Rapid aneuploidy detection with multiplex ligation-dependent probe amplification: a prospective study of 4000 amniotic fluid samples.
The introduction of prenatal screening requires rapid high-throughput diagnosis of common aneuploidies. Multiplex ligation-dependent probe amplification (MLPA) allows for quick, easily automated multiplex testing of these aneuploidies in one polymerase chain reaction. We performed a large prospective study using MLPA on 4000 amniotic fluid (AF) samples including all indications and compared its value to karyotyping and fluorescence in situ hybridization (FISH). MLPA can reliably determine common aneuploidies with 100% sensitivity and 100% specificity. Moreover, some mosaic cases and structural chromosome aberrations were detected as well. In cases of a male fetus, triploidies can be detected by an aberrant pattern of probe signals, which mimics maternal cell contamination (MCC). Macroscopic blood contamination was encountered in 3.2% of the AF samples. In 20% of these samples, an MLPA pattern was found consistent with MCC, although there were no false negatives of the most common aneuploidies. As the vast majority of inconclusive results (1.7%) is due to potential MCC, we designed a protocol in which we determine whether MLPA can be performed on blood-contaminated AF samples by testing if blood is of fetal origin. Then, the number of inconclusive results could be theoretically reduced to 0.05%. We propose an alternative interpretation of relative probe signals for rapid aneuploidy diagnosis (RAD). We discuss the value of MLPA for the detection of (submicroscopic) structural chromosome anomalies. MLPA is a reliable method that can replace FISH and could be used as a stand-alone test for RAD instead of karyotyping.


Four-hourly scheduling of temozolomide improves tumour growth delay but not therapeutic index in A375M melanoma xenografts.
To establish whether temozolomide is more effective against A375M human melanoma xenografts if given every 4 h rather than every 24 h, in order to exploit depletion of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) by prior doses of the drug. ATase depletion in A375M human melanoma xenografts was determined over 24 h after a single dose of temozolomide. The effect of different drug schedules (all of total dose 500 mg/kg) in delaying the growth of the xenografts was tested, and ATase depletion and DNA methylation damage assessed in tumour and normal tissue. Maximal depletion of ATase in tumour, to 2.52 +/- 0.23% of pretreatment levels, occurred 4-8 h after a single 100 mg/kg i.p. dose of temozolomide, with 23.0% recovery of protein levels at 24 h. Scheduling of temozolomide every 4 h increased tumour growth delay (33.6 +/- 1.39 days with temozolomide 100 mg/kg 4-hourly x versus 23.2 +/- 1.43 days with temozolomide 100 mg/kg once daily x 5; P < 0.0001) at the expense of increased toxicity (17.4 +/- 1.55% animal weight loss versus 10.6 +/- 1.27%. respectively). Temozolomide every 4 h did not increase ATase depletion compared with the 5-day schedule, but resulted in greater DNA 06-guanine methylation (29.0% more in tumour, 20.8% in liver and 56.0% in brain, comparing areas under the methylation-time curve). The 4-hourly schedule of temozolomide delayed tumour growth significantly more than the once-daily and 12-hourly schedules, probably as a result of greater DNA damage inflicted, but also increased toxicity. It remains to be seen if this regimen confers a net benefit over the standard schedule.


Self-oriented nanoparticles for site-selective immunoglobulin G recognition via epitope imprinting approach.
Molecular imprinting is a polymerization technique that provides synthetic analogs for template molecules. Molecularly imprinted polymers (MIPs) have gained much attention due to their unique properties such as selectivity and specificity for target molecules. In this study, we focused on the development of polymeric materials with molecular recognition ability, so molecular imprinting was combined with miniemulsion polymerization to synthesize self-orienting nanoparticles through the use of an epitope imprinting approach. Thus, L-lysine imprinted nanoparticles (LMIP) were synthesized via miniemulsion polymerization technique. Immunoglobulin G (IgG) was then bound to the cavities that specifically formed for L-lysine molecules that are typically found at the C-terminus of the Fc region of antibody molecules. The resulting nanoparticles makes it possible to minimize the nonspecific interaction between monomer and template molecules. In addition, the orientation of the entire IgG molecule was controlled, and random imprinting of the IgG was prevented. The optimum conditions were determined for IgG recognition using the imprinted nanoparticles. The selectivity of the nanoparticles against IgG molecules was also evaluated using albumin and hemoglobin as competitor molecules. In order to show the self-orientation capability of imprinted nanoparticles, human serum albumin (HSA) adsorption onto both the plain nanoparticles and immobilized nanoparticles by anti-human serum albumin antibody (anti-HSA antibody) was also carried out. Due to anti-HSA antibody immobilization on the imprinted nanoparticles, the adsorption capability of nanoparticles against HSA molecules vigorously enhanced. It is proved that the oriented immobilization of antibodies was appropriately succeeded.


A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells.
The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.


Artificial light at night desynchronizes strictly seasonal reproduction in a wild mammal.
Change in day length is an important cue for reproductive activation in seasonally breeding animals to ensure that the timing of greatest maternal investment (e.g. lactation in mammals) coincides with favourable environmental conditions (e.g. peak productivity). However, artificial light at night has the potential to interfere with the perception of such natural cues. Following a 5-year study on two populations of wild marsupial mammals exposed to different night-time levels of anthropogenic light, we show that light pollution in urban environments masks seasonal changes in ambient light cues, suppressing melatonin levels and delaying births in the tammar wallaby. These results highlight a previously unappreciated relationship linking artificial light at night with induced changes in mammalian reproductive physiology, and the potential for larger-scale impacts at the population level.


Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.
Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.


Identification of melamine/cyanuric acid-containing nephrolithiasis by infrared spectroscopy.
Melamine-contaminated milk formula caused infant nephrolithiasis in some areas of China. Its combination with cyanuric acid causes crystallization in renal tubules. Following this renal damage and even renal failure that require long-term hemodialysis has been reported. Therefore, correct and timely diagnosis of these complex diseases is critical. Melamine containing stone is a combination of equal molar ratios of common stone compositions that has been reported from previous animal studies. We have previously identified the compositions of urinary tract stones with infrared (IR) spectroscopy. We hypothesized that the absorbance of wavelength of IR can identify melamine/cyanuric acid in the presence of mixing human stone compositions. In this study, we made an artificial stone composition and examine under IR absorbance by mixing equal molar ratios of melamine/cyanuric acid with different types of human urinary stones, and established a reference of IR analysis for the identification of melamine/cyanuric acid-containing human urinary tract stones. Knowledge of the precise stone composition allowed institution of appropriate prophylactic dietary and medical therapy and this may help in the prevention of urinary stone recurrence. The results are promising that melamine and cyanuric acid can be identified clearly in a low percentile (approximately 1%) of stone mixture pellet. Therefore, IR seems to be an ideal tool for the identification of melamine/cyanuric acid-containing stones.


Large carbon dioxide fluxes from headwater boreal and sub-boreal streams.
Half of the world's forest is in boreal and sub-boreal ecozones, containing large carbon stores and fluxes. Carbon lost from headwater streams in these forests is underestimated. We apply a simple stable carbon isotope idea for quantifying the CO2 loss from these small streams; it is based only on in-stream samples and integrates over a significant distance upstream. We demonstrate that conventional methods of determining CO2 loss from streams necessarily underestimate the CO2 loss with results from two catchments. Dissolved carbon export from headwater catchments is similar to CO2 loss from stream surfaces. Most of the CO2 originating in high CO2 groundwaters has been lost before typical in-stream sampling occurs. In the Harp Lake catchment in Canada, headwater streams account for 10% of catchment net CO2 uptake. In the Krycklan catchment in Sweden, this more than doubles the CO2 loss from the catchment. Thus, even when corrected for aquatic CO2 loss measured by conventional methods, boreal and sub-boreal forest carbon budgets currently overestimate carbon sequestration on the landscape.


Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of SCA7.
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder caused by a CAG repeat expansion. To determine the mechanism of neurotoxicity, we produced transgenic mice and observed a cone-rod dystrophy. Nuclear inclusions were present, suggesting that the disease pathway involves the nucleus. When yeast two-hybrid assays indicated that cone-rod homeobox protein (CRX) interacts with ataxin-7, we performed further studies to assess this interaction. We found that ataxin-7 and CRX colocalize and coimmunoprecipitate. We observed that polyglutamine-expanded ataxin-7 can dramatically suppress CRX transactivation. In SCA7 transgenic mice, electrophoretic mobility shift assays indicated reduced CRX binding activity, while RT-PCR analysis detected reductions in CRX-regulated genes. Our results suggest that CRX transcription interference accounts for the retinal degeneration in SCA7 and thus may provide an explanation for how cell-type specificity is achieved in this polyglutamine repeat disease.


Mind the gap: resources required to receive, process and interpret research-returned whole genome data.
Most genotype-phenotype studies have historically lacked population diversity, impacting the generalizability of findings and thereby limiting the ability to equitably implement precision medicine. This well-documented problem has generated much interest in the ascertainment of new cohorts with an emphasis on multiple dimensions of diversity, including race/ethnicity, gender, age, socioeconomic status, disability, and geography. The most well known of these new cohort efforts is arguably All of Us, formerly known as the Precision Medicine Cohort Initiative Program. All of Us intends to ascertain at least one million participants in the United States representative of the multiple dimensions of diversity. As an incentive to participate, All of Us is offering the return of research results, including whole genome sequencing data, as well as the opportunity to contribute to the scientific process as non-scientists. The scale and scope of the proposed return of research results are unprecedented. Here, we briefly review possible return of genetic data models, including the likely data file formats and modes of data transfer or access. We also review the resources required to access and interpret the genetic or genomic data once received by the average participant, highlighting the nuanced anticipated barriers that will challenge both the digitally, computationally literate and illiterate participant alike. This inventory of resources required to receive, process, and interpret return of research results exposes the potential for access disparities and warns the scientific community to mind the gap so that all participants have equal access and understanding of the benefits of human genetic research.


