An in situ atomic force microscope for normal-incidence nanofocus X-ray experiments.

A compact high-speed X-ray atomic force microscope has been developed for in situ use in normal-incidence X-ray experiments on synchrotron beamlines, allowing for simultaneous characterization of samples in direct space with nanometric lateral resolution while employing nanofocused X-ray beams. In the present work the instrument is used to observe radiation damage effects produced by an intense X-ray nanobeam on a semiconducting organic thin film. The formation of micrometric holes induced by the beam occurring on a timescale of seconds is characterized.

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αß sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting.

IGRP and insulin vaccination induce CD8+ T cell-mediated autoimmune diabetes in the RIP-CD80GP mouse.

Autoimmune diabetes is characterized by autoantigen-specific T cell-mediated destruction of pancreatic islet beta cells, and CD8(+) T cells are key players during this process. We assessed whether the bitransgenic RIP-CD80 x RIP-LCMV-GP (RIP-CD80GP) mice may be a versatile antigen-specific model of inducible CD8(+) T cell-mediated autoimmune diabetes. Antigen-encoding DNA, peptide-loaded dendritic cells and antigen plus incomplete Freund's adjuvant were used for vaccination. Of 14 pancreatic proteins tested by DNA vaccination, murine pre-proinsulin 2 (100% of mice; median time after vaccination, 60 days) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (77%, 58 days) could induce diabetes. Vaccination with DNA encoding for zinc transporter 8, Ia-2, Ia-2ß, glutamic acid decarboxylase 67 (Gad67), chromogranin A, insulinoma amyloid polypeptide and homeobox protein Nkx-2.2 induced diabetes development in 25-33% of mice. Vaccination with DNA encoding for Gad65, secretogranin 5, pancreas/duodenum homeobox protein 1 (Pdx1), carboxyl ester lipase, glucagon and control hepatitis B surface antigen (HBsAg) induced diabetes in <20% of mice. Diabetes induction efficiency could be increased by DNA vaccination with a vector encoding a ubiquitin-antigen fusion construct. Diabetic mice had florid T cell islet infiltration. CD8(+) T cell targets of IGRP were identified with a peptide library-based enzyme-linked immunospot assay, and diabetes could also be induced by vaccination with major histocompatibility complex (MHC) class I-restricted IGRP peptides loaded on mature dendritic cells. Vaccination with antigen plus incomplete Freund's adjuvant, which can prevent diabetes in other models, led to rapid diabetes development in the RIP-CD80GP mouse. We conclude that RIP-CD80GP mice are a versatile model of antigen specific autoimmune diabetes and may complement existing mouse models of autoimmune diabetes for evaluating CD8(+) T cell-targeted prevention strategies.

The effect of gestation and fetal mismatching on the development of autoimmune diabetes in non-obese diabetic mice.

The impact of gestation and fetal-maternal interactions on pre-existent autoimmune beta cell destruction is widely unknown. The aim of this study was to investigate the influence of gestation per se and fetal mismatching on the onset of autoimmune diabetes in female non-obese diabetic (NOD) mice. We examined cumulative diabetes frequencies of NOD dams mated to syngeneic NOD, haploidentical CByB6F1/J and fully mismatched C57BL/6J male mice. Pregnancy from NOD males neither increased nor accelerated the diabetes onset of NOD dams (71% by age 28 weeks) compared to unmated female NOD mice (81% by age 28 weeks; P = 0·38). In contrast, delayed diabetes onset was observed when NOD dams were mated at 10 weeks of age with major histocompatibility complex (MHC) haploidentical CByB6F1/J male mice (38% at age 28 weeks; P = 0·01). Mating with fully MHC mismatched C57BL/6J male mice (72% diabetes by age 28 weeks; P = 0·22) or mating with the haploidentical males at the later time-point of age 13 weeks (64% versus 91% in unmated litter-matched controls; P = 0·13) did not delay diabetes significantly in NOD females. Because infusion of haploidentical male mouse splenocytes was found previously to prevent diabetes in NOD mice we looked for, but found no evidence of, persistent chimeric lymphocytes from haploidentical paternal origin within the dams' splenocytes. Gestation per se appears to have no aggravating or ameliorating effects on pre-existent autoimmune beta cell destruction, but pregnancy from MHC partially mismatched males delays diabetes onset in female NOD mice.

A low level of extragalactic background light as revealed by gamma-rays from blazars.

