Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents.
We investigated the relationship between DNA cytosine methylation and the expression of two genes associated with resistance to DNA methylation damage. Variants of RajiMex- cells acquired resistance to N-methyl-N-nitrosourea by either reactivating a previously silent O6-methylguanine-DNA methyltransferase (MGMT) gene or by repressing the hMSH6 mismatch repair gene. DNA sequencing and measurements of mRNA and enzyme levels revealed that MGMT activity was not correlated with methylation of the core MGMT promoter. Treatment with the demethylating agent 5-azadeoxycytidine reduced MGMT mRNA and enzyme levels, indicating that methylation of some nonpromoter sequences may be required for MGMT gene expression. In contrast, both hMSH6 mRNA and protein levels were increased by 5-azadeoxycytidine treatment of an N-methyl-N-nitrosourea-resistant variant that did not express detectable hMSH6, which implies that this gene was transcriptionally silenced by cytosine methylation. This could be substantiated by in vitro modification of the CpG sites in the hMSH6 promoter with restriction methylase M.SssI, which abolished the transcription of a reporter gene under its control in a transient transfection assay. Taken together, our data show that treatment with chemical methylating agents alters gene expression patterns through increased CpG methylation of genomic DNA, and thereby permits the emergence and selection of clones that are resistant to these agents due to increased repair or tolerance of O6-methylguanine.


Effort versus Reward: Preparing Samples for Fungal Community Characterization in High-Throughput Sequencing Surveys of Soils.
Next generation fungal amplicon sequencing is being used with increasing frequency to study fungal diversity in various ecosystems; however, the influence of sample preparation on the characterization of fungal community is poorly understood. We investigated the effects of four procedural modifications to library preparation for high-throughput sequencing (HTS). The following treatments were considered: 1) the amount of soil used in DNA extraction, 2) the inclusion of additional steps (freeze/thaw cycles, sonication, or hot water bath incubation) in the extraction procedure, 3) the amount of DNA template used in PCR, and 4) the effect of sample pooling, either physically or computationally. Soils from two different ecosystems in Minnesota, USA, one prairie and one forest site, were used to assess the generality of our results. The first three treatments did not significantly influence observed fungal OTU richness or community structure at either site. Physical pooling captured more OTU richness compared to individual samples, but total OTU richness at each site was highest when individual samples were computationally combined. We conclude that standard extraction kit protocols are well optimized for fungal HTS surveys, but because sample pooling can significantly influence OTU richness estimates, it is important to carefully consider the study aims when planning sampling procedures.


Predictors of response to intramyocardial bone marrow cell treatment in patients with refractory angina and chronic myocardial ischemia.
We previously showed that intramyocardial bone marrow cell (BMC) injection in patients with refractory angina and chronic myocardial ischemia improves myocardial perfusion, cardiac function and disease-related complaints. Treatment effect varied between patients, but the predictors of response remain to be identified. Therefore, the aim of the present study was to assess whether patient characteristics, procedural data and baseline measurements influence the response to intramyocardial BMC treatment in a large cohort of refractory angina patients. In 120 patients (64 ± 9 years, 88% men) with refractory angina, 97 ± 13 × 10(6) BMCs were injected intramyocardially in regions with stress-inducible ischemia as assessed by single photon emission computed tomography (SPECT). Canadian Cardiovascular Society angina (CCS) class, quality-of-life score, exercise testing, SPECT and magnetic resonance imaging were performed at baseline and at 3 months follow-up demonstrating significant improvements in CCS class, quality-of-life, exercise capacity, myocardial perfusion and left ventricular function (all variables P<0.001). Multivariate analysis was performed to evaluate the influence of patient characteristics, procedural data and baseline measurements on BMC treatment response. Based on the improvement of myocardial perfusion at stress, diabetes and a large number of ischemic segments at baseline were shown to be independently associated with a large response to BMC therapy. The present study demonstrates that diabetes and a large number of ischemic segments are predictors of a large response to intramyocardial BMC injection in refractory angina and chronic ischemia. Furthermore, the safety and efficacy results of previous trials are now confirmed in a larger study population.


High-Resolution Integrated Transport Model for Studying Surface Water-Groundwater Interaction.
Transport processes that lead to exchange of mass between surface water and groundwater play a significant role for the ecological functioning of aquatic systems, for hydrological processes and for biogeochemical transformations. In this study, we present a novel integral modeling approach for flow and transport at the sediment-water interface. The model allows us to simultaneously simulate turbulent surface and subsurface flow and transport with the same conceptual approach. For this purpose, a conservative transport equation was implemented to an existing approach that uses an extended version of the Navier-Stokes equations. Based on previous flume studies which investigated the spreading of a dye tracer under neutral, losing and gaining flow conditions the new solver is validated. Tracer distributions of the experiments are in close agreement with the simulations. The simulated flow paths are significantly affected by in- and outflowing groundwater flow. The highest velocities within the sediment are found for losing condition, which leads to shorter residence times compared to neutral and gaining conditions. The largest extent of the hyporheic exchange flow is observed under neutral condition. The new solver can be used for further examinations of cases that are not suitable for the conventional coupled models, for example, if Reynolds numbers are larger than 10. Moreover, results gained with the integral solver provide high-resolution information on pressure and velocity distributions at the rippled streambed, which can be used to improve flow predictions. This includes the extent of hyporheic exchange under varying ambient groundwater flow conditions.


SKP2 overexpression is associated with increased serine 10 phosphorylation of p27 (pSer10p27) in triple-negative breast cancer.
S-phase kinase-associated protein 2 (SKP2) is an important cell cycle regulator, targeting the cyclin-dependent kinase (CDK) inhibitor p27 for degradation, and is frequently overexpressed in breast cancer. p27 regulates G1 /S transition by abrogating the activity of cyclin/CDK complexes. p27 can undergo phosphorylation at serine 10 (pSer10p27). This phosphorylation event is associated with increased cell proliferation and poor prognosis in patients with glioma. The relationship between SKP2 and pSer10p27 in breast cancer has not been previously investigated. Immunohistochemistry (IHC) of SKP2, p27, pSer10p27, and other genes involved in this pathway, was analyzed in 188 breast tumors and 50 benign reduction mammoplasty samples. IHC showed SKP2 to be more highly expressed in estrogen receptor α (ERα)-negative breast cancers and demonstrated that triple-negative tumors were more likely to have high expression of SKP2 than were non-triple negative, ERα-negative tumors. A significant positive relationship was discovered for SKP2 and pSer10p27. High levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple-negative than in ERα-positive breast cancers. Use of the triple-negative TMX2-28 breast cancer cell line to address the role of SKP2 in cell cycle progression confirmed that SKP2 contributes to a more rapid cell cycle progression and may regulates pSer10p27 levels. Together, the results indicate that presence of high SKP2 plus high pSer10p27 levels in triple-negative breast cancers is associated with aggressive growth, and highlight the validity of using SKP2 inhibitors as a therapeutic approach for treating this subset of breast cancers.


Cardiac outcome in classic infantile Pompe disease after 13 years of treatment with recombinant human acid alpha-glucosidase.
Cardiac failure is the main cause of death in untreated classic infantile Pompe disease, an inheritable metabolic myopathy characterized by progressive hypertrophic cardiomyopathy. Since the introduction of enzyme replacement therapy (ERT), survival has increased significantly due to reduced cardiac hypertrophy and improved cardiac function. However, little is known about ERT's long-term effects on the heart. Fourteen patients were included in this prospective study. Cardiac dimensions, function, conduction and rhythm disturbances were evaluated at baseline and at regular intervals thereafter. Treatment duration ranged from 1.1 to 13.9 years (median 4.8 years). At baseline, all patients had increased left ventricular mass index (LVMI) (median LVMI 226 g/m2, range 98 to 599 g/m2, Z-score median 7, range 2.4-12.4). During the first four weeks, LVMI continued to increase in six patients. Normalization of LVMI was observed in 13 patients (median 30 weeks; range 3 to 660 weeks). After clinical deterioration, LVMI increased again slightly in one patient. At baseline, PR interval was shortened in all patients; it normalized in only three. A delta-wave pattern on ECG was seen in six patients and resulted in documented periods of supraventricular tachycardias (SVTs) in three patients, two of whom required medication and/or ablation. One patient had severe bradycardia (35 beats/min). This study shows that ERT significantly reduced LVMI, and sustained this effect over a period of 13.9 years. The risk for rhythm disturbances remains. Regular cardiac evaluations should be continued, also after initially good response to ERT.


Correlation of actin crosslinker and capper expression levels with stereocilia growth phases.
During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development.


Eliminating dual-task costs by minimizing crosstalk between tasks: The role of modality and feature pairings.
We tested the independent influences of two content-based factors on dual-task costs, and on the parallel processing ability: The pairing of S-R modalities and the pairing of relevant features between stimuli and responses of two tasks. The two pairing factors were realized across four dual-task groups. Within each group the two tasks comprised two different stimulus modalities (visual and auditory), two different relevant stimulus features (spatial and verbal) and two response modalities (manual and vocal). Pairings of S-R modalities (standard: visual-manual and auditory-vocal, non-standard: visual-vocal and auditory-manual) and feature pairings (standard: spatial-manual and verbal-vocal, non-standard: spatial-vocal and verbal-manual) varied across groups. All participants practiced their respective dual-task combination in a paradigm with simultaneous stimulus onset before being transferred to a psychological refractory period paradigm varying stimulus-onset asynchrony. A comparison at the end of practice revealed similar dual-task costs and similar pairing effects in both paradigms. Dual-task costs depended on modality and feature pairings. Groups training with non-standard feature pairings (i.e., verbal stimulus features mapped to spatially separated response keys, or spatial stimulus features mapped to verbal responses) and non-standard modality pairings (i.e., auditory stimulus mapped to manual response, or visual stimulus mapped to vocal responses) had higher dual-task costs than respective standard pairings. In contrast, irrespective of modality pairing dual-task costs virtually disappeared with standard feature pairings after practice in both paradigms. The results can be explained by crosstalk between feature-binding processes for the two tasks. Crosstalk was present for non-standard but absent for standard feature pairings. Therefore, standard feature pairings enabled parallel processing at the end of practice.