The treatment and outcome of postmeningitic subdural empyema in infants.
The management of subdural empyema (SDE) has been debated in the literature for decades. Craniotomy and bur hole drainage have been shown to achieve a favorable outcome. However, there is a lack of comparative data for these modes of management of SDE subsequent to meningitis in infants. The authors conducted a retrospective review of 33 infants identified with SDE due to meningitis at the Department of Neurosurgery, Chang Gung Memorial Hospital between 2000 and 2006. Preoperative clinical presentation, duration of symptoms, radiological investigations, CSF data, and postoperative outcome were analyzed and compared between these 2 surgical groups. At diagnosis, there were no differences between the groups in age, weight, degree of consciousness, CSF analysis, or duration of fever. The outcome data showed no difference in the number of days until afebrile, number of days of postsurgical antibiotic treatment, neurological outcome, recurrence rate, or complication rate. There was only 1 death in the series. Subdural empyema due to meningitis in infants is unique with respect to the pathophysiology, presentation, and treatment of SDE. Early detection and removal of SDE provide a favorable outcome in both surgical intervention groups. Bur hole drainage is less invasive, and it is possible to expect a clinical outcome as good as with craniotomy in postmeningitic SDE.


Rapid microbial response to the presence of an ancient relic in the Antarctic Dry Valleys.
The extreme cold and aridity of the Antarctic McMurdo Dry Valleys have led to the longstanding belief that metabolic rates of soil microbiota are negligible, and that ecosystem changes take place over millennia. Here we report the first direct experimental evidence that soil microbial communities undergo rapid and lasting changes in response to contemporary environmental conditions. Mummified seals, curious natural artifacts found scattered throughout Dry Valleys, alter their underlying soil environment by stabilizing temperatures, elevating relative humidity and reducing ultraviolet exposure. In a unique, multi-year mummified seal transplantation experiment, we found that endemic Dry Valley microbial communities responded to these changes within 3 years, resulting in a sevenfold increase in CO(2) flux and a significant reduction in biodiversity. These findings challenge prevailing ideas about Antarctic Dry Valley ecosystems and indicate that current and future environmental conditions may strongly influence the ecology of the dominant biota in the Dry Valleys.


Utility of immunophenotypic and immunogenotypic analysis in the study of necrotic lymph nodes.
We report a case of complete lymph node necrosis. No specific aetiology could be determined by morphology, but a B lymphoid population and clonal rearrangement of the immunoglobulin heavy chain gene were demonstrated in immunophenotypic and immunogenotypic studies performed using DNA extracted from paraffin embedded necrotic tissue. In the setting of lymph node necrosis, we suggest that immunohistochemical and gene rearrangement studies may provide additional diagnostic information.


Choice of control groups in the appraisal of sympathetic nervous activity in essential hypertension.
1. Plasma noradrenaline concentrations were similar in normotensive and hypertensive outpatients, but were significantly lower in laboratory control subjects. 2. Standing plasma noradrenaline concentrations were similar in all three groups. 3. Urinary vanillyl mandelic acid, catecholamines and metanephrines were also similar in the normotensive and hypertensive groups. 4. Laboratory controls, possibly because of familiarity with the techniques of sphygmomanometry and blood sampling, may attain a 'basal' resting level of sympathetic nervous discharge more readily and rapidly than subjects who are unfamiliar with such procedures. 5. After orthostatic stimulation by standing for 2 min, the activity of the sympathetic nervous system, as determined by pulse rate and plasma noradrenaline concentrations, was similar in the three groups, despite the lower starting values in the laboratory staff. 6. The absence of differences in plasma noradrenaline or urinary catecholamine and metabolite concentrations does not support the hypothesis of excessive sympathetic nervous activity in essential hypertension.


Neural Activity Reveals Preferences Without Choices.
We investigate the feasibility of inferring the choices people would make (if given the opportunity) based on their neural responses to the pertinent prospects when they are not engaged in actual decision making. The ability to make such inferences is of potential value when choice data are unavailable, or limited in ways that render standard methods of estimating choice mappings problematic. We formulate prediction models relating choices to "non-choice" neural responses and use them to predict out-of-sample choices for new items and for new groups of individuals. The predictions are sufficiently accurate to establish the feasibility of our approach.


Drug use evaluation of cephalosporins in a tertiary care hospital.
Infectious disease burden in India is among the largest in the world. Cephalosporins are being used extensively in the current scenario, both empirically and as definitive treatment. With this information, we tried to evaluate the prescription pattern of drugs for infections in medicine and general surgical wards of a tertiary care hospital and evaluate the utilization of cephalosporins in the same. The study was conducted for a duration of 3 months in a tertiary care hospital after approval from the Institutional Ethics Committee, and permission of the respective heads of the surgery and medicine departments was obtained. After satisfying the inclusion criteria, participants' demographic details and the prescription notes by the treating doctor were noted and analyzed. The WHO prescription indicators were analyzed and the prescriptions were evaluated for the completeness of them. The utilization of cephalosporins was evaluated based on the institutional standard treatment guideline (STG) - Guidelines for Antimicrobial Therapy and Prophylaxis, 2014. Data were analyzed using descriptive statistics. A total of 600 patients were recruited, of which 350 were male and 250 were female. A total of 4341 drugs were prescribed. On an average, 7 drugs per prescription were found. The generic drugs prescribed were 27% (1163). Among the drugs prescribed, 19% (850) were antibiotics, of which 36.94% (314) were cephalosporins and 81% (3491) were other drugs. Ninety-four percent (565) prescriptions were incomplete (in terms of dose, frequency, duration, or dosage form). After referring to the STG, we found that cephalosporins were prescribed empirically in 40% (126) cases, of which medicine prescriptions accounted for it the most. Cephalosporins are extensively prescribed in medicine and surgery wards of the tertiary care hospital.


The BioGRID interaction database: 2013 update.
The Biological General Repository for Interaction Datasets (BioGRID: http//thebiogrid.org) is an open access archive of genetic and protein interactions that are curated from the primary biomedical literature for all major model organism species. As of September 2012, BioGRID houses more than 500 000 manually annotated interactions from more than 30 model organisms. BioGRID maintains complete curation coverage of the literature for the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and the model plant Arabidopsis thaliana. A number of themed curation projects in areas of biomedical importance are also supported. BioGRID has established collaborations and/or shares data records for the annotation of interactions and phenotypes with most major model organism databases, including Saccharomyces Genome Database, PomBase, WormBase, FlyBase and The Arabidopsis Information Resource. BioGRID also actively engages with the text-mining community to benchmark and deploy automated tools to expedite curation workflows. BioGRID data are freely accessible through both a user-defined interactive interface and in batch downloads in a wide variety of formats, including PSI-MI2.5 and tab-delimited files. BioGRID records can also be interrogated and analyzed with a series of new bioinformatics tools, which include a post-translational modification viewer, a graphical viewer, a REST service and a Cytoscape plugin.


Frequent retinal ganglion cell damage after acute optic neuritis.
To identify the extent of ganglion cell damage after first-time optic neuritis (ON) using the inter-ocular difference between affected and fellow eyes, and whether this approach is able to detect more patients suffering from ganglion cell damage than using absolute values. Thirty-four patients with first-time unilateral ON were followed for a median 413 days. Patients underwent optical coherence tomography testing to determine ganglion cell plus inner plexiform layer thickness (GCIP). Ganglion cell loss was quantified as GCIP difference between ON-affected and fellow eyes (inter-GCIP) and was compared against measurements from 93 healthy controls (HC). Visual function was assessed with high contrast visual acuity; and standard automated perimetry-derived measures of mean deviation and foveal threshold. At clinical presentation after median 19 days from symptom onset, 47.1% of patients showed early GCIP thinning in the ON-affected eye based on inter-GCIP. At the last visit acute ON was associated with 16.1 ± 10.0 µm GCIP thinning compared to fellow eyes (p = 3.669e-06). Based on inter-GCIP, 84.9% of ON patients sustained GCIP thinning in their affected eye at the last visit, whereas using absolute values only 71.0% of patients suffered from GCIP thinning (p = 0.002076). Only 32.3% of these patients had abnormal visual function. The best predictor of GCIP thinning as a measure of ON severity at the last visit was worse visual field mean deviation at clinical presentation. Inter-ocular GCIP identifies significantly more eyes suffering damage from ON than absolute GCIP, visual fields or visual acuity loss. Effective interventional options are needed to prevent ganglion cell loss.


Carotid endarterectomy at the millennium: what interventional therapy must match.
Carotid endarterectomy (CEA) is supported by level 1 evidence as the standard treatment of severe carotid stenosis in both symptomatic and asymptomatic patients. As interventional techniques are emerging for treatment of this disease, this study was undertaken to provide a contemporary surgical standard for comparison to carotid stenting. During the interval 1989 to 1999, 2236 isolated CEAs were performed on 1897 patients (62% male, 36% symptomatic, 4.6% reoperative procedures). Study endpoints included perioperative events, patient survival, late incidence of stroke, anatomic durability of CEA, and resource utilization changes during the study. Variables associated with complications, long-term and stroke free survival, restenosis, and resource utilization were analyzed by univariate and multivariate analysis. Perioperative complications occurred in 5.5% of CEA procedures, including any stroke/death (1.4%), neck hematoma (1.7%), cardiac complications (0.5%), and cranial nerve injury (0.4%). Actuarial survival at 5 and 10 years was 72.4% (95% confidence interval [CI] 69.3-73.5) and 44.7% (95% CI 41.7-47.9) respectively, with coronary artery disease (P < 0.0018), chronic obstructive pulmonary disease (P < 0.00018) and diabetes mellitus (P < 0.0011) correlating with decreased longevity. The age- and sex-adjusted incidence of any stroke during follow-up was reduced by 22% (upper 0.35, lower 0.08) of predicted with the patient classification of hyperlipidemia (P < 0.0045) as the only protective factor. Analysis of CEA anatomic durability during a median follow-up period of 5.9 years identified a 7.7% failure rate (severe restenosis/occlusion, 4.5%; or reoperative CEA, 3.2%) with elevated serum cholesterol (P < 0.017) correlating with early restenosis. Resource utilization diminished (first versus last 2-year interval periods) for average hospital length of stay from 10.3 +/- 1.5 days to 4.3 +/- 0.7 days (P < 0.01) and preoperative contrast angiography from 87% +/- 1.4% to 10.3% +/- 4%. These data delineate the safety, durability, and effectiveness in long-term stroke prevention of CEA. They provide a standard to which emerging catheter-based therapies for carotid stenosis should be compared.