The diffuse extragalactic background light consists of the sum of the starlight emitted by galaxies through the history of the Universe, and it could also have an important contribution from the 'first stars', which may have formed before galaxy formation began. Direct measurements are difficult and not yet conclusive, owing to the large uncertainties caused by the bright foreground emission associated with zodiacal light. An alternative approach is to study the absorption features imprinted on the gamma-ray spectra of distant extragalactic objects by interactions of those photons with the background light photons. Here we report the discovery of gamma-ray emission from the blazars H 2356 - 309 and 1ES 1101 - 232, at redshifts z = 0.165 and z = 0.186, respectively. Their unexpectedly hard spectra provide an upper limit on the background light at optical/near-infrared wavelengths that appears to be very close to the lower limit given by the integrated light of resolved galaxies. The background flux at these wavelengths accordingly seems to be strongly dominated by the direct starlight from galaxies, thus excluding a large contribution from other sources-in particular from the first stars formed. This result also indicates that intergalactic space is more transparent to gamma-rays than previously thought.

Discovery of very-high-energy gamma-rays from the Galactic Centre ridge.

The source of Galactic cosmic rays (with energies up to 10(15) eV) remains unclear, although it is widely believed that they originate in the shock waves of expanding supernova remnants. At present the best way to investigate their acceleration and propagation is by observing the gamma-rays produced when cosmic rays interact with interstellar gas. Here we report observations of an extended region of very-high-energy (> 10(11) eV) gamma-ray emission correlated spatially with a complex of giant molecular clouds in the central 200 parsecs of the Milky Way. The hardness of the gamma-ray spectrum and the conditions in those molecular clouds indicate that the cosmic rays giving rise to the gamma-rays are likely to be protons and nuclei rather than electrons. The energy associated with the cosmic rays could have come from a single supernova explosion around 10(4) years ago.

Downregulation of Sef, an inhibitor of receptor tyrosine kinase signaling, is common to a variety of human carcinomas.

Carcinomas are tumors of epithelial origin accounting for over 80% of all human malignancies. A substantial body of evidence implicates oncogenic signaling by receptor tyrosine kinases (RTKs) in carcinoma development. Here we investigated the expression of Sef, a novel inhibitor of RTK signaling, in normal human epithelial tissues and derived malignancies. Human Sef (hSef) was highly expressed in normal epithelial cells of breast, prostate, thyroid gland and the ovarian surface. By comparison, substantial downregulation of hSef expression was observed in the majority of tumors originating from these epithelia. Among 186 primary carcinomas surveyed by RNA in situ hybridization, hSef expression was undetectable in 116 cases including 72/99 (73%) breast, 11/16 (69%) thyroid, 16/31 (52%) prostate and 17/40 (43%) ovarian carcinomas. Moderate reduction of expression was observed in 17/186, and marked reduction in 40/186 tumors. Only 13/186 cases including 12 low-grade and one intermediate grade tumor retained high hSef expression. The association of hSef downregulation and tumor progression was statistically significant (P<0.001). Functionally, ectopic expression of hSef suppressed proliferation of breast carcinoma cells, whereas inhibition of endogenous hSef expression accelerated fibroblast growth factor and epidermal growth factor-dependent proliferation of cervical carcinoma cells. The inhibitory effect of hSef on cell proliferation combined with consistent downregulation in human carcinoma indicates a tumor suppressor-like role for hSef, and implicates loss of hSef expression as a common mechanism in epithelial neoplasia.

The Elicitation of Ethylene Biosynthesis by a Trichoderma Xylanase Is Not Related to the Cell Wall Degradation Activity of the Enzyme.

A [beta]-1,4-endoxylanase (EIX) isolated from Trichoderma viride elicits plant defense responses in certain tobacco (Nicotiana tabacum L.) cultivars in addition to its xylan degradation activity. It was not clear whether elicitation occurs by cell wall fragments released by the enzymic activity or by the xylanase protein interacting directly with the plant cells. We used protoplasts isolated from tobacco leaves to test whether the cell wall is required for the stimulation of ethylene biosynthesis by EIX. Protoplasts of tobacco (cv Xanthi) responded to treatment with the EIX, as indicated by an increased production of ethylene and the loss of protoplast viability. Protoplasts prepared from ethylene-pretreated leaves produced more ethylene and had higher rates of cell death in response to EIX than protoplasts prepared from nonethylene-treated leaves. Protoplasts of an EIX-insensitive cultivar of tobacco (Hicks) were insensitive to high concentrations of EIX. The addition of a crude cell wall preparation to protoplasts during incubation with EIX did not enhance the induction of ethylene biosynthesis by nonsaturating as well as saturating concentrations of EIX. These data indicate that the xylanase activity of EIX is unrelated to the elicitation of ethylene biosynthesis through the production of some cell wall fragment, since the protein per se appears capable of eliciting ethylene biosynthesis in protoplasts.

