Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension.
Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function.


Population-Based Long-Term Cardiac-Specific Mortality Among 34 489 Five-Year Survivors of Childhood Cancer in Great Britain.
Increased risks of cardiac morbidity and mortality among childhood cancer survivors have been described previously. However, little is known about the very long-term risks of cardiac mortality and whether the risk has decreased among those more recently diagnosed. We investigated the risk of long-term cardiac mortality among survivors within the recently extended British Childhood Cancer Survivor Study. The British Childhood Cancer Survivor Study is a population-based cohort of 34 489 five-year survivors of childhood cancer diagnosed from 1940 to 2006 and followed up until February 28, 2014, and is the largest cohort to date to assess late cardiac mortality. Standardized mortality ratios and absolute excess risks were used to quantify cardiac mortality excess risk. Multivariable Poisson regression models were used to evaluate the simultaneous effect of risk factors. Likelihood ratio tests were used to test for heterogeneity and trends. Overall, 181 cardiac deaths were observed, which was 3.4 times that expected. Survivors were 2.5 times and 5.9 times more at risk of ischemic heart disease and cardiomyopathy/heart failure death, respectively, than expected. Among those >60 years of age, subsequent primary neoplasms, cardiac disease, and other circulatory conditions accounted for 31%, 22%, and 15% of all excess deaths, respectively, providing clear focus for preventive interventions. The risk of both overall cardiac and cardiomyopathy/heart failure mortality was greatest among those diagnosed from 1980 to 1989. Specifically, for cardiomyopathy/heart failure deaths, survivors diagnosed from 1980 to 1989 had 28.9 times the excess number of deaths observed for survivors diagnosed either before 1970 or from 1990 on. Excess cardiac mortality among 5-year survivors of childhood cancer remains increased beyond 50 years of age and has clear messages in terms of prevention strategies. However, the fact that the risk was greatest in those diagnosed from 1980 to 1989 suggests that initiatives to reduce cardiotoxicity among those treated more recently may be having a measurable impact.


Nutritional intervention in COPD: a systematic overview.
We conducted a systematic overview of randomized controlled trials (RCTs) to clarify the contribution of nutritional supplementation for patients with stable COPD. RCTs were identified from several sources, including the Cochrane Airways Group register of RCTs, a hand search of abstracts presented at international meetings, and consultation with experts. Two reviewers independently selected trials for inclusion, assessed quality, and extracted the data. Twenty-one reports were classified according to the type, duration of supplementation, and the presence of anabolic substances. High carbohydrate meals were associated with an increase in carbon dioxide production and a decrease in exercise capacity. Short-term crossover studies in which diets of various compositions were administered supported the notion that high carbohydrate loads increase the stress on the ventilatory system. The influence of longer-term supplementation (> 2 weeks) on weight, anthropometry, and exercise capacity varied, without there being a consistent effect. Lean body weight was only occasionally reported and health-related quality of life too rarely to be included as an outcome. The influence of recombinant human growth hormone was disappointing. Anabolic steroids increased body weight and lean body mass, but had little influence on exercise capacity. This systematic overview in patients with COPD supports the notion that those with marginal ventilatory reserve might benefit from a dietary regimen in which a high percentage of calories are supplied by fat. Although there are reports of the benefits of nutritional repletion, trials of > 2 weeks failed to show consistent benefit on body weight. Evaluating nutritional repletion is hampered by the absence of information regarding body composition, exercise, and health-related quality of life. Growth hormone has not been shown to be useful. Further studies are needed to refine the beneficial effects of anabolic steroids as adjunctive agents together with nutritional support and exercise.


Positive reinforcement mediated by midbrain dopamine neurons requires D1 and D2 receptor activation in the nucleus accumbens.
The neural basis of positive reinforcement is often studied in the laboratory using intracranial self-stimulation (ICSS), a simple behavioral model in which subjects perform an action in order to obtain exogenous stimulation of a specific brain area. Recently we showed that activation of ventral tegmental area (VTA) dopamine neurons supports ICSS behavior, consistent with proposed roles of this neural population in reinforcement learning. However, VTA dopamine neurons make connections with diverse brain regions, and the specific efferent target(s) that mediate the ability of dopamine neuron activation to support ICSS have not been definitively demonstrated. Here, we examine in transgenic rats whether dopamine neuron-specific ICSS relies on the connection between the VTA and the nucleus accumbens (NAc), a brain region also implicated in positive reinforcement. We find that optogenetic activation of dopaminergic terminals innervating the NAc is sufficient to drive ICSS, and that ICSS driven by optical activation of dopamine neuron somata in the VTA is significantly attenuated by intra-NAc injections of D1 or D2 receptor antagonists. These data demonstrate that the NAc is a critical efferent target sustaining dopamine neuron-specific ICSS, identify receptor subtypes through which dopamine acts to promote this behavior, and ultimately help to refine our understanding of the neural circuitry mediating positive reinforcement.


Ferrous-iron-dependent uptake of L-glutamate by a mesophilic, mixotrophic iron-oxidizing bacterium strain OKM-9.
Strain OKM-9 is a mesophilic, mixotrophic iron-oxidizing bacterium that absolutely requires ferrous iron as its energy source and L-amino acids (including L-glutamate) as carbon sources for growth. The properties of the L-glutamate transport system were studied with OKM-9 resting cells, plasma membranes, and actively reconstituted proteoliposomes. L-Glutamate uptake into resting cells was totally dependent on ferrous iron that was added to the reaction mixture. Potassium cyanide, an iron oxidase inhibitor, completely inhibited the activity at 1 mM. The optimum pH for Fe2+-dependent uptake activity of L-glutamate was 3.5-4.0. Uptake activity was dependent on the concentration of the L-glutamate. The Km and Vmax for L-glutamate were 0.4 mM and 11.3 nmol x min(-1) x mg(-1), respectively. L-Aspartate, D-aspartate, D-glutamate, and L-cysteine strongly inhibited L-glutamate uptake. L-Aspartate competitively inhibited the activity, and the apparent Ki for this amino acid was 75.9 microM. 2,4-Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, gramicidin D, valinomycin, and monensin did not inhibit Fe2+-dependent L-glutamate uptake. The OKM-9 plasma membranes had approximately 40% of the iron-oxidizing activity of the resting cells and approximately 85% of the Fe2+-dependent uptake activity. The glutamate transport system was solubilized from the membranes with 1% n-octyl-beta-D-glucopyranoside and reconstituted into a lecithin liposome. The L-glutamate transport activity of the reconstituted proteoliposomes was 8-fold than that of the resting cells. The Fe2+-dependent L-glutamate uptake observed here seems to explain the mixotrophic nature of this strain, which absolutely requires Fe2+ oxidation when using amino acids as carbon sources.