Searching for the evidence in pre-hospital care: a review of randomised controlled trials. On behalf of the Ambulance Response Time Sub-Group of the National Ambulance Advisory Committee.
To identify randomised controlled trials (RCTs) which evaluate aspects of pre-hospital care; to perform categorisation by theme; to compare the sensitivity and precision of the search databases. August 1997 updates of MEDLINE and EMBASE databases, using the Datastar online system. Papers published in 1987 or later were included, with no language restrictions. A trial was eligible for inclusion if it was judged, by two independent and blinded assessors, that participants followed up in the trial were definitely or possibly assigned prospectively to one of two or more alternative forms of healthcare with random allocation or a quasi-random method of allocation. The literature search retrieved 849 papers, of which 569 (67%) were in MEDLINE and 486 (57%) in EMBASE. Forty one (5%) were confirmed as reports of RCTs or quasi-RCTs, and the total number of individual trials was 38. Ten of these trials dealt with thrombolytic drugs; 14 were concerned with other drugs, 12 with equipment, and two with other interventions. Four trials were based on a sample size of more than 1000, and seven reported a statistically significant effect on mortality. All 41 papers were in EMBASE, and all but one were also in MEDLINE. Evidence based policy making with respect to the organisation of pre-hospital services cannot depend on RCTs. In the current relative absence of such evidence, practitioners and decision makers must use alternative information sources. A future review could examine a broader range of literature and be based on a wider search of published and unpublished material.


Patients with rectal cancer receiving adjuvant chemotherapy have an increased survival: a population-based longitudinal study.
The aim of this study was to investigate whether or not the use of adjuvant chemotherapy in stage III rectal cancer varies between regions and over time, and if this has had an effect on survival rates. Patients from the Uppsala/Örebro region below 75 years-of-age, operated 1995-2002 and registered in the Swedish Rectal Cancer Register, were monitored between 1995 and September 2008. A multivariate Cox proportional hazard regression model was used for analysis. Overall survival was described using the Kaplan-Meier method. Four hundred and thirty-six patients with stage III rectal cancer were included. Adjuvant chemotherapy was given to 42% of the patients (proportions varying from 13% to 77% among counties), and there were substantial increases over time. The 5-year overall survival was 65.8% [95% confidence interval (CI) 50-84] for patients having adjuvant chemotherapy compared with 45.6% (95% CI 39-52) for patients not treated with chemotherapy. The multivariate hazard ratio for death was 0.65 (95% CI 0.5-0.8) for patients treated with adjuvant chemotherapy. The use of adjuvant chemotherapy for rectal cancer has increased, but varies considerably between hospitals/counties. In this cohort, those having adjuvant chemotherapy had a longer overall survival.


Prenatal bisphenol a exposure and dysregulation of infant hypothalamic-pituitary-adrenal axis function: findings from the APrON cohort study.
Animal models show that prenatal bisphenol A (BPA) exposure leads to sexually dimorphic disruption of the neuroendocrine system in offspring, including the hypothalamic-pituitary-adrenal (HPA) neuroendocrine system, but human data are lacking. In humans, prenatal BPA exposure is associated with sex-specific behavioural problems in children, and HPA axis dysregulation may be a biological mechanism. The objective of the current study was to examine sex differences in associations between prenatal maternal urinary BPA concentration and HPA axis function in 3 month old infants. Mother-infant pairs (n = 132) were part of the Alberta Pregnancy Outcomes and Nutrition study, a longitudinal birth cohort recruited (2010-2012) during pregnancy. Maternal spot urine samples collected during the 2nd trimester were analyzed for total BPA and creatinine. Infant saliva samples collected prior to and after a blood draw were analyzed for cortisol. Linear growth curve models were used to characterize changes in infant cortisol as a function of prenatal BPA exposure. Higher maternal BPA was associated with increases in baseline cortisol among females (β = 0.13 log μg/dL; 95% CI: 0.01, 0.26), but decreases among males (β = -0.22 log μg/dL; 95% CI: -0.39, -0.05). In contrast, higher BPA was associated with increased reactivity in males (β = .30 log μg/dL; 95% CI: 0.04, 0.56) but decreased reactivity in females (β = -0.15 log μg/dL; 95% CI: -0.35, 0.05). Models adjusting for creatinine yielded similar results. Prenatal BPA exposure is associated with sex-specific changes in infant HPA axis function. The biological plausibility of these findings is supported by their consistency with evidence in rodent models. Furthermore, these data support the hypotheses that sexually dimorphic changes in children's behaviour following prenatal BPA exposure are mediated by sexually dimorphic changes in HPA axis function.


Prenatal treatment prevents learning deficit in Down syndrome model.
Down syndrome is the most common genetic cause of mental retardation. Active fragments of neurotrophic factors release by astrocyte under the stimulation of vasoactive intestinal peptide, NAPVSIPQ (NAP) and SALLRSIPA (SAL) respectively, have shown therapeutic potential for developmental delay and learning deficits. Previous work demonstrated that NAP+SAL prevent developmental delay and glial deficit in Ts65Dn that is a well-characterized mouse model for Down syndrome. The objective of this study is to evaluate if prenatal treatment with these peptides prevents the learning deficit in the Ts65Dn mice. Pregnant Ts65Dn female and control pregnant females were randomly treated (intraperitoneal injection) on pregnancy days 8 through 12 with saline (placebo) or peptides (NAP 20 µg +SAL 20 µg) daily. Learning was assessed in the offspring (8-10 months) using the Morris Watermaze, which measures the latency to find the hidden platform (decrease in latency denotes learning). The investigators were blinded to the prenatal treatment and genotype. Pups were genotyped as trisomic (Down syndrome) or euploid (control) after completion of all tests. two-way ANOVA followed by Neuman-Keuls test for multiple comparisons, P<0.05 was used to denote statistical significance. Trisomic mice who prenatally received placebo (Down syndrome-placebo; n = 11) did not demonstrate learning over the five day period. DS mice that were prenatally exposed to peptides (Down syndrome-peptides; n = 10) learned significantly better than Down syndrome-placebo (p<0.01), and similar to control-placebo (n = 33) and control-peptide (n = 30). In conclusion prenatal treatment with the neuroprotective peptides (NAP+SAL) prevented learning deficits in a Down syndrome model. These findings highlight a possibility for the prevention of sequelae in Down syndrome and suggest a potential pregnancy intervention that may improve outcome.


Thermal requirements for growth, survival and aerobic performance of weatherfish larvae Misgurnus fossilis.
Thermal requirements of larval weatherfish Misgurnus fossilis were investigated in terms of growth, survival and aerobic performance. Growth and survival of M. fossilis larvae acclimated to five temperatures (11, 15, 19, 23 and 27° C) were measured over 25 days. In the upper temperature treatments (19, 23 and 27° C), survival of larvae was stable throughout the entire rearing period (>75%), whereas 11 and 15° C resulted in severe declines in survival (to <10%). Growth of larvae (expressed as dry mass and total length) was highest at 19 and 23° C, but significantly decreased at 27° C. Routine metabolic rate of 3 days post-hatch larvae was estimated as oxygen consumption rate (ṀO2 ) during acute exposure (30 min to 1 h) to seven temperatures (11, 15, 19, 23, 27, 31 and 35° C). Larval oxygen uptake increased with each consecutive temperature step from 11 to 27° C, until a plateau was reached at temperatures >27° C. All larvae of the 35° C regime, however, died within the ṀO2 measurement period. M. fossilis larvae show greater than expected tolerance of high temperatures. On the other hand, low temperatures that are within the range of likely habitat conditions are critical because they might lead to high mortality rates when larvae are exposed over periods >10 days. These findings help to improve rearing conditions and to identify suitable waters for stocking and thus support the management of re-introduction activities for endangered M. fossilis.


