94 genes in 717 BC/OC patients from Germany and Lithuania. Method: Inclusion criteria for the patients in this study were defined by the German Consortium for Breast and Ovarian Cancer. Next generation sequencing (NGS) was performed on an Illumina MiSeq sequencer, with 150 bp paired end sequencing chemistry and an average base coverage of 300 fold. Target enrichment was performed with the Illumina TruSight cancer panel, which includes 94 genes associated with both common (e.g., breast, colorectal) and rare cancers. Results: In 19.7% of the patients, BRCA1 or BRCA2 variations have been found. These were either clearly pathogenic loss-of-function mutations (43%) or very rare, unclassified missense variations with high probability of a deleterious effect (57%). In 17.9% of the patients we found null-mutations and rare, unclassified missense variants in the acknowledged BC/OC susceptibility genes ATM, CDH1, CHEK2, NBN, PALB2, RAD51C/D and TP53. Analysis of the non-BC/OC genes on the NGS panel identified the “excision repair cross-complementing rodent repair deficiency, complementation group 2” gene (ERCC2 or XPD) as a promising BC/OC predisposition candidate: we found 3 frame-shift mutations and 1 splice-site mutation in four independent BC/OC families. Additionally we found 20 rare, unclassified sequence variations in ERCC2. These variants have a cumulative allele frequency of 2.9% in our BC/OC cohort, which is 14.5-fold overrepresentation compared to the “exome aggregation consortium” (ExAC) cohort (61486 exomes). In all affected families tested so far, the ERCC2 mutations co-segregate with the occurrence of BC and/or OC. Functional assays testing the ERCC2 variants have been initiated. First results show that at least some of the missense variants (e.g. NM_000400.3:p.Val536Met) have lost their DNA repair ability. Conclusion: Deleterious mutations and probably pathogenic missense variations in ERCC2, which are significantly overrepresented in our BC/OC families and co-segregate with the affected individuals, define ERCC2 clearly as a susceptibility gene for BC/OC predisposition. Functional assays will be continued in order to identify missense variations that diminish the DNA-repair capacity of ERCC2. Affected individuals with excluded mutations in the known BC/OC predisposition genes should be tested for mutations in ERCC2. Citation Format: Evelin Schrock, Anna Benet-Pagès, Steffen Schubert, Ramūnas Janavicius, Karl Hackmann, Elitza Betcheva-Krajcir, Luisa Mackenroth, Janin Lehmann, AM Nissen, Janine Altmueller, Holger Thiele, Nataliya Di Donato, Barbara Klink, Jan D. Kuhlmann, Andreas Tzschach, Karin Kast, Pauline Wimberger, Elke Holinski-Feder, Alfons Meindl, Steffen Emmert, Andreas Rump. Germline mutations in patients with hereditary breast and ovarian cancer establish ERCC2 as a cancer susceptibility gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-044. doi:10.1158/1538-7445.AM2015-LB-044

Background: Craniofacial birth defects result from defects in cranial neural crest (NC) patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1) signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified. Results: Through screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage. Conclusion: This work represents a systematic identification of approximately 25% of the essential genes required for craniofacial development. The identification of zebrafish models for two human disease syndromes indicates that homologs to the other genes are likely to also be relevant for human craniofacial development. The initial characterization of wdr68 suggests an important role in craniofacial development for the highly conserved Wdr68-Dyrk1 protein complexes., National Institutes of Health (U.S.)

Summary The present in vitro experiments were carried out in order to study whether variations in the bovine growth hormone (bGH)/insulin-like growth factor (IGF)-I axis induced by plane of nutrition and bGH treatment of heifer calves caused variations in serum-induced proliferation of C2C12 myoblasts. Serum was obtained from two groups each of six heifer calves (195 ± 8 kg) before (d −1) and after treatment with 15 mg/day of bGH for 6 days (d 6) fed either a low (GHL) or a high plane (GHH) of nutrition. Preceding the experiment all 12 heifer calves were fed at the low plane of nutrition. At d 6, serum concentrations of insulin and IGF-I were increased while that of IGF-binding proteins (IGFBP)-2 was decreased in GHH, but unchanged in GHL calves. Serum-induced proliferation of C2C12 myoblasts, was elevated at d 6 by GHH treatment. Especially human IGFBP-3 but also bovine IGFBP-2 added to cell cultures inhibited the rate of proliferation of C2C12 myoblasts stimulated by human IGF-I. The present results showed that GH treatment causes changes in the GH/IGF axis, which leads to changes in serum-induced growth of C2C12 muscle cells dependent on the plane of nutrition that mimic in vivo effects of GH treatment, which indicate an endocrine contribution of the IGF system. However, drawbacks of this suggestion are discussed.