Choroidal melanoma overlying scleral buckle evading detection treated with proton beam radiotherapy.
to present a case of a choroidal melanoma located along a scleral buckle contour and was successfully treated with proton beam radiotherapy, without disturbing the scleral buckle. A 60-year-old woman presented with photopsias and history of retinal detachment repair with scleral buckle 36 years prior. She had annual dilated exams with her ophthalmologist over the last several years. While initially diagnosis of choroidal detachment, hemorrhage and intraocular buckle intrusion were suspected, the patient was referred to an ophthalmic oncology center and choroidal melanoma was confirmed. The patient underwent tantalum marker placement, trans-retinal biopsy and proton beam radiotherapy. Despite no evidence of metastatic disease on systemic imaging, prognostic genetic testing of the tumor revealed high-risk alterations for future metastasis, and the patient enrolled in an adjuvant clinical trial. The location of this choroidal melanoma along a scleral buckle contour presented a unique therapeutic challenge. We successfuly treated the tumor with proton beam radiotherapy without disturbing the scleral buckle. chorioretinal elevations adjacent a scleral buckle may be presumed to be due to the buckle element; ultrasound can be helpful in distinguishing an intraocular tumor. Proton beam radiotherapy has the advantage over plaque brachytherapy in this setting as removing the scleral buckle is not needed and placing a plaque over the scleral buckle risks altering the effective radiotherapy dose.


Genomic Classification and Prognosis in Acute Myeloid Leukemia.
Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice. We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes. We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials. The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).


Modular prophage interactions driven by capsule serotype select for capsule loss under phage predation.
Klebsiella species are able to colonize a wide range of environments and include worrisome nosocomial pathogens. Here, we sought to determine the abundance and infectivity of prophages of Klebsiella to understand how the interactions between induced prophages and bacteria affect population dynamics and evolution. We identified many prophages in the species, placing these taxa among the top 5% of the most polylysogenic bacteria. We selected 35 representative strains of the Klebsiella pneumoniae species complex to establish a network of induced phage-bacteria interactions. This revealed that many prophages are able to enter the lytic cycle, and subsequently kill or lysogenize closely related Klebsiella strains. Although 60% of the tested strains could produce phages that infect at least one other strain, the interaction network of all pairwise cross-infections is very sparse and mostly organized in modules corresponding to the strains' capsule serotypes. Accordingly, capsule mutants remain uninfected showing that the capsule is a key factor for successful infections. Surprisingly, experiments in which bacteria are predated by their own prophages result in accelerated loss of the capsule. Our results show that phage infectiousness defines interaction modules between small subsets of phages and bacteria in function of capsule serotype. This limits the role of prophages as competitive weapons because they can infect very few strains of the species complex. This should also restrict phage-driven gene flow across the species. Finally, the accelerated loss of the capsule in bacteria being predated by their own phages, suggests that phages drive serotype switch in nature.


Significance of FHIT expression in chronic myelogenous leukemia.
Loss or reduced expression of the fragile histidine triad (FHIT) gene, a tumor suppressor gene localized at chromosome 3p14.2, is common in several solid and hematological cancers and has been associated with tumor progression and worse prognosis. The role of the FHIT gene in the pathogenesis of chronic myelogenous leukemia (CML) or its progression from a chronic phase to the accelerated and blastic phases is not known. The aim of this study was to evaluate whether Fhit protein expression is altered in CML, and whether it plays any role in CML progression, disease responsiveness to therapy, or prognosis. A total of 195 patients with Philadelphia chromosome-positive CML were evaluated, including 129 patients in early chronic phase (time from diagnosis to study, 12 months or less), 30 patients in late chronic phase, and 36 patients in the accelerated and blastic phases. The levels of cellular Fhit protein expression were determined using Western blot analysis and solid-phase RIA and compared to the levels in 31 normal marrows. The median Fhit expression in normal marrows was assigned a value of 1, and the levels in CML samples were normalized to the median of the normal control. Fhit levels in CML samples were evaluated in relation to CML phase and patient characteristics and prognosis in the early chronic phase. The median Fhit value in CML samples was 0.89 (range, 0.34-2.62). Eight of the 195 (4%) CML samples showed Fhit levels <0.5 and lacked detectable Fhit protein by Western blot. There was no difference in the levels of Fhit expression by different CML phases. In early chronic phase, reduced Fhit expression tended to be associated with leukocytosis (P = 0.04) and lower platelet counts (P = 0.01), but not with poorer-risk groups. No differences in response to IFN-alpha therapy or in survival were observed by different Fhit levels. Lack of Fhit protein expression was detected in 4% of CML cases, and reduced expression occurred in a subpopulation of patients. However, reduced Fhit expression is not associated with progression, response to therapy, or prognosis in CML.


Treatment of male partners and recurrence of bacterial vaginosis: a randomised trial.
To test the efficacy of treatment with clindamycin of a partner on the recurrence rate of bacterial vaginosis in women within 3 months from diagnosis. Eligible for the study were sexually active women with one current sexual partner, who were aged 18-45 years, with a clinical diagnosis of bacterial vaginosis and whose partner agreed to be treated. A double blind, randomised, controlled trial was conducted comparing the effect of treating the partner with either clindamycin capsules or placebo on the reduction of the recurrence rate of bacterial vaginosis. Women were treated with clindamycin 2% vaginal cream, administered intravaginally once daily at bedtime for 7 consecutive days. The partners were randomly allocated to clindamycin hydrochloride capsules, 150 g by mouth four times daily for 7 consecutive days, or a placebo. A total of 139 couples were randomised--69 were treated with clindamycin vaginal cream group and 70 with placebo. One, 4, and 12 weeks after the end of treatment the patients and their partners were examined; vaginal discharges were examined to check for clue cells, vaginal pH was determined, and a KOH test carried out. Overall, 131 women out of the 139 who entered the study were cured (94.2%, lower 95% confidence interval 79.8, based on Poisson's approximation). There was no difference in the cure rate among women whose partner received clindamycin or placebo (chi(2) p = not significant). A total of 55 couples (26 in the clindamycin and 29 in the placebo group) withdrew from the study during the follow up period. Of the 69 women whose partner received clindamycin, 22 (31.9%) reported "recurrence" or persistence. The corresponding number was 21 (30%) of the 70 women whose partner received placebo (chi(2) p = not significant). Of the 84 couples in which the woman was cured by the first week's visit and who completed the study; there were five recurrences (11.6%) among the 43 women whose partner received clindamycin and nine (22.0%) of the 41 whose partner received placebo (chi(2) p = not significant). This study indicates that vaginal clindamycin is effective and safe in the treatment of bacterial vaginosis, but it does not support the suggestion that male treatment markedly reduces the short term recurrence rate.


