Reversible transition of extracellular field potential recordings to intracellular recordings of action potentials generated by neurons grown on transistors

The employment of standard CMOS technology to produce semiconductor chips for recording neuronal activity or for its future use to link neurons and transistors under in vivo conditions, suffers from a low signal to noise ratio. Using Aplysia neurons cultured on CMOS floating gate field effect transistors, we report here that minor mechanical pressure applied to restricted neuronal compartment that face the sensing pad induces two independent alterations: (a) increase in the seal resistance formed between the neuron's membrane and the sensing pad, and (b) increase the conductance of the membrane patch that faces the sensing pad. These alterations (from approximately 0.5 to approximately 1.2 MOmega and 75 to approximately 600 nS correspondingly), are sufficient to transform the low capacitive coupling between a neuron and a transistor to Ohmic coupling, which is manifested by semi-intracellular recordings of APs with amplitudes of up to 30 mV. The semi-intracellular recordings could be maintained for hours. As a number of compression and decompression cycles could be applied to a single cell without causing significant alterations in its excitable properties, we conclude that the mechanical damage inflicted to the neurons by local compression are reversible. Based on these observations, we suggest that the application of minimal local pressure or suction forces could be used to transform conventional extracellular field potential recordings into quasi-intracellular recording, and thereby dramatically improve both the signal to noise ratio and the quality of recordings from neurons cultured on CMOS semiconductors chips.     

Baseline Correlates of Insulin Resistance in Inner City High‐BMI African‐American Children

To characterize the influence of diet‐, physical activity–, and self‐esteem‐related factors on insulin resistance in 8–10‐year‐old African‐American (AA) children with BMI greater than the 85th percentile who were screened to participate in a community‐based type 2 diabetes mellitus (T2DM) prevention trial. In 165 subjects, fasting glucose‐ and insulin‐derived values for homeostasis model assessment of insulin resistance (HOMA‐IR) assessed insulin resistance. Body fatness was calculated following bioelectrical impedance analysis, and fitness was measured using laps from a 20‐m shuttle run. Child questionnaires assessed physical activity, dietary habits, and self‐esteem. Pubertal staging was assessed using serum levels of sex hormones. Parent questionnaires assessed family demographics, family health, and family food and physical activity habits. Girls had significantly higher percent body fat but similar anthropometric measures compared with boys, whereas boys spent more time in high‐intensity activities than girls. Scores for self‐perceived behavior were higher for girls than for boys; and girls desired a more slender body. Girls had significantly higher insulin resistance (HOMA‐IR), compared with boys (P < 0.01). Adjusting for age, sex, pubertal stage, socioeconomic index (SE index), and family history of diabetes, multivariate regression analysis showed that children with higher waist circumference (WC) (P < 0.001) and lower Harter's scholastic competence (SC) scale (P = 0.044) had higher insulin resistance. WC and selected self‐esteem parameters predicted insulin resistance in high‐BMI AA children. The risk of T2DM may be reduced in these children by targeting these factors.     

Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model

Introduction Apoptosis is a central mechanism of cell death following reperfusion of the ischemic liver. Recombinant human erythropoietin (rhEPO) have an important role in the treatment of myocardial ischemia/reperfusion (I/R) injury, by preventing apoptosis. The aim of the study was to investigate the effect of different regimens of rhEPO in preventing apoptosis following I/R-induced hepatic injury. Material and methods Isolated mouse livers were randomly divided into five groups: (1) control group, perfused for the whole study period (105 min); (2) 30-min perfusion followed by 90 min of ischemia and 15 min of reperfusion; (3), (4) and (5) like group 2, but with administration of rhEPO 5,000 units/kg i.p. at 30 min, 24 h, or both 30 min and 24 h respectively, before induction of ischemia. Perfusate liver enzyme levels and intrahepatic caspase-3 activity were measured, and apoptotic cells were identified by morphological criteria, TUNEL assay, and immunohistochemistry for caspase-3. Using immunoblot the expression of the proapoptotic JNK and inhibitor of NFκB (IκBα) were also evaluated. von Willebrand factor (vWF) immunohistochemistry was used as a marker of endothelial cells. Results Compared to the I/R livers, all 3 rhEPO pretreated groups showed: a significant reduction in liver enzyme levels (P < 0.05) and intrahepatic caspase-3 activity (P < 0.05), fewer apoptotic hepatocytes (P < 0.05) and positive vWF staining in numerous endothelial cells lining the sinusoids. EPO decreased JNK phosphorylation and the degradation of the inhibitor of NFκB (IκBα) during I/R. There was no added benefit of the multiple- over the single-dose rhEPO regimen. Conclusion Pretreatment with one dose of rhEPO can attenuate post-I/R hepatocyte apoptotic liver damage. NFκB and JNK activation is likely to play a pivotal role in the pathophysiology of I/R hepatic injury and might have a key role in EPO-mediated protective effects. This effect is associated with the increase in sinusoidal vWF immunostaining suggests an additional effect of rhEPO in liver angiogenesis recovery. These findings have important implications for the potential use of rhEPO in I/R injury during liver transplantation. Edith Hochhauser and Orit Pappo are first two coauthors.     

