Heterogeneity of G protein activation by the calcium-sensing receptor.
The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G protein subfamilies (Gαq/11, Gαi/o, Gα12/13, Gαs), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messengers or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G protein activation directly, we took advantage of a recently described NanoBiT G protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca2+e stimulation activates all Gαq/11 family and several Gαi/o family proteins, although Gαz was not activated. CaSR stimulated dissociation of Gα12/13 and Gαs from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα11, Gαz, Gαi1 and Gα13 genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα11 and Gαi1 in parathyroids. In kidney and pancreas, the majority of G proteins were similarly expressed, suggesting CaSR may activate multiple G proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.


Methods to decrease postoperative infections following posterior cervical spine surgery.
To decrease surgical site infections, we initiated a protocol of preliminary preparation of the skin and surrounding plastic drapes with alcohol foam, and the placement of a suprafascial drain in addition to a subfascial drain in obese patients in 2004. In 2008, we additionally placed 500 mg of vancomycin powder into the wound prior to closure. The purpose of this study was to analyze the infection rates for three groups: Group C (control that received standard perioperative intravenous antibiotics alone), Group AD (alcohol foam and drain), and Group VAD (vancomycin with alcohol foam and drain). A consecutive series of 1001 all-posterior cervical spine surgical procedures performed at one institution by the senior author from 1995 to 2010 was retrospectively reviewed. These surgical procedures included foraminotomy, laminectomy, laminoplasty, arthrodesis, instrumentation, and/or osteotomies. There were 483 patients in Group C, 323 in Group AD, and 195 in Group VAD. In Group C, nine (1.86%) of the 483 patients had an acute postoperative deep infection, in which methicillin-resistant Staphylococcus aureus was the most common pathogen. A significantly higher rate of infection was found in patients with an active smoking history (p = 0.008; odds ratio = 2.6 [95% confidence interval, 1.0 to 7.1]), rheumatoid arthritis (p = 0.005; odds ratio = 4.0 [95% confidence interval, 1.4 to 7.9]), and a body mass index of ≥30 kg/m2 (p = 0.005; odds ratio = 4.1 [95% confidence interval, 1.5 to 7.7]). Group AD (n = 323) had one infection, a significant decrease compared with Group C (p = 0.047). In Group VAD, none of the 195 patients had infections, which was also a significant decrease compared with Group C (p = 0.048). In this study, preliminary preparation with alcohol foam and the placement of suprafascial drains for deep wounds resulted in one postoperative deep infection in 323 surgical procedures. The addition of intrawound vancomycin powder in 195 consecutive posterior cervical spine surgical procedures resulted in no infections and no adverse effects. To our knowledge, this is the first description of a technique for significantly decreasing postoperative cervical spine infections.


Means-end comprehension in four parrot species: explained by social complexity.
A comparative approach is required to investigate the evolutionary origins of cognitive abilities. In this paper, we compare the performance of four parrot species, spectacled parrotlets (Forpus conspicillatus), rainbow lorikeets (Trichoglossus haematodus), green-winged macaws (Ara chloroptera) and sulphur-crested cockatoos (Cacatua galerita triton) in standardized string-pulling and string-choice paradigms. We varied the spatial relationship between the strings, the presence of a reward and the physical contact between the string and the reward to test different cognitive skills requiring means-end comprehension. The species tested showed a high individual and inter-specific variation in their ability to solve the tasks. Spectacled parrotlets performed best among the four species and solved the most complex choice tasks, namely crossed-string task and broken-string task, spontaneously. In contrast, macaws and cockatoos failed to identify the correct string in these two tasks. The rainbow lorikeets were outperformed by the parrotlets, but outperformed in turn the macaws and the cockatoos. The findings can be best explained by the variation in social complexity among species, rather than in their ecology.


Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.
Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.


TGF-β3 stimulates stromal matrix assembly by human corneal keratocyte-like cells.
We have previously shown that TGF-β3 (T3) stimulates extracellular matrix (ECM) assembly while maintaining antifibrotic characteristics in a model using human corneal fibroblasts (HCFs). This model, however, requires non-physiological levels of serum. In the current study, we tested whether T3 could stimulate human corneal keratocytes (HCKs) in vitro to assemble a functional ECM, while maintaining their characteristics. Human corneal keratocytes and HCFs were isolated and cultured using 1% or 10% serum, respectively ±T3. The constructs were processed for indirect immunofluorescence (IF), transmission electron microscopy (TEM), and qRT-PCR, analyzing for keratocyte marker, keratocan, and ECM components, collagen (col) types I, III, and V. Quantitative reverse transcriptase PCR data showed that keratocan, col I, and V were all upregulated in HCKs compared with HCFs, whereas col III was expressed at low levels in HCKs. Transforming growth factor beta 3 stimulation further enhanced the level of change. Without T3, HCK constructs were very thin, approximately 5 μm; however, as with HCFs, upon stimulation with T3, HCK constructs increased in thickness by approximately 5-fold. Cell counts and ECM production revealed that HCKs assembled more ECM per unit area compared with HCFs, and IF revealed downregulation of fibrotic markers, col III, and thrombospondin-1, with T3 stimulation. Transmission electron microscopy data revealed aligned ECM with long fibrils for all conditions except HCK Controls. Human corneal keratocytes+T3 also showed denser collagen fibrils with more consistent fibril diameter. Overall, the data suggests that it is possible to stimulate matrix secretion and assembly by HCKs in vitro by using a single growth factor, T3.


The effect of sex and age on isokinetic hip-abduction torques.
As high school female athletes demonstrate a rate of noncontact anterior cruciate ligament (ACL) injury 3-6 times higher than their male counterparts, research suggests that sagittal-plane hip strength plays a role in factors associated with ACL injuries. To determine if gender or age affect hip-abductor strength in a functional standing position in young female and male athletes. Prospective cohort design. Biomechanical laboratory. Over a 3-y time period, 852 isokinetic hip-abduction evaluations were conducted on 351 (272 female, 79 male) adolescent soccer and basketball players. Before testing, athletes were secured in a standing position, facing the dynamometer head, with a strap secured from the uninvolved side and extending around the waist just above the iliac crest. The dynamometer head was positioned in line with the body in the coronal plane by aligning the axis of rotation of the dynamometer with the center of hip rotation. Subjects performed 5 maximum-effort repetitions at a speed of 120°/s. The peak torque was recorded and normalized to body mass. All test trials were conducted by a single tester to limit potential interrater test error. Standing isokinetic hip-abduction torque. Hip-abduction torque increased in both males and females with age (P < .001) on both the dominant and nondominant sides. A significant interaction of gender and age was observed (P < .001), which indicated that males experienced greater increases in peak torque relative to body weight than did females as they matured. Males exhibit a significant increase in normative hip-abduction strength, while females do not. Future study may determine if the absence of similar increased relative hip-abduction strength in adolescent females, as they age, may be related to their increased risk of ACL injury compared with males.


Why we need observational studies to evaluate the effectiveness of health care.
The view is widely held that experimental methods (randomised controlled trials) are the "gold standard" for evaluation and that observational methods (cohort and case control studies) have little or no value. This ignores the limitations of randomised trials, which may prove unnecessary, inappropriate, impossible, or inadequate. Many of the problems of conducting randomised trials could often, in theory, be overcome, but the practical implications for researchers and funding bodies mean that this is often not possible. The false conflict between those who advocate randomised trials in all situations and those who believe observational data provide sufficient evidence needs to be replaced with mutual recognition of the complementary roles of the two approaches. Researchers should be united in their quest for scientific rigour in evaluation, regardless of the method used.


Human cytomegalovirus (HCMV) glycoprotein gB promotes virus entry in trans acting as the viral fusion protein rather than as a receptor-binding protein.
ABSTRACT Human cytomegalovirus (HCMV) glycoproteins gB and gH/gL are both necessary and sufficient for cell-cell fusion. However, it is not clear what roles these glycoproteins play in virus entry, whether acting directly in membrane fusion or in binding receptors. With other herpesviruses, it appears that gB is the fusion protein and is triggered by gH/gL, which, in some cases, binds receptors. However, for HCMV, there is published evidence that gB binds cellular ligands necessary to promote virus entry into or signaling of cells. Most mechanistic information on herpesvirus fusion proteins involves cell-cell fusion assays, which do not allow a determination of whether gB or gH/gL in the virion envelope must be oriented toward cellular membranes that contain receptors. Here, we showed that HCMV virions lacking gB were unable to enter normal cells but entered cells that expressed gB. Analyses of gB mutants lacking the cytoplasmic domain or with substitutions in putative "fusion loops" provided evidence that gB fusion activity was required for this "entry in trans." In gB-mediated entry in trans, gB is oriented toward the virion envelope that apparently lacks receptors, arguing against an essential role for gB in binding receptors or signaling molecules. In contrast, particles lacking gH/gL did not enter cells expressing gH/gL, apparently because gH/gL must be oriented toward cellular membranes (which have receptors). Coupled with our previous interference studies, in which gH/gL expressed in cells blocked HCMV entry, our findings here support the hypothesis that HCMV gH/gL binds cellular receptors before triggering gB, which acts as the fusion protein. IMPORTANCE Human cytomegalovirus (HCMV) produces major disease in neonates and immunosuppressed transplant patients. As with other herpesviruses, HCMV requires two membrane glycoproteins, gB and gH/gL, to enter host cells. However, it has not been clear how gB and gH/gL function in two steps of the HCMV entry pathway, i.e., (i) binding of cellular receptors and (ii) fusion of the virion envelope with cellular membranes. There are studies that suggest that HCMV gB is required for receptor binding and other studies suggesting that gH/gL is the receptor binding protein and gB is the fusion protein. Here, we show that HCMV virions lacking gB can enter cells that express gB in cellular membranes. In contrast, virus particles lacking gH/gL could not enter cells expressing gH/gL. Our study supports the hypothesis that gB is the fusion protein and gH/gL acts upstream of gB to bind receptors and then activate gB for fusion.


