Ploidy and nuclear area as a predictive factor of histologic grade in primary breast cancer

OBJECTIVE: To compare ploidy and nuclear area with histologic grade in breast cancer using cytologic samples. STUDY DESIGN: Fine needle aspirates from 85 patients with primary breast cancer were analyzed to identify ploidy and nuclear area. The Feulgen technique was used to stain the material. We used the SAMBA 4000 image analysis system (Grenoble, France) for analyzing ploidy and nuclear area. Each patient underwent a biopsy, and the histologic grade was analyzed. RESULTS: A significant association was found between ploidy and nuclear area, between histologic grade and nuclear area, and between ploidy and histologic grade. As ploidy became aneuploid and polyploid and nuclear area became larger, histologic grade became higher. CONCLUSION: A reliable and rapid evaluation of variables for breast cancer can be achieved using cytologic preparations by measuring ploidy and nuclear area of malignant cells with an image analysis system. Ploidy and nuclear area have a significant association with histologic grade.     

A Synechococcus PglnA::luxAB fusion for estimation of nitrogen bioavailability to freshwater cyanobacteria

In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp. strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 micro M for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.     

Glycoprotein IIb/IIIa receptor antagonists in acute myocardial infarction and primary coronary stenting: Not always effective

Glycoprotein IIb/IIIa antagonists have been shown to be effective in reducing thrombotic complications prior to high-risk coronary interventions. Some studies have reported improved coronary flow after abciximab in slow or no-reflow phenomenon. We report a case in which abciximab did not clear the thrombotic occlusion or restore artery flow. Further studies are needed into the refractory no-flow phenomenon.     

GTP enhances the degradation of the photosystem II D1 protein irrespective of its conformational heterogeneity at the Q(B) site

The light exposure history and/or binding of different herbicides at the Q(B) site may induce heterogeneity of photosystem II acceptor side conformation that affects D1 protein degradation under photoinhibitory conditions. GTP was recently found to stimulate the D1 protein degradation of photoinactivated photosystem II (Spetea, C. , Hundal, T., Lohmann, F., and Andersson, B. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6547-6552). Here we report that GTP enhances the cleavage of the D1 protein D-E loop following exposure of thylakoid membranes to either high light, low light, or repetitive single turnover flashes but not to trypsin. GTP does not stimulate D1 protein degradation in the presence of herbicides known to affect the accessibility of the cleavage site to proteolysis. However, GTP stimulates degradation that can be induced even in darkness in some photosystem II conformers following binding of the PNO8 herbicide (Nakajima, Y., Yoshida, S., Inoue, Y., Yoneyama, K., and Ono, T. (1995) Biochim. Biophys. Acta 1230, 38-44). Both the PNO8- and the light-induced primary cleavage of the D1 protein occur in the grana membrane domains. The subsequent migration of photosytem II containing the D1 protein fragments to the stroma domains for secondary proteolysis is light-activated. We conclude that the GTP effect is not confined to a specific photoinactivation pathway nor to the conformational state of the photosystem II acceptor side. Consequently, GTP does not interact with the site of D1 protein cleavage but rather enhances the activity of the endogenous proteolytic system.     

Similarities and seasonal variations in bacterial communities from the blood of rodents and from their flea vectors

Vector-borne microbes are subject to the ecological constraints of two distinct microenvironments: that in the arthropod vector and that in the blood of its vertebrate host. Because the structure of bacterial communities in these two microenvironments may substantially affect the abundance of vector-borne microbes, it is important to understand the relationship between bacterial communities in both microenvironments and the determinants that shape them. We used pyrosequencing analyses to compare the structure of bacterial communities in Synosternus cleopatrae fleas and in the blood of their Gerbillus andersoni hosts. We also monitored the interindividual and seasonal variability in these bacterial communities by sampling the same individual wild rodents during the spring and again during the summer. We show that the bacterial communities in each sample type (blood, female flea or male flea) had a similar phylotype composition among host individuals, but exhibited seasonal variability that was not directly associated with host characteristics. The structure of bacterial communities in male fleas and in the blood of their rodent hosts was remarkably similar and was dominated by flea-borne Bartonella and Mycoplasma phylotypes. A lower abundance of flea-borne bacteria and the presence of Wolbachia phylotypes distinguished bacterial communities in female fleas from those in male fleas and in rodent blood. These results suggest that the overall abundance of a certain vector-borne microbe is more likely to be determined by the abundance of endosymbiotic bacteria in the vector, abundance of other vector-borne microbes co-occurring in the vector and in the host blood and by seasonal changes, than by host characteristics.     

Integrated Microfluidics for Protein Modification Discovery

Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system''s potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.Protein post-translational modifications (PTMs)¹ vastly diversify eukaryotic proteomes and are integrated in essentially all cellular processes (1). Proteomic approaches, such as mass spectrometry (MS), have been instrumental in monitoring global molecular dynamics for research and clinical applications (2–5). However, even in this modern era, large-scale analyses of PTMs by MS is challenging because of the limited number of modified peptides derived from proteins that, by themselves, may not be abundant. Moreover, comprehensive PTM analysis by MS often requires significant amounts of biological material that may not be available. PTM analysis using protein arrays can overcome these limitations because of the equimolar amount of the arrayed proteins (6, 7). Large-scale protein arrays have been successfully integrated into PTM research (8, 9). However, this technology relies on pre-purified proteins that are arrayed on a surface and thus, incompatible with biochemically challenging proteins, let alone insoluble proteins. Moreover, the production of recombinant protein arrays is impractical in-house. Therefore, such arrays cannot be used fresh, and they are inherently limited to certain designs, protein compositions, and model organisms of high commercial value. To overcome the abovementioned limitations, we designed a modular integrated microfluidic platform for PTM analysis (IMPA).     

De Novo Loss-of-Function Mutations in SETD5, Encoding a Methyltransferase in a 3p25 Microdeletion Syndrome Critical Region,Cause Intellectual Disability

To identify further Mendelian causes of intellectual disability (ID), we screened a cohort of 996 individuals with ID for variants in 565 known or candidate genes by using a targeted next-generation sequencing approach. Seven loss-of-function (LoF) mutations—four nonsense (c.1195A>T [p.Lys399^∗], c.1333C>T [p.Arg445^∗], c.1866C>G [p.Tyr622^∗], and c.3001C>T [p.Arg1001^∗]) and three frameshift (c.2177_2178del [p.Thr726Asnfs^∗39], c.3771dup [p.Ser1258Glufs^∗65], and c.3856del [p.Ser1286Leufs^∗84])—were identified in SETD5, a gene predicted to encode a methyltransferase. All mutations were compatible with de novo dominant inheritance. The affected individuals had moderate to severe ID with additional variable features of brachycephaly; a prominent high forehead with synophrys or striking full and broad eyebrows; a long, thin, and tubular nose; long, narrow upslanting palpebral fissures; and large, fleshy low-set ears. Skeletal anomalies, including significant leg-length discrepancy, were a frequent finding in two individuals. Congenital heart defects, inguinal hernia, or hypospadias were also reported. Behavioral problems, including obsessive-compulsive disorder, hand flapping with ritualized behavior, and autism, were prominent features. SETD5 lies within the critical interval for 3p25 microdeletion syndrome. The individuals with SETD5 mutations showed phenotypic similarity to those previously reported with a deletion in 3p25, and thus loss of SETD5 might be sufficient to account for many of the clinical features observed in this condition. Our findings add to the growing evidence that mutations in genes encoding methyltransferases regulating histone modification are important causes of ID. This analysis provides sufficient evidence that rare de novo LoF mutations in SETD5 are a relatively frequent (0.7%) cause of ID.