Pituitary hypoplasia in Pttg-/- mice is protective for Rb+/- pituitary tumorigenesis

Pituitary tumor transforming gene (Pttg) is induced in pituitary tumors and associated with increased tumor invasiveness. Pttg-null mice do not develop tumors, but exhibit pituitary hypoplasia, whereas mice heterozygous for the retinoblastoma (Rb) deletion develop pituitary tumors with high penetrance. Pttg-null mice were therefore cross-bred with Rb+/- mice to test the impact of pituitary hypoplasia on tumor development. Before tumor development, Rb+/-Pttg-/- mice have smaller pituitary glands with fewer cycling pituitary cells and exhibit induction of pituitary p21 levels. Pttg silencing in vitro with specific short hairpin interfering RNA in AtT20 mouse corticotrophs led to a marked induction of p21 mRNA and protein levels, decreased RB phosphorylation, and subsequent 24% decrease in S-phase cells. Eighty-six percent of Rb+/-Pttg+/+ mice develop pituitary adenomas by 13 months, in contrast to 30% of double-crossed Rb+/-Pttg-/- animals (P < 0.01). Pituitary hypoplasia, associated with suppressed cell proliferation, prevents the high penetrance of pituitary tumors in Rb+/- animals, and is therefore a protective determinant for pituitary tumorigenesis.     

Combinations of polyclonal or monoclonal antibodies to proteins of the outer membranes of the two infectious forms of vaccinia virus protect mice against a lethal respiratory challenge

Previous studies demonstrated that antibodies to live vaccinia virus infection are needed for optimal protection against orthopoxvirus infection. The present report is the first to compare the protective abilities of individual and combinations of specific polyclonal and monoclonal antibodies that target proteins of the intracellular (IMV) and extracellular (EV) forms of vaccinia virus. The antibodies were directed to one IMV membrane protein, L1, and to two outer EV membrane proteins, A33 and B5. In vitro studies showed that the antibodies to L1 neutralized IMV and that the antibodies to A33 and B5 prevented the spread of EV in liquid medium. Prophylactic administration of individual antibodies to BALB/c mice partially protected them against disease following intranasal challenge with lethal doses of vaccinia virus. Combinations of antibodies, particularly anti-L1 and -A33 or -L1 and -B5, provided enhanced protection when administered 1 day before or 2 days after challenge. Furthermore, the protection was superior to that achieved with pooled immune gamma globulin from human volunteers inoculated with live vaccinia virus. In addition, single injections of anti-L1 plus anti-A33 antibodies greatly delayed the deaths of severe combined immunodeficiency mice challenged with vaccinia virus. These studies suggest that antibodies to two or three viral membrane proteins optimally derived from the outer membranes of IMV and EV, may be beneficial for prophylaxis or therapy of orthopoxvirus infections.     

Mediterranean fruit fly as a potential vector of bacterial pathogens

The Mediterranean fruit fly (Ceratitis capitata) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit flies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female flies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit fly to serve as a vector for transmission of human pathogens to fruits. Addition of green fluorescent protein (GFP)-tagged Escherichia coli to a Mediterranean fruit fly feeding solution resulted in a dose-dependent increase in the fly's bacterial load. Flies exposed to fecal material enriched with GFP-tagged E. coli were similarly contaminated and were capable of transmitting E. coli to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the E. coli. Flies inoculated with E. coli harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of fluorescent bacteria were confined along the pseudotrachea in the labelum edge of the fly proboscis. Wild flies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identification of E. coli was made. These findings support the hypothesis that the common Mediterranean fruit fly is a potential vector of human pathogens to fruits.     