Health and functioning among baby boomers approaching 60.
To investigate whether the health and functioning of the Baby Boom generation are better or worse than those of previous cohorts in middle age. Trend analysis of vital statistics and self-reports from the National Health Interview Survey for the 40-59 population. Specific outcomes (years of data): mortality (1982-2004); poor or fair health (1982-2006); nine conditions (1997-2006); physical functional limitations (1997-2006); and needing help with personal care, routine needs, or either (1997-2006). In 2005, the mortality rate of 59-year-olds, the leading edge of the Baby Boom, was 31% lower than that of 59-year-olds in 1982 (8.3 vs. 12.1 per 1,000). There was a similar proportional decline in poor/fair health, but the decline reversed in the last decade. From 1997 to 2006, the prevalence of reports of four conditions increased significantly, but this trend may reflect improvements in diagnosis and treatment. Functional limitations and need for help with routine needs were stable, but the need for help with personal care, while quite low, increased. Trends varied by indicator, period, and age. It is surprising that, given the socioeconomic, medical, and public health advantages of Baby Boomers throughout their lives, they are not doing considerably better on all counts.


Intranasal infusion of enilconazole for treatment of sinonasal aspergillosis in dogs.
To determine effectiveness of infusion of 1 and 2% enilconazole for treatment of nasal and sinusal aspergillosis, respectively, in dogs. Case series. 26 client-owned dogs with aspergillosis. All dogs had typical clinical signs of aspergillosis and rhinoscopically visible intrasinusal or intranasal fungal plaques associated with turbinate destruction. During rhinoscopy, affected nasal cavities and frontal sinuses were debrided meticulously. Nineteen dogs (group A) were treated with 1% enilconazole by use of a modified noninvasive infusion procedure. Seven dogs (group B) were treated with 2% enilconazole via catheters that were placed via endoscopic guidance into the frontal sinuses. All dogs underwent follow-up rhinoscopy for determination of further treatment until cure was established. Age, disease duration, clinical score, and rhinoscopic score were similar for both groups before treatment. In group A, 17 of 19 dogs were cured; 9, 6, and 2 dogs were cured after 1, 2, or 3 treatments, respectively. The remaining 2 dogs were euthanatized before the end of the treatment protocol. In group B, all dogs were cured; 6 dogs and 1 dog were cured after 1 or 2 treatments, respectively. Only minor adverse effects such as nasal discharge, epistaxis, and sneezing developed. After extensive rhinoscopic debridement, 1 and 2% enilconazole infused into the nasal cavities and the frontal sinuses, respectively, were effective for treatment of aspergillosis in dogs. Intrasinusal administration via endoscopically placed catheters appeared to require fewer infusions for success. Follow-up rhinoscopy is strongly advised.


Development and application of an ethically and epidemiologically advantageous assay for the multi-locus microsatellite analysis of Schistosoma mansoni.
Non-availability of adult worms from living hosts remains a key problem in population genetic studies of schistosomes. Indirect sampling involving passage through laboratory animals presents significant ethical and practical drawbacks, and may result in sampling biases such as bottlenecking processes and/or host-induced selection pressures. The novel techniques reported here for sampling, storage and multi-locus microsatellite analysis of larval Schistosoma mansoni, allowing genotyping of up to 7 microsatellite loci from a single larva, circumvent these problems. The utility of these assays and the potential problems of laboratory passage, were evaluated using 7 S. mansoni population isolates collected from school-children in the Hoima district of Uganda, by comparing the associated field-collected miracidia with adult worms and miracidia obtained from a single generation in laboratory mice. Analyses of laboratory-passaged material erroneously indicated the presence of geographical structuring in the population, emphasizing the dangers of indirect sampling for population genetic studies. Bottlenecking and/or other sampling effects were demonstrated by reduced variability of adult worms compared to their parent field-collected larval samples. Patterns of heterozygote deficiency were apparent in the field-collected samples, which were not evident in laboratory-derived samples, potentially indicative of heterozygote advantage in establishment within laboratory hosts. Genetic distance between life-cycle stages in the majority of isolates revealed that adult worms and laboratory-passaged miracidia clustered together whilst segregating from field miracidia, thereby further highlighting the utility of this assay.


The problem with Dr Bob's alternative vaccine schedule.
In October 2007, Dr Robert Sears, in response to growing parental concerns about the safety of vaccines, published The Vaccine Book: Making the Right Decision for Your Child. Sears' book is enormously popular, having sold >40000 copies. At the back of the book, Sears includes "Dr Bob's Alternative Vaccine Schedule," a formula by which parents can delay, withhold, separate, or space out vaccines. Pediatricians now confront many parents who insist that their children receive vaccines according to Sears' schedule, rather than that recommended by the American Academy of Pediatrics, the Centers for Disease Control and Prevention, and the American Academy of Family Physicians. This article examines the reasons for the popularity of Sears' book, deconstructs the logic and rationale behind its recommendations, and describes how Sears' misrepresentation of vaccine science misinforms parents trying to make the right decisions for their children.


Modulation of immunodominant sites in influenza hemagglutinin compromise antigenic variation and select receptor-binding variant viruses.
The regions of antigenic variation in influenza hemagglutinin (HA) are located on surface-accessible regions in the three-dimensional structure of the HA1 monomer. The aim of this study was to establish whether a novel variant virus, IMUT4, in which we had mutated specific amino acid residues (HA1 63, 144, 158, and 193) in these regions, previously shown to be immunodominant for CBA/Ca mice, would either (a) establish holes in the antibody (ab) repertoire or (b) preclude further antigenic variation in IMUT4. CBA/Ca mice were able to mount a neutralizing ab response to IMUT4 infection and molecular recognition sites were established by sequencing of the HA genes of monoclonal antibody (mAb)-selected laboratory variants of wild-type X31 virus (HA1 131, 145, 155, and 196). However, each of these mAbs failed to select further antigenic variants of IMUT4, in ovo, but rather a receptor binding mutant (HA1 190 Glu-->Asp; 226 Leu-->Gln) that was still recognized by the selecting mAb, specific for HA1 155 of X31 virus. The facility for antigenic variation in influenza would appear to be compromised, therefore, by targeted mutation of immunodominant sites, as initially proposed by S. Fazekas de St. Groth (Fazekas de St. Groth, S. 1977. Antigenic, adaptive and adsorptive variants of the influenza haemagglutinin. In Topics in Infectious Diseases. Vol. 3. R.G. Laver, H. Bachmayer, and R. Weil, editors. Springer-Verlag, Vienna. 25-48.). It is interesting to note that recent isolates of the H3 subtype, (e.g., A/Beijing/92) obtained between 1991 and 1993, contain the same substitutions at HA1 190 and 226, which may indicate similar constraints to immune evasion and the relevance of our findings to antigenic variation in the human population.


The phenotype-fitness map in experimental evolution of phages.
Evolutionary biologists commonly interpret adaptations of organisms by reference to a phenotype-fitness map, a model of how different states of a phenotype affect fitness. Notwithstanding the popularity of this approach, it remains difficult to directly test these mappings, both because the map often describes only a small subset of phenotypes contributing to total fitness and because direct measures of fitness are difficult to obtain and compare to the map. Both limitations can be overcome for bacterial viruses (phages) grown in the experimental condition of unlimited hosts. A complete accounting of fitness requires 3 easily measured phenotypes, and total fitness is also directly measurable for arbitrary genotypes. Yet despite the presumed transparency of this system, directly estimated fitnesses often differ from fitnesses calculated from the phenotype-fitness map. This study attempts to resolve these discrepancies, both by developing a more exact analytical phenotype-fitness map and by exploring the empirical foundations of direct fitness estimates. We derive an equation (the phenotype-fitness map) for exponential phage growth that allows an arbitrary distribution of lysis times and burst sizes. We also show that direct estimates of fitness are, in many cases, plausibly in error because the population has not attained stable age distribution and thus violates the model underlying the phenotype-fitness map. In conjunction with data provided here, the new understanding appears to resolve a discrepancy between the reported fitness of phage T7 and the substantially lower value calculated from its phenotype-fitness map.


A Novel Phellinidium sp. Causes Laminated Root Rot on Qilian Juniper (Sabina przewalskii) in Northwest China.
A laminated root rot on Qilian juniper (Sabina przewalskii) caused by a species of Phellinidium (Basidiomycota) was observed in northwest China. Seventeen fungal samples collected from Qinghai Province during 2012 and 2013 were used for taxonomic and pathogenicity tests. The fungal pathogen was identified by morphological characters and phylogenetic analyses based on nLSU sequences. A new fungus is described herein as Phellinidium qilianense sp. nov. One-year-old Qilian juniper seedlings were wound-inoculated under controlled conditions to test pathogenicity of the fungal species. The fungus was successfully reisolated from decayed tissue of tested seedlings. P. qilianense is a new forest pathogen on coniferous trees in China.


Functional overlap between eIF4G isoforms in Saccharomyces cerevisiae.
Initiation factor eIF4G is a key regulator of eukaryotic protein synthesis, recognizing proteins bound at both ends of an mRNA to help recruit messages to the small (40S) ribosomal subunit. Notably, the genomes of a wide variety of eukaryotes encode multiple distinct variants of eIF4G. We found that deletion of eIF4G1, but not eIF4G2, impairs growth and global translation initiation rates in budding yeast under standard laboratory conditions. Not all mRNAs are equally sensitive to loss of eIF4G1; genes that encode messages with longer poly(A) tails are preferentially affected. However, eIF4G1-deletion strains contain significantly lower levels of total eIF4G, relative to eIF4G2-delete or wild type strains. Homogenic strains, which encode two copies of either eIF4G1 or eIF4G2 under native promoter control, express a single isoform at levels similar to the total amount of eIF4G in a wild type cell and have a similar capacity to support normal translation initiation rates. Polysome microarray analysis of these strains and the wild type parent showed that translationally active mRNAs are similar. These results suggest that total eIF4G levels, but not isoform-specific functions, determine mRNA-specific translational efficiency.