Notch-Induced Myeloid Reprogramming in Spontaneous Pancreatic Ductal Adenocarcinoma by Dual Genetic Targeting.
Despite advances in our understanding of the genetics of pancreatic ductal adenocarcinoma (PDAC), the efficacy of therapeutic regimens targeting aberrant signaling pathways remains highly limited. Therapeutic strategies are greatly hampered by the extensive desmoplasia that comprises heterogeneous cell populations. Notch signaling is a contentious pathway exerting opposite roles in tumorigenesis depending on cellular context. Advanced model systems are needed to gain more insights into complex signaling in the multilayered tumor microenvironment. In this study, we employed a dual recombinase-based in vivo strategy to modulate Notch signaling specifically in myeloid cells to dissect the tumorigenic role of Notch in PDAC stroma. Pancreas-specific KrasG12D activation and loss of Tp53 was induced using a Pdx1-Flp transgene, whereas Notch signaling was genetically targeted using a myeloid-targeting Lyz2-Cre strain for either activation of Notch2-IC or deletion of Rbpj. Myeloid-specific Notch activation significantly decreased tumor infiltration by protumorigenic M2 macrophages in spontaneous endogenous PDAC, which translated into significant survival benefit. Further characterization revealed upregulated antigen presentation and cytotoxic T effector phenotype upon Notch-induced M2 reduction. This approach is the first proof of concept for genetic targeting and reprogramming of myeloid cells in a complex disease model of PDAC and provides evidence for a regulatory role of Notch signaling in intratumoral immune phenotypes.Significance: This study provides insight into the role of myeloid-dependent NOTCH signaling in PDAC and accentuates the need to dissect differential roles of signaling pathways in different cellular components within the tumor microenvironment. Cancer Res; 78(17); 4997-5010. ©2018 AACR.


Mechanism-based combinations with Pim kinase inhibitors in cancer treatments.
Proviral integration site for Moloney murine leukemia virus (Pim) kinases is a potential therapeutic target in both hematological and solid tumors, and is up-regulated in various cancer types. In certain cases, their expression levels are positively correlated with poor clinical outcome. A number of selective Pim kinase inhibitors are under development and a few are in clinical trials. Investigations of the mechanism of actions of these drugs have demonstrated that by inhibiting Pim kinases, processes such as transcription, translation, cell cycle progression, cell survival and drug resistance are affected. Pim kinases can be upregulated by multiple growth factors, cytokines, and chemokines, which also activate redundant pathways such as phosphatidylinositide 3-kinases/protein kinase B/mammalian targets of rapamycin, and mitogen-activated protein kinases. Interestingly, Pim kinases also share substrates with these parallel pathways. To overcome this challenge, Pim kinase inhibitors were tested in combination with other therapeutic agents based on their unique mechanism of actions. Based on existing literature, we identified studies where Pim kinase inhibitors were part of the combination strategies that used targeted agents or broad-spectrum chemotherapeutic drugs (including FDA-approved agents). The addition of Pim kinase inhibitors to these treatment strategies leads to additive to synergistic cytotoxic effect in cancer cells. Depending on the compound, combination results in sequential or complementary blockage or downregulation of oncogenic pathway. In summary, these studies provide evidence for developing mechanism-based combination therapies with Pim kinase inhibitors to treat cancers.


All-Cause Mortality Following Bariatric Surgery in Smokers and Non-smokers.
Bariatric surgery is associated with lower all-cause mortality, but many studies exclude smokers. We sought to determine if the association of mortality and bariatric surgery differs between smokers and non-smokers. We conducted a retrospective cohort study in a large Israeli integrated payer/provider health care organization. A total of 7747 adult patients who underwent bariatric surgery between January 1, 2005, and December 31, 2014, were selected and compared with non-surgical patients (and were matched on age, sex, diabetes, and BMI using a sequential/simultaneous stratification matching). A total of 30,742 patients with a median follow-up of 4.3 years were included in this study with less than 1% lost to follow-up. The type of bariatric surgery (gastric banding, Roux-en-Y gastric bypass, or sleeve gastrectomy) and smoking status were determined from electronic health records. The rate of all-cause mortality in matched surgical and non-surgical patients was compared in smoking and non-smoking subgroups, adjusted for key potential confounders. There was a statistically significantly higher mortality associated with not having bariatric surgery in both smoking (HR, 1.99; 95% CI, 1.54-2.56) and non-smoking (HR, 1.93; 95% CI, 1.12-3.34) subgroups. Although smokers had higher rates of mortality overall (2.6% in smokers compared with 1.7% in non-smokers), the mortality hazard ratio (comparing matched non-surgical patients to surgical patients) did not differ significantly between smokers and non-smokers (p for interaction = .67). Bariatric surgery was associated with significantly lower mortality in both smokers and non-smokers.


Salicylic acid regulates basal resistance to Fusarium head blight in wheat.
Fusarium head blight (FHB) is a destructive disease of cereal crops such as wheat and barley. Previously, expression in wheat of the Arabidopsis NPR1 gene (AtNPR1), which encodes a key regulator of salicylic acid (SA) signaling, was shown to reduce severity of FHB caused by Fusarium graminearum. It was hypothesized that SA signaling contributes to wheat defense against F. graminearum. Here, we show that increased accumulation of SA in fungus-infected spikes correlated with elevated expression of the SA-inducible pathogenesis-related 1 (PR1) gene and FHB resistance. In addition, FHB severity and mycotoxin accumulation were curtailed in wheat plants treated with SA and in AtNPR1 wheat, which is hyper-responsive to SA. In support of a critical role for SA in basal resistance to FHB, disease severity was higher in wheat expressing the NahG-encoded salicylate hydroxylase, which metabolizes SA. The FHB-promoting effect of NahG was overcome by application of benzo (1,2,3), thiadiazole-7 carbothioic acid S-methyl ester, a synthetic functional analog of SA, thus confirming an important role for SA signaling in basal resistance to FHB. We further demonstrate that jasmonate signaling has a dichotomous role in wheat interaction with F. graminearum, constraining activation of SA signaling during early stages of infection and promoting resistance during the later stages of infection.


Colonic bacterial metabolism of corticosteroids.
The aim of this study was to investigate the stability of four corticosteroids in the presence of human colonic bacteria to understand better their luminal behaviour when delivered orally in the treatment of inflammatory bowel disease. The stability of prednisolone, budesonide, beclometasone (17, 21) dipropionate (BDP) and its active metabolite, beclometasone-17-monopropionate (17-BMP), were investigated at three different concentrations following incubation in a mixed faecal inoculum (simulated human colonic fluid) under anaerobic conditions. Prednisolone, at all three concentrations, was completely degraded within 3 h. The degradation of budesonide progressed at a slower rate, with complete degradation occurring within 7h; the degradation of the S epimer of budesonide was faster than the R epimer. BDP degraded completely within 2 h while its active metabolite 17-BMP was comparatively stable. In contrast to the results in the faecal inoculum, all molecules were stable in the simulated colonic fluid in the absence of human faeces (control). This study demonstrates that prednisolone, BDP and budesonide are completely metabolized in simulated human colonic fluid and confirms the role of colonic bacteria in the metabolism of corticosteroids.


Association between the Serum Metabolic Profile and Lung Function in Chronic Obstructive Pulmonary Disease.
Although dietary patterns are known to modulate disease severity in patients with chronic obstructive pulmonary disease (COPD), the relationship between the circulating lipid profile and lung function in COPD has not been studied extensively. There were 43 COPD patients with a history of smoking and 39 patients with a history of biomass fuel exposure recruited in this study, along with 43 age-matched healthy controls. All participants underwent complete lung function profiling, and their glucose and lipid profiles were measured. The association between the metabolic profile and lung function was assessed using the Spearman's rank-order correlation coefficient. 52.4% of the COPD patients were smokers compared to the healthy group (46.5%). We found an inverse correlation between triglyceride and functional residual capacity (p=-0.21, p=0.05) and a positive association between serum cholesterol and overall airway resistance (R5) (p=0.24, p=0.04) and central airway resistance (R20) (p=0.32, p=0.004). Low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) ratio and LDL/HDL ratio were also found to correlate with R5 (p=0.25, 0.23, and 0.22, respectively) and R20 (p=0.31, 0.24, and 0.24, respectively). No significant association was observed between other metabolites and either spirometric or plethysmographic lung function indices. High serum triglyceride and cholesterol may increase the resistance in the airways, which may lead to increased airway obstruction. Therefore, monitoring of lipid profile should be considered in the diagnosis and management of COPD.


Carnitine palmitoyltransferase in cardiac ischemia. A potential site for altered fatty acid metabolism.
The sensitivity of carnitine palmitoyl coenzyme A (CoA) transferase I to inhibition of its activity by malonyl-CoA is progressively reduced in mitochondria isolated from ischemic cardiac cells as blood flow decreases to 30% or less of the preocclusion flow. The activity of carnitine palmitoyl-CoA transferase I in mitochondria isolated from nonischemic cardiac cells demonstrates incomplete inhibition, even at high concentrations of malonyl-CoA. Kinetic analyses of these data gave results most consistent with the expression of two overt enzyme activities: one activity that is sensitive to inhibition by malonyl-CoA and one activity that demonstrates little or no sensitivity to such inhibition. The decrease in malonyl-CoA-sensitive activity associated with ischemia results from a 13% decrease in the activity of the sensitive component and a corresponding 13% increase in the activity of the insensitive component. Decreased sensitivity of ischemic carnitine palmitoyl-CoA transferase I to inhibition by malonyl-CoA, together with potential fluctuations in the content of malonyl-CoA in tissue, would increase the synthesis of palmitoylcarnitine during ischemia and facilitate return to the use of fatty acid as a preferred metabolic fuel on reperfusion. This apparent conversion occurs concomitantly with a decrease in the free protein thiol content of the mitochondrial membranes isolated from ischemic cardiac cells. Treatment of the mitochondria from ischemic cardiac cells with dithiothreitol in vitro partially reverses the loss in sensitivity to malonyl-CoA, suggesting the possible role of thiol oxidation in the altered metabolism of ischemic mitochondria. Western blot analysis of these mitochondria using an antibody against carnitine palmitoyltransferase II purified from beef heart demonstrates a 68-kDa protein, which under ischemic conditions apparently is decreased by 2 kDa. These results are more indicative of a modification in protein folding of carnitine palmitoyltransferase than proteolytic changes during ischemia.