Ubiquitin Ligases and Posttranslational Regulation of Energy in the Heart: The Hand that Feeds.
Heart failure (HF) is a costly and deadly syndrome characterized by the reduced capacity of the heart to adequately provide systemic blood flow. Mounting evidence implicates pathological changes in cardiac energy metabolism as a contributing factor in the development of HF. While the main source of fuel in the healthy heart is the oxidation of fatty acids, in the failing heart the less energy efficient glucose and glycogen metabolism are upregulated. The ubiquitin proteasome system plays a key role in regulating metabolism via protein-degradation/regulation of autophagy and regulating metabolism-related transcription and cell signaling processes. In this review, we discuss recent research that describes the role of the ubiquitin-proteasome system (UPS) in regulating metabolism in the context of HF. We focus on ubiquitin ligases (E3s), the component of the UPS that confers substrate specificity, and detail the current understanding of how these E3s contribute to cardiac pathology and metabolism. © 2017 American Physiological Society. Compr Physiol 7:841-862, 2017.


Cell membrane expression of cardiac sodium channel Na(v)1.5 is modulated by alpha-actinin-2 interaction.
Cardiac sodium channel Na(v)1.5 plays a critical role in heart excitability and conduction. The molecular mechanism that underlies the expression of Na(v)1.5 at the cell membrane is poorly understood. Previous studies demonstrated that cytoskeleton proteins can be involved in the regulation of cell surface expression and localization of several ion channels. We performed a yeast two-hybrid screen to identify Na(v)1.5-associated proteins that may be involved in channel function and expression. We identified alpha-actinin-2 as an interacting partner of the cytoplasmic loop connecting domains III and IV of Na(v)1.5 (Na(v)1.5/LIII-IV). Co-immunoprecipitation and His(6) pull-down assays confirmed the physical association between Na(v)1.5 and alpha-actinin-2 and showed that the spectrin-like repeat domain is essential for binding of alpha-actinin-2 to Na(v)1.5. Patch-clamp studies revealed that the interaction with alpha-actinin-2 increases sodium channel density without changing their gating properties. Consistent with these findings, coexpression of alpha-actinin-2 and Na(v)1.5 in tsA201 cells led to an increase in the level of expression of Na(v)1.5 at the cell membrane as determined by cell surface biotinylation. Lastly, immunostaining experiments showed that alpha-actinin-2 was colocalized with Na(v)1.5 along the Z-lines and in the plasma membrane. Our data suggest that alpha-actinin-2, which is known to regulate the functional expression of the potassium channels, may play a role in anchoring Na(v)1.5 to the membrane by connecting the channel to the actin cytoskeleton network.


Late collapse of distal radius fractures after K-wire removal: is it significant?
The aim of this study was to find out whether distal radius fractures treated by Kirschner wire (K wire) fixation loose reduction after wire removal and analyze the variables may influence this. Patients who underwent K wire fixation for unstable fractures of distal radius over a period of 3 years were included in this retrospective study. Fractures were classified according to AO classification. Radiographs taken just prior to removal of K wires and radiographs taken at least 1 month after wire removal were analyzed to study three radiological parameters; Palmar or dorsal tilt, radial inclination and ulnar variance. Loss of these angles was analyzed statistically against variables like age, sex, AO classification and duration of fixation. 59 fractures were analyzed with mean age of 56 years and male to female ratio of 1:2. Average loss of radial tilt was 2.6 degrees , loss of palmar tilt was 2.6 degrees and loss of ulnar variance was 1.3 mm. We found that distal radius fractures treated by percutaneous K wire fixation, did not suffer significant loss of reduction of fracture position after removal of wires. This remains true regardless of age, sex, fracture type according to AO type or duration of wire fixation.


Connecting empirical phenomena and theoretical models of biological coordination across scales.
Coordination in living systems-from cells to people-must be understood at multiple levels of description. Analyses and modelling of empirically observed patterns of biological coordination often focus either on ensemble-level statistics in large-scale systems with many components, or on detailed dynamics in small-scale systems with few components. The two approaches have proceeded largely independent of each other. To bridge this gap between levels and scales, we have recently conducted a human experiment of mid-scale social coordination specifically designed to reveal coordination at multiple levels (ensemble, subgroups and dyads) simultaneously. Based on this experiment, the present work shows that, surprisingly, a single system of equations captures key observations at all relevant levels. It also connects empirically validated models of large- and small-scale biological coordination-the Kuramoto and extended Haken-Kelso-Bunz (HKB) models-and the hallmark phenomena that each is known to capture. For example, it exhibits both multistability and metastability observed in small-scale empirical research (via the second-order coupling and symmetry breaking in extended HKB) and the growth of biological complexity as a function of scale (via the scalability of the Kuramoto model). Only by incorporating both of these features simultaneously can we reproduce the essential coordination behaviour observed in our experiment.