The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili

Although the phenomenology and mechanics of swimming are very similar in eubacteria and archaeabacteria (e.g. reversible rotation, helical polymorphism of the filament and formation of bundles), the dynamic flagellar filaments seem completely unrelated in terms of morphogenesis, structure and amino acid composition. Archeabacterial flagellar filaments share important features with type IV pili, which are components of retractable linear motors involved in twitching motility and cell adhesion. The archeabacterial filament is unique in: (1) having a relatively smooth surface and a small diameter of approximately 100A as compared to approximately 240A of eubacterial filaments and approximately 50A of type IV pili; (2) being glycosylated and sulfated in a pattern similar to the S-layer; (3) being synthesized as pre-flagellin with a signal-peptide cleavable by membrane peptidases upon transport; and (4) having an N terminus highly hydrophobic and homologous with that of the olygomerization domain of pilin.The synthesis of archeabacterial flagellin monomers as pre-flagellin and their post-translational, extracellular glycosylation suggest a different mode of monomer transport and polymerization at the cell-proximal end of the filament, similar to pili rather than to eubacterial flagellar filaments. The polymerization mode and small diameter may indicate the absence of a central channel in the filament.Using low-electron-dose images of cryo-negative-stained filaments, we determined the unique symmetry of the flagellar filament of the extreme halophile Halobacterium salinarum strain R1M1 and calculated a three-dimensional density map to a resolution of 19A. The map is based on layer-lines of order n=0, +10, -7, +3, -4, +6, and -1. The cross-section of the density map has a triskelion shape and is dominated by seven outer densities clustered into three groups, which are connected by lower-density arms to a dense central core surrounded by a lower-density shell. There is no evidence for a central channel. On the basis of the homology with the oligomerization domain of type IV pilin and the density distribution of the filament map, we propose a structure for the central core.     

Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration

Nitric oxide (NO), produced by distinct nitric oxide synthase (NOS) isoforms, and prostaglandins generated by expression of cyclooxygenases are important mediators in tumor progression. Previous studies have shown that NO can influence the formation of prostaglandin E2 (PGE2). We provide evidence that NO, derived from iNOS and eNOS activity in LMM3 murine mammary adenocarcinoma cell line, is involved in tumor angiogenesis and in tumor cell migration. LMM3 cells that also stimulate their neovascularization activity and migration liberate high basal amounts of PGE2. There is large amount of evidence that postulates positive regulatory interactions between NOS and cyclooxygenase (COX) isoforms. We here show that, in the LMM3 cell line, while PGE2 exerts a positive modulation on NOS activity, NO closes the loop with a negative feed back on COX activity. We also provide evidence of a positive regulatory effect of protein tyrosine kinases on NOS as well as on COX enzymatic functions affecting tumor induced angiogenesis and cell migration.     

Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability

Fluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times.     

Infection of glioma cells with Sindbis virus induces selective activation and tyrosine phosphorylation of protein kinase C delta. Implications for Sindbis virus-induced apoptosis

Sindbis virus (SV) is an alpha virus used as a model for studying the role of apoptosis in virus infection. In this study, we examined the role of protein kinase C (PKC) in the apoptosis induced by SVNI, a virulent strain of SV. Infection of C6 cells with SVNI induced a selective translocation of PKCdelta to the endoplasmic reticulum and its tyrosine phosphorylation. The specific PKCdelta inhibitor rottlerin and a PKCdelta kinase-dead mutant increased the apoptosis induced by SVNI. To examine the role of the tyrosine phosphorylation of PKCdelta in the apoptosis induced by SVNI we used a PKCdelta mutant in which five tyrosine residues were mutated to phenylalanine (PKCdelta5). PKCdelta5-overexpressing cells exhibited increased apoptosis in response to SVNI as compared with control cells and to cells overexpressing PKCdelta. SVNI also increased the cleavage of caspase 3 in cells overexpressing PKCdelta5 but did not induce cleavage of PKCdelta or PKCdelta5. Using single tyrosine mutants, we identified tyrosines 52, 64, and 155 as the phosphorylation sites associated with the apoptosis induced by SVNI. We conclude that PKCdelta exerts an inhibitory effect on the apoptosis induced by SV and that phosphorylation of PKCdelta on specific tyrosines is required for this function.