Zinc potentiates neuronal nicotinic receptors by increasing burst duration.
Micromolar zinc potentiates neuronal nicotinic acetylcholine receptors (nAChRs) in a subtype-dependent manner. Zinc potentiates receptor function even at saturating agonist concentrations, without altering the receptor desensitization rate. Potentiation could occur through an increase in the number of available receptors, an increase in single-channel current amplitude, or an increase in single-channel open probability. To distinguish among these possibilities, we examined rat neuronal nAChRs expressed in Xenopus oocytes. Blockade of a large fraction of ACh activated alpha4beta4 or alpha4beta2 receptors by the open channel blocker hexamethonium failed to change the extent of potentiation by zinc, suggesting that zinc does not change the number of available receptors. The single-channel amplitudes of ACh (1 microM) activated alpha4beta4 receptors in outside-out patches were similar in the absence and the presence of 100 microM zinc (3.0 +/- 0.1 and 2.9 +/- 0.1 pA, respectively). To determine the effect of zinc on single-channel open probability, we examined alpha4beta4 receptors in cell-attached patches. The open probability at 100 nM ACh (0.011 +/- 0.002) was increased 4.5-fold by 100 microM zinc (0.050 +/- 0.008), accounting for most of the potentiation observed at the whole cell level. The increase in open probability was due to an increase in burst duration, which increased from 207 +/- 38 ms in the absence of zinc to 830 +/- 189 ms in the presence of zinc. Our results suggest that potentiation of neuronal nAChRs by zinc is due to a stabilization of the bursting states of the receptor.


Frontotemporal dementia-amyotrophic lateral sclerosis syndrome locus on chromosome 16p12.1-q12.2: genetic, clinical and neuropathological analysis.
Numerous families exhibiting both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) have been described, and although many of these have been shown to harbour a repeat expansion in C9ORF72, several C9ORF72-negative FTD-ALS families remain. We performed neuropathological and genetic analysis of a large European Australian kindred (Aus-12) with autosomal dominant inheritance of dementia and/or ALS. Affected Aus-12 members developed either ALS or dementia; some of those with dementia also had ALS and/or extrapyramidal features. Neuropathology was most consistent with frontotemporal lobar degeneration with type B TDP pathology, but with additional phosphorylated tau pathology consistent with corticobasal degeneration. Aus-12 DNA samples were negative for mutations in all known dementia and ALS genes, including C9ORF72 and FUS. Genome-wide linkage analysis provided highly suggestive evidence (maximum multipoint LOD score of 2.9) of a locus on chromosome 16p12.1-16q12.2. Affected individuals shared a chromosome 16 haplotype flanked by D16S3103 and D16S489, spanning 37.9 Mb, with a smaller suggestive disease haplotype spanning 24.4 Mb defined by recombination in an elderly unaffected individual. Importantly, this smaller region does not overlap with FUS. Whole-exome sequencing identified four variants present in the maximal critical region that segregate with disease. Linkage analysis incorporating these variants generated a maximum multipoint LOD score of 3.0. These results support the identification of a locus on chromosome 16p12.1-16q12.2 responsible for an unusual cluster of neurodegenerative phenotypes. This region overlaps with a separate locus on 16q12.1-q12.2 reported in an independent ALS family, indicating that this region may harbour a second major locus for FTD-ALS.


Neuropeptide signaling regulates the susceptibility of developing C. elegans to anoxia.
Inadequate delivery of oxygen to organisms during development can lead to cell dysfunction/death and life-long disabilities. Although the susceptibility of developing cells to low oxygen conditions changes with maturation, the cellular and molecular pathways that govern responses to low oxygen are incompletely understood. Here we show that developing Caenorhabditis elegans are substantially more sensitive to anoxia than adult animals and that this sensitivity is controlled by nervous system generated hormones (e.g., neuropeptides). A screen of neuropeptide genes identified and validated nlp-40 and its receptor aex-2 as a key regulator of anoxic survival in developing worms. The survival-promoting action of impaired neuropeptide signaling does not rely on five known stress resistance pathways and is specific to anoxic insult. Together, these data highlight a novel cell non-autonomous pathway that regulates the susceptibility of developing organisms to anoxia.


Problem effectiveness in a course using problem-based learning.
Problem-based learning (PBL) emphasizes active generation of learning issues by students. Both students and teachers, however, tend to worry that not all important knowledge will be acquired. To explore this question, problem effectiveness (i.e., for each problem, the degree of correspondence between student-generated learning issues and present faculty objectives) was examined in three interdependent studies. The three studies used the same participants: about 120 second-year students and 12 faculty tutors in a six-week course on normal pregnancy, delivery, and child development at the medical school of the University of Limburg in The Netherlands, 1990-91. The participants were randomly assigned to 12 tutorial groups that were each given the same 12 problems; the problems were based on 51 faculty objectives; the tutors were asked to record all learning issues generated by their groups. Study 1 addressed this question: To what degree are faculty objectives reflected by student-generated learning issues? Study 2: To what extent do students miss certain objectives, and are these objectives classifiable? Study 3: Do students generate learning issues not expected by the faculty, and are these issues relevant to course content, and finally, why do students generate these issues? To help answer these questions, the studies employed expert raters and a teacher familiar with the course content. Study 1: For the set of 12 problems, the average overlap between learning issues and faculty objectives was 64.2% with the percentages for individual problems ranging from 27.7% to 100%. Study 2: Of the 51 objectives, 30 were not identified by at least one tutorial group; these objectives were grouped into three categories; on average, each group failed to identify 7.4 objectives (15%). Study 3: Of 520 learning issues, 32 (6%) were unexpected; 15 of these were judged to be at least fairly relevant to course content; they were grouped into four categories. The students' learning activities covered an average of 64% of the intended course content; in addition, the students generated learning issues not expected by the faculty, and half of these issues were judged relevant to the course content. Thus, PBL seems to permit students to adapt learning activities to their own needs and interests.


Complete imperforate tranverse vaginal septum with septate uterus: A rare anomaly.
The isolated, complete, transverse vaginal septum is one of the most infrequent anomalies of the female genital tract, and when it coexists with a septate uterus, it is even rarer. This report describes a case of transverse vaginal septum with septate uterus. A 12-year-old girl sought medical assessment because of severe cyclic lower abdominal cramping and pelvic pain. Local examination revealed a blind vaginal pouch of 2 cm and on rectal examination a tender pelvic mass was noted. Radiological examination showed transverse vaginal septum in the lower vagina with bicornuate uterus. Surgical resection of the vaginal septum was done under laparoscopic guidance. Hysteroscopy revealed presence of uterine septum which was resected by a resectoscope. Post-operative dilatation of vagina was done to prevent restenosis. Laparoscopic guided abdominoperineal approach is better in such a case as multiple mullerian anomalies may coexist with each other.


Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint.
Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint. We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase epsilon, essential for leading-strand DNA replication and for the checkpoint. In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C). Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding, but not Pol2C interaction. Mrc1 is required to stabilize Pol2 at replication forks stalled in HU. The bimodal Mrc1/Pol2 interaction may be an additional step in regulating the S phase checkpoint response to DNA damage on the leading strand. We propose that Mrc1, which also interacts with the MCMs, may modulate coupling of polymerization and unwinding at the replication fork.


A short-range gradient of histone H3 acetylation and Tup1p redistribution at the promoter of the Saccharomyces cerevisiae SUC2 gene.
Chromatin immunoprecipitation assays are used to map H3 and H4 acetylation over the promoter nucleosomes and the coding region of the Saccharomyces cerevisiae SUC2 gene, under repressed and derepressed conditions, using wild type and mutant strains. In wild type cells, a high level of H3 acetylation at the distal end of the promoter drops sharply toward the proximal nucleosome that covers the TATA box, a gradient that become even steeper on derepression. In contrast, substantial H4 acetylation shows no such gradient and extends into the coding region. Overall levels of both H3 and H4 acetylation rise on derepression. Mutation of GCN5 or SNF2 lead to substantially reduced SUC2 expression; in gnc5 there is no reduction in basal H3 acetylation, but large reductions occur on derepression. SNF2 mutation has little effect on H3 acetylation, so SAGA and SWI/SNF recruitment seem to be independent events. H4 acetylation is little affected by either GCN5 or SNF2 mutation. In a double snf2/gcn5 mutant (very low SUC2 expression), H3 acetylation is at the minimal level, but H4 acetylation remains largely unaffected. Transcription is thus linked to H3 but not H4 acetylation. Chromatin immunoprecipitation assays show that Tup1p is evenly distributed over the four promoter nucleosomes in repressed wild type cells but redistributes upstream on derepression, a movement probably linked to its conversion from a repressor to an activator.


Glucagon-like peptide 2 and its beneficial effects on gut function and health in production animals.
Numerous endocrine cell subtypes exist within the intestinal mucosa and produce peptides contributing to the regulation of critical physiological processes including appetite, energy metabolism, gut function, and gut health. The mechanisms of action and the extent of the physiological effects of these enteric peptides are only beginning to be uncovered. One peptide in particular, glucagon-like peptide 2 (GLP-2) produced by enteroendocrine L cells, has been fairly well characterized in rodent and swine models in terms of its ability to improve nutrient absorption and healing of the gut after injury. In fact, a long-acting form of GLP-2 recently has been approved for the management and treatment of human conditions like inflammatory bowel disease and short bowel syndrome. However, novel functions of GLP-2 within the gut continue to be demonstrated, including its beneficial effects on intestinal barrier function and reducing intestinal inflammation. As knowledge continues to grow about GLP-2's effects on the gut and its mechanisms of release, the potential to use GLP-2 to improve gut function and health of food animals becomes increasingly more apparent. Thus, the purpose of this review is to summarize: (1) the current understanding of GLP-2's functions and mechanisms of action within the gut; (2) novel applications of GLP-2 (or stimulators of its release) to improve general health and production performance of food animals; and (3) recent findings, using dairy calves as a model, that suggest the therapeutic potential of GLP-2 to reduce the pathogenesis of intestinal protozoan infections.