Diagnosis and Treatment of Ankyloglossia in Newborns and Infants: A Review.
The influence of tongue tie, or ankyloglossia, on breastfeeding is the subject of growing debate. Restriction of tongue mobility from the frenulum varies greatly among newborns and infants (hereinafter referred to as infants). Controversies about whether an infant has ankyloglossia and which infants need treatment are evident with wide variations in medical practice and a lack of high-quality clinical studies that provide guidance. Diagnosis and management of ankyloglossia in infants can be a source of confusion and frustration for clinicians and families. Frenotomy is a low-risk procedure that is likely to be beneficial with careful patient selection, but the natural history of untreated ankyloglossia is not well documented. The variability in presentation and treatment outcomes of ankyloglossia indicate that the complexity of infant feeding and tongue development is not fully encapsulated in a simplistic ankyloglossia etiologic framework. Consistent terminology with emphasis on symptomatic ankyloglossia and a uniform grading system, such as the Hazelbaker Assessment Tool for Lingual Frenulum Function and Coryllos grading, are needed to improve the quality of research in the future. The ability to make definitive practice guidelines is limited with our current understanding of ankyloglossia. Additional research is needed to better understand the complexity of infant feeding and the role of ankyloglossia.


The GABA(A) receptor alpha1 subtype in the ventral pallidum regulates alcohol-seeking behaviors.
We investigated the potential role of the alpha1-containing GABA(A) receptor in regulating the reinforcing properties of alcohol. To accomplish this, we developed 3-propoxy-beta-carboline hydrochloride (3-PBC), a mixed agonist-antagonist benzodiazepine site ligand with binding selectivity at the alpha1 receptor. We then tested the capacity of 3-PBC to block alcohol-maintained responding in the ventral pallidum (VP), a novel alcohol reward substrate, which primarily expresses the alpha1-receptor isoform. Our results demonstrated that bilateral microinfusion of 3-PBC (0.5-40 microg) in the anterior and medial VP produced marked reductions in alcohol-maintained responding in a genetically selected rodent model of alcohol drinking. The VP infusions showed both neuroanatomical and reinforcer specificity because no effects were seen in sites dorsal to the VP (e.g., nucleus accumbens, caudate putamen). The saccharin-maintained responding was reduced only with the highest dose (40 microg). Parenteral injections of 3-PBC (1-20 mg/kg) also showed a similar selectivity on alcohol-maintained responding. Complementary in vitro studies revealed that 3-PBC exhibited a low partial agonist efficacy profile at recombinant diazepam-sensitive receptors (e.g., alpha1beta3gamma2, alpha2beta3gamma, and alpha3beta3gamma2). The selective suppression of 3-PBC on alcohol-maintained responding after central and parenteral administrations, together with its low-efficacy agonist profile, suggest that the reduction in alcohol-maintained behaviors was not attributable to a general suppression on consummatory behaviors. These results demonstrate that the alpha1-containing GABA(A) receptors in both the anterior and medial VP are important in regulating the reinforcing properties of alcohol. These receptors represent novel targets in the design and development of pharmacotherapies for alcohol-dependent subjects.


Cloning human fetal gamma globin and mouse alpha-type globin DNA: characterization and partial sequencing.
Two globin-related clones isolated from collections of bacteriophages containing unfractionated Eco RI fragments of human and mouse DNA were characterized. Charon3AHs51.1Hbgamma includes 2.7 kilobase pairs of human DNA containing a large part of a fetal gamma globin chain structural gene; Charon 3AMm30.5 includes 4.7 kilobase pairs of mouse DNA related to alpha globin. The human fetal gamma globin gene has within its coding region two intervening sequences of noncoding DNA, IVS 1 and IVS 2, of approximately 1-0 and 900 base pairs. Sequence IVS 1 is located at the position of one of the two intervening sequences occurring in adult globin genes; IVS 2 is located at the position of the other.


Soluble epoxide hydrolase as a therapeutic target for pain, inflammatory and neurodegenerative diseases.
Eicosanoids are biologically active lipid signaling molecules derived from polyunsaturated fatty acids. Many of the actions of eicosanoid metabolites formed by cyclooxygenase and lipoxygenase enzymes have been characterized, however, the epoxy-fatty acids (EpFAs) formed by cytochrome P450 enzymes are newly described by comparison. The EpFA metabolites modulate a diverse set of physiologic functions that include inflammation and nociception among others. Regulation of EpFAs occurs primarily via release, biosynthesis and enzymatic transformation by the soluble epoxide hydrolase (sEH). Targeting sEH with small molecule inhibitors has enabled observation of the biological activity of the EpFAs in vivo in animal models, greatly contributing to the overall understanding of their role in the inflammatory response. Their role in modulating inflammation has been demonstrated in disease models including cardiovascular pathology and inflammatory pain, but extends to neuroinflammation and neuroinflammatory disease. Moreover, while EpFAs demonstrate activity against inflammatory pain, interestingly, this action extends to blocking chronic neuropathic pain as well. This review outlines the role of modulating sEH and the biological action of EpFAs in models of pain and inflammatory diseases.


Increasing corn for biofuel production reduces biocontrol services in agricultural landscapes.
Increased demand for corn grain as an ethanol feedstock is altering U.S. agricultural landscapes and the ecosystem services they provide. From 2006 to 2007, corn acreage increased 19% nationally, resulting in reduced crop diversity in many areas. Biological control of insects is an ecosystem service that is strongly influenced by local landscape structure. Here, we estimate the value of natural biological control of the soybean aphid, a major pest in agricultural landscapes, and the economic impacts of reduced biocontrol caused by increased corn production in 4 U.S. states (Iowa, Michigan, Minnesota, and Wisconsin). For producers who use an integrated pest management strategy including insecticides as needed, natural suppression of soybean aphid in soybean is worth an average of $33 ha(-1). At 2007-2008 prices these services are worth at least $239 million y(-1) in these 4 states. Recent biofuel-driven growth in corn planting results in lower landscape diversity, altering the supply of aphid natural enemies to soybean fields and reducing biocontrol services by 24%. This loss of biocontrol services cost soybean producers in these states an estimated $58 million y(-1) in reduced yield and increased pesticide use. For producers who rely solely on biological control, the value of lost services is much greater. These findings from a single pest in 1 crop suggest that the value of biocontrol services to the U.S. economy may be underestimated. Furthermore, we suggest that development of cellulosic ethanol production processes that use a variety of feedstocks could foster increased diversity in agricultural landscapes and enhance arthropod-mediated ecosystem services.


Sequential Tc-99m MDP bone scans after cementless total hip arthroplasty in asymptomatic patients.
To evaluate the distribution and degree of radioactivity on sequential Tc-99m methylene diphosphonate (Tc-99m MDP) scans in the diagnosis of postoperative loosening in asymptomatic patients who underwent cementless total hip replacement. Tc-99m MDP scans were obtained every three months where possible, for one year in those patients who underwent total hip replacement using a Harris Galante cementless prosthesis. Eighty-three scans from 45 patients were obtained. Uptake was assessed within the five periprosthetic zones (zone 1: greater trochanter; 2: lateral stem of the prosthesis; 3: tip of the prosthesis; 4: medial stem of the prosthesis; and 5: lesser trochanter) using an arbitrary 5-point grading system. Grade 1 indicated normal uptake and Grade 5 indicated highest uptake. The grade of uptake within each zone was then analyzed against their postoperative ages. The radioactivity regressed to Grade 1 or 2 from Grade 4 or 5 within 12 months after cementless total hip replacement in 87.5% and 93.8% of patients in zones 2 and 4, respectively. Even though the radioactivity within zones 1, 3, and 5 tended to decrease, persistent elevation of activity (over grade 2) lasted up to 12 months after surgery in 31% or 37% of the patients. The radioactivity after cementless total hip replacement tended to regress in proportion to the postimplant age. The regression rate varied depending on the periprosthetic zones.


Socio-demographic and AIDS-related factors associated with tuberculosis stigma in southern Thailand: a quantitative, cross-sectional study of stigma among patients with TB and healthy community members.
Tuberculosis (TB) remains one of the most important infectious diseases worldwide. A comprehensive approach towards disease control that addresses social factors including stigma is now advocated. Patients with TB report fears of isolation and rejection that may lead to delays in seeking care and could affect treatment adherence. Qualitative studies have identified socio-demographic, TB knowledge, and clinical determinants of TB stigma, but only one prior study has quantified these associations using formally developed and validated stigma scales. The purpose of this study was to measure TB stigma and identify factors associated with TB stigma among patients and healthy community members. A cross-sectional study was performed in southern Thailand among two different groups of participants: 480 patients with TB and 300 healthy community members. Data were collected on socio-demographic characteristics, TB knowledge, and clinical factors. Scales measuring perceived TB stigma, experienced/felt TB stigma, and perceived AIDS stigma were administered to patients with TB. Community members responded to a community TB stigma and community AIDS stigma scale, which contained the same items as the perceived stigma scales given to patients. Stigma scores could range from zero to 30, 33, or 36 depending on the scale. Three separate multivariable linear regressions were performed among patients with TB (perceived and experience/felt stigma) and community members (community stigma) to determine which factors were associated with higher mean TB stigma scores. Only low level of education, belief that TB increases the chance of getting AIDS, and AIDS stigma were associated with higher TB stigma scores in all three analyses. Co-infection with HIV was associated with higher TB stigma among patients. All differences in mean stigma scores between index and referent levels of each factor were less than two points, except for incorrectly believing that TB increases the chance of getting AIDS (mean difference of 2.16; 95% CI: 1.38, 2.94) and knowing someone who died from TB (mean difference of 2.59; 95% CI: 0.96, 4.22). These results suggest that approaches addressing the dual TB/HIV epidemic may be needed to combat TB stigma and that simply correcting misconceptions about TB may have limited effects.