Isolation of an ompC-like outer membrane protein gene from Salmonella typhi.

We have isolated the structural gene for an outer membrane protein of Salmonella typhi, from a genomic library constructed in bacteriophage lambda 1059, using the Escherichia coli ompC gene as a heterologous probe. E. coli ompC codes for an outer membrane pore protein (porin) that is induced preferentially at high osmolarity and high temperature. The S. typhi ompC-like gene was subcloned in pBR322 and introduced into E. coli HB101 and into P678-54, a minicell-producing strain. In both strains it expressed a 38.5-kDa protein, which was incorporated into the outer membrane envelope and comigrated with an S. typhi outer membrane protein which was expressed both at low and high osmolarity in vivo.

Septohippocampal cholinergic changes after destruction of the A10-septal dopaminergic pathways.

Mice were injected bilaterally into the septum with 6-hydroxydopamine and 6 weeks later the hippocampi were assayed for activity of choline acetyltransferase, muscarinic receptor binding capabilities and for formation of inositol phosphate in response to direct (carbachol) or presynaptically elicited (K+) stimulation of the postsynaptic receptors. Levels of dopamine in the septum were reduced by 70% in the lesioned animals and hippocampal choline acetyltransferase was elevated by the same amount. The Bmax of muscarinic binding was significantly reduced without changes in Kd; nevertheless, carbachol-induced stimulation of formation of inositol phosphate was unaffected. The response to K+ was markedly elevated in the 6-hydroxydopamine-treated animals. Thus, the regulatory effect of A10-septal dopaminergic pathways on the septohippocampal cholinergic innervations is both on the presynaptic and postsynaptic levels.

Hippocampal cholinergic alterations and related behavioral deficits after early exposure to ethanol.

The present study was designed to ascertain septohippocampal cholinergic alterations and their related behavioral deficits after early exposure to ethanol. Mouse pups were exposed to ethanol, 3 g/kg by daily subcutaneous injection on postnatal days 2-14. At age 50 days, the ethanol-exposed mice had significant reductions from control levels in eight-arm maze performance. For example, on the fourth testing day, the number of correct entries in the ethanol group was 21% below control levels (P < 0.05) and the number of trials needed to enter all arms was 48% above control (P < 0.001). It took the ethanol-exposed mice twice the time to reach criterion than it did control (P < 0.01). A 33% increase from control level in muscarinic receptor number (Bmax) was found in the treated mice of age 22 days and a 64% increase at age 50 days (P < 0.001). However, no differences between control and treated groups could be detected in the presynaptic component of the cholinergic innervation, choline acetyltransferase activity. The results suggest that early ethanol exposure acts on hippocampal function similarly to phenobarbital, probably via alterations in postsynaptic processes in the septohippocampal cholinergic pathways.

Alterations in septohippocampal cholinergic innervations and related behaviors after early exposure to heroin and phencyclidine.

Mice were exposed to diacetylmorphine (heroin) or phencyclidine (PCP) prenatally or neonatally. At a later age, they were tested for hippocampus-related behavioral deficits and concomitant alterations in the septohippocampal cholinergic innervations. Actually, this is an application of the previously established phenobarbital neuroteratogenicity model to heroin and PCP. Prenatal exposure was accomplished transplacentally by injecting the mother 10 mg/kg heroin or PCP on gestation days 9-18. Neonatal administrations were applied directly by injections of 10 mg/kg of either drug to the pups between neonatal days 2-21. At the age of 50 days, mice exposed to heroin and PCP prenatally exhibited a 107% and 159% increase in their muscarinic cholinergic receptors Bmax, respectively. Neonatal exposure to heroin or PCP caused an 83% and 76% increase in the receptors respectively. On the behavioral level, both prenatal and neonatal exposure to heroin or PCP reduced performance in the hippocampus related eight-arm maze and Morris mazes. Depending on the drug, the test and the period of drug administration, the reduction ranged between 10% and 75%. The results suggest that heroin and PCP induce alterations in the septohippocampal cholinergic innervations and in related behavioral performance. Further studies are necessary in order to connect the biochemical and behavioral events in causal relationships.

Reversal of early phenobarbital-induced cholinergic and related behavioral deficits by neuronal grafting.