We have developed a new model to describe endogenous glucose kinetics during a labeled (hot) intravenous glucose tolerance test (IVGTT) to derive a time profile of endogenous glucose production (EGP). We reanalyzed data from a previously published study (P. Vicini, J. J. Zachwieja, K. E. Yarasheski, D. M. Bier, A. Caumo, and C. Cobelli. Am J Physiol Endocrinol Metab 276: E285–E294, 1999), in which insulin-modified [6,6-2H2]glucose-labeled IVGTTs (0.33 g/kg glucose) were performed in 10 normal subjects. In addition, a second tracer ([U-13C]glucose) was infused in a variable rate to clamp the endogenous glucose tracer-to-tracee ratio (TTR). Our new model describing endogenous glucose kinetics was incorporated into the two-compartment hot minimal-model structure. The model gave estimates of glucose effectiveness [1.54 ± 0.31 (SE) ml·kg−1·min−1], insulin sensitivity (37.74 ± 5.23 104 dl·kg−1·min−1·μU−1·ml), and a new parameter describing the sensitivity of EGP to the inhibitory effect of insulin (IC50 = 0.0195 ± 0.0046 min−1). The model additionally provided an estimate of the time course of EGP showing almost immediate inhibition, followed by a secondary inhibitory effect caused by infusion of insulin, and a large overshoot as EGP returns to its basal value. Our estimates show very good agreement with those obtained via deconvolution and the model-independent TTR clamp technique. These results suggest that the new integrated model can serve as a simple one-step approach to obtain metabolic indexes while also providing a parametric description of EGP.

Prenatal growth is very complex and a highly integrated process. Both maternal nutrition and the maternal somatotropic axis play a significant role in coordinating nutrient partitioning and utilization between maternal, placental and fetal tissues. Maternal nutrition may alter the nutrient concentrations and in turn the expression of growth regulating factors such as IGFs and IGFBPs in the blood and tissues, while GH acts in parallel via changing IGFs/IGFBPs and nutrient availability. The similarity in the target components implies that maternal nutrition and the somatotropic axis are closely related to each other and may induce similar effects on placental and fetal growth. Severe restriction of nutrients throughout gestation has a permanent negative effect on fetal and postnatal growth, whereas the effects of both temporary restriction and feeding above requirements during gestation seem to be of transitional character. Advantages in fetal growth gained by maternal growth hormone treatment during early to mid-gestation are not maintained to term, whereas treatment during late or greatest part of gestation increases progeny size at birth, which could be of advantage for postnatal growth. This review summarizes the available knowledge on the effects of different maternal feeding strategies and maternal GH administration during pregnancy and their interactions on metabolic and hormonal (especially IGFs/IGFBPs) status in the feto-maternal unit, skeletal muscle development and growth of the offspring in pigs.

We identified four insertional alleles of foxi one ( foo ), an embryonic lethal mutation in zebrafish that displays defects in both otic placode and the jaw. In foo/foo embryos the otic placode is split into two smaller placodes and mutant embryos show a dorsoventral (DV) cartilage defect manifested as a reduced hyomandibular and reduced third and fourth branchial arches. We identified foxi one ( foo ), the zebrafish ortholog of Foxi1 (FREAC6, FKHL10, HFH-3, Fkh10) and a member of the forkhead domain transcriptional regulator family, as the gene mutated in foo/foo embryos. foo is expressed in otic placode precursor cells, and foo/foo embryos lack placodal pax8 expression and have disorganized otic expression of pax2.1 and dlx3 . Third stream neural crest cell migration, detected by dlx2 and krox20 expression, is aberrant in that it invades the otic placode territory. foo is expressed in pharyngeal pouch endoderm and is required for pouch expression of pax8 and proper patterning of other markers in the pouch such as nkx2.3 . In foo/foo embryos, we observed a failure to maintain fgf3 expression in the pouches, followed by apoptosis of neural crest cells in adjacent arches. We conclude that foo expression is essential for pax8 expression probably downstream of Fgf signaling in a conserved pathway jointly required for integrity of patterning in the otic placode and pharyngeal pouches. We propose that correct placement of survival/proliferation cues is essential for shaping the pharyngeal cartilages and that evolutionary links between jaw and ear formation can be traced to Fgf-Foxi1-Pax8 pathways.