Light-switchable polymer from cationic to zwitterionic form: synthesis, characterization, and interactions with DNA and bacterial cells.
A novel cationic polymer poly(N,N-dimethyl-N-[3-(methacroylamino) propyl]-N-[2-[(2-nitrophenyl)methoxy]-2-oxo-ethyl]ammonium chloride) is synthesized by free-radical polymerization of N-[3-(dimethylamino)propyl] methacrylamide and subsequent quaternization with o-nitrobenzyl 2-chloroacetate. The photolabile o-nitrobenzyl carboxymethyl pendant moiety is transformed to the zwitterionic carboxybetaine form upon the irradiation at 365 nm. This feature is used to condense and, upon the light irradiation, to release double-strand DNA tested by gel electrophoresis and surface plasmon resonance experiments as well as to switch the antibacterial activity to non-toxic character demonstrated for Escherichia coli bacterial cells in solution and at the surface using the self-assembled monolayers.


Increased prokineticin 2 expression in gut inflammation: role in visceral pain and intestinal ion transport.
Prokineticin 2 (PROK2) is an inflammatory cytokine-like molecule expressed predominantly by macrophages and neutrophils infiltrating sites of tissue damage. Given the established role of prokineticin signaling on gastrointestinal function, we have explored Prok2 gene expression in inflammatory conditions of the gastrointestinal tract and assessed the possible consequences on gut physiology. Prokineticin expression was examined in normal and colitic tissues using qPCR and immunohistochemistry. Functional responses to PROK2 were studied using calcium imaging and a novel antagonist, Compound 3, used to determine the role of PROK2 and prokineticin receptors in inflammatory visceral pain and ion transport. Prok2 gene expression was up-regulated in biopsy samples from ulcerative colitis patients, and similar elevations were observed in rodent models of inflammatory colitis. Prokineticin receptor 1 (PKR1) was localized to the enteric neurons and extrinsic sensory neurons, whereas Pkr2 expression was restricted to sensory ganglia. In rats, PROK2-increased intracellular calcium levels in cultured enteric and dorsal root ganglia neurons, which was blocked by Compound 3. Moreover, PROK2 acting at prokineticin receptors stimulated intrinsic neuronally mediated ion transport in rat ileal mucosa. In vivo, Compound 3 reversed intracolonic mustard oil-induced referred allodynia and TNBS-induced visceral hypersensitivity, but not non-inflammatory, stress-induced visceral pain. Elevated Prok2 levels, as a consequence of gastrointestinal tract inflammation, induce visceral pain via prokineticin receptors. This observation, together with the finding that PROK2 can modulate intestinal ion transport, raises the possibility that inhibitors of PROK2 signaling may have clinical utility in gastrointestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease.


Dispersion of TiO2 nanoparticles improves burn wound healing and tissue regeneration through specific interaction with blood serum proteins.
Burn wounds are one of the most important causes of mortality and especially morbidity around the world. Burn wound healing and skin tissue regeneration remain thus one of the most important challenges facing the mankind. In the present study we have addressed this challenge, applying a solution-stabilized dispersion TiO2 nanoparticles, hypothesizing that their ability to adsorb proteins will render them a strong capacity in inducing body fluid coagulation and create a protective hybrid material coating. The in vitro study of interaction between human blood and titania resulted at enhanced TiO2 concentrations in formation of rather dense gel composite materials and even at lower content revealed specific adsorption pattern initiating the cascade response, promising to facilitate the regrowth of the skin. The subsequent in vivo study of the healing of burn wounds in rats demonstrated formation of a strongly adherent crust of a nanocomposite, preventing infection and inflammation with quicker reduction of wound area compared to untreated control. The most important result in applying the TiO2 dispersion was the apparently improved regeneration of damaged tissues with appreciable decrease in scar formation and skin color anomalies.


Enhanced metabolism and selection of pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte).
Western corn rootworm (WCR) pyrethroid resistance has been previously reported in the United States (US) western Corn Belt, and cross-resistance and synergism studies suggested that both target site insensitivity and enhanced metabolism may be conferring WCR resistance to pyrethroids. The present study aimed to investigate the potential mechanisms of WCR pyrethroid resistance and to estimate the heritability of the resistance trait. Biochemical assays using model substrates and spectrophotometry revealed 2-4-fold higher activity of P450s and esterases in pyrethroid-resistant WCR populations, whereas the biological activity of glutathione S-transferase was similar between populations tested. No mutation in the voltage-gated sodium channel was detected in pyrethroid-resistant WCR individuals by sequencing PCR products containing the para-homologous L1014, T929, and M918 amino acid positions that are commonly associated with target site mutations in other pyrethroid-resistant insects. A pilot estimation of pyrethroid resistance heritability obtained during laboratory selection of a WCR population suggested a major genetic component of the resistance trait and predicted a 10-fold increase in WCR bifenthrin resistance within ~7 generations of insecticide lethal exposure. Results support earlier indirect evidence that enhanced metabolism may be contributing to WCR resistance to pyrethroids and illustrates the potential of WCR pyrethroid resistance evolution.


New modular manganese(I) tricarbonyl complexes as PhotoCORMs: in vitro detection of photoinduced carbon monoxide release using COP-1 as a fluorogenic switch-on probe.
Five manganese(i) tricarbonyl complexes of the general formulae [Mn(bpea(N=CHC6H4R))(CO)3]PF6 and [Mn(bpea(NHCH2C6H4R))(CO)3]PF6 based on the tridentate bis(pyrazolyl)ethylamine (bpea) ligand, each containing a pendant 4-substituted phenyl group with R = H, I, and C≡C-H, were synthesized and fully characterized, including X-ray structure analysis for three compounds. All complexes are stable in the dark in aqueous buffer for an extended period of time. However, CO-release could be triggered by illumination at 365 nm, establishing these compounds as novel photoactivatable CO-releasing molecules (PhotoCORMs). The influence of the imine vs. amine group in the ligands on the electronic structure and the photophysical behavior was investigated with the aid of DFT and TDDFT calculations. Solution IR studies on selected compounds allowed identification of intermediates resulting from the photoreaction. Finally, light-induced CO release from a model compound was demonstrated both in PBS buffer and in vitro in human umbilical vein endothelial cells (HUVECs) using COP-1 as a fluorescent switch-on probe.


The Leprosy Post-Exposure Prophylaxis (LPEP) programme: update and interim analysis.
Innovative approaches are required to further enhance leprosy control, reduce the number of people developing leprosy, and curb transmission. Early case detection, contact screening, and chemoprophylaxis currently is the most promising approach to achieve this goal. The Leprosy Post-Exposure Prophylaxis (LPEP) programme generates evidence on the feasibility of integrating contact tracing and single-dose rifampicin (SDR) administration into routine leprosy control activities in different settings. The LPEP programme is implemented within the leprosy control programmes of Brazil, Cambodia, India, Indonesia, Myanmar, Nepal, Sri Lanka and Tanzania. Focus is on three key interventions: tracing the contacts of newly diagnosed leprosy patients; screening the contacts for leprosy; and administering SDR to eligible contacts. Country-specific protocol adaptations refer to contact definition, minimal age for SDR, and staff involved. Central coordination, detailed documentation and rigorous supervision ensure quality evidence. Around 2 years of field work had been completed in seven countries by July 2017. The 5,941 enrolled index patients (89·4% of the registered) identified a total of 123,311 contacts, of which 99·1% were traced and screened. Among them, 406 new leprosy patients were identified (329/100,000), and 10,883 (8·9%) were excluded from SDR for various reasons. Also, 785 contacts (0·7%) refused the prophylactic treatment with SDR. Overall, SDR was administered to 89·0% of the listed contacts. Post-exposure prophylaxis with SDR is safe; can be integrated into the routines of different leprosy control programmes; and is generally well accepted by index patients, their contacts and the health workforce. The programme has also invigorated local leprosy control.


CLIPick: a sensitive peak caller for expression-based deconvolution of HITS-CLIP signals.
High-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP, also called CLIP-Seq) has been used to map global RNA-protein interactions. However, a critical caveat of HITS-CLIP results is that they contain non-linear background noise-different extent of non-specific interactions caused by individual transcript abundance-that has been inconsiderately normalized, resulting in sacrifice of sensitivity. To properly deconvolute RNA-protein interactions, we have implemented CLIPick, a flexible peak calling pipeline for analyzing HITS-CLIP data, which statistically determines the signal-to-noise ratio for each transcript based on the expression-dependent background simulation. Comprising of streamlined Python modules with an easy-to-use standalone graphical user interface, CLIPick robustly identifies significant peaks and quantitatively defines footprint regions within which RNA-protein interactions were occurred. CLIPick outperforms other peak callers in accuracy and sensitivity, selecting the largest number of peaks particularly in lowly expressed transcripts where such marginal signals are hard to discriminate. Specifically, the application of CLIPick to Argonaute (Ago) HITS-CLIP data were sensitive enough to uncover extended features of microRNA target sites, and these sites were experimentally validated. CLIPick enables to resolve critical interactions in a wide spectrum of transcript levels and extends the scope of HITS-CLIP analysis. CLIPick is available at: http://clip.korea.ac.kr/clipick/.


Interaction of eye protein kinase C and INAD in Drosophila. Localization of binding domains and electrophysiological characterization of a loss of association in transgenic flies.
Drosophila eye-specific protein kinase C (eye-PKC) is involved in light adaptation and deactivation. eye-PKC, NORPA (phospholipase Cbeta), and transient-receptor-potential (TRP) (calcium channel) are integral components of a signal transduction complex organized by INAD, a protein containing five PDZ domains. We previously demonstrated the direct association between the third PDZ domain of INAD with TRP in addition to the carboxyl-terminal half of INAD with the last three residues of NORPA. In this work, the molecular interaction between eye-PKC and INAD is defined via the yeast two-hybrid and ligand overlay assays. We show that the second PDZ domain of INAD interacts with the last three residues in the carboxyl-terminal tail of eye-PKC, Thr-Ile-Ile. The association between eye-PKC and INAD is disrupted by an amino acid substitution (Ile-700 to Asp) at the final residue of eye-PKC. In flies lacking endogenous eye-PKC (inaCp215), normal visual physiology is restored upon expression of wild-type eye-PKC, whereas the eye-PKCI700D mutant is completely inactive. Flies homozygous for inaCp209 and InaDp215, a mutation that causes a loss of the INAD-TRP association, were generated. These double mutants display a more severe response inactivation than either of the single mutants. Based on these findings, we conclude that the in vivo activity of eye-PKC depends on its association with INAD and that the sensitivity of photoreceptors is cooperatively regulated by the presence of both eye-PKC and TRP in the signaling complex.