Gut disruption impairs rehabilitation in patients curatively operated for pancreaticoduodenal cancer - a qualitative study.
How patients recover and resume everyday life after curative hepato-pancreato-biliary (HPB) surgery with intestinal reconstruction has, to our knowledge, not previously been investigated. We wanted to explore the patient experience in order to develop our capability to support their rehabilitation and identify interventional gaps in the current post-surgical care of these patients. Therefore, the aim of the present study was to explore patients' experiences of their gut, digestion, recovery and uptake of everyday life after HPB surgery with intestinal reconstruction. A qualitative explorative study with semi-structured interviews with 12 patients. We analysed data using qualitative content analysis with an inductive approach. Two main themes with six sub-themes emerged from the analysis: 1. "Disrupted gut" covering the sub-themes: the weakened body; fighting cachexia; re-aligning to the altered body. 2. "Recovery work" with the sub-themes: the value of municipal rehabilitation programmes; reclaiming the sociality of meals; going back to work. The patients described overarching digestive changes, predominantly diarrhea and nausea. Diarrhea and nausea challenged rehabilitation efforts and limited patients' participation in social activities. Patients toiled to regain strength and every-day life as it was before surgery. Current municipal rehabilitation programmes facilitated these efforts. The patients articulated an overarching experience of gut disruption, predominantly presenting as nausea, diarrhea and difficulty eating. This challenged their recovery work and uptake of every-day life. Specialised follow-up at expert centres might mitigate the sequelae of gut disruption after HPB surgery. We suggest that follow-up programmes systematically monitor the experienced symptoms of gut disruption with HPB-specific PROMS. Furthermore, research into the pathophysiology of cachexia and novel interventions for reducing cachexia and weakness after curative HPB surgery is relevant.


Functional recombinant MHC class II molecules and high-throughput peptide-binding assays.
Molecules of the class II major histocompability complex (MHC-II) specifically bind and present exogenously derived peptide epitopes to CD4+ T helper cells. The extreme polymorphism of the MHC-II hampers the complete analysis of peptide binding. It is also a significant hurdle in the generation of MHC-II molecules as reagents to study and manipulate specific T helper cell responses. Methods to generate functional MHC-II molecules recombinantly, and measure their interaction with peptides, would be highly desirable; however, no consensus methodology has yet emerged. We generated alpha and beta MHC-II chain constructs, where the membrane-spanning regions were replaced by dimerization motifs, and the C-terminal of the beta chains was fused to a biotinylation signal peptide (BSP) allowing for in vivo biotinylation. These chains were produced separately as inclusion bodies in E. coli , extracted into urea, and purified under denaturing and non-reducing conditions using conventional column chromatography. Subsequently, diluting the two chains into a folding reaction with appropriate peptide resulted in efficient peptide-MHC-II complex formation. Several different formats of peptide-binding assay were developed including a homogeneous, non-radioactive, high-throughput (HTS) binding assay. Binding isotherms were generated allowing the affinities of interaction to be determined. The affinities of the best binders were found to be in the low nanomolar range. Recombinant MHC-II molecules and accompanying HTS peptide-binding assay were successfully developed for nine different MHC-II molecules including the DPA1*0103/DPB1*0401 (DP401) and DQA1*0501/DQB1*0201, where both alpha and beta chains are polymorphic, illustrating the advantages of producing the two chains separately. We have successfully developed versatile MHC-II resources, which may assist in the generation of MHC class II -wide reagents, data, and tools.


Dietary intake of pterostilbene, a constituent of blueberries, inhibits the beta-catenin/p65 downstream signaling pathway and colon carcinogenesis in rats.
Stilbenes are phytochemicals present in grapes, berries, peanuts and red wine. A widely studied stilbene, resveratrol (trans-3,5,4'-trihydroxystilbene), has been shown to exert antioxidant, anti-inflammatory, chemopreventive and antiaging effects in a number of biological systems. We reported earlier that pterostilbene (trans-3,5-dimethoxy-4'-hydroxystilbene), a structurally related stilbene found in blueberries, was effective in reducing the incidence and multiplicity of aberrant crypt foci formation in the colon of rats injected with azoxymethane (AOM). Our present study was to identify the chemopreventive potential of pterostilbene with colonic tumor formation as an end point and further to evaluate the mechanistic action of pterostilbene during colon carcinogenesis. F344 rats were given two AOM injections subcutaneously when they were 7 and 8 weeks old and continuously fed the control or 40 p.p.m. pterostilbene diet for 45 weeks. Overall analyses indicated that pterostilbene reduced colon tumor multiplicity of non-invasive adenocarcinomas, lowered proliferating cell nuclear antigen and downregulated the expression of beta-catenin and cyclin D1. Pterostilbene decreased mucosal levels of the proinflammatory cytokines, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-4. Colon tumors from pterostilbene-fed animals showed reduced expression of inflammatory markers as well as nuclear staining for phospho-p65, a key molecule in the nuclear factor-kappaB pathway. In HT-29 cells, pterostilbene reduced the protein levels of beta-catenin, cyclin D1 and c-MYC, altered the cellular localization of beta-catenin and inhibited the phosphorylation of p65. Our data with pterostilbene in suppressing colon tumorigenesis, cell proliferation as well as key inflammatory markers in vivo and in vitro suggest the potential use of pterostilbene for colon cancer prevention.


Evidence for the coincident initiation of homolog pairing and synapsis during the telomere-clustering (bouquet) stage of meiotic prophase.
To improve knowledge of the prerequisites for meiotic chromosome segregation in higher eukaryotes, we analyzed the spatial distribution of a pair of homologs before and during early meiotic prophase. Three-dimensional images of fluorescence in situ hybridization (FISH) were used to localize a single pair of homologs in diploid nuclei of a chromosome-addition line of oat, oat-maize9b. The system provided a robust assay for pairing based on cytological colocalization of FISH signals. Using a triple labeling scheme for simultaneous imaging of chromatin, telomeres and the homolog pair, we determined the timing of pairing in relation to the onset of three sequential hallmarks of early meiotic prophase: chromatin condensation (the leptotene stage), meiotic telomere clustering (the bouquet stage) and the initiation of synapsis (the zygotene stage). We found that the two homologs were mostly unpaired up through middle leptotene, at which point their spherical cloud-like domains began to transform into elongated and stretched-out domains. At late leptotene, the homologs had completely reorganized into long extended fibers, and the beginning of the bouquet stage was conspicuously marked by the de novo clustering of telomeres at the nuclear periphery. The homologs paired and synapsed during the bouquet stage, consistent with the timing of pairing observed for several oat 5S rDNA loci. In summary, results from analysis of more than 100 intact nuclei lead us to conclude that pairing and synapsis of homologous chromosomes are largely coincident processes, ruling out a role for premeiotic pairing in this system. These findings suggest that the genome-wide remodeling of chromatin and telomere-mediated nuclear reorganization are prerequisite steps to the DNA sequence-based homology-search process in higher eukaryotes.


Overexpression of microRNAs from the miR-17-92 paralog clusters in AIDS-related non-Hodgkin's lymphomas.
Individuals infected by HIV are at an increased risk for developing non-Hodgkin's lymphomas (AIDS-NHL). In the highly active antiretroviral therapy (HAART) era, there has been a significant decline in the incidence of AIDS-associated primary central nervous system lymphoma (PCNSL). However, only a modest decrease in incidence has been reported for other AIDS-NHL subtypes. Thus, AIDS-NHLs remain a significant cause of morbidity and mortality in HIV infected individuals. Recently, much attention has been directed toward the role of miRNAs in cancer, including NHL. Several miRNAs, including those encoded by the miR-17-92 polycistron, have been shown to play significant roles in B cell tumorigenesis. However, the role of miRNAs in NHL in the setting of HIV infection has not been defined. We used quantitative realtime PCR to assess the expression of miRNAs from three different paralog clusters, miR-17-92, miR-106a-363, and miR-106b-25 in 24 cases of AIDS-NHLs representing four tumor types, Burkitt's lymphoma (BL, n = 6), diffuse large B-cell lymphoma (DLBCL, n = 8), primary central nervous system lymphoma (PCNSL, n = 5), and primary effusion lymphoma (PEL, n = 5). We also used microarray analysis to identify a differentiation specific miRNA signature of naïve, germinal center, and memory B cell subsets from tonsils (n = 4). miRNAs from the miR-17-92 paralog clusters were upregulated by B cells, specifically during the GC differentiation stage. We also found overexpression of these miRNA clusters in all four AIDS-NHL subtypes. Finally, we also show that select miRNAs from these clusters (miR-17, miR-106a, and miR-106b) inhibited p21 in AIDS-BL and DLBCL cases, thus providing a mechanistic role for these miRNAs in AIDS-NHL pathogenesis. Dysregulation of miR-17-92 paralog clusters is a common feature of AIDS-associated NHLs.