Two distinct serum mannose-binding lectins function as beta inhibitors of influenza virus: identification of bovine serum beta inhibitor as conglutinin.
Normal bovine and mouse sera contain a component, termed beta inhibitor, that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. We have previously shown these beta inhibitors to be mannose-binding lectins that apparently act by binding to carbohydrate on the viral hemagglutinin, blocking access of the receptor-binding site to receptors on host cells (E. M. Anders, C. A. Hartley, and D. C. Jackson, Proc. Natl. Acad. Sci. USA 87:4485-4489, 1990). For the H3-subtype virus A/Memphis/1/71 x A/Bel/42 (H3N1), sensitivity to beta inhibitors is determined by the oligosaccharide at residue 165 of the hemagglutinin, this glycosylation site being lost in a resistant mutant selected by growth in the presence of bovine serum. In the present study, we sequenced the hemagglutinin genes of additional bovine serum-resistant mutants derived from influenza viruses A/Philippines/2/82 (H3N2) and A/Brazil/11/78 (H1N1). The results confirm the importance of carbohydrate at residue 165 for inhibitor sensitivity of H3 viruses and implicate carbohydrate at residue 87 (94a in the H3 numbering system) as an important determinant in the sensitivity of H1-subtype viruses to the bovine inhibitor. Unlike the two H3 mutants, which had also gained resistance to hemagglutination inhibition by mouse serum, the H1 bovine serum-resistant mutant remained sensitive to the mouse beta inhibitor, suggesting that inhibition by the two types of sera is mediated by distinct mannose-binding lectins. In support of this hypothesis, the beta inhibitors in bovine and mouse sera were shown to differ in their pattern of inhibition by monosaccharides and in their sensitivity to 2-mercaptoethanol. In these and other properties, the bovine inhibitor closely resembled conglutinin, a Ca(2+)-dependent N-acetylglucosamine- and mannose-binding lectin present in bovine serum but absent from the serum of other species. Furthermore, polyclonal and monoclonal anticonglutinin antibodies abrogated the hemagglutination-inhibiting activity of bovine serum. Direct binding of conglutinin to the parent viruses and reduced binding to their respective mutants were confirmed by radioimmunoassay.


Escalating weekly doses of cetuximab and correlation with skin toxicity: a phase I study.
Cetuximab is a chimeric monoclonal antibody targeting the epidermal growth factor receptor (EGFR). The recommended dosage is an initial load of 400 mg/m² intravenously (IV) followed by a weekly maintenance dose of 250 mg/m². It has been reported retrospectively that cetuximab efficacy was correlated with dose-related severity of skin rash. This study was prospectively designed to examine the safety and feasibility of escalating weekly doses of cetuximab, testing the hypothesis of the relationship of dose-dependent skin toxicity and efficacy. Methods Four dose levels were tested: Cetuximab 400 mg/m² IV loading dose and 250, 300, 350, 400 mg/m² weekly IV maintenance. There was no intra-patient dose escalation. Standard dose limiting toxicity criteria were used. Rash was evaluated using two additional validated dermatology methods: global acne grading scale (GAGS) and acne lesion counting (ALC). Tumor specimens and blood samples were obtained for correlative analyses. Twenty seven patients with solid tumors were enrolled: five head and neck, three pancreas, four gall bladder, two each of prostate, breast, colorectal, lung, and esophagus, and five others. Planned dose escalation was completed without reaching dose-limiting toxicity (DLT) or the maximum tolerated dose (MTD). The highest dose level was expanded to a total of 17 patients. Gr 3/4 toxicities included: lymphopenia (2), fatigue (2), and hypomagnesemia (2). One patient experienced a grade 3 rash (350 mg/m²). Sixty five percent of pts had a ≥ Gr 2 rash that was not dose dependent. In 22 evaluable patients, there was one partial response (PR) in a patient with cholangiocarcinoma (400 mg/m²) and seven patients had stable disease (SD). ALC and GAGS demonstrated no correlation with dose or response. Correlative studies evaluating k-ras, EGFR FISH status and immunologic correlatives were conducted on available tumor samples. Cetuximab administered at 400 mg/m² IV as a loading dose with weekly maintenance dose of 400 mg/m² is feasible and well tolerated. There was no direct correlation of the grade of rash with dose in this group of patients with heterogenous solid tumors.


O-GlcNAcylation alters the selection of mRNAs for translation and promotes 4E-BP1-dependent mitochondrial dysfunction in the retina.
Diabetes promotes the posttranslational modification of proteins by O-linked addition of GlcNAc (O-GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is O-GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O-GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O-GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs (i.e. mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, ∼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of O-GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O-GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O-GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O-GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation.


Does this patient have temporal arteritis?
Clinicians must be able to confidently diagnose temporal arteritis (TA), since failure to make a correct diagnosis may lead to irreversible visual loss as well as inappropriate evaluation and treatment of headache, fatigue, and other potential presenting symptoms. The diagnostic value of particular signs and symptoms among patients with suspected TA is unknown. To determine the accuracy of historical features, physical examination, and erythrocyte sedimentation rate (ESR) in diagnosis of TA. We performed a MEDLINE search of English-language articles published between January 1966 and July 2000 and a hand search of bibliographies of retrieved articles, previous reviews, monographs, and textbooks. Studies that provided detailed clinical information on patients who had been referred for temporal artery biopsy. Of 114 studies retrieved, 41 met our inclusion criteria; 21 included both biopsy-positive and biopsy-negative patients and formed the core of our review. Both authors independently reviewed each study to determine eligibility, abstracted data using a standardized instrument, and classified study quality using predetermined criteria. The prevalence of TA in the general population is less than 1%. However, in our 21 core studies, 39% of patients referred for temporal artery biopsy had positive results. The only 2 historical features that substantially increased the likelihood of TA among patients referred for biopsy were jaw claudication (positive likelihood ratio [LR], 4.2; 95% confidence interval [CI], 2.8-6.2) and diplopia (positive LR, 3.4; 95% CI, 1.3-8.6). The absence of any temporal artery abnormality was the only clinical factor that modestly reduced the likelihood of disease (negative LR, 0.53; 95% CI, 0.38-0.75). Predictive physical findings included temporal artery beading (positive LR, 4.6; 95% CI, 1.1-18.4), prominence (positive LR, 4.3; 95% CI, 2.1-8.9), and tenderness (positive LR, 2.6; 95% CI, 1.9-3.7). Normal ESR values indicated much less likelihood of disease (negative LR for abnormal ESR, 0.2; 95% CI, 0.08-0.51). A small number of clinical features are helpful in predicting the likelihood of a positive temporal artery biopsy among patients with a clinical suspicion of disease; the most useful finding is a normal ESR, which makes TA unlikely.


The neural basis of categorical face perception: graded representations of face gender in fusiform and orbitofrontal cortices.
Face gender, like many other things, is perceived categorically: Subjective perceptions are distorted toward the categories, male or female, and the objective gradiency inherent across faces is partially lost. The neural basis of such categorical face perception remains virtually unknown. Participants passively viewed faces whose sexually dimorphic content was morphed monotonically from male to female while neural activity was measured using functional magnetic resonance imaging. Subjective perceptions revealed strong nonlinearity despite monotonic linear changes in face gender, consistent with categorical perception. Neuroimaging results indicated that the lateral fusiform gyrus, bilaterally, and the fusiform face area linearly encoded graded parameters of objective face gender, but these regions correlated substantially less with subjective perceptions (which were nonlinear and affected by categorical perception effects). Such subjective perceptions, however, were represented in the orbitofrontal cortex, but this region correlated substantially less with objective parameters. The attention-independent graded representations of face gender in fusiform and orbitofrontal cortices reveal how objective face parameters are encoded and transformed into subjective categorically warped perceptions in the human brain.


Evaluation of peripheral blood mononuclear cell processing and analysis for Survival Motor Neuron protein.
Survival Motor Neuron (SMN) protein levels may become key pharmacodynamic (PD) markers in spinal muscular atrophy (SMA) clinical trials. SMN protein in peripheral blood mononuclear cells (PBMCs) can be quantified for trials using an enzyme-linked immunosorbent assay (ELISA). We developed protocols to collect, process, store and analyze these samples in a standardized manner for SMA clinical studies, and to understand the impact of age and intraindividual variability over time on PBMC SMN signal. Several variables affecting SMN protein signal were evaluated using an ELISA. Samples were from healthy adults, adult with respiratory infections, SMA patients, and adult SMA carriers. Delaying PBMCs processing by 45 min, 2 hr or 24 hr after collection or isolation allows sensitive detection of SMN levels and high cell viability (>90%). SMN levels from PBMCs isolated by EDTA tubes/Lymphoprep gradient are stable with processing delays and have greater signal compared to CPT-collected samples. SMN signal in healthy individuals varies up to 8x when collected at intervals up to 1 month. SMN signals from individuals with respiratory infections show 3-5x changes, driven largely by the CD14 fraction. SMN signal in PBMC frozen lysates are relatively stable for up to 6 months. Cross-sectional analysis of PBMCs from SMA patients and carriers suggest SMN protein levels decline with age. The sources of SMN signal variability in PBMCs need to be considered in the design and of SMA clinical trials, and interpreted in light of recent medical history. Improved normalization to DNA or PBMC subcellular fractions may mitigate signal variability and should be explored in SMA patients.


Indications and administration practices amongst medical cannabis healthcare providers: a cross-sectional survey.
The medical use of cannabis has been legislatively restricted for decades in the US and abroad. In recent years, changing local and national policies have given rise to a community of healthcare providers who may be recommending the medical use of cannabis without the benefit of formal clinical practice guidelines or sufficient training and education. In addition, a citizen science movement has emerged whereby unlicensed and untrained individuals are acting as healthcare provider proxies, offering cannabis-specific clinical care to "patients". This study sought to characterize the clinical practice characteristics of these provider groups. An anonymous, online survey was designed to describe levels of cannabis-specific education, practice characteristics, indications for medical use, dose, administration forms and adverse effects related to cannabis use. The questionnaire was disseminated via professional medical cannabis associations and by word-of-mouth. It was accessed between June 31-December 31, 2018. A self-selecting sample of respondents (n = 171) completed the survey. Formal education or training in the medical use of cannabis was significantly more common among licensed respondents than unlicensed respondents (95.5% vs 76.9% respectively, OR, 6.3, 95% CI, 1.2-32.3, p = 0.03). The vast majority (n = 74, 83.15%) of licensed respondents reported having recommended cannabis as an adjunct to an existing prescription drug. Almost two-thirds (n = 64, 71.9%) reported having recommended it as a substitute. When delta-9-tetrahydrocannabinol (THC) is the principal therapeutic constituent of interest, vaporization is the most common method of administration recommended (n = 94 responses, 71.4% of respondents). In contrast, when cannabidiol (CBD) is the principal therapeutic constituent of interest, oral administration (sublingual or oromucosal absorption) is the most common method (n = 70 responses, 71.4% of respondents). Individuals who recommend the medical use of cannabis appear to be self-generating a community standard of practice in the absence of formal clinical guidelines on dosing, interactions and other characteristics. Reducing barriers to clinical research on cannabis products is needed, not only to better understand their risks and benefits, but also to augment the evidence-base for informing clinical practice.