Improvement of serum amino acid profile in hepatic failure with the bioartificial liver using multicellular hepatocyte spheroids.
We designed a bioartificial liver support system in which encapsulated multicellular spheroids of rat hepatocytes were utilized as a bioreactor in a hollow fiber cartridge. The spheroids, formed in a positively charged polystyrene dish that contained hormonally defined medium, were encapsulated into microdroplets of agarose that contained about 9 x 10(7) rat hepatocytes. The medium, including 150 mL reservoir volume, was circulated in a closed circuit in which the cartridge was inserted. The pH and levels of dissolved oxygen were monitored and automatically regulated so that they were maintained within a constant range for 72 h. Albumin accumulated in the circuit at the rate of 2.0 mg/L/h in this system. When the bioreactor cells in the system were replaced with Hep G2 cells, a human hepatoblastoma cell line, albumin accumulated at the rate of 0.15 mg/L/h. The spheroids of primary culture hepatocytes had 13 times higher albumin-producing capacity than the aggregates of Hep G2. The serum of a patient with fulminant hepatic failure was circulated in this system with the spheroids of primary culture hepatocytes. The concentration of branched amino acid (BCAA) in the circuit significantly increased during the 48 h circulation, while the concentration of aromatic amino acid (AAA) and methionine decreased. The ratio of BCAA/AAA increased from 0.640 to 0.772, indicating that the hepatocyte spheroids had improved the imbalance of the amino acid profile in the serum. These findings indicate that this system may be a useful model for an artificial liver support. (c) 1996 John Wiley & Sons, Inc.


[Intraperitoneal dissemination of testicular tumor: a case report].
A 42-year-old male presented with bilateral scrotal swelling and sense of abdominal fullness. Ultrasonography and computed tomographic (CT) scan showed right testicular tumor, left scrotal hydrocele and excessive ascites, but no distant metastases. The serum lactic acid dehydrogenase (LDH) value was as high as 4,060 IU/L and beta human chorionic gonadotropin (hCG) value was 4.0 ng/ml while alpha fetoprotein (AFP) value was within the normal range. Right high orchiectomy was performed as well as the ascites and the left scrotal fluid sampling. Histological examinations revealed anaplastic seminoma in the right testis, and similar cells were found in the ascites and the left scrotal fluid. The patient received three courses of BEP chemotherapy consisting of bleomycin, VP-16 and cisplatin. The ascites and tumor markers (LDH and beta hCG) decreased markedly. Intraperitoneal dissemination of the tumor cells seems to be caused by invasion along the right spermatic cord, but the route of dissemination into the left hydrocele fluid is not known. To the best of our knowledge, this is the first case report of testicular tumor that showed intraperitoneal dissemination without any evidence of metastasis to the lymph nodes or distant organs.


Management of paediatric Lyme disease in non-endemic and endemic areas: data from the Registry of the Italian Society for Pediatric Infectious Diseases.
The purpose of this investigation was to determine how specialists in paediatric infectious diseases (PIDs) manage children with suspected Lyme disease (LD) by comparing their approaches in Italian endemic and non-endemic areas. A cross-sectional survey of the PID specialists participating in the Italian Society for Pediatric Infectious Disease (SITIP) Registry of LD was carried out between 1 January and 30 April 2012. A total of 160 children (80 living in endemic areas and 80 living in non-endemic areas) were diagnosed as having LD between 1 January 2005 and 31 December 2011. The clinical manifestations were erythema migrans in 130 cases (81.3 %), arthritis in 24 (15.0 %) and neuroborreliosis in six (3.8 %). Significant differences from the recommendations concerning serology and the tests to undertake were mainly observed in the children with erythema migrans, especially those living in non-endemic areas (p < 0.05). The children with erythema migrans who lived in non-endemic areas were treated with antibiotics significantly less frequently than those living in endemic areas (p < 0.05), and significantly fewer children with erythema migrans or arthritis living in non-endemic areas were treated with amoxicillin in comparison with those living in endemic regions (p < 0.05). The duration of antimicrobial therapy was significantly shorter than recommended in the children with erythema migrans or arthritis, especially those living in non-endemic areas (p < 0.05). Paediatric LD is also present in areas of Italy in which it is not considered endemic, but knowledge concerning its management is generally poor among PID specialists and characterised by enormous gaps in non-endemic areas.


Scientists' warning to humanity: microorganisms and climate change.
In the Anthropocene, in which we now live, climate change is impacting most life on Earth. Microorganisms support the existence of all higher trophic life forms. To understand how humans and other life forms on Earth (including those we are yet to discover) can withstand anthropogenic climate change, it is vital to incorporate knowledge of the microbial 'unseen majority'. We must learn not just how microorganisms affect climate change (including production and consumption of greenhouse gases) but also how they will be affected by climate change and other human activities. This Consensus Statement documents the central role and global importance of microorganisms in climate change biology. It also puts humanity on notice that the impact of climate change will depend heavily on responses of microorganisms, which are essential for achieving an environmentally sustainable future.


Inhibitory responses in Aplysia pleural sensory neurons act to block excitability, transmitter release, and PKC Apl II activation.
Expression of the 5-HT(1Apl(a)) receptor in Aplysia pleural sensory neurons inhibited 5-HT-mediated translocation of the novel PKC Apl II in sensory neurons and prevented PKC-dependent synaptic facilitation at sensory to motoneuron synapses (Nagakura et al. 2010). We now demonstrate that the ability of inhibitory receptors to block PKC activation is a general feature of inhibitory receptors and is found after expression of the 5-HT(1Apl(b)) receptor and with activation of endogenous dopamine and FMRFamide receptors in sensory neurons. Pleural sensory neurons are heterogeneous for their inhibitory response to endogenous transmitters, with dopamine being the most prevalent, followed by FMRFamide, and only a small number of neurons with inhibitory responses to 5-HT. The inhibitory response is dominant, reduces membrane excitability and synaptic efficacy, and can reverse 5-HT facilitation at both naive and depressed synapses. Indeed, dopamine can reverse PKC translocation during the continued application of 5-HT. Reversal of translocation can also be seen after translocation mediated by an analog of diacylglycerol, suggesting inhibition is not through blockade of diacylglycerol production. The effects of inhibition on PKC translocation can be rescued by phosphatidic acid, consistent with the inhibitory response involving a reduction or block of production of this lipid. However, phosphatidic acid could not recover PKC-dependent synaptic facilitation due to an additional inhibitory effect on the non-L-type calcium flux linked to synaptic transmission. In summary, we find a novel mechanism downstream of inhibitory receptors linked to inhibition of PKC activation in Aplysia sensory neurons.


Simultaneous quantitative evaluation of in-plane and out-of-plane deformations by use of a multidirectional spatial carrier.
An optical system for the parallel evaluation of in-plane and out-of-plane deformations is described. The object is illuminated from two different directions and imaged onto a CCD sensor. Each illumination interferes with its corresponding reference beam. This produces two sensitivity vectors. The references have different directions in order to produce two-directional spatial carriers. Two separate interferograms of an object under test in its undeformed and deformed states are recorded. The Fourier method is used for the quantitative evaluation. The measurements along different sensitivity vectors are separated in the Fourier domain. The phases of the two interferograms are obtained from the complex amplitudes, and the two-dimensional deformation is calculated from the phases. Two different arrangements (with and without a lens system) are presented together with some experimental results.


Conditional survival after surgical treatment of melanoma: an analysis of the Surveillance, Epidemiology, and End Results database.
Survival curves following surgical treatment of cutaneous melanoma are heavily influenced by early deaths. Therefore, survival estimates may be misleading for long-term cancer survivors. We examined whether conditional survival (CS) is more accurate in predicting long-term melanoma survival. We used the Surveillance, Epidemiology, and End Results database (1992-2005) to identify patients who underwent surgical treatment for melanoma. We included patients with T2-T4 disease and with known nodal status. Patients were stratified into low-risk (T2-3N0M0) and high-risk (T4N0M0 or T2-4N1-3M0) categories. We defined CS as time-specific estimates conditioned on living to a certain point in follow-up, and calculated 10-year cancer-specific survival curves conditioned on annual survival. We adjusted for potential confounders using a Cox proportional hazards regression model (alpha = 0.05). A total of 8647 patients met inclusion criteria (low-risk, 5987 [69.2%]; high-risk, 2660 [30.8%]). At diagnosis, low-risk patients had a significantly better 10-year survival rate (low-risk, 79.6%; high-risk, 41.2%; P < 0.001). On CS analysis, survival differences remained until 8 years after treatment, after which 10-year cancer-specific survival rates were no longer significantly different (P = 0.51) for low-risk (95.4%) and high-risk (91.7%) groups. Multivariate analysis demonstrated that age, gender, location, and ulceration (initial predictors of survival) were no longer predictive after 8 years of survival. For patients who survive 8 years after surgical treatment of melanoma, CS data become discordant with traditionally used estimates. Our findings have important implications for patient counseling, as high-risk melanoma survivors may require no more intensive surveillance than low-risk survivors 8 years after treatment.