The present experiment was performed to assess the possible restoration of normal maze behavior, as well as parallel muscarinic receptor binding capabilities, in mice pre- or neonatally exposed to phenobarbital. Mice were exposed to phenobarbital prenatally by feeding the mother phenobarbital (3 gkg milled food) on gestation days 9-18 (PreB mice), or neonatally, by daily injections of 50 mg/kg Na phenobarbital to the pups on days 2-21 (NeoB). At adulthood, PreB and NeoB mice were 61.3% and 65% deficient, respectively, in the hippocampus-related Morris maze behavior, as compared to control. Both groups had a 58% increase in their hippocampal muscarinic receptors maximal binding (Bmax) (p < 0.001); the dissociation constant (Kd) was not affected by the phenobarbital exposure. Treated animals and their respective controls received septal cholinergic embryonic graft into the hippocampus. The viability of the transplants was confirmed by AChE histochemistry. Nine weeks later the grafted mice showed significant improvement in the Morris maze (52% for both PreB and NeoB (p < 0.001)). Their Bmax was also reduced from early phenobarbital exposed animals' levels by 15% for PreB and by 25% for NeoB (p < 0.001). The results suggest that early phenobarbital-induced behavioral deficit and their related biochemical alterations can be partially corrected by the appropriate neural grafting, and thus provide further support to the apparent relationship between the early phenobarbital-induced septohippocampal cholinergic alterations and the hippocampus-related behavioral deficits.

Alterations in hippocampal cholinergic receptors and hippocampal behaviors after early exposure to nicotine.

Mice were exposed to nicotine prenatally by injecting the mother with 1.5 mg/kg nicotine SC twice daily on gestation days 9-18 (PreN mice) or neonatally by daily SC injections of 1.5 mg/kg nicotine on postnatal days 2-21 (NeoN mice). At age 50 days, hippocampal muscarinic receptors Bmax of PreN and NeoN mice were 58% and 79% above control, respectively (p less than 0.01); Kd was unaffected by early nicotine exposure. Eight-arm maze performance of nicotine-exposed animals fell behind control level. Both PreN and NeoN made approximately 10% less correct responses in the first eight trials than controls throughout the test period (p less than 0.01). By the last day of testing, PreN needed 23% and NeoN 31% more trials than controls to enter all arms (p less than 0.001). In addition, PreN needed 35 and NeoN 42% more days than controls to reach criterion (p less than 0.05). Similarly, while 61% of controls reached criterion by day 6 only 17% of PreN and 25% of NeoN reached criterion (p less than 0.01). In the Morris maze, PreN needed from 43-119% more time to reach the platform (p less than 0.001). In the spatial probe test, PreN animals made 35% fewer crosses over the area of the missing platform (p less than 0.001). The study suggests that nicotine administered to the fetus or neonate alters septohippocampal chemistry and induces deficits in hippocampus-related behaviors. The possible reversal of the behavioral changes by manipulating the cholinergic innervations should be the subject of future investigations.

Hippocampal cholinergic alterations and related behavioral deficits after early exposure to phenobarbital.

Mice were exposed to phenobarbital (PhB) prenatally and neonatally. Prenatal exposure was accomplished by feeding the mother PhB (3 g/kg milled food) on gestation days 9-18. Neonatal exposure was accomplished by daily injections of 50 mg/kg sodium PhB directly to the pups on days 2-21. Long-term biochemical alterations in the pre- and postsynaptic septohippocampal system, as well as related behavioral deficits, were assessed in the treated animals. Significant increase in B(max) values for binding of [3H]QNB to muscarinic cholinergic receptors was obtained on both ages 22 and 50 in prenatally (40-90%, respectively, p less than 0.001) and neonatally exposed (58-89%, p less than 0.001) mice whereas Kd remained normal. Similarly, a significant increase of inositol phosphate (IP) formation in response to carbachol was found after both prenatal and neonatal exposure to PhB (p less than 0.05). No alterations in choline acetyltransferase (ChAT) activity were observed in the prenatally or neonatally treated animals. The early exposed mice showed deficits in the performance in Morris water maze, a behavior related to the septohippocampal pathway. The results suggest that early exposure to PhB induces alterations in postsynaptic components of the hippocampal cholinergic system and concomitantly to impairment in hippocampus-related behavior.

Neural and behavioral alterations after early exposure to phenobarbital.