Bronchodilators in Tobacco-Exposed Persons with Symptoms and Preserved Lung Function.
Many persons with a history of smoking tobacco have clinically significant respiratory symptoms despite an absence of airflow obstruction as assessed by spirometry. They are often treated with medications for chronic obstructive pulmonary disease (COPD), but supporting evidence for this treatment is lacking. We randomly assigned persons who had a tobacco-smoking history of at least 10 pack-years, respiratory symptoms as defined by a COPD Assessment Test score of at least 10 (scores range from 0 to 40, with higher scores indicating worse symptoms), and preserved lung function on spirometry (ratio of forced expiratory volume in 1 second [FEV1] to forced vital capacity [FVC] ≥0.70 and FVC ≥70% of the predicted value after bronchodilator use) to receive either indacaterol (27.5 μg) plus glycopyrrolate (15.6 μg) or placebo twice daily for 12 weeks. The primary outcome was at least a 4-point decrease (i.e., improvement) in the St. George's Respiratory Questionnaire (SGRQ) score (scores range from 0 to 100, with higher scores indicating worse health status) after 12 weeks without treatment failure (defined as an increase in lower respiratory symptoms treated with a long-acting inhaled bronchodilator, glucocorticoid, or antibiotic agent). A total of 535 participants underwent randomization. In the modified intention-to-treat population (471 participants), 128 of 227 participants (56.4%) in the treatment group and 144 of 244 (59.0%) in the placebo group had at least a 4-point decrease in the SGRQ score (difference, -2.6 percentage points; 95% confidence interval [CI], -11.6 to 6.3; adjusted odds ratio, 0.91; 95% CI, 0.60 to 1.37; P = 0.65). The mean change in the percent of predicted FEV1 was 2.48 percentage points (95% CI, 1.49 to 3.47) in the treatment group and -0.09 percentage points (95% CI, -1.06 to 0.89) in the placebo group, and the mean change in the inspiratory capacity was 0.12 liters (95% CI, 0.07 to 0.18) in the treatment group and 0.02 liters (95% CI, -0.03 to 0.08) in the placebo group. Four serious adverse events occurred in the treatment group, and 11 occurred in the placebo group; none were deemed potentially related to the treatment or placebo. Inhaled dual bronchodilator therapy did not decrease respiratory symptoms in symptomatic, tobacco-exposed persons with preserved lung function as assessed by spirometry. (Funded by the National Heart, Lung, and Blood Institute and others; RETHINC ClinicalTrials.gov number, NCT02867761.).


Low frequency mutation of the Ephrin receptor A3 gene in hepatocellular carcinoma.
EphA3 is a component of the Eph/ephrin tyrosine kinase system, which participates in vasculature development. This receptor/ligand system is associated with various signaling pathways related to cell growth and viability, cytoskeletal organization, cell migration, and anti-apoptosis. Accumulated evidence suggests that aberrant regulation of EphA3 and its genetic alterations are implicated in the development and progression of various cancers. However, despite a high incidence of EphA3 over-expression, no such investigation has been performed in hepatocellular carcinoma. Thus, we investigated genetic alterations of the EphA3 gene in 73 cases of hepatocellular carcinoma by single-strand conformational polymorphism and sequencing. One novel D219V missense mutation was found in the extracellular domain of EphA3, and two genetic alterations in the intracellular sterile-alpha-motif (SAM) domain of EphA3 appeared to be polymorphisms. Although the functional assessments of this mutant are incomplete, it is believed that this novel EphA3 mutation may contribute to the development of hepatocellular carcinoma.


Optogenetic activation of intracellular antibodies for direct modulation of endogenous proteins.
Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and β2-adrenergic receptor (β2AR) optobodies suppressed endogenous gelsolin activity and β2AR signaling, respectively.


Replacement manifolds: a method to uniformize semiclassical wave functions.
We present a semiclassical technique that relies on replacing complicated classical manifold structure with simpler manifolds, which are then evaluated by the usual semiclassical rules. Under circumstances where the original manifold structure gives poor or useless results semiclassically the replacement manifolds can yield remarkable accuracy. We give several working examples to illustrate the theory presented here.


Assessing the assessments: Taking stock of learning outcomes data in India.
Data on learning outcomes is essential for tracking progress in achieving education goals, understanding what education policies work (and don't work), and holding public officials accountable. We assess the accuracy and reliability of India's two nationally representative surveys on learning outcomes, ASER and NAS, so that users of these datasets may better understand when, and for what purposes, these two datasets can reasonably be used. After restricting our sample to maximize comparability between the two datasets, we find that NAS state averages are significantly higher than ASER state averages and averages from an independently conducted and nationally representative survey (IHDS). In addition, state rankings based on NAS data display almost no correlation with state rankings based on ASER, IHDS, or net state domestic product per capita. We conclude that NAS state averages are likely artificially high and contain little information about states' relative performance. The presence of severe bias in the NAS data suggests that this data should be used carefully or not at all for comparisons between states, constructing learning profiles, or any other purpose. We then analyze the internal reliability of ASER data using variance decomposition methods. We find that while ASER data is mostly reliable for comparing state averages, it is less reliable for looking at district averages, or changes in district and state averages over time. We conclude that analysts may use ASER data with confidence for comparisons between states in a single year, constructing learning profiles, and assessing learning inequality but should exercise caution when comparing changes in state scores and avoid using ASER district-level data.


GaAs-Based Superluminescent Light-Emitting Diodes with 290-nm Emission Bandwidth by Using Hybrid Quantum Well/Quantum Dot Structures.
A high-performance superluminescent light-emitting diode (SLD) based upon a hybrid quantum well (QW)/quantum dot (QD) active element is reported and is assessed with regard to the resolution obtainable in an optical coherence tomography system. We report on the appearance of strong emission from higher order optical transition from the QW in a hybrid QW/QD structure. This additional emission broadening method contributes significantly to obtaining a 3-dB linewidth of 290 nm centered at 1200 nm, with 2.4 mW at room temperature.


Changes in job stressors in the Canadian working population.
To determine the changes in levels of work stressors in a nationally representative sample of Canadian workers from 1994/95 to 2000/01. We compared responses for an abbreviated version of the Job Content Questionnaire in two waves of the National Population Health Survey (NPHS). Other items and scales related to work and health were also analyzed. Data were transformed to range from 0 to 10. Comparisons of the 2000/01 data were also made with the Canadian Community Health Survey (CCHS) conducted in 2000. There were only very small absolute differences between NPHS 2000/01 data and CCHS 2000 data. The NPHS comparison from 1994/95 to 2000/01 showed an increase in job security (change in means = 0.49, 95% CI 0.39 to 0.58) and a decrease in job physical demands (change in means = 0.45, 95% CI 0.35 to 0.54). Other changes in work characteristics were small in absolute value. The combined "overall work stressors" index dropped by 0.12 (95% CI 0.08 to 0.15). Levels of work stressors did not increase over the period. Some subscales showed an improvement.


The role of intent in serosorting behaviors among men who have sex with men sexual partnerships.
Serosorting is increasingly assessed in studies of men who have sex with men (MSM). Most research studies have measured serosorting by combining reported unprotected anal intercourse (UAI) and the occurrence of participant and partner same HIV status (seroconcordance). The Centers for Disease Control and Prevention's definition of serosorting also incorporates intent to be in such a partnership, although few studies incorporate both intent and behavior into their measures. Using data from a national, online survey of 3519 US MSM, we assessed the role of intention in seroconcordant partnerships, as measured by participant rating of the importance of shared serostatus when selecting a sex partner. For HIV+ men, 30% partnerships were seroconcordant; of these, 48% reported intent to be in such a partnership (intentional seroconcordance). For HIV- men, 64% partnerships were seroconcordant; of these, 80% reported intentional seroconcordance. Intentional seroconcordance was associated with UAI for HIV+ partnerships [odds ratio (OR): 1.9; 95% confidence interval (CI): 1.3 to 2.9] but not significant for HIV- partnerships (OR: 1.1; CI: 0.99 to 1.3). In separate models where intent was not considered, seroconcordance was associated with UAI for HIV+ partnerships (OR: 3.2; 95% CI: 2.2 to 4.6) and for HIV- partnerships (OR: 1.2; 95% CI: 1.0 to 1.3; P = 0.03). Regardless of intentionality, seroconcordance was strongly associated with UAI for HIV+ men and weakly associated with UAI for HIV- men. Intentional seroconcordance was not associated with UAI more strongly than was seroconcordance in absence of consideration of intent. Intentionality may not be a critical element of the relationship between seroconcordance and UAI.