Timed performance weaknesses on computerized tasks in pediatric brain tumor survivors: A comparison with sibling controls.
With more children surviving a brain tumor, insight into the late effects of the disease and treatment is of high importance. This study focused on profiling the neurocognitive functions that might be affected after treatment for a pediatric brain tumor, using a broad battery of computerized tests. Predictors that may influence neurocognitive functioning were also investigated. A total of 82 pediatric brain tumor survivors (PBTSs) aged 8-18 years (M = 13.85, SD = 3.15, 49% males) with parent-reported neurocognitive complaints were compared to a control group of 43 siblings (age M = 14.27, SD = 2.44, 40% males) using linear mixed models. Neurocognitive performance was assessed using measures of attention, processing speed, memory, executive functioning, visuomotor integration (VMI), and intelligence. Tumor type, treatment, tumor location, hydrocephalus, gender, age at diagnosis, and time since diagnosis were entered into regression analyzes as predictors for neurocognitive functioning. The PBTSs showed slower processing speeds and lower intelligence (range effect sizes .71-.82, p < .001), as well as deficits in executive attention, short-term memory, executive functioning, and VMI (range effect sizes .40-.57, p < .05). Older age at assessment was associated with better neurocognitive functioning (B = .450, p < .001) and younger age at diagnosis was associated with lower intelligence (B = .328, p < .05). Medical risk factors, e.g., hydrocephalus, did not show an association with neurocognitive functioning. Late effects in PBTSs include a broad range of neurocognitive deficits. The results suggest that even PBTSs that were traditionally viewed as low risk for neurocognitive problems (e.g., surgery only, no hydrocephalus) may suffer from decreased neurocognitive functioning.


Melatonin and pancreatic cancer: Current knowledge and future perspectives.
Pancreatic cancer has a high mortality rate due to the absence of early symptoms and subsequent late diagnosis; additionally, pancreatic cancer has a high resistance to radio- and chemotherapy. Multiple inflammatory pathways are involved in the pathophysiology of pancreatic cancer. Melatonin an indoleamine produced in the pineal gland mediated and receptor-independent action is the pancreas and other where has both receptors. Melatonin is a potent antioxidant and tissue protector against inflammation and oxidative stress. In vivo and in vitro studies have shown that melatonin supplementation is an appropriate therapeutic approach for pancreatic cancer. Melatonin may be an effective apoptosis inducer in cancer cells through regulation of a large number of molecular pathways including oxidative stress, heat shock proteins, and vascular endothelial growth factor. Limited clinical studies, however, have evaluated the role of melatonin in pancreatic cancer. This review summarizes what is known regarding the effects of melatonin on pancreatic cancer and the mechanisms involved.


NMDA receptor antagonism potentiates the L-DOPA-induced extracellular dopamine release in the subthalamic nucleus of hemi-parkinson rats.
Long term treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) is associated with several motor complications. Clinical improvement of this treatment is therefore needed. Lesions or high frequency stimulation of the hyperactive subthalamic nucleus (STN) in Parkinson's disease (PD), alleviate the motor symptoms and reduce dyskinesia, either directly and/or by allowing the reduction of the L-DOPA dose. N-methyl-D-aspartate (NMDA) receptor antagonists might have similar actions. However it remains elusive how the neurochemistry changes in the STN after a separate or combined administration of L-DOPA and a NMDA receptor antagonist. By means of in vivo microdialysis, the effect of L-DOPA and/or MK 801, on the extracellular dopamine (DA) and glutamate (GLU) levels was investigated for the first time in the STN of sham and 6-hydroxydopamine-lesioned rats. The L-DOPA-induced DA increase in the STN was significantly higher in DA-depleted rats compared to shams. MK 801 did not influence the L-DOPA-induced DA release in shams. However, MK 801 enhanced the L-DOPA-induced DA release in hemi-parkinson rats. Interestingly, the extracellular STN GLU levels remained unchanged after nigral degeneration. Furthermore, administration of MK 801 alone or combined with L-DOPA did not alter the STN GLU levels in both sham and DA-depleted rats. The present study does not support the hypothesis that DA-ergic degeneration influences the STN GLU levels neither that MK 801 alters the GLU levels in lesioned and non-lesioned rats. However, NMDA receptor antagonists could be used as a beneficial adjuvant treatment for PD by enhancing the therapeutic efficacy of l-DOPA at least in part in the STN.


Antimicrobial prophylaxis for urinary tract infection in persons with spinal cord dysfunction.
To assess the benefits and harms of antimicrobial prophylaxis to prevent urinary tract infections (UTIs) in persons with neurogenic bladders caused by spinal cord dysfunction. A broad search strategy with no language restriction was conducted of MEDLINE (1966-January 1998), EMBASE (1974-January 1998), and CINAHL (1982-July 1998) using the general search terms urinary tract, urinary tract infections, bacteriuria, paraplegia, quadriplegia, spinal cord injuries, multiple sclerosis, neurogenic bladder, and neuropathic bladder. Additional articles were identified by experts and by reviewing reference lists of articles obtained from searches. Criteria included human studies of adults and adolescents who had neurogenic bladder due to spinal cord dysfunction; the studies had to address antimicrobial prophylaxis of UTI and include bacteriuria or UTI as an outcome. We excluded any study that was clearly not a controlled trial or that only included children under the age of 13 years. Two reviewers independently abstracted data, and disagreements were resolved by consensus. Two reviewers independently abstracted data, and disagreements were resolved by consensus. Studies were graded by 1 project investigator according to quality criteria developed by Jadad and Schulz. The sizes of the effect of antimicrobial prophylaxis on weekly infection rates from 15 trials that met the inclusion criteria were pooled by using a random effects model. Antimicrobial prophylaxis did not significantly decrease symptomatic infections. Prophylaxis was associated with a reduction in asymptomatic bacteriuria among acute patients (<90d after spinal cord injury; P <.05); 1 patient would require 3.7 weeks of treatment on average to prevent 1 asymptomatic infection. For nonacute patients, the reduction approached statistical significance (P =.06). Prophylaxis resulted in an approximately twofold increase in antimicrobial-resistant bacteria. The regular use of antimicrobial prophylaxis for most patients who have neurogenic bladder caused by spinal cord dysfunction is not supported. A clinically important effect, however, has not been excluded. Future research should focus on randomized trials in those patients who have recurrent UTIs that limit their daily functioning and well-being.


The impact of E-learning in medical education.
The authors provide an introduction to e-learning and its role in medical education by outlining key terms, the components of e-learning, the evidence for its effectiveness, faculty development needs for implementation, evaluation strategies for e-learning and its technology, and how e-learning might be considered evidence of academic scholarship. E-learning is the use of Internet technologies to enhance knowledge and performance. E-learning technologies offer learners control over content, learning sequence, pace of learning, time, and often media, allowing them to tailor their experiences to meet their personal learning objectives. In diverse medical education contexts, e-learning appears to be at least as effective as traditional instructor-led methods such as lectures. Students do not see e-learning as replacing traditional instructor-led training but as a complement to it, forming part of a blended-learning strategy. A developing infrastructure to support e-learning within medical education includes repositories, or digital libraries, to manage access to e-learning materials, consensus on technical standardization, and methods for peer review of these resources. E-learning presents numerous research opportunities for faculty, along with continuing challenges for documenting scholarship. Innovations in e-learning technologies point toward a revolution in education, allowing learning to be individualized (adaptive learning), enhancing learners' interactions with others (collaborative learning), and transforming the role of the teacher. The integration of e-learning into medical education can catalyze the shift toward applying adult learning theory, where educators will no longer serve mainly as the distributors of content, but will become more involved as facilitators of learning and assessors of competency.


Impact of restaurant hygiene grade cards on foodborne-disease hospitalizations in Los Angeles County.
Although health departments routinely inspect restaurants to assess compliance with established hygienic standards, few data are available on the effectiveness of these efforts in preventing foodborne disease. The study reported here assessed the impact on foodborne-disease hospitalizations in Los Angeles County of a restaurant hygiene grading system that utilized publicly posted grade cards. The grading systm was introduced in January 1998. Hospital discharge data on foodborne-disease hospitalizations were analyzed for Los Angeles County and, as a control, for the rest of California during the period 1993-2000. Ordinary least-squares regression analysis was done to measure the effect of the grading progam on these hospitalizations. After baseline temporal and geographic trends were adjusted for, the restaurant hygiene grading program was associated with a 13.1 percent decrease (p < .01) in the number of foodborne-disease hospitalizations in Los Angeles County in the year following implementation the program (1998). This decrease was sustained over the next two years (1999-2000). The results suggest that restaurant hygiene grading with public posting of results is an effective intervention for reducing the burden of foodborne disease.


Measuring bioconcentration factors in fish using exposure to multiple chemicals and internal benchmarking to correct for growth dilution.
Modern chemical legislation requires measuring the bioconcentration factor (BCF) of large numbers of chemicals in fish. The BCF must be corrected for growth dilution, because fish growth rates vary between laboratories. Two hypotheses were tested: (1) that BCFs of multiple chemicals can be measured simultaneously in one experiment, and (2) that internal benchmarking using a conservative test substance in the chemical mixture can be used to correct for growth dilution. Bioconcentration experiments were conducted following major elements of the OECD 305 guideline. Fish were simultaneously exposed to 11 chemicals selected to cover a range of BCFs and susceptibility to biotransformation. A method was developed to calculate the growth-corrected elimination rate constant from the concentration ratio of the analyte and a benchmarking chemical for which growth dilution dominated other elimination mechanisms. This method was applied to the experimental data using hexachlorobenzene as the benchmarking chemical. The growth dilution correction lowered the apparent elimination rate constants by between 5% and a factor of four for eight chemicals, while for two chemicals the growth-corrected elimination rate constant was not significantly different from zero. The benchmarking method reduced the uncertainty in the elimination rate constant compared to the existing method for growth dilution correction. The BCFs from exposing fish to 10 chemicals at once were consistent with BCF values from single-chemical exposures from the literature, supporting hypothesis 1.