Influence of operative technique on anterior cruciate ligament reconstruction in patients older than 50 years.
A consequence of the steady growth in the worldwide population of elderly individuals who remain in good health and continue to engage in sports is an increase in the incidence of anterior cruciate ligament (ACL) rupture occurring after 50 years of age. ACL reconstruction was formerly reserved for young athletes but now seems to produce good outcomes in over 50s. The type of graft and graft fixation method were selected empirically until now, given the absence of investigations into potential relationships of these two parameters with the outcomes. The objective of this study was to assess associations linking the type of graft and the method of femoral graft fixation to outcomes in patients older than 50 years at ACL reconstruction. The operative technique is not associated with the clinical outcomes or differential laxity. A multicentre retrospective cohort of 398 patients operated between 1 January 2011 and 31 December 2015 and a multicentre prospective cohort of 228 patients operated between 1 January 2016 and 30 June 2017 were conducted. Mean follow-up was 42.7 months in the retrospective cohort and 14.2 months in the prospective cohort. The primary evaluation criterion was the clinical outcome as assessed using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and the Tegner Activity Score (TAS). Differential laxity was the secondary evaluation criterion. The Wilcoxon rank sum test and Kruskal-Wallis test were used to compare groups, and p-values<0.05 were considered significant. In the retrospective and prospective cohorts, hamstring tendons were used in 269 (67.6%) and 197 (86.4%) patients and extensor apparatus tendons in 124 (31.2%) and 31 (13.6%) patients. Femoral fixation in the retrospective cohort was cortical in 81 (20.4%) cases, by press-fit in 112 (28.1%) cases, and by interference screw in 205 (51.5%) cases; corresponding figures in the prospective cohort were 135 (59.2%), 17 (7.5%), and 76 (33.3%). The multivariate analysis of the retrospective data identified no significant associations of graft type or femoral fixation type with the KOOS, TAS, or differential laxity values. In the prospective cohort, hamstring grafts were associated with 0.6mm of additional laxity (p=0.007); compared to cortical fixation, press-fit fixation of patellar tendon grafts was associated with 0.3mm of additional laxity (p=0.029) and a 0.5-point lower TAS value (p=0.033), with no difference in KOOS values. None of these differences were clinically significant. The various ACL reconstruction techniques used in patients older than 50 years produce similar outcomes. The technique can be chosen based on surgeon preference without regard for patient age. IV.


Allosuppressor and allohelper T cells in acute and chronic graft-vs.-host disease. II. F1 recipients carrying mutations at H-2K and/or I-A.
By induction of a graft-vs.-host reaction (GVHR) in nonirradiated H-2-different F1 mice, one can induce stimulatory pathological symptoms, such as lymphadenopathy and hypergammaglobulinemia, combined with the production of autoantibodies characteristic of systemic lupus erythematosus (SLE). Alternatively, the GVHR can lead to the suppressive pathological symptoms, such as pancytopenia and hypogammaglobulinemia, characteristic of acute GVH disease (GVHD). Whether stimulatory or suppressive symptoms are induced by a GVHR depends, in our view (2-4), on the functional subset of donor T cells activated in the F1 host. The purpose of the present study was to investigate whether class I and/or class II H-2 alloantigens can selectively trigger, out of a pool of unselected donor T cells, those subpopulations of T cells responsible for the stimulatory and suppressive GVH symptoms, respectively. For the induction of the GVHR, 10(8) lymphoid cells from C57BL/6 (B6) donors were injected into three kinds of F1 hybrid mice, which had been bred from H-2 mutant strains on a B6 background. Whereas the I-A-disparate (B6 X bm12)F1 recipients exclusively developed stimulatory GVH symptoms, including SLE-like autoantibodies and immune complex glomerulonephritis, the K locus-disparate (B6 X bm1)F1 recipients showed neither clearly stimulatory nor clearly suppressive GVH symptoms. In marked contrast, the (bm1 X bm12)F1 recipients, which differ from the B6 donor strain by mutations at both K and I-A locus, initially developed stimulatory GVH symptoms, but rapidly thereafter showed the suppressive pathological symptoms of acute GVHD and died. Moreover, spleen cells obtained from (B6 X bm12)F1 mice injected with B6 donor cells helped the primary anti-sheep erythrocyte (SRBC) response of normal (B6 X bm12)F1 spleen cells in vitro, whereas spleen cells (bm1 X bm12)F1 mice injected with B6 donor cells strongly suppressed the primary anti-SRBC response of normal (bm1 X bm12)F1 spleen cells. Spleen cells from the K locus-disparate (B6 X bm1)F1 recipients also suppressed the primary anti-SRBC of normal (B6 X bm1)F1 spleen cells; this suppression, however, was weak when compared with the suppression induced by spleen cells from GVH (bm1 X bm12)F1 mice. Taken together, these findings indicate that a small class II (I-A) antigenic difference suffices to trigger the alloreactive donor T helper cells causing SLE-like GVHD. In contrast, both class I (H-2K) and class II (I-A) differences are required to trigger the subsets of donor T cells responsible for acute GVHD. It appears that alloreactive donor T helper cells induce the alloreactive T suppressor cells, which then act as the suppressor effector cells causing the pancytopenia of acute GVHD. These findings may help to understand the variability of GVH-like diseases caused by a given etiologic agent, their cellular pathogenesis, and association with certain HLA loci.


Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus.
Foot-and-mouth disease viruses from serotypes O, A and C have been crystallized. The particular strains studied include O1K, A10(61), A22 Iraq 24/64, A24 Cruzeiro and C-S8c1. In addition, crystals have been grown of G67, a monoclonal antibody neutralization escape mutant derived from O1K, and of virus R100, recovered after the establishment of a persistent infection in baby hamster kidney cells with C-S8c1. Empty particles, capsids which lack the RNA genome, have also been crystallized for subtypes A22 Iraq 24/64 and A10(61). In almost all cases, crystals suitable for high resolution structure determination were obtained from (NH4)2SO4 or mixtures of polyethylene glycol and NH4Cl.


Off-Label Use of Second-generation Antipsychotics in Bipolar Disorder: A Survey of Italian Psychiatrists.
Bipolar disorder (BD) is characterized by recurrent depressive and manic episodes. Lithium, valproate, lamotrigine, and some second-generation antipsychotics (SGAs) are the most typical pharmacological treatments for BD, the main goal being mood stabilization. However, despite these treatments, most patients continue to experience recurrent mood episodes and residual symptoms. Findings from several studies suggest that some SGAs may be beneficial beyond approved indications. The goal of the survey presented in this article was to examine Italian psychiatrists' attitudes concerning the off-label use of SGAs in depressive and maintenance phases of BD. A questionnaire about the off-label prescription of SGAs was e-mailed to 300 psychiatrists from Northern, Central, and Southern Italy affiliated with the Italian Society of Psychopharmacology (SINPF) to investigate the frequency of and motivation for off-label use of SGAs and evaluate the psychiatrists' attitude toward use of specific SGAs in BD; 202 questionnaires were completed. The respondents were equally distributed in terms of sex, and the mean age of respondents was 44.1 years. The majority of the sample reported use of SGAs for off-label indications either very often (16.7%), often (33.7%), or occasionally (34.7%). The main motivation for off-label use of the SGAs was the presence of published evidence (51.5%), followed by patients' nonresponse to previous treatment (37.1%). With regard to the use of specific SGAs in BD, off-label aripiprazole was considered appropriate for depressive episodes by 46% of the psychiatrists, followed by olanzapine which was considered appropriate by 33.7%. For maintenance treatment of BD, off-label asenapine was considered appropriate by 45% of the psychiatrists, followed by long-acting aripiprazole and olanzapine pamoate, which were considered appropriate by 37.1% and 23.8%, respectively. In summary, ~50% of Italian psychiatrists frequently (very often or often) prescribe SGAs for off-label indications. Given the relatively limited number of indicated effective treatments for BD, the use of some SGAs off-label may be considered appropriate when dealing with patients whose BD is resistant to medications with labeled indications for BD.


Comparative assessment of substrates and activity based probes as tools for non-invasive optical imaging of cysteine protease activity.
Recent advances in the field of non-invasive optical imaging have included the development of contrast agents that report on the activity of enzymatic targets associated with disease pathology. In particular, proteases have proven to be ideal targets for development of optical sensors for cancer. Recently developed contrast agents for protease activity include both small peptides and large polymer-based quenched fluorescent substrates as well as fluorescently labeled activity based probes (ABPs). While substrates produce a fluorescent signal as a result of processing by a protease, ABPs are retained at the site of proteolysis due to formation of a permanent covalent bond with the active site catalytic residue. Both methods have potential advantages and disadvantages yet a careful comparison of substrates and ABPs has not been performed. Here we present the results of a direct comparison of commercially available protease substrates with several recently described fluorescent ABPs in a mouse model of cancer. The results demonstrate that fluorescent ABPs show more rapid and selective uptake into tumors as well as overall brighter signals compared to substrate probes. These data suggest that the lack of signal amplification for an ABP is offset by the increased kinetics of tissue uptake and prolonged retention of the probes once bound to a protease target. Furthermore, fluorescent ABPs can be used as imaging reagents with similar or better results as the commercially available protease substrates.


Major Histocompatibility Complex (MHC) markers in conservation biology.
Human impacts through habitat destruction, introduction of invasive species and climate change are increasing the number of species threatened with extinction. Decreases in population size simultaneously lead to reductions in genetic diversity, ultimately reducing the ability of populations to adapt to a changing environment. In this way, loss of genetic polymorphism is linked with extinction risk. Recent advances in sequencing technologies mean that obtaining measures of genetic diversity at functionally important genes is within reach for conservation programs. A key region of the genome that should be targeted for population genetic studies is the Major Histocompatibility Complex (MHC). MHC genes, found in all jawed vertebrates, are the most polymorphic genes in vertebrate genomes. They play key roles in immune function via immune-recognition and -surveillance and host-parasite interaction. Therefore, measuring levels of polymorphism at these genes can provide indirect measures of the immunological fitness of populations. The MHC has also been linked with mate-choice and pregnancy outcomes and has application for improving mating success in captive breeding programs. The recent discovery that genetic diversity at MHC genes may protect against the spread of contagious cancers provides an added impetus for managing and protecting MHC diversity in wild populations. Here we review the field and focus on the successful applications of MHC-typing for conservation management. We emphasize the importance of using MHC markers when planning and executing wildlife rescue and conservation programs but stress that this should not be done to the detriment of genome-wide diversity.