Evaluating parameter availability for physiologically based pharmacokinetic (PBPK) modeling of perfluorooctanoic acid (PFOA) in zebrafish.
Physiologically based pharmacokinetic (PBPK) models are considered useful tools to describe the absorption, distribution, metabolism and excretion of xenobiotics. For accurate predictions, PBPK models require species-specific and compound-specific parameters. Zebrafish are considered an appropriate vertebrate model for investigating the toxicity of a wide variety of compounds. However, no specific mechanistic model exists for the pharmacokinetics of perfluoroalkyl acids (PFAAs) in zebrafish, despite growing concern about this class of ubiquitous environmental contaminants. The purpose of this study was to evaluate the current state of knowledge for the parameters that would be needed to construct such a model for zebrafish. We chose perfluorooctanoic acid (PFOA) as a model PFAA with greater data availability. We have updated a previous PBPK model for rainbow trout to simulate PFOA fate in zebrafish following waterborne exposure. For the first time, the model considers hepatobiliary circulation. In order to evaluate the availability of parameters to implement this model, we performed an extensive literature review to find zebrafish-specific parameters. As in previous approaches, we broadened our search to include mammalian and other fish studies when zebrafish-specific data were lacking. Based on the method used to measure or estimate parameters, or based on their species-specific origin, we scored and ranked the quality of available parameters. These scores were then used in Monte Carlo and partial rank correlation analyses to identify the most critical data gaps. The liver, where fatty acid binding proteins (FABPs) and plasma proteins are considered, represented the best model-data agreement. Lack of agreement in other tissues suggest better parameters are needed. The results of our study highlight the lack of zebrafish-specific parameters. Based on sensitivity and uncertainty analysis, parameters associated with PFAA-protein interactions and passive diffusion need further refinement to enable development of predictive models for these emerging chemicals in zebrafish.


Molecular Epidemiology of AY.28 and AY.104 Delta Sub-lineages in Sri Lanka.
The worst SARS-CoV-2 outbreak in Sri Lanka was due to the two Sri Lankan delta sub-lineages AY.28 and AY.104. We proceeded to further characterize the mutations and clinical disease severity of these two sub-lineages. 705 delta SARS-CoV-2 genomes sequenced by our laboratory from mid-May to November 2021 using Illumina and Oxford Nanopore were included in the analysis. The clinical disease severity of 440/705 individuals were further analyzed to determine if infection with either AY.28 or AY.104 was associated with more severe disease. Sub-genomic RNA (sg-RNA) expression was analyzed using periscope. AY.28 was the dominant variant throughout the outbreak, accounting for 67.7% of infections during the peak of the outbreak. AY.28 had three lineage defining mutations in the spike protein: A222V (92.80%), A701S (88.06%), and A1078S (92.04%) and seven in the ORF1a: R24C, K634N, P1640L, A2994V, A3209V, V3718A, and T3750I. AY.104 was characterized by the high prevalence of T95I (90.81%) and T572L (65.01%) mutations in the spike protein and by the absence of P1640L (94.28%) in ORF1a with the presence of A1918V (98.58%) mutation. The mean sgRNA expression levels of ORF6 in AY.28 were significantly higher compared to AY.104 (p < 0.0001) and B.1.617.2 (p < 0.01). Also, ORF3a showed significantly higher sgRNA expression in AY.28 compared to AY.104 (p < 0.0001). There was no difference in the clinical disease severity or duration of hospitalization in individuals infected with these sub lineages. Therefore, AY.28 and AY.104 appear to have a fitness advantage over the parental delta variant (B.1.617.2), while AY.28 also had a higher expression of sg-RNA compared to other sub-lineages. The clinical implications of these should be further investigated.


The Impact of the West Africa Ebola Outbreak on Obstetric Health Care in Sierra Leone.
As Sierra Leone celebrates the end of the Ebola Virus Disease (EVD) outbreak, we can begin to fully grasp its impact on already weak health systems. The EVD outbreak in West Africa forced many hospitals to close down or reduce their activity, either to prevent nosocomial transmission or because of staff shortages. The aim of this study is to assess the potential impact of EVD on nationwide access to obstetric care in Sierra Leone. Community health officers collected weekly data between January 2014-May 2015 on in-hospital deliveries and caesarean sections (C-sections) from all open facilities (public, private for-profit and private non-profit sectors) offering emergency obstetrics in Sierra Leone. This was compared to official data of EVD cases per district. Logistic and Poisson regression analyses were used to compute risk and rate estimates. Nationwide, the number of in-hospital deliveries and C-sections decreased by over 20% during the EVD outbreak. The decline occurred early on in the EVD outbreak and was mainly attributable to the closing of private not-for-profit hospitals rather than government facilities. Due to difficulties in collecting data in the midst of an epidemic, limitations of this study include some missing data points. Both the number of in-hospital deliveries and C-sections substantially declined shortly after the onset of the EVD outbreak. Since access to emergency obstetric care, like C-sections, is associated with decreased maternal mortality, many women are likely to have died due to the reduced access to appropriate care during childbirth. Future research on indirect health effects of health system breakdown should ideally be nationwide and continue also into the recovery phase. It is also important to understand the mechanisms behind the deterioration so that important health services can be reestablished.


Apoptotic Bodies Elicit Gas6-Mediated Migration of AXL-Expressing Tumor Cells.
Metastases are a major cause of cancer mortality. AXL, a receptor tyrosine kinase aberrantly expressed in many tumors, is a potent oncogenic driver of metastatic cell motility and has been identified as broadly relevant in cancer drug resistance. Despite its frequent association with changes in cancer phenotypes, the precise mechanism leading to AXL activation is incompletely understood. In addition to its ligand growth arrest specific-6 (Gas6), activation of AXL requires the lipid moiety phosphatidylserine (PS). Phosphatidylserine is only available to mediate AXL activation when it is externalized on cell membranes, an event that occurs during certain physiologic processes such as apoptosis. Here, it is reported that exposure of cancer cells to phosphatidylserine-containing vesicles, including synthetic liposomes and apoptotic bodies, contributes to enhanced migration of tumor cells via a PS-Gas6-AXL signaling axis. These findings suggest that anticancer treatments that induce fractional cell killing enhance the motility of surviving cells in AXL-expressing tumors, which may explain the widespread role of AXL in limiting therapeutic efficacy.Implications: This study demonstrates that motility behavior of AXL-expressing tumor cells can be elicited by Gas6-bearing apoptotic bodies generated from tumor treatment with therapeutics that produce killing of a portion of the tumor cells present but not all, hence generating potentially problematic invasive and metastatic behavior of the surviving tumor cells. Mol Cancer Res; 15(12); 1656-66. ©2017 AACR.


Influence of elevated temperatures on the immune response of abalone, Haliotis rubra.
Elevated water temperature can act as a stressor impacting the immune responses of molluscs, potentially increasing their susceptibility to microbial infections. Abalone are commercially important marine molluscs that have recently experienced disease outbreaks caused by a herpesvirus and Vibrio bacteria. Sampling of wild-caught Haliotis rubra showed a significant correlation between water temperature and both antiviral and antibacterial activity, with higher activity in summer than in winter months. However, antibacterial activity was compromised in favour of antiviral activity as the water temperatures peaked in summer. A controlled laboratory experiment was then used to investigate several immune responses of H. rubra, including total haemocyte count (THC), stimulated superoxide anion production (SO), antiviral activity against a model herpesvirus, herpes simplex virus type 1 and antibacterial activity against a representative pathogenic bacterium, Vibrio anguillarum, over one week after raising water temperature from 18 to 21 or 24 °C. THC and SO increased at day 1 and then dropped back to control levels by days 3 and 7. By comparison, the humoural immune parameters showed a delayed response with antibacterial and antiviral activity significantly increasing on days 3 and 7, respectively. Consistent with the field study, antibacterial activity became significantly depressed after prolonged exposure to elevated temperatures. A principal components analysis on the combined immune parameters showed a negative correlation between antiviral and antibacterial activity. SO was positively correlated to THC and neither of these cellular parameters were correlated to the humoural antimicrobial activity. Overall, this study indicates that abalone may have more resilience to viruses than bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.


Proteomic analysis of highly prevalent amyloid A amyloidosis endemic to endangered island foxes.
Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.


Oral use of Streptococcus salivarius K12 in children with secretory otitis media: preliminary results of a pilot, uncontrolled study.
Secretory otitis media (SOM) remains a common disease among children. Although its cause is not yet perfectly established, the pathology, often a sequel of acute otitis media (AOM), is mainly characterized by persistent fluid in the middle ear cavity. Twenty-two children with a diagnosis of SOM were treated daily for 90 days with an oral formulation containing the oral probiotic Streptococcus salivarius K12 (Bactoblis(®)). After treatment, the children were evaluated for AOM episodes and subjected to tone audiometry, tympanometry, endonasal endoscopy, otoscopy, and tonsillar examination. Subject compliance and probiotic tolerability and side effects have also been evaluated. Our results indicate a good safety profile, a substantial reduction of AOM episodes, and a positive outcome from the treatment for all of the clinical outcomes tested. We conclude that strain K12 may have a role in reducing the occurrence and/or severity of SOM in children. From our perspective, this study constitutes a starting point toward the organization of a more extensive placebo-controlled study aimed at critically appraising our preliminary observations.