Abstract 2185 Objective: The Adenylate Kinase (AK) gene family consists of 7 different members (AK1-7) that contribute to energy metabolism of the cells by converting ATP (or GTP) and free AMP to ADP (or GDP) and free ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues and are generally involved in a broad range of cellular functions. Among AKs, AK2 is uniquely located in the mitochondrial intermembrane space and has been implicated in Caspase 10-mediated apoptosis, although the published data remain controversial. More recently, it was demonstrated that mutations of the AK2 gene cause reticular dysgenesis, an autosomal recessive form of severe combined immunodeficiency (SCID). Reticular dysgenesis is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation resulting in overwhelming infections and high lethality in affected patients. Moreover, patients commonly present with bilateral sensorineural deafness. The mechanisms underlying the biological consequences of AK2-defieincy remain unclear and the generation and characterization study of model systems is expected to provide useful insigths. Ak2 gene-targeted mice have not been reported in the literature, but lines carrying homozygous inactivating retroviral insertions have been shown to be embryonically lethal (our unpublished observations). Because of the known advantages of zebrafish as model system for developmental studies and the similarities of hematopoiesis in zebrafish and higher vertebrates, we set out to investigate the function of the zebrafish ak2 gene in development, with particular emphasis on hematopoiesis. Results: Similar to humans, we found that two different alternatively spliced isoforms of the ak2 gene (Isoform A and Isoform B) are expressed in zebrafish. By Real-Time PCR and In situ Hybridization (ISH) we analyzed the expression of both ak2 isoforms during embryo development. Preliminary data indicate that Isoform A is more abundantly represented than Isoform B during embryo development. ISH analysis showed that the two isoforms have different spatial expression patterns. These data suggest different functionalities for ak2 isoforms during embryo development. To explore such hypothesis, we injected two different morpholinos (MOs) targeting the ak2 isoforms. Downregulation of both ak2 isoforms phenocopied the human disease and resulted in a strong reduction of developing lymphocytes. Moreover we observed a hypochromic phenotype that also suggested impairment of the erythroid lineage. ISH experiments are underway to better define the affected hematopoietic lineages. Interestingly, ak2 MOs-injected embryos showed also developmental defects beyond the hematopoietic system, such as abnormal jaw development. Future studies will focus on the characterization of the specific function of the alternatively spliced ak2 isoforms. Conclusions: We show that the transcription features of the AK2 gene are conserved in zebrafish. The observed differential expression patterns of the zebrafish ak2 isoforms may provide new insights into the function of AK2 in the development of the hematopoietic system, as well as other organs and offers prospects for the understanding of the molecular mechanisms involved in reticular dysgenesis. Disclosures: No relevant conflicts of interest to declare.

Introduction An association between paternal age and children9s health was suggested by Penrose as early as 1955. More recently, relationships have been suggested between paternal age and specific diseases and fetal death. The association is mainly put down to the increased mutation rate in male germ cells. The aim of this study was to investigate the relationship between paternal age and under 5-year mortality. Methods Based on data from Danish population-covering registers, we investigated the relationship between paternal age and under 5-year mortality, including cause-specific mortality, taking maternal age, parity and parental educational levels into account. A total of 1 140 689 live born children were included in the study. Cox regression models were used to estimate HRs for death during the first 5 years of life. Results Compared with children born to fathers aged 30–34 years an excess risk was found for children born to fathers aged 45+ years (HR 1.22; 95% CI 1.05 to 1.42). When only 1–5 years olds were included the RR rose to 1.70; 95% CI 1.23 to 2.34. The excess risk for children of fathers aged 45 years or more was primarily attributed to an elevated risk of dying from congenital malformations and malignancies. Conclusion Children with a father aged 45 years or more have an increased risk of dying before the age of 5 years. The findings are compatible with the hypothesis suggesting increased frequency of point mutations in the fertilising sperm cells from men of advanced age.

A multifocal cavernous hemangioma was found in the renal pelvis of a young male, in whom intermittent macroscopic hematuria developed. Selective angiography was found to be normal on two occasions. By means of preoperative ureterorenoscopy a presumptive diagnosis of hemangioma was made. Nephrectomy was performed because the location of the lesion precluded partial nephrectomy.

On the basis of a retrospective study of 61 cases of carcinoma of the prostate, it is attempted to evaluate the prognosis, either by way of a clinical classification into stages or by histological grading. Both types showed a clear-cut correlation with the prognosis. Collation of the clinical classification into stages and the histological grading of the lesions in 33 patients showed that the two methods represented two unrelated types of classification.

It has been previously reported that actinomycin D (act D) increases the thyroid serum 131I– concentration ratio (T/S) in rats. Such an effect could be due to enhanced thyroidal clearance (k1) -of 131I–, a diminished rate constant (IC2) for the exit of m I– from the thyroid, or changes in both k1 and k2. Sixteen hr after the administration of act D, kinetic analysis of thyroidal I– transport in terms of a 2-compartment model, done with the use of a digital computer, revealed no change in k1 as compared with control rats, but a substantial reduction in k2. Act D therefore increases thyroidal accumulation of 131I– by diminishing its loss from the gland, not by enhancing its clearance from plasma by the thyroid. In contrast, other factors which raise the T/S, for example TSH, do so by elevating k1. (Endocrinology 84: 943, 1969)