Greek-origin royal jelly improves the lipid profile of postmenopausal women.
Menopause transition is associated with chronic conditions such as osteoporosis and cardiovascular disease. Concerns about the long-term safety of menopausal hormone therapy make alternative natural methods an appealing approach to management. The aim of this study was to examine the effect of royal jelly (RJ) on cardiovascular and bone turnover markers in clinically healthy postmenopausal women. A total of 36 postmenopausal healthy women were studied in a prospective follow-up study. Participants received 150 mg of RJ daily for three months. Circulating cardiovascular risk markers [lipid profile, antithrombin-III (ATIII), Protein C, Protein S, Plasminogen Activator Inhibitor-1 (PAI-1)] and bone turnover parameters [Total calcium, phosphate (P), parathormone (PTH), total type-1 Procollagen N-terminal (P1NP), Osteocalcin and serum collagen type 1 cross-linked C-telopeptide (CTX)] were compared between the baseline and the three-month visit. The RJ used in this study was particularly rich in medium chain fatty acids, compounds with hypolipidemic properties, which comprised 63% of the dry weight fatty content. RJ treatment resulted in a significant increase in high density lipoprotein - cholesterol (HDL-C 60.2 mg/dL ± 12.3 versus 64.7 mg/dL ± 13.9, 7.7% increase, p = 0.0003), as well as in a significant decrease in low density lipoprotein - cholesterol (LDL-C, 143.9 ± 37.5 versus 136.2 ± 32, 4.1% decrease, p = 0.011) and in total cholesterol (224.4 ± 38.6 to 216.1 ± 36.5, 3.09% decrease, p = 0.018). No statistical significant changes were found in the remaining cardiovascular or the bone turnover parameters. The intake of RJ 150 mg for three months is associated with significant improvements of the lipid profile of postmenopausal women. RJ supplementation may offer an alternative method of controlling the menopause - associated dyslipidemia.


Early alcohol use and problem drinking among students in Zambia and Uganda.
Excessive alcohol use is a serious public health concern worldwide, but less attention has been given to the prevalence, risk and protective factors, and consequences of early alcohol use in low-income, developing countries. The purpose of this study was to determine the associations between early alcohol use, before age 13, and problem drinking among adolescents in Uganda and Zambia. Data from students in Zambia (n=2257; 2004) and Uganda (n=3215; 2003) were obtained from the cross-sectional Global School-Based Student Health Survey (GSHS). The self-administered questionnaires were completed by students primarily 13 to 16 years of age. Multiple statistical models were computed using logistic regression analyses to test the associations between early alcohol initiation and problem drinking, while controlling for possible confounding factors (e.g., current alcohol use, bullying victimization, sadness, lack of friends, missing school, lack of parental monitoring, and drug use). Results show that early alcohol initiation was associated with problem drinking in both Zambia (AOR=1.28; 95% CI:1.02-1.61) and Uganda (AOR=1.48; 95% CI: 1.11-1.98) among youth after controlling for demographic characteristics, risky behaviors, and other possible confounders.The study shows that there is a significant association between alcohol initiation before 13 years of age and problem drinking among youth in these two countries. These findings underscore the need for interventions and strict alcohol controls as an important policy strategy for reducing alcohol use and its dire consequences among vulnerable youth.


Comorbidity, age, race and stage at diagnosis in colorectal cancer: a retrospective, parallel analysis of two health systems.
Stage at diagnosis plays a significant role in colorectal cancer (CRC) survival. Understanding which factors contribute to a more advanced stage at diagnosis is vital to improving overall survival. Comorbidity, race, and age are known to impact receipt of cancer therapy and survival, but the relationship of these factors to stage at diagnosis of CRC is less clear. The objective of this study is to investigate how comorbidity, race and age influence stage of CRC diagnosis. Two distinct healthcare populations in the United States (US) were retrospectively studied. Using the Cancer Care Outcomes Research and Surveillance Consortium database, we identified CRC patients treated at 15 Veterans Administration (VA) hospitals from 2003-2007. We assessed metastatic CRC patients treated from 2003-2006 at 10 non-VA, fee-for-service (FFS) practices. Stage at diagnosis was dichotomized (non-metastatic, metastatic). Race was dichotomized (white, non-white). Charlson comorbidity index and age at diagnosis were calculated. Associations between stage, comorbidity, race, and age were determined by logistic regression. 342 VA and 340 FFS patients were included. Populations differed by the proportion of patients with metastatic CRC at diagnosis (VA 27% and FFS 77%) reflecting differences in eligibility criteria for inclusion. VA patients were mean (standard deviation; SD) age 67 (11), Charlson index 2.0 (1.0), and were 63% white. FFS patients were mean age 61 (13), Charlson index 1.6 (1.0), and were 73% white. In the VA cohort, higher comorbidity was associated with earlier stage at diagnosis after adjusting for age and race (odds ratio (OR) 0.76, 95% confidence interval (CI) 0.58-1.00; p = 0.045); no such significant relationship was identified in the FFS cohort (OR 1.09, 95% CI 0.82-1.44; p = 0.57). In both cohorts, no association was found between stage at diagnosis and either age or race. Higher comorbidity may lead to earlier stage of CRC diagnosis. Multiple factors, perhaps including increased interactions with the healthcare system due to comorbidity, might contribute to this finding. Such increased interactions are seen among patients within a healthcare system like the VA system in the US versus sporadic interactions which may be seen with FFS healthcare.


Gating a channel photomultiplier with a fast high-voltage switch: reduction of afterpulse rates in a laser-induced fluorescence instrument for measurement of atmospheric OH radical concentrations.
By employing a commercially available high-voltage switch in a time-gating circuit to drive a channel photomultiplier (CPM), the afterpulse rates are significantly reduced in the time window to collect fluorescence >200 ns after the pulsed laser excitation. The CPM, kept deactivated under normal conditions (normally off), is turned on immediately after the passage of the laser pulse by shifting the voltage applied to the photocathode by 150 V to collect the fluorescence. When the detection system is used as part of a laser-induced fluorescence instrument to measure atmospheric OH radicals with the photon-counting method, the background signal is reduced by more than a factor of 10 as compared with our previous case where a conventional dynode-gated photomultiplier tube (PMT) is used, while the sensitivity toward the fluorescence is almost unchanged. A detection limit as low as 2 x 10(5) radicals cm-3 or 0.008 parts per trillion by volume is achieved for OH, with an integration time of 1 min and a signal-to-noise ratio of 2, enabling sensitive detection of the important radical in the atmosphere. This system is a superior choice with higher sensitivity and cost effectiveness as compared with the gated PMITs utilizing a microchannel plate as an electron multiplier, and could also be used effectively in light detection and ranging (lidar) instruments, where a delayed scattering signal would be efficiently discriminated from afterpulses.


