Single institute study of <Emphasis Type="Italic">FLT3</Emphasis> mutation in acute myeloid leukemia with near tetraploidy in Serbia

Patients with de novo acute myeloid leukemia (AML) and near-tetraploid or completely tetraploid karyotype at presentation are rare. We present four patients with near-tetraploidy/tetraploidy in a cohort of 426 consecutive AML patients (0.98%) in respect to their cytogenetic findings, immunophenotype pattern, response to chemotherapy, course of disease and molecular analyses including tyrosine kinase receptor FLT3 gene, NRAS gene, and tumour suppressor gene, p53. We have found FLT3/ITD mutation only in one patient among the four with near-tetraploidy. The main finding is that these patients had a variable clinical course, with two having a long period of remission (36 and 12 months) and two died, not having achieved remission.     

Advice to a young scientist (by someone who doesn’t know how to give it)

While trying to extract original and general advice from the details of my career, I realized this might not be possible. My path, like those of so many others, had too many idiosyncratic twists and turns that had to work out just the way they did to be mined for generally useful strategies. So I abandon the conceit of advice and simply give you my story. There are many like it, but this one is mine. Take what you wish from it.     

Improved group‐specific primers based on the full SILVA 16S rRNA gene reference database

Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T‐RFLP) analysis, are well‐suited techniques for the examination of microbial community structures. The use of phylum‐ and class‐specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain‐specific primers. To date, several phylum‐ and class‐specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non‐target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T‐RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above‐mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics.     

The Rap1 Guanine Nucleotide Exchange Factor C3G Is Required for Preservation of Larval Muscle Integrity in Drosophila melanogaster

C3G is a guanine nucleotide exchange factor (GEF) and modulator of small G-protein activity, which primarily acts on members of the Rap GTPase subfamily. Via promotion of the active GTP bound conformation of target GTPases, C3G has been implicated in the regulation of multiple cellular and developmental events including proliferation, differentiation and apoptosis. The Drosophila C3G orthologue exhibits a domain organization similar to that of vertebrate C3G. Through deletion of the C3G locus, we have observed that loss of C3G causes semi-lethality, and that escaping adult flies are characterized by a reduction in lifespan and general fitness. In situ hybridization reveals C3G expression in the developing embryonic somatic and visceral muscles, and indeed analysis of C3G mutants suggests essential functions of C3G for normal body wall muscle development during larval stages. C3G mutants display abnormal muscle morphology and attachment, as well as failure to properly localize βPS integrins to muscle attachment sites. Moreover, we show that C3G stimulates guanine nucleotide exchange on Drosophila Rap GTPases in vitro. Taken together, we conclude that Drosophila C3G is a Rap1-specific GEF with important functions in maintaining muscle integrity during larval stages.     

Micro- and nanotechnology in cell separation

This review describes recent work in cell separation using micro- and nanoscale technologies. These devices offer several advantages over conventional, macroscale separation systems in terms of sample volumes, low cost, portability, and potential for integration with other analytical techniques. More importantly, and in the context of modern medicine, these technologies provide tools for point-of-care diagnostics, drug discovery, and chemical or biological agent detection. This review describes work in five broad categories of cell separation based on (1) size, (2) magnetic attraction, (3) fluorescence, (4) adhesion to surfaces, and (5) new emerging technologies. The examples in each category were selected to illustrate separation principles and technical solutions as well as challenges facing this rapidly emerging field.     

Oxygen gradients correlate with cell density and cell viability in engineered cardiac tissue

For clinical utility, cardiac grafts should be thick and compact, and contain physiologic density of metabolically active, differentiated cells. This involves the need to control the levels of nutrients, and most critically oxygen, throughout the construct volume. Most culture systems involve diffusional transport within the constructs, a situation associated with gradients of oxygen concentration, cell density, cell viability, and function. The goal of our study was to measure diffusional gradients of oxygen in statically cultured cardiac constructs, and to correlate oxygen gradients to the spatial distributions of cell number and cell viability. Using microelectrodes, we measured oxygen distribution in a disc-shaped constructs (3.6 mm diameter, 1.8 mm thickness) based on neonatal rat cardiomyocytes cultured on collagen scaffolds for 16 days in static dishes. To rationalize experimental data, a mathematical model of oxygen distribution was derived as a function of cell density, viability, and spatial position within the construct. Oxygen concentration and cell viability decreased linearly and the live cell density decreased exponentially with the distance from the construct surface. Physiological density of live cells was present only within the first 128 microm of the construct thickness. Medium flow significantly increased oxygen concentration within the construct, correlating with the improved tissue properties observed for constructs cultured in convectively mixed bioreactors.     

Mechanism of replication-coupled DNA interstrand crosslink repair

DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase zeta. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.     

Relation between lipid peroxidation and iron concentration in mouse liver after acute and subacute cadmium intoxication

In this study the effect of acute and subacute cadmium (Cd) intoxication on iron (Fe) concentration and lipid peroxidation (LPO) was investigated in the livers of Swiss mice. Animals were divided into two groups: the Cd group – mice intoxicated with Cd and controls. In acute time-response studies, Fe and malondialdehyde (MDA) levels were determined at 4, 6, 12, 24 and 48 h after a single oral dose of Cd (20 mg Cd/kg b.w.). In the subacute experiment, mice were given 10 mg Cd/kg b.w. orally every day for 14 days; Fe and MDA contents were determined in liver after 1 and 2 weeks. Acute Cd intoxication induced a significantly increased hepatic Fe content after 4 and 6 h, and a statistically significant increase in MDA 6, 12 and 24 h after Cd administration, although a significantly decreased MDA level was observed after 48 h. The results suggest development of early oxidative stress in livers of mice after acute intoxication with Cd. The decreased MDA observed after 48 h occurred presumably due to the adaptive response of the organism. Subacute Cd intoxication induced a significant decrease of hepatic Fe and MDA levels at both investigated time intervals compared with control. These results indicate a positive correlation between hepatic Fe and MDA content and suggest that prolonged Cd intoxication decreases hepatic LPO indirectly, by reducing the Fe content of mouse liver.     

An unexpected functional link between lysosomal thiol reductase and mitochondrial manganese superoxide dismutase

Gamma interferon-inducible thiol reductase (GILT) is an enzyme involved in the initial steps of antigen processing and presentation. Recently we have shown that GILT is also expressed in mouse T cells, where it exerts an inhibitory role on T cell activation. In this study, we identified mitochondrial manganese superoxide dismutase (SOD2) as one of the key intermediaries affected by GILT expression in fibroblasts. Expression and activity of SOD2 is reduced in the absence of GILT because of reduced SOD2 protein stability. The forced increase in SOD2 expression in the absence of GILT restores fibroblast proliferation to wild-type levels. Thus, GILT appears to have a fundamental role in cellular proliferation mediated through its influence on SOD2 protein activity and expression.