Holoprosencephaly: new models, new insights.
Holoprosencephaly (HPE) is a common congenital malformation that is characterised by a failure to divide the forebrain into left and right hemispheres and is usually accompanied by defects in patterning of the midline of the face. HPE exists in inherited, autosomal dominant (familial) forms and mutation-associated sporadic forms, but environmental factors are also implicated. There are several features of HPE that are not well understood, including the extremely variable clinical presentation, even among obligate carriers of familial mutations, and the restriction of structural anomalies to the ventral anterior midline, despite association with defects in signal transduction pathways that regulate development of many additional body structures. The new animal models described in this review may help unravel these puzzles. Furthermore, these model systems suggest that human HPE arises from a complex interaction between the timing and strength of developmental signalling pathways, genetic variation and exposure to environmental agents.


Proton pump inhibitor alone vs proton pump inhibitor plus mucosal protective agents for endoscopic submucosal dissection-induced ulcer: a systematic review and meta-analysis.
Mucosal protective agents may improve healing of patients with endoscopic submucosal dissection-induced ulcers. The present study systematically evaluated published clinical trials to determine whether combined therapeutic use of mucosal protective agents and proton pump inhibitors can improve the outcome of patients with endoscopic submucosal dissection-induced ulcers compared to treatment with proton pump inhibitors alone. PubMed, the Cochrane Library, and the Igaku-Chuo-Zasshi database were searched to identify eligible randomized trials for systematic review. We identified 11 randomized trials for inclusion in our study (1,160 patients). Pooled endoscopic submucosal dissection-induced ulcer healing rates were 45.8% and 34.4% for patients with or without mucosal protective agents, respectively. The odds ratio was 2.28 (95% confidence interval, 1.57-3.31) with no significant study heterogeneity. In conclusion, the systematic review and meta-analysis showed that the combined therapeutic use of proton pump inhibitors and mucosal protective agents improved healing rates of endoscopic submucosal dissection-induced ulcers compared to treatment with proton pump inhibitor monotherapy.


Liver transplantation in patients with alcoholic liver disease.
Although alcoholic liver disease (ALD) is one of the most common indications for liver transplantation (LT), there are still unresolved controversies about the goals of treatment, the referral, evaluation, and selection of patients with ALD for LT, and their care after LT. It is uncertain whether there is a large unmet need for LT among patients with ALD because of the unmeasured effects of recent drinking, relapse, and recovery with abstinence in this population. A careful assessment of the extrahepatic effects of alcohol-related end-organ damage is needed for ALD patients who are referred for an LT evaluation. Although there clearly is a relationship between the length of sobriety and future abstinence, the present methods for predicting future drinking are inexact. The survival of ALD patients after LT is as good as the survival of non-ALD patients, although patients with coincident ALD and hepatitis C virus have higher mortality and morbidity rates. After LT, ALD patients have an increased risk of developing malignancies and cardiovascular disease. These risks appear to be linked to cigarette smoking. Covert drinking occurs both before and after transplantation, and approximately 20% of patients return to harmful drinking after LT. Harmful drinking after LT (instead of slips) causes liver damage and reduces survival. Better therapies for controlling addictions to alcohol and nicotine are needed for ALD patients both before and after LT.


Social determinants of nutrient intake in smokers and non-smokers during pregnancy.
The aim was to investigate the effects of social factors (education, income, marital status, partners' employment status, housing tenure, social class), smoking, and maternal height on the dietary intake of pregnant women. The study was a prospective investigation on a two phase sample. The study involved women attending the antenatal clinic at a district general hospital. A group of pregnant Caucasian women, selected because they were heavy smokers (15+ cigarettes/day) (n = 94) and a randomly selected sample of never smokers (n = 112) were studied. Data on social factors were collected by interviewer administered questionnaire. A 7 day weighed intake method was used to determine dietary intake at 28 weeks gestation. In univariate analyses, income, housing tenure and social class had significant effects on intakes of both macro- and micronutrients, and maternal education and smoking had significant effects on intakes of micronutrients. Using a stepwise multivariate analysis with income, smoking and maternal education, income was a significant factor in the intake of most nutrients but this effect disappeared when social class and housing tenure factors were entered into the model. Only social class and housing tenure had any significant effect on intakes of macronutrients--energy, protein and fat. Smoking and maternal education were the most important determinants of quality of diet (nutrient density); other factors had only negligible effects. Income was the only significant factor in alcohol intake. It is suggested that the effects of social class and income are overlapping. Smoking, being renters of accommodation, and being of minimum education and low social class are risk factors for poor dietary intake. It is recommended that such higher risk groups be specifically targeted for nutritional advice in pregnancy.


Revisiting the relationship between adaptive smoothing and anisotropic diffusion with modified filters.
Anisotropic diffusion has been known to be closely related to adaptive smoothing and discretized in a similar manner. This paper revisits a fundamental relationship between two approaches. It is shown that adaptive smoothing and anisotropic diffusion have different theoretical backgrounds by exploring their characteristics with the perspective of normalization, evolution step size, and energy flow. Based on this principle, adaptive smoothing is derived from a second order partial differential equation (PDE), not a conventional anisotropic diffusion, via the coupling of Fick's law with a generalized continuity equation where a "source" or "sink" exists, which has not been extensively exploited. We show that the source or sink is closely related to the asymmetry of energy flow as well as the normalization term of adaptive smoothing. It enables us to analyze behaviors of adaptive smoothing, such as the maximum principle and stability with a perspective of a PDE. Ultimately, this relationship provides new insights into application-specific filtering algorithm design. By modeling the source or sink in the PDE, we introduce two specific diffusion filters, the robust anisotropic diffusion and the robust coherence enhancing diffusion, as novel instantiations which are more robust against the outliers than the conventional filters.


Contrasting roles of H3K4me3 and H3K9me3 in regulation of apoptosis and gemcitabine resistance in human pancreatic cancer cells.
Pancreas ductal adenocarcinoma (PDAC) has the most dismal prognosis among all human cancers since it is highly resistant to chemotherapy, radiotherapy and immunotherapy. The anticipated consequence of all therapies is induction of tumor apoptosis. The highly resistance nature of PDACs to all therapies suggests that the intrinsic tumor cell factors, likely the deregulated apoptosis pathway, are key mechanisms underlying PDAC non-response to these therapies, rather than the therapeutic agents themselves. The aim of this study is to test the hypothesis that epigenetic dysregulation of apoptosis mediators underlies PDAC resistance to gemcitabine, the standard chemotherapy for human PDAC. PDAC cells were analyzed for apoptosis sensitivity in the presence of a selective epigenetic inhibitor. The epigenetic regulation of apoptosis regulators was determined by Western Blotting and quantitative PCR. The specific epigenetic modification of apoptosis regulator promoter chromatin was determined by chromatin immunoprecipitation in PDAC cells. Inhibition of histone methyltransferase (HMTase) by a selective HMTase inhibitor, verticillin A, significantly increased human PDAC cell sensitivity to gemcitabine-induced growth suppression. Verticillin A treatment decreased FLIP, Mcl-1, Bcl-x and increased Bak, Bax and Bim protein level in the tumor cells, resulting in activation of caspases, elevated cytochrome C release and increased apoptosis as determined by upregulated PARP cleavage in tumor cells. Analysis of human PDAC specimens indicated that the expression levels of anti-apoptotic mediators Bcl-x, Mcl-1, and FLIP were significantly higher, whereas the expression levels of pro-apoptotic mediators Bim, Bak and Bax were dramatically lower in human PDAC tissues as compared to normal pancreas. Verticillin A downregulated H3K4me3 levels at the BCL2L1, CFLAR and MCL-1 promoter to decrease Bcl-x, FLIP and Mcl-1 expression level, and inhibited H3K9me3 levels at the BAK1, BAX and BCL2L11 promoter to upregulate Bak, Bax and Bim expression level. We determined that PDAC cells use H3K4me3 to activate Bcl-x, FLIP and Mcl-1, and H3K9me3 to silence Bak, Bax and Bim to acquire an apoptosis-resistant phenotype. Therefore, selective inhibition of H3K4me3 and H3K9me3 is potentially an effective approach to overcome PDAC cells resistance to gemcitabine.


Patient information sources when facing repair of abdominal aortic aneurysm.
Shared medical decision making is most important when there are competing options for repair such as in treatment of abdominal aortic aneurysm (AAA). We sought to understand the sources of patients' pre-existing knowledge about AAA to better inform treating physicians about patients' needs for preoperative counseling. We performed a multicenter survey of patients facing AAA repair at 20 Veterans Affairs hospitals across the United States as part of the Preferences for Open Versus Endovascular Repair of AAA study. A validated survey instrument was administered to examine the sources of information available and commonly used by patients to learn about their repair options. The survey was administered by study personnel before the patient had any interaction with the vascular surgeon because survey data were collected before the vascular clinic visit. Preliminary analysis of data from 99 patients showed that our cohort was primarily male (99%) and elderly (mean age 73 years). Patients commonly had a history of hypertension (86%), prior myocardial infarction (32%), diabetes (32%), and were overweight (58%). Patients arrived at their surgeon's office appointment with limited information. A majority of patients (52%) reported that they had not talked to their primary care physician at all about their options for AAA repair, and one-half (50%) reported that their view of the different surgical options had not been influenced by anyone. Slightly less than one-half of patients reported that they did not receive any information about open surgical aneurysm repair and endovascular aortic aneurysm repair (41% and 37%, respectively). Few patients indicated using the internet as their main source of information about open surgical aneurysm repair and endovascular aortic aneurysm repair (10% and 11%, respectively). Patients are commonly referred for AAA repair having little to no information regarding AAA pathology or repair options. Fewer than one in five patients searched the internet or had accessed other sources of information on their own. Most vascular surgeons should assume that patients will present to their first vascular surgery appointment with minimal understanding of the treatment options available to them.


Is it time for a new practice environment? An operational look at your practice.
In this era of reimbursement not keeping pace with overhead and healthcare reform an unknown, it is more important than ever to assess the use of the doctors' and mid-level providers' time in your practice. There could be a very large untapped revenue source in the misallocated time of these providers that could help your practice increase patient volume and therefore practice revenue. Accessing this untapped revenue source may require additional resources in space and staff. To determine the level of untapped revenue and the investment that will be required to access that revenue, an objective assessment of your current practice flow is required.