Magnetic resonance imaging anatomy of the female genitalia in premenopausal and postmenopausal women.
We used contrast enhanced magnetic resonance imaging (MRI) of the female genital organs to describe normal anatomy and differences between premenopausal and postmenopausal women. A total of 12 healthy premenopausal and 9 healthy postmenopausal women underwent MRI. A 1.5 Tesla system with phased array coils anterior and posterior to the pelvis was used to produce T1-weighted images before and after intravenous administration of MS-325 (Epix Medical, Cambridge, Massachusetts), a new gadolinium based blood pool contrast agent. Select structural dimensions were measured for reference. The clitoris and vestibular bulbs were well delineated on T1-weighted post-contrast images. The clitoral unit formed a brightly enhancing, wishbone-shaped structure lying just anterior to the inverted V of the bulbs, which surrounded the urethra and vagina. The urethral complex had a target-like appearance with layers that were discernible on T1 post-contrast images. The urethra, vagina and rectum formed a distinct complex within uniformly enhancing soft tissue. The vagina was well visualized in premenopausal subjects but without distinguishable mucosal rugae or clearly separate layers in postmenopausal subjects. Postmenopausal subjects were also observed to have smaller labia minora width, vestibular bulb width, vaginal width and wall thickness, and cervical diameter. Pelvic and genital structures were not well visualized on T1 noncontrast images. To our knowledge we describe detailed female genital anatomy for the first time using MRI with MS-325 contrast medium. The clitoris, vestibular bulbs, labia majora and minora, urethra, vagina, cervix and rectum are well visualized on T1 post-contrast images. The observed genital anatomy on MRI was consistent with descriptions in current anatomical texts. Differences in the female genitalia between premenopausal and postmenopausal women were discernible on MRI. These data are important for future studies using MRI for evaluating anatomical anomalies, postoperative changes and female sexual function.


MISC-1/OGC links mitochondrial metabolism, apoptosis and insulin secretion.
We identified MISC-1 (Mitochondrial Solute Carrier) as the C. elegans orthologue of mammalian OGC (2-oxoglutarate carrier). OGC was originally identified for its ability to transfer α-ketoglutarate across the inner mitochondrial membrane. However, we found that MISC-1 and OGC are not solely involved in metabolic control. Our data show that these orthologous proteins participate in phylogenetically conserved cellular processes, like control of mitochondrial morphology and induction of apoptosis. We show that MISC-1/OGC is required for proper mitochondrial fusion and fission events in both C. elegans and human cells. Transmission electron microscopy reveals that loss of MISC-1 results in a decreased number of mitochondrial cristae, which have a blebbed appearance. Furthermore, our pull-down experiments show that MISC-1 and OGC interact with the anti-apoptotic proteins CED-9 and Bcl-x(L), respectively, and with the pro-apoptotic protein ANT. Knock-down of misc-1 in C. elegans and OGC in mouse cells induces apoptosis through the caspase cascade. Genetic analysis suggests that MISC-1 controls apoptosis through the physiological pathway mediated by the LIN-35/Rb-like protein. We provide genetic and molecular evidence that absence of MISC-1 increases insulin secretion and enhances germline stem cell proliferation in C. elegans. Our study suggests that the mitochondrial metabolic protein MISC-1/OGC integrates metabolic, apoptotic and insulin secretion functions. We propose a novel mechanism by which mitochondria integrate metabolic and cell survival signals. Our data suggest that MISC-1/OGC functions by sensing the metabolic status of mitochondria and directly activate the apoptotic program when required. Our results suggest that controlling MISC-1/OGC function allows regulation of mitochondrial morphology and cell survival decisions by the metabolic needs of the cell.


NADiA ProsVue prostate-specific antigen slope is an independent prognostic marker for identifying men at reduced risk of clinical recurrence of prostate cancer after radical prostatectomy.
To validate the hypothesis that men displaying serum prostate-specific antigen (PSA) slopes ≤ 2.0 pg/mL/mo after prostatectomy, measured using a new immuno-polymerase chain reaction diagnostic test (NADiA ProsVue), have a reduced risk of clinical recurrence as determined by positive biopsy, imaging findings, or death from prostate cancer. From 4 clinical sites, we selected a cohort of 304 men who had been followed up for 17.6 years after prostatectomy for clinical recurrence. We assessed the prognostic value of a PSA slope cutpoint of 2.0 pg/mL/mo against established risk factors to identify men at low risk of clinical recurrence using uni- and multivariate Cox proportional hazards regression and Kaplan-Meier analyses. The univariate hazard ratio of a PSA slope >2.0 pg/mL/mo was 18.3 (95% confidence interval 10.6-31.8) compared with a slope ≤ 2.0 pg/mL/mo (P <.0001). The median disease-free survival interval was 4.8 years vs >10 years in the 2 groups (P <.0001). The multivariate hazard ratio for PSA slope with the covariates of preprostatectomy PSA, pathologic stage, and Gleason score was 9.8 (95% confidence interval 5.4-17.8), an 89.8% risk reduction for men with PSA slopes ≤ 2.0 pg/mL/mo (P <.0001). The Gleason score (<7 vs ≥ 7) was the only other significant predictor (hazard ratio 5.4, 95% confidence interval 2.1-13.8, P = .0004). Clinical recurrence after radical prostatectomy is difficult to predict using established risk factors. We have demonstrated that a NADiA ProsVue PSA slope of ≤ 2.0 pg/mL/mo after prostatectomy is prognostic for a reduced risk of prostate cancer recurrence and adds predictive power to the established risk factors.


Magnetomotive laser speckle imaging.
Laser speckle imaging (LSI) involves analysis of reflectance images collected during coherent optical excitation of an object to compute wide-field maps of tissue blood flow. An intrinsic limitation of LSI for resolving microvascular architecture is that its signal depends on relative motion of interrogated red blood cells. Hence, with LSI, small-diameter arterioles, venules, and capillaries are difficult to resolve due to the slow flow speeds associated with such vasculature. Furthermore, LSI characterization of subsurface blood flow is subject to blurring due to scattering, further limiting the ability of LSI to resolve or quantify blood flow in small vessels. Here, we show that magnetic activation of superparamagnetic iron oxide (SPIO) nanoparticles modulate the speckle flow index (SFI) values estimated from speckle contrast analysis of collected images. With application of an ac magnetic field to a solution of stagnant SPIO particles, an apparent increase in SFI is induced. Furthermore, with application of a focused dc magnetic field, a focal decrease in SFI values is induced. Magnetomotive LSI may enable wide-field mapping of suspicious tissue regions, enabling subsequent high-resolution optical interrogation of these regions. Similarly, subsequent photoactivation of intravascular SPIO nanoparticles could then be performed to induce selective photothermal destruction of unwanted vasculature.