Mice who were exposed to phenobarbital prenatally (B mice) had at adulthood deficits in the hippocampal eight-arm maze, spontaneous alternations, and water maze behaviors. Morphological studies revealed neuronal losses in the hippocampus. The surviving neurons had reductions from control in the number of dendritic branches, area and spine density, but wider fission angle than control. Neurochemical studies on the hippocampus revealed the following alterations: (a) decrease in NE level and the number of the NE cell bodies (b) no change in the serotonergic system (c) an increase in muscarinic receptors Bmax in the hippocampus; (d) no changes in GABA and benzodiazepine receptors. However, neonatal phenobarbital exposure caused an increase in the Bmax of GABA and benzodiazepine receptors. Transplantation of fetal septal cholinergic neurons into the hippocampus of B mice reversed most of the deficits in eight-arm maze behavior, while transplantation of noradrenergic cells did not affect the performance of B mice. In further studies on cholinergic mechanisms, the dopaminergic innervations in the septum (originating from A10), which are known to indirectly inhibit the activity of the septohippocampal cholinergic pathways, were destroyed by 6-OHDA. B mice treated with 6-OHDA had an increase in hippocampal ChAT activity and improved their eight-arm maze performance. Thus, understanding of the mechanism of a particular behavioral deficit enables one to correct it despite the nonspecific action of the neuroteratogen.

Spiral computerized tomography and video thoracoscopy in catamenial pneumothorax.

Catamenial pneumothorax is a rare disease that is usually diagnosed on clinical grounds. Delay in diagnosis can lead to considerable morbidity. We describe a case in which spiral CT scan and videothoracoscopy led to early objective diagnosis and management.

Examination of bacteriophage as a biocontrol method for salmonella on fresh-cut fruit: a model study.

The preparation and distribution of fresh-cut produce is a rapidly developing industry that provides the consumer with convenient and nutritious food. However, fresh-cut fruits and vegetables may represent an increased food safety concern because of the absence or damage of peel and rind, which normally help reduce colonization of uncut produce with pathogenic bacteria. In this study, we found that Salmonella Enteritidis populations can (i) survive on fresh-cut melons and apples stored at 5 degrees C, (ii) increase up to 2 log units on fresh-cut fruits stored at 10 degrees C, and (iii) increase up to 5 log units at 20 degrees C during a storage period of 168 h. In addition, we examined the effect of lytic, Salmonella-specific phages on reducing Salmonella numbers in experimentally contaminated fresh-cut melons and apples stored at various temperatures. We found that the phage mixture reduced Salmonella populations by approximately 3.5 logs on honeydew melon slices stored at 5 and 10 degrees C and by approximately 2.5 logs on slices stored at 20 degrees C, which is greater than the maximal amount achieved using chemical sanitizers. However, the phages did not significantly reduce Salmonella populations on the apple slices at any of the three temperatures. The titer of the phage preparation remained relatively stable on melon slices, whereas on apple slices the titer decreased to nondetectable levels in 48 h at all temperatures tested. Inactivation of phages, possibly by the acidic pH of apple slices (pH 4.2 versus pH 5.8 for melon slices), may have contributed to their inability to reduce Salmonella contamination in the apple slices. Higher phage concentrations and/or the use of low-pH-tolerant phage mutants may be required to increase the efficacy of the phage treatment in reducing Salmonella contamination of fresh-cut produce with a low pH.

Historical group debriefing after combat exposure.

BACKGROUND: Studies of group debriefing after extreme events have failed to show a significant long-term effect of this technique. However, the heterogeneity of the interventions studied and the length of time between debriefing and its assessment do not allow a proper interpretation of these findings. OBJECTIVE: This study evaluates the immediate effect of debriefing in Israeli soldiers exposed to combat. METHOD: Six small units (N = 39) have undergone historical group debriefing within 72 hours of exposure. State anxiety, self-efficacy, and combat evaluation were measured before and immediately after the sessions. RESULTS: Debriefing was followed by reduction in anxiety, improvement in self-efficacy, and increased homogeneity of the group. CONCLUSION: These effects may be attributable to enhancing group cohesion or to a proper beneficial effect of debriefing.

Auditory startle reflex in post-traumatic stress disorder patients treated with clonazepam.

Manifestations of acute hyperarousal in the Post-Traumatic Stress Disorder (PTSD) have been assimilated with panic attacks. This study examines the auditory startle reflex in PTSD patients treated with clonazepam, a benzodiazepine with anti-panic properties. PTSD patients treated with clonazepam did not differ from drug-free PTSD patients in the magnitude and the habituation rate of their responses to an auditory startle, with both groups showing abnormally slow habituation. It is suggested that the mechanisms underlying phasic hyperarousal in PTSD might differ from those which have been implied in the genesis of panic attacks.