Prevalence of Oral Premalignant Lesions and Its Risk Factors among the Adult Population in Udupi Taluk of Coastal Karnataka, India
Objective: Globally oral cancer is one of the ten most common cancers with prevalence being high in Central and South East Asian countries. This survey was conducted to estimate the prevalence of oral pre-malignant lesions (OPML) and to identify their risk factors. Methods: A community based cross-sectional study was carried out among 2033 individuals aged ≥18 years. A questionnaire was administered to collect socio-demographic characteristics, various risk factors for oral cancer and presence of its symptoms. Oral cavity of all the participants was examined in detail by the study investigator as per WHO guidelines for the early diagnosis of oral neoplasia. Result: The prevalence of OPML was found to be 3.73%. Among those with OPML, all were ever tobacco consumers and had poor oral hygiene. A significant association was observed between OPML and younger age group (OR=2.56, 95% CI 1.08-6.02), males (OR=26.76, 95% CI 8.40-85.19) and low socio-economic status (OR = 1.91, 95% CI 1.20-3.02). Tobacco (p<0.001), alcohol (OR= 7.92, 95% CI 4.77-13.14) and areca nut consumption (OR = 5.48, 95% CI 3.42-8.77) were strongly associated with OPML. On multivariate analysis among ever tobacco users, OPML was associated with younger individuals, males and those using smokeless forms of tobacco (p <0.05). The study showed that the participants with OPML were more likely to be never married (OR=1.6, 95% CI 0.92-2.96),to be unskilled workers (OR= 1.45, 95% CI 0.61-3.43), to have suffered from oral trauma (OR =1.30, 95% CI 0.75-2.26), to have consumed hot and spicy food frequently (OR=1.53, 95% CI 0.96-2.24), to have consumed fruits infrequently (OR=1.53, 95% CI 0.90-2.59) and to report family history of any cancer (OR = 1.29, 95% CI 0.58-2.87) . However, these associations were statistically insignificant. Conclusion: The study reinforces that use of substances such as tobacco, alcohol and areca nut are the modifiable risk factors for OPML.


Top-down influences mediate hand bias in spatial attention.
Spatial attention can be biased to locations near the hand. Some studies have found facilitated processing of targets appearing within hand-grasping space. In this study, we investigated how changing top-down task priorities alters hand bias during visual processing. In Experiment 1, we used a covert orienting paradigm with nonpredictive cues and emphasized the location of the hand relative to the target. Hands or visual anchors (boards) were placed next to potential target locations, and responses were made with the contralateral hand. Results indicated a hand-specific processing bias: Hand location, but not board location, speeded responses to targets near the hand. This pattern of results replicated previous studies using covert orienting paradigms with highly predictive cues. In Experiment 2, we used the same basic paradigm but emphasized the location of the response hand. Results now showed speeded responses to targets near response locations. Together these experiments demonstrated that top-down instructional sets (i.e., what is considered to be most relevant to task performance) can change the processing priority of hand location by influencing the strength of top-down, as compared with bottom-up, inputs competing for attention resources.


Expression of Bcl-2 protein and Bcl-2 mRNA in normal and neoplastic lymphoid tissues.
The bcl-2 gene is a unique proto-oncogene that blocks apoptosis; its product is localized on the inner mitochondrial membrane. In non neoplastic human lymphoid tissues, bcl-2 protein is strongly expressed in the small recirculating lymphocytes of the follicular mantle zone; it is expressed less intensely in T-cell areas, and is almost absent from germinal center cells. Bcl-2 mRNA, in contrast to bcl-2 protein, is strongly expressed on most of the latter cells, a similar phenomenon also being observed in peripheral blood lymphocytes (PBL). Resting PBL express both bcl-2 mRNA and protein, while most lymphoblasts in mitogen-stimulated PBL cultures lose bcl-2 protein and become apoptotic, despite expressing higher levels of mRNA. Posttranscriptional regulation of the bcl-2 gene may cause this paradoxical down-regulation of bcl-2 protein and may play an important role in the clonal selection of lymphocytes. Bcl-2 protein is frequently expressed in follicular lymphomas bearing the t(14;18) chromosomal translocation, but it is also widely expressed in many other B- and T-cell lymphomas without bcl-2 rearrangement, showing that mechanisms other than t(14;18) translocation may deregulate bcl-2 expression. Many lymphoid and myeloid cell lines also express bcl-2 protein with no correlation being shown with differentiation stage. Thus, it is conceivable that bcl-2 protein may play a role in the oncogenesis of many hematolymphoid malignancies by interfering with programmed cell death, in concert with other oncogenes or tumor suppressor genes.


The mannose 6-phosphate receptor targeted with porphyrin-based periodic mesoporous organosilica nanoparticles for rhabdomyosarcoma theranostics.
Porphyrin-based periodic mesoporous organosilica nanoparticles (PMO) synthesized from a large functional octatriethoxysilylated porphyrin precursor and allowing two-photon excitation photodynamic therapy (TPE-PDT) and NIR imaging were synthesized. These PMO were grafted with polyethylene glycol (PEG) moieties and an analogue of mannose 6-phosphate functionalized at the anomeric position (AMFA). AMFAs are known to efficiently target mannose 6-phosphate receptors (M6PRs) which are over-expressed in various cancers. Here, we demonstrated for the first time that M6PRs were over-expressed in rhabdomyosarcoma (RMS) cells and could be efficiently targeted with PMO-AMFA allowing TPE imaging and TPE-PDT of RMS cells. The comparison with healthy myoblasts demonstrated an absence of biological effects, suggesting a cancer cell specificity in the biomedical action observed.


Current status and perspectives of neuroprotection in ischemic stroke treatment.
In developed countries, ischemic stroke is one of the leading causes of death and neurological impairment. The two most important therapeutic approaches in patients with acute cerebral ischemia consist of improving cerebral blood flow and blocking the biochemical and metabolic changes at the ischemic cascade level. The significant advances made in the past decade in the knowledge of the physiopathological mechanisms of cerebral ischemia, and the development of new drugs have given rise to true expectations regarding treatment and the rejection of nihilist attitudes. In the past 15 years, based on the excellent results obtained in experimental models of ischemia, many clinical trials have been conducted with different neuroprotective drugs. The results obtained in most studies have been negative, or the studies were terminated early owing to side effects. However, some drugs (citicoline, clomethiazole, piracetam and ebselen) have shown a certain degree of clinical efficacy, limited to subgroups of patients, and with a narrow therapeutic window, longer-lasting in the case of citicoline. The design of new clinical trials with neuroprotective drugs requires adequate preclinical assessment and the use of the new magnetic resonance techniques for the selection of patients and the assessment of the efficacy of treatment. The new trends in neuroprotection in focal cerebral ischemia and the results of the clinical trials published to date are reviewed.


Predictive factors for institutionalization of the elderly: a case-control study.
To identify predictive indicators of institutionalization of the elderly. A case-control study was carried out with 991 elderly individuals in the city of Pelotas, (Southern Brazil), from 2007 to 2008. The cases of institutionalized elderly adults (n = 393) were detected using a census of all long-stay institutions for the elderly in the city. The population controls (n = 598) were randomly selected using a comprehensive health survey. Pearson's chi-square test and linear trends were used to compare groups in the crude analysis; and the binary logistic regression model of the adjusted analysis, with the effects expressed as odds ratios. Institutionalization was more frequent in females (OR = 1.96, 95%CI 1.31, 2.95). Elderly with advanced age (OR = 3.23 and OR = 9.56 for age groups 70-79 and > 80 years, respectively), those who lived without a partner (single, divorced or widowed), and those who had no formal schooling or had a functional disability preventing them from performing basic activities for daily living were more likely to be institutionalized. An inverse trend between the incidence of elderly institutionalization and the level of physical activity was observed, where somewhat active and inactive subjects were more likely to be institutionalized (OR = 1.71 and OR = 4.73, respectively). Of the factors examined, age > 80 years, living without a partner and being physically inactive were the indicators most strongly associated with institutionalization. The encouragement of informal care through cultural and educational activities focused on the role of the family in caring for the elderly can prevent the institutionalization of these individuals.


Expression of the nirS, hzsA, and hdh Genes in Response to Nitrite Shock and Recovery in Candidatus Kuenenia stuttgartiensis.
In this study, Candidatus Kuenenia stuttgartiensis were subjected to distinct nitrite shocks (66 (control), 200, 300, 400, and 500 mg N/L), and the responses of mRNA levels of cytochrome cd1 nitrite/nitric oxide oxidoreductase (nirS), hydrazine synthase (hzsA), and hydrazine dehydrogenase (hdh) were assessed. Changes in the hydrazine dehydrogenase (HDH) protein level were monitored. At 200 mg NO2(-)-N/L, the normalized specific anaerobic ammonium-oxidizing activity (nSAA) slightly increased relative to the control despite a significant decrease in nirS, hzsA, and hdh mRNA levels. When nitrite increased to 300 and 400 mg N/L, increased nirS, hzsA, and hdh mRNA levels were observed, but the nSAA decreased, relative to the 200 mg NO2(-)-N/L exposure. HDH protein detection revealed that Candidatus Kuenenia stuttgartiensis attempted to yield high enzyme levels by stimulating mRNA synthesis to resist the nitrite-induced stress. On 500 mg NO2(-)-N/L shock, the nirS, hzsA, and hdh mRNA levels decreased, alongside decreased nSAA and HDH levels. Although the mRNA levels did not always coincide with activities, our findings advance understanding of the mechanisms that anammox bacteria use to cope with nitrite inhibition at the transcriptional and translational levels, which will improve the diagnostic accuracy of bioreactor failures when nitrite accumulation occurs.