Nuclear localization of Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM complex assembly.
The minichromosome maintenance (MCM) proteins MCM2-MCM7 are conserved eukaryotic replication factors that assemble in a heterohexameric complex. In fission yeast, these proteins are nuclear throughout the cell cycle. In studying the mechanism that regulates assembly of the MCM complex, we analyzed the cis and trans elements required for nuclear localization of a single subunit, Mcm2p. Mutation of any single mcm gene leads to redistribution of wild-type MCM subunits to the cytoplasm, and this redistribution depends on an active nuclear export system. We identified the nuclear localization signal sequences of Mcm2p and showed that these are required for nuclear targeting of other MCM subunits. In turn, Mcm2p must associate with other MCM proteins for its proper localization; nuclear localization of MCM proteins thus requires assembly of MCM proteins in a complex. We suggest that coupling complex assembly to nuclear targeting and retention ensures that only intact heterohexameric MCM complexes remain nuclear.


A nonsense mutation in the COL7A1 gene causes epidermolysis bullosa in Vorderwald cattle.
The widespread use of individual sires for artificial insemination promotes the propagation of recessive conditions. Inadvertent matings between unnoticed carriers of deleterious alleles may result in the manifestation of fatal phenotypes in their progeny. Breeding consultants and farmers reported on Vorderwald calves with a congenital skin disease. The clinical findings in affected calves were compatible with epidermolysis bullosa. Pedigree analysis indicated autosomal recessive inheritance of epidermolysis bullosa in Vorderwald cattle. We genotyped two diseased and 41 healthy animals at 41,436 single nucleotide polymorphisms and performed whole-genome haplotype-based association testing, which allowed us to map the locus responsible for the skin disease to the distal end of bovine chromosome 22 (P = 8.0 × 10-14). The analysis of whole-genome re-sequencing data of one diseased calf, three obligate mutation carriers and 1682 healthy animals from various bovine breeds revealed a nonsense mutation (rs876174537, p.Arg1588X) in the COL7A1 gene that segregates with the disease. The same mutation was previously detected in three calves with dystrophic epidermolysis bullosa from the Rotes Höhenvieh cattle breed. We show that diseased animals from Vorderwald and Rotes Höhenvieh cattle are identical by descent for an 8.72 Mb haplotype encompassing rs876174537 indicating they inherited the deleterious allele from a recent common ancestor. Autosomal recessive epidermolysis bullosa in Vorderwald and Rotes Höhenvieh cattle is caused by a nonsense mutation in the COL7A1 gene. Our findings demonstrate that deleterious alleles may segregate across cattle populations without apparent admixture. The identification of the causal mutation now enables the reliable detection of carrier animals. Genome-based mating strategies can avoid inadvertent matings of carrier animals thereby preventing the birth of homozygous calves that suffer from a painful skin disease.


Myocardium Segmentation From DE MRI Using Multicomponent Gaussian Mixture Model and Coupled Level Set.
Objective: In this paper, we propose a fully automatic framework for myocardium segmentation of delayed-enhancement (DE) MRI images without relying on prior patient-specific information. Methods: We employ a multicomponent Gaussian mixture model to deal with the intensity heterogeneity of myocardium caused by the infarcts. To differentiate the myocardium from other tissues with similar intensities, while at the same time maintain spatial continuity, we introduce a coupled level set (CLS) to regularize the posterior probability. The CLS, as a spatial regularization, can be adapted to the image characteristics dynamically. We also introduce an image intensity gradient based term into the CLS, adding an extra force to the posterior probability based framework, to improve the accuracy of myocardium boundary delineation. The prebuilt atlases are propagated to the target image to initialize the framework. Results: The proposed method was tested on datasets of 22 clinical cases, and achieved Dice similarity coefficients of 87.43 ± 5.62% (endocardium), 90.53 ± 3.20% (epicardium) and 73.58 ± 5.58% (myocardium), which have outperformed three variants of the classic segmentation methods. Conclusion: The results can provide a benchmark for the myocardial segmentation in the literature. Significance: DE MRI provides an important tool to assess the viability of myocardium. The accurate segmentation of myocardium, which is a prerequisite for further quantitative analysis of myocardial infarction (MI) region, can provide important support for the diagnosis and treatment management for MI patients.Objective: In this paper, we propose a fully automatic framework for myocardium segmentation of delayed-enhancement (DE) MRI images without relying on prior patient-specific information. Methods: We employ a multicomponent Gaussian mixture model to deal with the intensity heterogeneity of myocardium caused by the infarcts. To differentiate the myocardium from other tissues with similar intensities, while at the same time maintain spatial continuity, we introduce a coupled level set (CLS) to regularize the posterior probability. The CLS, as a spatial regularization, can be adapted to the image characteristics dynamically. We also introduce an image intensity gradient based term into the CLS, adding an extra force to the posterior probability based framework, to improve the accuracy of myocardium boundary delineation. The prebuilt atlases are propagated to the target image to initialize the framework. Results: The proposed method was tested on datasets of 22 clinical cases, and achieved Dice similarity coefficients of 87.43 ± 5.62% (endocardium), 90.53 ± 3.20% (epicardium) and 73.58 ± 5.58% (myocardium), which have outperformed three variants of the classic segmentation methods. Conclusion: The results can provide a benchmark for the myocardial segmentation in the literature. Significance: DE MRI provides an important tool to assess the viability of myocardium. The accurate segmentation of myocardium, which is a prerequisite for further quantitative analysis of myocardial infarction (MI) region, can provide important support for the diagnosis and treatment management for MI patients.


