Mutation Analysis of BRCA1, BRCA2, PALB2 and BRD7 in a Hospital-Based Series of German Patients with Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive form of breast carcinoma with a poor prognosis. Recent evidence suggests that some patients with TNBC harbour germ-line mutations in DNA repair genes which may render their tumours susceptible to novel therapies such as treatment with PARP inhibitors. In the present study, we have investigated a hospital-based series of 40 German patients with TNBC for the presence of germ-line mutations in BRCA1, BRCA2, PALB2, and BRD7 genes. Microfluidic array PCR and next-generation sequencing was used for BRCA1 and BRCA2 analysis while conventional high-resolution melting and Sanger sequencing was applied to study the coding regions of PALB2 and BRD7, respectively. Truncating mutations in BRCA1 were found in six patients, and truncating mutations in BRCA2 and PALB2 were detected in one patient each, whereas no truncating mutation was identified in BRD7. One patient was a double heterozygote for the PALB2 mutation, c.758insT, and a BRCA1 mutation, c.927delA. Our results confirm in a hospital-based setting that a substantial proportion of German TNBC patients (17.5%) harbour germ-line mutations in genes involved in homology-directed DNA repair, with a preponderance of BRCA1 mutations. Triple-negative breast cancer should be considered as an additional criterion for future genetic counselling and diagnostic sequencing.     

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis,Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P₂. In N. crassa hyphae, PtdIns(4,5)P₂ localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P₂ that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.     

A 35-Year Trend Analysis for Back Pain in Austria: The Role of Obesity

Background

The prevalence of back pain is constantly increasing and a public health problem of high priority. In Austria there is a lack of empirical evidence for the development of back pain and its related factors. The present study aims to investigate trends in the prevalence of back pain across different subpopulations (sex, age, obesity).

Methods

A secondary data analysis based on five nationally representative cross-sectional health surveys (1973–2007) was carried out. Face-to-face interviews were conducted in private homes in Austria. Subjects aged 20 years and older were included in the study sample (n = 178,818). Obesity was defined as BMI≥30 kg/m² and adjusted for self-report bias. Back pain was measured as the self-reported presence of the disorder.

Results

The age-standardized prevalence of back pain was 32.9% in 2007; it was higher among women than men (p<0.001), higher in older than younger subjects (p<0.001) and higher in obese than non-obese individuals (p<0.001). During the investigation period the absolute change in the prevalence of back pain was +19.4%. Among all subpopulations the prevalence steadily increased. Obese men showed the highest increase of and the greatest risk for back pain.

Conclusion

These results help to understand the development of back pain in Austria and can be used to plan controlled promotion programs. Further monitoring is recommended in order to control risk groups and plan target group-specific prevention strategies. In Austria particular emphasis should be on obese individuals. We recommend conducting prospective studies to confirm our results and investigate causal relationships.     

Molecular and morphological delimitation of Australian <Emphasis Type="Italic">Triops</Emphasis> species (Crustacea: Branchiopoda: Notostraca)—large diversity and little morphological differentiation

Australia has a very rich and diverse large branchiopod fauna with approximately 140 described or provisionally delimited species, but only one species of Triops, Triops australiensis (Spencer and Hall 1895), is currently recognized. Previous studies identified extensive genetic diversity within T. australiensis that suggested the presence of cryptic species. Herein, we employed an integrative approach to taxonomy to delimit putative species, integrating COI and EF1α sequence data and morphological data. Putative species were initially delimited based on COI by two computational approaches (GMYC and ABGD). The results were interpreted in the light of several species concepts, with particular emphasis on reproductive isolation. Twenty to 27 genetic lineages were delimited. Of these, up to 26 represent species following an evolutionary or phylogenetic species concept. Eighteen are biological species, though reproductive isolation could not be unambiguously established for allopatric species or species without known males. The level of co-occurrences was exceptionally high for Triops, with up to three syntopic and six sympatric species. Species delimitation was impeded by extensive overlap between intraspecific variability and interspecific variation in the genetic as well as morphological datasets. Without prior delimitation of putative species via COI, morphological delimitation would have been impossible. A potential explanation for the morphological variability is the retention of ancestral polymorphisms over long periods of time and across multiple speciation events without subsequent differentiation.     

Spatial variation of microtubule depolymerization in large asters

Microtubule plus-end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared with the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and microtubule-associated proteins (MAPs) in the interior cytosol compared with that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.