A Subset of Patients with Acute Myeloid Leukemia Has Leukemia Cells Characterized by Chemokine Responsiveness and Altered Expression of Transcriptional as well as Angiogenic Regulators.
Acute myeloid leukemia (AML) is an aggressive and heterogeneous bone marrow malignancy, the only curative treatment being intensive chemotherapy eventually in combination with allogeneic stem cell transplantation. Both the AML and their neighboring stromal cells show constitutive chemokine release, but chemokines seem to function as regulators of AML cell proliferation only for a subset of patients. Chemokine targeting is therefore considered not only for immunosuppression in allotransplanted patients but also as a possible antileukemic strategy in combination with intensive chemotherapy or as part of disease-stabilizing treatment at least for the subset of patients with chemokine-responsive AML cells. In this study, we characterized more in detail the leukemia cell phenotype of the chemokine-responsive patients. We investigated primary AML cells derived from 79 unselected patients. Standardized in vitro suspension cultures were used to investigate AML cell proliferation, and global gene expression profiles were compared for chemokine responders and non-responders identified through the proliferation assays. CCL28-induced growth modulation was used as marker of chemokine responsiveness, and 38 patients were then classified as chemokine-responsive. The effects of exogenous CCL28 (growth inhibition/enhancement/no effect) thus differed among patients and was also dependent on the presence of exogenous hematopoietic growth factors as well as constitutive AML cell cytokine release. The effect of CCR1 inhibition in the presence of chemokine-secreting mesenchymal stem cells also differed among patients. Chemokine-responsive AML cells showed altered expression of genes important for (i) epigenetic transcriptional regulation, particularly lysine acetylation; (ii) helicase activity, especially DExD/H RNA helicases; and (iii) angioregulatory proteins important for integrin binding. Thus, chemokine responsiveness is part of a complex AML cell phenotype with regard to extracellular communication and transcriptional regulation. Chemokine targeting in chemokine-responsive patients may thereby alter AML cell trafficking and increase their susceptibility toward antileukemic treatment, e.g., conventional chemotherapy or targeting of other phenotypic characteristics of the chemokine-responsive cells.


Site-specific release of nascent chains from ribosomes at a sense codon.
"2A" oligopeptides are autonomous elements containing a D(V/I)EXNPGP motif at the C terminus. Protein synthesis from an open reading frame containing an internal 2A coding sequence yields two separate polypeptides, corresponding to sequences up to and including 2A and those downstream. We show that the 2A reaction occurs in the ribosomal peptidyltransferase center. Ribosomes pause at the end of the 2A coding sequence, over the glycine and proline codons, and the nascent chain up to and including this glycine is released. Translation-terminating release factors eRF1 and eRF3 play key roles in the reaction. On the depletion of eRF1, a greater proportion of ribosomes extend through the 2A coding sequence, yielding the full-length protein. In contrast, impaired eRF3 GTPase activity leads to many ribosomes failing to translate beyond 2A. Further, high-level expression of a 2A peptide-containing protein inhibits the growth of cells compromised for release factor activity and leads to errors in stop codon recognition. We propose that the nascent 2A peptide interacts with ribosomes to drive a highly unusual and specific "termination" reaction, despite the presence of a proline codon in the A site. After this, the majority of ribosomes continue translation, generating the separate downstream product.


Mitochondrial stop and go: signals that regulate organelle movement.
In order to satisfy the metabolic and ion homeostasis demands of neurons, mitochondria must be transported to appropriate locations within cells. Although it is well established that much of this trafficking occurs on microtubules and, to a lesser extent, actin, the mechanisms by which the trafficking of mitochondria is controlled are poorly understood. A recent study by Chada and Hollenbeck shows that nerve growth factor halts the movement of mitochondria in axons by means of a mechanism that depends on activation of phosphatidylinositol 3-kinase. These studies provide important new insights into the mechanisms that regulate mitochondrial movement and control mitochondrial docking. These insights are critical to the understanding of the factors that control the distribution, location, and function of mitochondria in both healthy and injured neurons.


Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.
Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.


Linking new paradigms in protein chemistry to reversible membrane-protein interactions.
Amphitrophic proteins are soluble, globular proteins that may - under certain conditions - interact reversibly with a plasma membrane. How this apparent duality in the properties of a protein is achieved has been a relatively little-studied subject until recently. In this review we aim to summarize the current knowledge regarding some important amphitrophic systems in which the interaction with the membrane does not require post-translational functional groups, but is an intrinsic property of the protein. We discuss mechanisms and driving forces involved in membrane binding in the context of two related concepts in protein folding and function that appear to have implications for understanding the association of proteins with membranes; first, the existence of some proteins with low-energy barrier heights for protein folding. Low folding barriers and the ability of proteins to form stable molten globule states are rationales that can explain how a protein can gain access to an ensemble (or continuum) of non-native conformations that are competent membrane binders. Second, the focus on order-disorder and disorder-order transitions to explain protein function, a concept which has been mainly developed within the novel protein trinity paradigm. Here, protein function can arise from any of three thermodynamic states: a solid, crystal-like state; a dense fluid state; and an extended disordered state. Together these concepts aid to understand amphitrophic mechanism and to unify interpretations of protein behaviour with respect to the degree of 8 (un)unfolding of the membrane-bound proteins.


p53 is not mutated in hepatocellular carcinomas from Alaska Natives.
Hepatocellular carcinoma is common among Alaska Natives. The known risk factor in this population is hepatitis B viral infection; fungal toxins, including aflatoxin B1, have not been detected in foodstuffs. In this series of 14 patients (including 4 siblings and 2 second cousins), 3 patients were less than 12 years old at diagnosis of hepatocellular carcinoma, 8 patients were 13-24 years old, and 3 patients were more than 60 years old. Since p53 mutations occur in 29% of hepatocellular carcinomas worldwide, we tested the tumors for p53 mutations and serum samples for anti-p53 antibodies. Serum samples from these 14 patients did not contain detectable levels of anti-p53 antibodies. Loss of heterozygosity within the p53 locus was not detected in any of 9 informative cases. Immunohistochemical analysis for p53 protein accumulation was negative in all of 11 tumors. DNA sequence analysis of 12 tumor samples showed no evidence of p53 mutation in the highly conserved regions included in exons 5-8. These data, combined with one case from a previous report, indicate a mutation frequency of 0 of 13, which differs significantly from the worldwide frequency of 29% (chi 2 3.9; P = 0.048). These results indicate that liver carcinogenesis among Alaska Natives occurs independently of a traditional p53 pathway. The familial clustering and early onset in this population strongly suggest an inherited genetic predisposition to develop liver cancer. Germline mutations in a tumor suppressor or a cancer susceptibility gene are likely. Future studies of these samples should include investigations of candidate suppressor or susceptibility genes which map to chromosomal regions commonly deleted in liver cancers.


Five strategies of successful part-time work.
Nearly one in ten professionals now works part-time. But all too often, part-time work creates as many problems as it solves. At best, many part-timers work more hours than they intended. At worst, they see their importance to their organizations dwindle. Two generations have wrestled with such arrangements, and today some part-time professionals have found ways to overcome the challenges, with shining results. Drawing on two years of research investigating part-time engineers, financial analysts, IT specialists, and consultants, the authors present five strategies used by successful part-timers to make their unique position work for themselves and their companies. To begin with, successful part-time professionals take pains to make their work-life priorities, their schedules, and their plans for the future transparent to the organization. Second, they broadcast the business case for their arrangement, being careful to demonstrate that the arrangement has not disrupted the business and may even have a positive impact. Third, they establish routines to protect their time at work and rituals to protect their time at home. Fourth, they cultivate champions in senior management who protect them from skeptics and advocate for their arrangements up and down the ranks. And last, they gently but firmly remind their colleagues that, despite their part-time status, they're still major players in the organization who cannot be ignored. Taken together, these strategies not only help the part-timer deal with the organization but also make the organization itself more receptive to the possibilities of part-time work.


Valproic acid silencing of ascl1b/Ascl1 results in the failure of serotonergic differentiation in a zebrafish model of fetal valproate syndrome.
Fetal valproate syndrome (FVS) is caused by in utero exposure to the drug sodium valproate. Valproate is used worldwide for the treatment of epilepsy, as a mood stabiliser and for its pain-relieving properties. In addition to birth defects, FVS is associated with an increased risk of autism spectrum disorder (ASD), which is characterised by abnormal behaviours. Valproate perturbs multiple biochemical pathways and alters gene expression through its inhibition of histone deacetylases. Which, if any, of these mechanisms is relevant to the genesis of its behavioural side effects is unclear. Neuroanatomical changes associated with FVS have been reported and, among these, altered serotonergic neuronal differentiation is a consistent finding. Altered serotonin homeostasis is also associated with autism. Here we have used a chemical-genetics approach to investigate the underlying molecular defect in a zebrafish FVS model. Valproate causes the selective failure of zebrafish central serotonin expression. It does so by downregulating the proneural gene ascl1b, an ortholog of mammalian Ascl1, which is a known determinant of serotonergic identity in the mammalian brainstem. ascl1b is sufficient to rescue serotonin expression in valproate-treated embryos. Chemical and genetic blockade of the histone deacetylase Hdac1 downregulates ascl1b, consistent with the Hdac1-mediated silencing of ascl1b expression by valproate. Moreover, tonic Notch signalling is crucial for ascl1b repression by valproate. Concomitant blockade of Notch signalling restores ascl1b expression and serotonin expression in both valproate-exposed and hdac1 mutant embryos. Together, these data provide a molecular explanation for serotonergic defects in FVS and highlight an epigenetic mechanism for genome-environment interaction in disease.


Air conditioning and television as protective factors in arboviral encephalitis risk.
In California, the advent of television and air conditioned housing has coincided with a general decrease in mosquito-borne viral encephalitis cases in humans in the past 25 years. During this same period, levels of vector populations, virus activity in vectors, birds, and horses has been high at times. Air conditioning and television encourage persons to remain indoors during summer evenings, the primary time when infected Culex tarsalis transmit western equine encephalomyelitis and St. Louis encephalitis viruses. The attack rates for these 2 diseases in 33 counties in the central valley of California, 1945-1982, were compared with the prevalence of household air conditioning and television. Encephalitis rates were negatively associated with television ownership in both cross-sectional and longitudinal analysis. Moreover, individual counties which experienced the most rapid increase in household television ownership had the greatest decline in encephalitis rates. A telephone survey conducted in Kern County revealed that air conditioner and television utilization times corresponded closely to the feeding times of Cx. tarsalis, and respondents indicated a preference to remain indoors during this time because of these appliances. It is concluded that changed behavioral patterns may protect from vector-borne diseases and be complementary to vector control programs.