Rural-urban differences in preventable hospitalizations among community-dwelling veterans with dementia.
Alzheimer's patients living in rural communities may face significant barriers to effective outpatient medical care. We sought to examine rural-urban differences in risk for ambulatory care sensitive hospitalizations (ACSH), an indicator of access to outpatient care, in community-dwelling veterans with dementia. Medicare and Veteran Affairs inpatient claims for 1,186 US veterans with dementia were linked to survey data from the 1998 National Longitudinal Caregiver Survey. ACSH were identified in inpatient claims over a 1-year period following collection of independent variables. Urban Influence Codes were used to classify care recipients into 4 categories of increasing county-level rurality: large metropolitan; small metropolitan; micropolitan; and noncore rural counties. We used the Andersen Behavioral Model of Health Services to identify veteran, caregiver, and community factors that may explain urban-rural differences in ACSH. Thirteen percent of care recipients had at least 1 ACSH. The likelihood of an ACSH was greater for patients in noncore rural counties versus large metropolitan areas (22.6% vs 12.8%, unadjusted odds ratio [OR]= 1.99; P < .01). The addition of other Andersen behavioral model variables did not eliminate the disparity (adjusted OR = 1.97; P < .05). We found that dementia patients living in the most rural counties were more likely to have an ACSH; this disparity was not explained by differences in caregiver, care recipient, or community factors. Furthermore, the annual rate of ACSH was higher in community-dwelling dementia patients compared to previous reports on the general older adult population. Dementia patients in rural areas may face particular challenges in receiving timely, effective ambulatory care.


Understanding and caring for the distressed patient with multiple medically unexplained symptoms.
Although physicians commonly encounter patients with complicated medical problems, some have a mix of unexplained medical symptoms and distress that can seem overwhelming to patient and physician alike. We describe a model for thinking about and helping these complex patients. The model was developed from our personal clinical experience as primary care psychiatric consultants, as well as from a review of the literature using such key words as "difficult," "frustrating," and "somatization." The model involves understanding predisposing factors that render the patient vulnerable to the development of a disabling condition, precipitating factors that initiate an episode of illness, and perpetuating factors that maintain maladaptive illness behaviors with time. Awareness of these factors allows the clinician not only to tolerate and gradually unravel the complicated interactions that create and sustain distress in patients with multiple medically unexplained symptoms, but also devise practical and effective management strategies for these complex patients.


Water quality modeling of fertilizer management impacts on nitrate losses in tile drains at the field scale.
Nitrate losses from subsurface tile drained row cropland in the Upper Midwest U.S. contribute to hypoxia in the Gulf of Mexico. Strategies are needed to reduce nitrate losses to the Mississippi River. This paper evaluates the effect of fertilizer rate and timing on nitrate losses in two (East and West) commercial row crop fields located in south-central Minnesota. The Agricultural Drainage and Pesticide Transport (ADAPT) model was calibrated and validated for monthly subsurface tile drain flow and nitrate losses for a period of 1999-2003. Good agreement was found between observed and predicted tile drain flow and nitrate losses during the calibration period, with Nash-Sutcliffe modeling efficiencies of 0.75 and 0.56, respectively. Better agreements were observed for the validation period. The calibrated model was then used to evaluate the effects of rate and timing of fertilizer application on nitrate losses with a 50-yr climatic record (1954-2003). Significant reductions in nitrate losses were predicted by reducing fertilizer application rates and changing timing. A 13% reduction in nitrate losses was predicted when fall fertilizer application rate was reduced from 180 to 123 kg/ha. A further 9% reduction in nitrate losses can be achieved when switching from fall to spring application. Larger reductions in nitrate losses would require changes in fertilizer rate and timing, as well as other practices such as changing tile drain spacings and/or depths, fall cover cropping, or conversion of crop land to pasture.


Bmp7 functions via a polarity mechanism to promote cloacal septation.
During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood. We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse. Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.


An ultra-high density genetic linkage map of perennial ryegrass (Lolium perenne) using genotyping by sequencing (GBS) based on a reference shotgun genome assembly.
High density genetic linkage maps that are extensively anchored to assembled genome sequences of the organism in question are extremely useful in gene discovery. To facilitate this process in perennial ryegrass (Lolium perenne L.), a high density single nucleotide polymorphism (SNP)- and presence/absence variant (PAV)-based genetic linkage map has been developed in an F2 mapping population that has been used as a reference population in numerous studies. To provide a reference sequence to which to align genotyping by sequencing (GBS) reads, a shotgun assembly of one of the grandparents of the population, a tenth-generation inbred line, was created using Illumina-based sequencing. The assembly was based on paired-end Illumina reads, scaffolded by mate pair and long jumping distance reads in the range of 3-40 kb, with >200-fold initial genome coverage. A total of 169 individuals from an F2 mapping population were used to construct PstI-based GBS libraries tagged with unique 4-9 nucleotide barcodes, resulting in 284 million reads, with approx. 1·6 million reads per individual. A bioinformatics pipeline was employed to identify both SNPs and PAVs. A core genetic map was generated using high confidence SNPs, to which lower confidence SNPs and PAVs were subsequently fitted in a straightforward binning approach. The assembly comprises 424 750 scaffolds, covering 1·11 Gbp of the 2·5 Gbp perennial ryegrass genome, with a scaffold N50 of 25 212 bp and a contig N50 of 3790 bp. It is available for download, and access to a genome browser has been provided. Comparison of the assembly with available transcript and gene model data sets for perennial ryegrass indicates that approx. 570 Mbp of the gene-rich portion of the genome has been captured. An ultra-high density genetic linkage map with 3092 SNPs and 7260 PAVs was developed, anchoring just over 200 Mb of the reference assembly. The combined genetic map and assembly, combined with another recently released genome assembly, represent a significant resource for the perennial ryegrass genetics community.