Quantitative distinctions of active site molecular recognition by P-glycoprotein and cytochrome P450 3A4.
The bulk of characterized xenobiotic defense and disposition is conferred by the abundant enzymes cytochrome P450 3A4 and P-glycoprotein. Although expressed in many tissues, these enzymes are most abundant in the liver and intestine and seem to share most substrates and inhibitors, with the apparent synergy between these two promiscuous enzymes asserted because of their extensive overlap of substrates and shared tissue location. Since the broad-spectrum tolerance to lipophilic compounds of various sizes naturally results in a similar pattern of substrate/inhibitor recognition, the cause or mechanism of many drug/drug and drug/herb interactions can be difficult to determine. These two seemingly indiscriminate enzymes, however, do not share some unique inhibitor selectivity. Particularly, we show various potent CYP3A4 inhibitors that do not affect P-gp active transport function. Remarkably, we have also identified several compounds-valinomycin, norverapamil, reserpine, nobiletin, emetine, gallopamil, fluphenazine-that uniquely inhibit P-gp function with affinities comparable to benchmark P-gp inhibitors despite a lack of effect on CYP3A4 function at physiologically relevant concentrations. Indeed, valinomycin inhibits P-gp with an IC(50) similar to cyclosporin A yet apparently does not affect CYP3A4 function, and emetine and nobiletin are also specific for interaction with P-gp. Additionally, norverapamil and reserpine have, respectively, a 60- and 40-fold preference for inhibition of P-gp over CYP3A4. Some striking structural analogies among these compounds are discussed. These distinguishing qualities of substrate recognition between CYP3A4 and P-gp should reveal nuances of active-site architecture unique to each and could serve as tools to probe for the specific discernment of P-gp-mediated drug/drug or drug/herb interactions. Learning more about binding distinctions and quantitative activity relationships of substrate/inhibitor interactions with these two enzymes and the differences between them may indicate how they recognize such a wide variety of molecules as substrates (and/or inhibitors). Moreover, identification of specific inhibitors will allow the determination of which enzyme is responsible for drug interactions and/or the extent of contribution in a multiple exposure situation.


TRIP8b is required for maximal expression of HCN1 in the mouse retina.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. HCN1 channels expressed in photoreceptors participate in keeping light responses transient and are required for normal mesopic vision. The subcellular localization of HCN1 varies among cell types. In photoreceptors HCN1 is concentrated in the inner segments while in other retinal neurons, HCN1 is evenly distributed though the cell. This is in contrast to hippocampal neurons where HCN1 is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b). Multiple splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate the surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in the retina and to test if loss of TRIP8b alters HCN1 expression or trafficking. We found that TRIP8b colocalizes with HCN1 in multiple retina neurons and all major splice isoforms of TRIP8b are expressed in the retina. Photoreceptors express three different isoforms. In TRIP8b knockout mice, the ability of HCN1 to traffic to the surface of retinal neurons is unaffected. However, there is a large decrease in the total amount of HCN1. We conclude that TRIP8b in the retina is needed to achieve maximal expression of HCN1.


Association of testosterone levels with socio-demographic characteristics in a sample of Ugandan men.
Testosterone, a male reproductive hormone, affects several physiological processes, such as sperm production, energy, strength, sexual behavior, sleep and the general well being of men. Normal levels of testosterone are necessary to effect these physiological processes. The objective of this study was to study the association between testosterone levels in a sample of Ugandan men with socio-demographic characteristics, and compare the testosterone levels of Ugandan men with that of men in other countries. Eighty men were enrolled from the medical campus at Makerere University. Blood samples were drawn from 7.00 - 8.00 a.m. and total testosterone was measured using radioimmunoassay. The free and bioavailable testosterone was calculated after measuring sex hormone binding globulin (SHBG) and albumin in the blood samples. Self-administered questionnaires were used to obtain socio-demographic characteristics of the subjects. Biometric measurements including weight, height and waist circumference were also recorded. Serum testosterone levels of Ugandan men were within the normal physiological ranges. Married participants and those with dependents had lower testosterone than unmarried participants and those without dependents respectively. Sexually active participants had lower testosterone levels than those who were not sexually active. Testosterone levels were lower in association with several socio-demographic characteristics including being married, having dependents, and daily coital frequency. Further research is warranted into the relationship between testosterone levels and contributory sexual behavior that may be important in understanding the spread of HIV/AIDS, given its high prevalence in sub-Saharan Africa.


Peptide retention time prediction.
Most methods for interpreting data from shotgun proteomics experiments are to large degree dependent on being able to predict properties of peptide-ions. Often such predicted properties are limited to molecular mass and fragment spectra, but here we put focus on a perhaps underutilized property, a peptide's chromatographic retention time. We review a couple of different principles of retention time prediction,and their applications within computational proteomics. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:615-623, 2017.


Memory training interventions for older adults: a meta-analysis.
A systematic review and meta-analysis of memory training research was conducted to characterize the effect of memory strategies on memory performance among cognitively intact, community-dwelling older adults, and to identify characteristics of individuals and of programs associated with improved memory. The review identified 402 publications, of which 35 studies met criteria for inclusion. The overall effect size estimate, representing the mean standardized difference in pre-post change between memory-trained and control groups, was 0.31 standard deviations (SD; 95% confidence interval (CI): 0.22, 0.39). The pre-post training effect for memory-trained interventions was 0.43 SD (95% CI: 0.29, 0.57) and the practice effect for control groups was 0.06 SD (95% CI: 0.05, 0.16). Among 10 distinct memory strategies identified in studies, meta-analytic methods revealed that training multiple strategies was associated with larger training gains (p=0.04), although this association did not reach statistical significance after adjusting for multiple comparisons. Treatment gains among memory-trained individuals were not better after training in any particular strategy, or by the average age of participants, session length, or type of control condition. These findings can inform the design of future memory training programs for older adults.


Generating genome-scale candidate gene lists for pharmacogenomics.
A critical task in pharmacogenomics is identifying genes that may be important modulators of drug response. High-throughput experimental methods are often plagued by false positives and do not take advantage of existing knowledge. Candidate gene lists can usefully summarize existing knowledge, but they are expensive to generate manually and may therefore have incomplete coverage. We have developed a method that ranks 12,460 genes in the human genome on the basis of their potential relevance to a specific query drug and its putative indications. Our method uses known gene-drug interactions, networks of gene-gene interactions, and available measures of drug-drug similarity. It ranks genes by building a local network of known interactions and assessing the similarity of the query drug (by both structure and indication) with drugs that interact with gene products in the local network. In a comprehensive benchmark, our method achieves an overall area under the curve of 0.82. To showcase our method, we found novel gene candidates for warfarin, gefitinib, carboplatin, and gemcitabine, and we provide the molecular hypotheses for these predictions.


Tumor vascular permeability to a nanoprobe correlates to tumor-specific expression levels of angiogenic markers.
Vascular endothelial growth factor (VEGF) receptor-2 is the major mediator of the mitogenic, angiogenic, and vascular hyperpermeability effects of VEGF on breast tumors. Overexpression of VEGF and VEGF receptor-2 is associated with the degree of pathomorphosis of the tumor tissue and unfavorable prognosis. In this study, we demonstrate that non-invasive quantification of the degree of tumor vascular permeability to a nanoprobe correlates with the VEGF and its receptor levels and tumor growth. We designed an imaging nanoprobe and a methodology to detect the intratumoral deposition of a 100 nm-scale nanoprobe using mammography allowing measurement of the tumor vascular permeability in a rat MAT B III breast tumor model. The tumor vascular permeability varied widely among the animals. Notably, the VEGF and VEGF receptor-2 gene expression of the tumors as measured by qRT-PCR displayed a strong correlation to the imaging-based measurements of vascular permeability to the 100 nm-scale nanoprobe. This is in good agreement with the fact that tumors with high angiogenic activity are expected to have more permeable blood vessels resulting in high intratumoral deposition of a nanoscale agent. In addition, we show that higher intratumoral deposition of the nanoprobe as imaged with mammography correlated to a faster tumor growth rate. This data suggest that vascular permeability scales to the tumor growth and that tumor vascular permeability can be a measure of underlying VEGF and VEGF receptor-2 expression in individual tumors. This is the first demonstration, to our knowledge, that quantitative imaging of tumor vascular permeability to a nanoprobe represents a form of a surrogate, functional biomarker of underlying molecular markers of angiogenesis.


Relationship of Human Immunodeficiency Virus Viral Load in Cerebrospinal Fluid and Plasma in Patients Co-infected With Cryptococcal Meningitis.
We measured human immunodeficiency virus (HIV) ribonucleic acid (RNA) in paired cerebrospinal fluid (CSF) and plasma samples in a prospective study of 91 HIV-infected, antiretroviral therapy-naive patients with cryptococcal meningitis. Cerebrospinal fluid HIV RNA was lower than in plasma (median 4.7 vs 5.2 log10 copies/mL, P < .0001) and positively correlated with plasma HIV RNA, peripheral CD4+ T-cell percentage, and CSF CXCL10. Plasma/CSF ratio of HIV RNA ranged widely from 0.2 to 265.5 with a median of 2.6. Cerebrospinal fluid quantitative cryptococcal culture positively correlated with CSF CCL2 and CCL3. CSF-plasma viral discordance was not associated with cryptococcal-associated immune reconstitution inflammatory syndrome.