Gene Duplication and Gain in the Trematode Atriophallophorus winterbourni Contributes to Adaptation to Parasitism.
Gene duplications and novel genes have been shown to play a major role in helminth adaptation to a parasitic lifestyle because they provide the novelty necessary for adaptation to a changing environment, such as living in multiple hosts. Here we present the de novo sequenced and annotated genome of the parasitic trematode Atriophallophorus winterbourni and its comparative genomic analysis to other major parasitic trematodes. First, we reconstructed the species phylogeny, and dated the split of A. winterbourni from the Opisthorchiata suborder to approximately 237.4 Ma (±120.4 Myr). We then addressed the question of which expanded gene families and gained genes are potentially involved in adaptation to parasitism. To do this, we used hierarchical orthologous groups to reconstruct three ancestral genomes on the phylogeny leading to A. winterbourni and performed a GO (Gene Ontology) enrichment analysis of the gene composition of each ancestral genome, allowing us to characterize the subsequent genomic changes. Out of the 11,499 genes in the A. winterbourni genome, as much as 24% have arisen through duplication events since the speciation of A. winterbourni from the Opisthorchiata, and as much as 31.9% appear to be novel, that is, newly acquired. We found 13 gene families in A. winterbourni to have had more than ten genes arising through these recent duplications; all of which have functions potentially relating to host behavioral manipulation, host tissue penetration, and hiding from host immunity through antigen presentation. We identified several families with genes evolving under positive selection. Our results provide a valuable resource for future studies on the genomic basis of adaptation to parasitism and point to specific candidate genes putatively involved in antagonistic host-parasite adaptation.


Supravital and vital staining of diseased corneal endothelium in whole-mount preparations.
Whereas specular microscopy may provide valuable preoperative information about the status of the endothelial cells in clear corneas, in many diseased corneas that come to penetrating keratoplasty, stromal opacities or epithelial edema prevents determinations about the condition of the endothelium. A method has been developed wherein diseased-host penetrating keratoplasty specimens are maintained postoperatively in McCarey-Kaufman medium and are thereafter stained as whole mounts with alizarin red S and trypan blue to evaluate the status of the endothelial monolayer. Seventy diseased corneas were studied with this method. The alizarin red S stained the intercellular junctions, while the trypan blue stained nuclei and provided information about the viability of the endothelial cells that were studied. Unusual findings were the presence of binucleated endothelial cells in decompensated cases of iris clip pseudophakos and scattered iris stromal melanocytes on barred Descemet's membrane in cases of chemical injury, trauma, and congenital glaucoma. The method was found to be rapid, reliable, and inexpensive, particularly when compared with alternative methods of evaluation, such as scanning electron microscopy.


Prediction of the total and standardized ileal digestible amino acid contents from the chemical composition of soybean meals of different origin in broilers.
The objective of this experiment was to determine total amino acid (TAA) content, standardized ileal digestibility (SID) of crude protein, and standardized ileal amino acid digestibility in 9 sources of soybean meal (SBM) of different origin and to subsequently establish equations for predicting the TAA content and concentration of standardized ileal digestible amino acids (SIDAA) based on their protein content and other proximate components. Concentration of SIDAA of the samples was also predicted using TAA values. A total of 160 1-day-old male broiler chicks were randomly assigned to 10 dietary treatments consisted of 9 semipurified diets containing one SBM (200 g of crude protein/kg) as the only source of dietary amino acid (AA) and one N-free diet to determine endogenous ileal AA flow. The birds were fed with a standard diet from 0 to 18 D of age, and experimental diets were fed from 19 to 24 D of age. The fitness of the models of the study was tested using the adjusted coefficient of determination (R2) value, P-value regression and coefficients, and standard error of prediction (SEP). The coefficient of SID for Lys and Cys among SBM varied from 86.7 to 96.3 and 74.1 to 89.3, respectively, with significant difference (P < 0.05). In equations based on protein content, the adjusted R2 value ranged from 40.7 (Ile) to 99.6 (Met) and 37.2 (Met + Cys) to 99.6 (Met) for TAA content and concentration of SIDAA, respectively. Inclusion of other proximate components of test samples (e.g., crude fiber, neutral detergent fiber, acid detergent fiber, ash, gross energy, and so on) into the regression equation increased the adjusted R2 value and decreased the SEP. The results of linear regression revealed that it is possible to satisfactorily estimate the TAA content and concentration of SIDAA of SBM through its protein content and other proximate components, but the prediction equations based on other proximate components were more accurate in terms of reflecting the measured results; however, additional time and costs were associated with this approach. It is also possible to estimate the concentration of SIDAA through TAA values with reasonable accuracy and lower SEP.


Atovaquone versus trimethoprim-sulfamethoxazole as Pneumocystis jirovecii pneumonia prophylaxis following renal transplantation.
Pneumocystis pneumonia (PCP) is associated with significant morbidity and mortality in renal transplant recipients (RTR). Trimethoprim-sulfamethoxazole (TMP-SMZ) is considered the prophylactic agent-of-choice. Some patients require an alternative owing to TMP-SMZ intolerance. This is the first evaluation of full-dose atovaquone vs. TMP-SMZ for PCP prevention in RTR. One hundred and eighty-five RTR were evaluated in this single-center, retrospective analysis. Patients received either single-strength TMP-SMZ daily (group I; n = 160) or 1500 mg/d of atovaquone and of a fluoroquinolone for one month (group II; n = 25). The primary endpoint was the incidence of PCP at 12 months post-transplant. There were no cases of PCP in either group. There were comparable rates of infections from bacterial pathogens and cytomegalovirus, but rates of BK viremia were significantly higher in group I (22.5%) vs. group II (4%; p = 0.03). The incidence of leukopenia was similar in both groups. Higher mean potassium levels were seen in group I at three months post-transplant but were comparable at all other time points. The need for dose reduction and/or premature discontinuation of therapy secondary to adverse events was more prevalent in TMP-SMZ-treated patients. In our experience, atovaquone appears to be effective in preventing PCP post-renal transplant and also demonstrates good tolerability.


What to give the patient who has everything? A qualitative study of prescribing for multimorbidity in primary care.
Using clinical guidelines in the management of patients with multimorbidity can lead to the prescription of multiple and sometimes conflicting medications. To explore how GPs make decisions when prescribing for multimorbid patients, with a view to informing intervention design. In-depth qualitative interviews incorporating chart-stimulated recall with purposively sampled GPs in the Republic of Ireland. Grounded theory analysis with iterative theory development. Twenty GPs were interviewed about 51 multimorbid cases. In these cases, GPs integrated information from multiple sources including the patient, specialists, and evidence-based medicine. Difficulties arose when recommendations or preferences conflicted, to which GPs responded by 'satisficing': accepting care that they deemed satisfactory and sufficient for a particular patient. Satisficing was manifest as relaxing targets for disease control, negotiating compromise with the patient, or making 'best guesses' about the most appropriate course of action to take. In multimorbid patients perceived as stable, GPs preferred to 'maintain the status quo' rather than rationalise medications, even in cases with significant polypharmacy. Proactive changes in medications were facilitated by continuity of care, sufficient consultation time, and open lines of communication with the patient, other healthcare professionals, and other GPs. GPs respond to conflicts in the management of multimorbid patients by making compromises between patient-centred and evidence-based care. These findings will be used to inform interventions that aim to care in multimorbidity.


Risk of suicide during treatment with venlafaxine, citalopram, fluoxetine, and dothiepin: retrospective cohort study.
To compare the risk of suicide in adults using the antidepressant venlafaxine compared with citalopram, fluoxetine, and dothiepin. Retrospective cohort study. UK General Practice Research Database. 219,088 patients, aged 18-89 years, who were prescribed venlafaxine, citalopram, fluoxetine, or dothiepin from 1995 to 2005. Completed suicide and attempted suicide. Venlafaxine users had a higher burden of risk factors for suicide, including previous suicide attempts and proxies for severe depression or depression that was difficult to treat. In the analysis for completed suicides, unadjusted and adjusted hazard ratios for venlafaxine compared with citalopram were 2.44 (95% confidence interval 1.12 to 5.31) and 1.70 (0.76 to 3.80), for venlafaxine compared with fluoxetine were 2.85 (1.37 to 5.94) and 1.63 (0.74 to 3.59), and for venlafaxine compared with dothiepin were 2.54 (1.07 to 6.02) and 1.31 (0.53 to 3.25). Compared with other study drugs, venlafaxine was also associated with an increased risk of attempted suicide, but adjustment for measured confounders substantially reduced the hazard ratios. Venlafaxine use was consistently associated with higher risk of suicide compared with citalopram, fluoxetine, and dothiepin. Venlafaxine users had a higher burden of suicide risk factors, however, and adjustment for measured confounders substantially reduced the excess risks. Since the secondary data used in this analysis allowed only indirect and partial measurements of potential confounders, it is possible that residual confounding explains much, if not all, of the observed excess risk.


Insights into RNA binding by the anticancer drug cisplatin from the crystal structure of cisplatin-modified ribosome.
Cisplatin is a widely prescribed anticancer drug, which triggers cell death by covalent binding to a broad range of biological molecules. Among cisplatin targets, cellular RNAs remain the most poorly characterized molecules. Although cisplatin was shown to inactivate essential RNAs, including ribosomal, spliceosomal and telomeric RNAs, cisplatin binding sites in most RNA molecules are unknown, and therefore it remains challenging to study how modifications of RNA by cisplatin contributes to its toxicity. Here we report a 2.6Å-resolution X-ray structure of cisplatin-modified 70S ribosome, which describes cisplatin binding to the ribosome and provides the first nearly atomic model of cisplatin-RNA complex. We observe nine cisplatin molecules bound to the ribosome and reveal consensus structural features of the cisplatin-binding sites. Two of the cisplatin molecules modify conserved functional centers of the ribosome-the mRNA-channel and the GTPase center. In the mRNA-channel, cisplatin intercalates between the ribosome and the messenger RNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA-translocation. Our structure provides an insight into RNA targeting and inhibition by cisplatin, which can help predict cisplatin-binding sites in other cellular RNAs and design studies to elucidate a link between RNA modifications by cisplatin and cisplatin toxicity.