Extraction Methods Affect the Structure of Goji (Lycium barbarum) Polysaccharides.
Polysaccharides are considered to be the most important active substances in Goji. However, the structure of polysaccharides varies according to the extraction methods applied, and the solution used to prepare Goji polysaccharides (LBPs) were limited. Thus, it is important to clarify the connection between extraction methods and structure of Goji polysaccharide. In view of the complex composition of cell wall polysaccharides and the various forms of interaction, different extraction methods will release different parts of the cell wall. The present study compared the effects of different extraction methods, which have been used to prepare different types of plant cell wall polysaccharides based on various sources, on the structure of cell-wall polysaccharides from Goji, by the single separate use of hot water, hydrochloric acid (0.4%) and sodium hydroxide (0.6%), at both high and low temperatures. Meanwhile, in order to explore the limitations of single extraction, sequential extraction methods were applied. Structural analysis including monosaccharide analysis, GPC-MALLS, AFM and 1H-NMR suggested the persistence of more extensively branched rhamnogalacturonan I (RG-I) domains in the procedures involving low-temperature-alkali, while procedures prepared by high-temperature-acid contains more homogalacturonan (HG) regions and results in the removal of a substantial part of the side chain, specifically the arabinan. A kind of acidic heteropolysaccharide was obtained by hot water extraction. SEC-MALLS and AFM confirmed large-size polymers with branched morphologies in alkali-extracted polysaccharides. Our results provide new insight into the extraction of Goji polysaccharides, which differ from the hot water extraction used by traditional Chinese medicine.


Histologic inflammation is a risk factor for progression to colorectal neoplasia in ulcerative colitis: a cohort study.
Although inflammation is presumed to contribute to colonic neoplasia in ulcerative colitis (UC), few studies have directly examined this relationship. Our aim was to determine whether severity of microscopic inflammation over time is an independent risk factor for neoplastic progression in UC. A cohort of patients with UC undergoing regular endoscopic surveillance for dysplasia was studied. Degree of inflammation at each biopsy site had been graded as part of routine clinical care using a highly reproducible histologic activity index. Progression to neoplasia was analyzed in proportional hazards models with inflammation summarized in 3 different ways and each included as a time-changing covariate: (1) mean inflammatory score (IS-mean), (2) binary inflammatory score (IS-bin), and (3) maximum inflammatory score (IS-max). Potential confounders were analyzed in univariate testing and, when significant, in a multivariable model. Of 418 patients who met inclusion criteria, 15 progressed to advanced neoplasia (high-grade dysplasia or colorectal cancer), and 65 progressed to any neoplasia (low-grade dysplasia, high-grade dysplasia, or colorectal cancer). Univariate analysis demonstrated significant relationships between histologic inflammation over time and progression to advanced neoplasia (hazard ration (HR), 3.0; 95% CI: 1.4-6.3 for IS-mean; HR, 3.4; 95% CI: 1.1-10.4 for IS-bin; and HR, 2.2; 95% CI: 1.2-4.2 for IS-max). This association was maintained in multivariable proportional hazards analysis. The severity of microscopic inflammation over time is an independent risk factor for developing advanced colorectal neoplasia among patients with long-standing UC.


What principles should guide visiting primary health care services in rural and remote communities? Lessons from a systematic review.
Visiting health services are a feature of health care delivery in rural and remote contexts. These services are often described as 'fly-in fly-out' or 'drive-in drive-out'. Posing the question 'What are the different types of visiting models of primary health care being used in rural and remote communities?', the objective of this article was to describe a typology of models of health services that visit remote communities. A systematic review of peer-reviewed literature from established databases was undertaken. Data were extracted from 20 papers (16 peer-reviewed papers and four from other sources), which met the inclusion criteria. From the available evidence, it was difficult to develop a typology of services. The central feature of service providers visiting rural and remote districts on a regular basis was consistent, although the service provider's geographical base varied and the extent to which the same service provider should be providing the service was not consistently endorsed. While a clear typology did not emerge from the systematic review, it became apparent that a set of guiding principles might be more helpful to service providers and planners. Focusing policy and decision-making on important principles of visiting services, rather than their typological features, is likely to be of ultimately more benefit to the health outcomes of people who live in rural and remote communities.


Cardiovascular Disease in Korean Americans: A Systematic Review.
Despite Korean Americans being one of the fastest growing immigrant groups in the United States, little is known about their cardiovascular health or cardiovascular disease risk factors. The purpose of this report is to describe the prevalence of cardiovascular disease risks and their contributing factors in Korean Americans and recommend future directions for the development of cardiovascular disease prevention or management research to meet the unique needs of this ethnic group. We conducted a systematic review using databases of PubMed, CINAHL, PsycINFO, Web of Science, and the Cochrane Database of Systematic Reviews and identified 27 studies that reported the prevalence of cardiovascular disease or its risk factors in Korean Americans, published in English between 2000 and 2016. We found high rates of unhealthy behaviors (eg, consumption of a high-sodium diet, physical inactivity, smoking) and risk factors (eg, hypertension, diabetes) for cardiovascular disease. Moreover, they were less likely to receive counseling about their diseases from healthcare providers and modify their lifestyle (eg, reduce their diet sodium intake, control their weight) to manage their diseases than were other ethnic populations. Individual-, interpersonal-, community-, and societal-level influences contributed to the high prevalence of cardiovascular risk factors. Data on subgroups of Asian Americans indicate that Korean Americans have significant lifestyle-related cardiovascular disease risks, which could be a critical agenda for researchers and clinicians to better understand cardiovascular health disparities in the United States.


Inhibition of cyclooxygenase-2 activity enhances steroidogenesis and steroidogenic acute regulatory gene expression in MA-10 mouse Leydig cells.
To study the mechanism for the regulatory effect of arachidonic acid (AA) on steroidogenesis, the role of cyclooxygenase (COX) in steroid production and steroidogenic acute regulatory (StAR) gene expression was investigated. Although stimulation with 0.05 mM dibutyryl cAMP (Bt(2)cAMP) did not increase StAR protein or progesterone in MA-10 mouse Leydig cells, the addition of 1 microM of the COX inhibitor indomethacin increased StAR protein expression and progesterone production by 5.7-fold and 34.3-fold, respectively. In the presence of indomethacin, the level of Bt(2)cAMP required for maximal steroidogenesis was reduced from 1.0 mM to 0.25 mM. Similar results were obtained in studies on StAR promoter activity and in Northern blot analyses of StAR mRNA expression, suggesting that inhibition of COX activity enhanced StAR gene transcription. COX2 (an inducible isoform of COX) was constitutively detected in MA-10 cells. Although SC560, a selective COX1 inhibitor, did not affect steroidogenesis, the COX2 inhibitor NS398 significantly enhanced Bt(2)cAMP-stimulated StAR protein expression and steroid production. Overexpression of the COX2 gene in COS-1 cells significantly inhibited StAR promoter activity. The results of the present study suggest that inhibition of COX2 activity increases the sensitivity of steroidogenesis to cAMP stimulation in MA-10 Leydig cells.


Iron acquisition by Helicobacter pylori: importance of human lactoferrin.
Helicobacter pylori is known to be an etiologic agent of gastritis and peptic ulcer disease in humans. However, the mechanism by which this organism acquires iron has not been studied. For this investigation, H. pylori was grown in iron-restricted medium. Siderophore production was not detected by chemical assays, and the strains were unable to use enterochelin and pyochelin for growth in low-iron media. Human lactoferrin supported full growth of the bacteria in media lacking other iron sources, but neither human transferrin, bovine lactoferrin, nor hen ovotransferrin served as a source for iron. Since lactoferrin was found in significant amounts in human stomach resections with superficial or atrophic gastritis, the iron acquisition system of H. pylori by the human lactoferrin receptor system may play a major role in the virulence of H. pylori infection.


Introduction to the EQIPD quality system.
While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.


Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli.
To continue our mutagenesis study of the polymerase active site of the Klenow fragment of DNA polymerase I, we have characterized new mutants with substitutions at Asp705, Glu710, and Glu883, and have investigated further the mutations in residues Arg668, Gln849, and Asp882, which showed strong Kcat effects in our previous study. To determine which step of the reaction is rate-limiting for each mutant protein, we measured the effect on the reaction rate of an alpha-thio-substituted dNTP. One group of mutants showed a substantial sulfur elemental effect, while a second group, like wild-type Klenow fragment, was unaffected by the phosphorothioate substitution. Consistent with earlier data, these results imply that, between formation of the enzyme-DNA-dNTP ternary complex and completion of phosphodiester bond formation, there are two kinetically distinct steps, only one of which is sensitive to sulfur substitution. The rather complex elemental effect data obtained with the mutant derivatives of Klenow fragment are more consistent with the elemental effect being the result of a steric clash in one of the transition states than with explanations based on electronegativity differences between sulfur and oxygen. The data suggest that the side chain of Asp882 is involved in the proposed steric clash, and that Gln849 and Glu883 participate in the sulfur-sensitive step of the reaction. Based on our results, and comparisons with other phosphoryl transfer enzymes, possible mechanisms for the polymerase reaction are discussed.


Implications for the practice of a patient expectation and satisfaction survey, at a teaching hospital in Karachi, Pakistan.
To study the expectations and satisfaction of patients visiting Family Practice Clinics, at the Aga Khan University Hospital, in Karachi, Pakistan. A cross sectional survey of 316 patients was carried out. A questionnaire, based on the study objectives was developed and administered. The participating patient signed a consent form, after assurance of confidentiality was provided. Data on the demographic profile of the patients was collected. The mean age was 33.81 years, with 105 (33.2%) women, and 211 (66.8%) men. The majority were married, with education above intermediate level and were in private or government service or were housewives. The median for the patient waiting time was 30 minutes, against an expectation of 12.69 minutes. Reading newspaper, watching television, reading magazine, reading Quran and listening to music were quoted as ways to lessen the burden of waiting to see a physician. The average consultation time with the physician was 13.89 minutes, against an expectation of 16.37 minutes. Patient expectation in terms of listening by the doctor with patience, explanation of the diagnosis and treatment, prescription of medicines, ordering of investigations and specialist referral has been documented. Objections to the presence of medical student, nursing student, resident doctor, nurse and an observer, in the consultation room have also been documented. Reasons quoted for the objection include issues of privacy/confidentiality, lack of justification, discomfort, and interference with the consultation process. The expected average cost for doctor's consultation was Pakistani Rs. 124, while 196 (61.8%) of the respondents were satisfied with the consultation based on Rs. 70. We have collected important information to improve the services offered at our Family Practice clinics.


Inflammation in atherosclerosis: transition from theory to practice.
Inflammation drives the formation, progression, and rupture of atherosclerotic plaques. Experimental studies have demonstrated that an inflammatory subset of monocytes/macrophages preferentially accumulate in atherosclerotic plaque and produce proinflammatory cytokines. T lymphocytes can contribute to inflammatory processes that promote thrombosis by stimulating production of collagen-degrading proteinases and the potent procoagulant tissue factor. Recent data link obesity, inflammation, and modifiers of atherosclerotic events, a nexus of growing clinical concern given the worldwide increase in the prevalence of obesity. Modulators of inflammation derived from visceral adipose tissue evoke production of acute phase reactants in the liver, implicated in thrombogenesis and clot stability. Additionally, C-reactive protein levels rise with increasing levels of visceral adipose tissue. Adipose tissue in obese mice contains increased numbers of macrophages and T lymphocytes, increased T lymphocyte activation, and increased interferon-gamma (IFN-gamma) expression. IFN-gamma deficiency in mice reduces production of inflammatory cytokines and inflammatory cell accumulation in adipose tissue. Another series of in vitro and in vivo mouse experiments affirmed that adiponectin, an adipocytokine, the plasma levels of which drop with obesity, acts as an endogenous antiinflammatory modulator of both innate and adaptive immunity in atherogenesis. Thus, accumulating experimental evidence supports a key role for inflammation as a link between risk factors for atherosclerosis and the biology that underlies the complications of this disease. The recent JUPITER trial supports the clinical utility of an assessment of inflammatory status in guiding intervention to limit cardiovascular events. Inflammation is thus moving from a theoretical concept to a tool that provides practical clinical utility in risk assessment and targeting of therapy.


Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs.
To provide context for the diversification of archosaurs--the group that includes crocodilians, dinosaurs, and birds--we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs.


Context-specific sensitization of cocaine-induced locomotor activity and associated neuronal ensembles in rat nucleus accumbens.
Repeated cocaine administration to rats outside their home cage induces behavioral sensitization that is strongly modulated by the drug administration environment. We hypothesized that stimuli in the drug administration environment activate specific sets of striatal neurons, called neuronal ensembles, for further cocaine-enhanced activation, and that repeated activation of these neuronal ensembles underlies context-specific sensitization. In the present study, we repeatedly administered cocaine or saline to rats on alternate days in two distinct environments outside the home cage, one paired with cocaine and the other with saline. On test day, cocaine challenge injections in the cocaine-paired environment produced strongly enhanced levels of locomotor activity, while cocaine challenge injections in the saline-paired environment did not. The corresponding record of past neuronal activation in nucleus accumbens and caudate-putamen during repeated drug administration was assessed using FosB immunohistochemistry, while acute neuronal activation on test day was assessed using c-fos in situ hybridization. Although only 2% of striatal neurons were FosB labeled, 87% of these FosB-labeled neurons were co-labeled with c-fos when cocaine was injected in the cocaine-paired environment. The degree of co-labeling was significantly less following cocaine or saline challenge injections in the saline-paired environment. Furthermore, the total number of c-fos-labeled neurons was greater with either cocaine or saline challenge injections in the cocaine-paired environment than in the saline-paired environment. These findings demonstrate that the drug administration environment partly determines which striatal neuronal ensembles are activated, and to what extent, following context-specific sensitization to cocaine.


Sleep patterns and sleep-impairing factors of persons providing informal care for people with cancer: a critical review of the literature.
Sleep is increasingly recognized as an area of functioning that may be greatly affected in persons who are practically and emotionally involved in the care of patients with cancer. Clinician awareness is required to ensure that effective care for informal caregivers with sleep problems is provided. A 2-fold critical review of the published literature was conducted, which aimed at summarizing and critically analyzing evidence regarding sleep patterns of informal caregivers of adults with cancer and contributing factors to sleep-wake disturbances. Using a wide range of key terms and synonyms, 3 electronic databases (MEDLINE, CINAHL, EMBASE) were systematically searched for the period between January 1990 and July 2011. Based on prespecified selection criteria, 44 articles were pooled to provide evidence on sleep-impairing factors in the context of informal caregiving, 17 of which specifically addressed sleep patterns of caregivers of people with cancer. At least 4 of 10 caregivers may report at least 1 sleep problem. Short sleep duration, nocturnal awakenings, wakefulness after sleep onset, and daytime dysfunction seem to be the areas most affected irrespective of stage or type of disease, yet circadian activity remains understudied. In addition, despite a wide spectrum of potential sleep-impairing factors, underlying causal pathways are yet to be explored. More longitudinal, mixed-methods, and comparison studies are warranted to explore caregiver sleep disorders in relation to the gravity of the caregiving situation in the context of diverse types of cancer and disease severity.


The Relationship between Life Course Socioeconomic Conditions and Objective and Subjective Memory in Older Age.
While objective memory performance in older adults was primarily shown to be affected by education as indicator of life course socioeconomic conditions, other life course socioeconomic conditions seem to relate to subjective memory complaints. However, studies differ in which life course stages were investigated. Moreover, studies have explored these effects in an isolated way, but have not yet investigated their unique effect when considering several stages of the life course simultaneously. This study, therefore, examined the respective influence of socioeconomic conditions from childhood up to late-life on prospective memory (PM) performance as an objective indicator of everyday memory as well as on subjective memory complaints (SMC) in older age using structural equation modeling. Data came from two waves of the Vivre-Leben-Vivere aging study (n=993, Mage=80.56). The results indicate that only socioeconomic conditions in adulthood significantly predicted late-life PM performance. PM performance was also predicted by age and self-rated health. In contrast, SMC in older age were not predicted by socioeconomic conditions at any stage of the life course but were predicted by level of depression. In line with the cognitive reserve hypothesis, present results highlight the significance of education and occupation (adulthood socioeconomic conditions) for cognitive functioning in later life.


Bioactive compounds, antioxidant and antimicrobial activities of extracts from different plant parts of two Ziziphus Mill. species.
Ziziphus lotus L. (Lam.) and Z. mauritiana Lam., as a widespread species in Tunisia, are well known for their medicinal and food uses. The aim of the present study was to screen the content of total polyphenols, flavonoids, and condensed tannins together with the radical scavenging capacity and the antimicrobial activity of leaves, fruits and seeds extracts of Z. lotus and Z. mauritiana from different localities. Results showed that leaves extracts presented the highest phenolic compounds content for both species. Furthermore, LC-ESI-MS analysis allowed the identification of 28 bioactive compounds regardless of species and organs, with the predominance of quinic acid and rutin. Leaves extract of Z. mauritiana possessed the highest total antioxidant capacity. The antimicrobial tests showed that leaves extracts of Z. mauritiana and Z. lotus from Oued Esseder exhibited the highest activity against four bacterial strains (Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Escherichia coli). The main results showed that the studied species of Ziziphus genus are an excellent source of natural bioactive molecules that could be an interesting material for industrial and food purposes.


Structural and biochemical approaches uncover multiple evolutionary trajectories of plant quinate dehydrogenases.
Quinate is produced and used by many plants in the biosynthesis of chlorogenic acids (CGAs). Chlorogenic acids are astringent and serve to deter herbivory. They also function as antifungal agents and have potent antioxidant properties. Quinate is produced at a branch point of shikimate biosynthesis by the enzyme quinate dehydrogenase (QDH). However, little information exists on the identity and biochemical properties of plant QDHs. In this study, we utilized structural and bioinformatics approaches to establish a QDH-specific primary sequence motif. Using this motif, we identified QDHs from diverse plants and confirmed their activity by recombinant protein production and kinetic assays. Through a detailed phylogenetic analysis, we show that plant QDHs arose directly from bifunctional dehydroquinate dehydratase-shikimate dehydrogenases (DHQD-SDHs) through different convergent evolutionary events, illustrated by our findings that eudicot and conifer QDHs arose early in vascular plant evolution whereas Brassicaceae QDHs emerged later. This process of recurrent evolution of QDH is further demonstrated by the fact that this family of proteins independently evolved NAD+ and NADP+ specificity in eudicots. The acquisition of QDH activity by these proteins was accompanied by the inactivation or functional evolution of the DHQD domain, as verified by enzyme activity assays and as reflected in the loss of key DHQD active site residues. The implications of QDH activity and evolution are discussed in terms of plant growth and development.