Glutamine Administration in Early or Late Septic Phase Downregulates Lymphocyte PD-1/PD-L1 Expression and the Inflammatory Response in Mice With Polymicrobial Sepsis.
Sepsis is a severe inflammatory response to infection. Excessive compensation to inflammation leads to dysregulated immune response that ultimately results in organ damage and lethality of sepsis. This study administered glutamine (GLN) in the early or late phase of sepsis to investigate its effects on regulating leukocyte programmed cell death 1 (PD-1) and its ligand (programmed cell death ligand 1 [PD-L1]) expression, macrophage function, inflammation, and acute kidney injury in sepsis. Mice were randomly assigned to cecal ligation and puncture (CLP) or sham-operated groups. Septic mice were respectively injected once with saline or 0.75 g GLN/kg body weight at 3 or 10 hours post-CLP via tail vein. All mice were sacrificed 24 hours after CLP. Sepsis enhanced the percentage of interferon-γ-expressing and interleukin (IL)-17A-expressing CD4+ T cells, expression of PD-1 on T cells, and PD-L1 on B cells and monocytes. Inflammatory mediator messenger RNA (mRNA) expression in kidney tissues and proapoptotic caspase-3 mRNA expression in mesenteric lymph nodes were also upregulated. GLN administration decreased plasma IL-6 level, downregulated the percentage of IL-17A-expressing CD4+ T cells, attenuated macrophage dysfunction, decreased caspase-3 mRNA expression, and reduced PD-1/PD-L1 expression by T and B cells. Histological findings also showed that kidney damage was attenuated. GLN administered at 3 and 10 hours after CLP offered nearly equal effects on PD-1/PD-L1 and inflammatory mediator expression after CLP. These findings suggest that a single dose of GLN administration in either the early or late phase during sepsis promotes a more balanced immune regulation and reduced systemic and kidney inflammatory responses in mice.


Late Pleistocene climate change and population dynamics of Japanese Myodes voles inferred from mitochondrial cytochrome b sequences.
The Japanese archipelago is comprised of four main islands-Hokkaido, Honshu, Shikoku, and Kyushu-which contain high mountainous areas that likely allowed for lineage differentiation and population genetic structuring during the climatic changes of the late Pleistocene. Here, we assess the historical background of the evolutionary dynamics of herbivorous red-backed voles (Myodes) in Japan, examining the evolutionary trends of mitochondrial cytochrome b gene (Cytb) sequence variation. Four apparent signals from rapid expansion events were detected in three species, M. rufocanus and M. rutilus from Hokkaido and M. smithii from central Honshu. Taken together with results from previous studies on Japanese wood mice (Apodemus spp.), three of the expansion events were considered to be associated with predicted bottleneck events at the marine isotope stage (MIS) 4 period, in which glaciers are thought to have expanded extensively, especially at higher elevations. In the late Pleistocene, the possible candidates are transitions MIS 6/5, MIS 4/3, and MIS 2/1, which can be characterized by the cold periods of the penultimate glacial maximum, MIS 4, and the last glacial maximum, respectively. Our data further reveal the genetic footprints of repeated range expansion and contraction in the northern and southern lineages of the vole species currently found in central Honshu, namely M. andersoni and M. smithii, in response to climatic oscillation during the late Pleistocene. The time-dependent evolutionary rates of the mitochondrial Cytb presented here would provide a possible way for assessing population dynamics of cricetid rodents responding to the late Pleistocene environmental fluctuation.


Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation.
In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.


Experimental evolution of the Caenorhabditis elegans sex determination pathway.
Sex determination is a critical developmental decision with major ecological and evolutionary consequences, yet a large variety of sex determination mechanisms exist and we have a poor understanding of how they evolve. Theoretical and empirical work suggest that compensatory adaptations to mutations in genes involved in sex determination may play a role in the evolution of these pathways. Here, we directly address this problem using experimental evolution in Caenorhabditis elegans lines fixed for a pair of mutations in two key sex-determining genes that jointly render sex determination temperature-sensitive and cause intersexual (but still weakly to moderately fertile) phenotypes at intermediate temperatures. After 50 generations, evolved lines clearly recovered toward wild-type phenotypes. However, changes in transcript levels of key sex-determining genes in evolved lines cannot explain their partially (or in some cases, nearly completely) rescued phenotypes, implying that wild-type phenotypes can be restored independently of the transcriptional effects of these mutations. Our findings highlight the microevolutionary flexibility of sex determination pathways and suggest that compensatory adaptation to mutations can elicit novel and unpredictable evolutionary trajectories in these pathways, mirroring the phylogenetic diversity, and macroevolutionary dynamics of sex determination mechanisms.


Better risk assessment with glycated hemoglobin instead of cholesterol in CVD risk prediction charts.
Traditional risk charts for the prediction of cardiovascular disease (CVD) include cholesterol parameters. We evaluated how models predict fatal CVD when cholesterol is replaced by glucose parameters. We used data from NHANES III, a US survey conducted 1988-1994 (follow-up until 2006); 15,454 participants (1,716 CVD deaths) were included. Based on the ESC SCORE method, we used age, sex, blood pressure, smoking and either of the following: (1) total cholesterol, (2) total-to-HDL-cholesterol, (3) glucose, (4) glycated hemoglobin (A1C). Scaled Brier score (BS), Nagelkerke's R(2) (NR) and integrated discrimination improvement (IDI) were used for model comparison. The ranking (best to worst) was: A1C (BS = 11.62 %; NR = 0.0865; IDI = 0.0091), glucose (11.16 %; 0.0734; 0.0067), total-to-HDL-cholesterol (9.97 %; 0.0547; 0.0010), cholesterol (9.75 %; 0.0484; 0, reference). Differences between models with cholesterol and glucose or A1C were statistically significant. This study suggests the use of A1C instead of cholesterol parameters in charts to assess CVD risk.


Dual Barriers: Examining Digital Access and Travel Burdens to Hospital Maternity Care Access in the United States, 2020.
Policy Points The White House Blueprint for Addressing the Maternal Health Crisis report released in June 2022 highlighted the need to enhance equitable access to maternity care. Nationwide hospital maternity unit closures have worsened the maternal health crisis in underserved communities, leaving many birthing people with few options and with long travel times to reach essential care. Ensuring equitable access to maternity care requires addressing travel burdens to care and inadequate digital access. Our findings reveal socioeconomically disadvantaged communities in the United States face dual barriers to maternity care access, as communities located farthest away from care facilities had the least digital access. With the increases in nationwide hospital maternity unit closures, there is a greater need for telehealth services for the supervision, evaluation, and management of prenatal and postpartum care. However, challenges in digital access persist. We examined associations between driving time to hospital maternity units and digital access to understand whether augmenting digital access and telehealth services might help mitigate travel burdens to maternity care. This cross-sectional study used 2020 American Hospital Association Annual Survey data for hospital maternity unit locations and 2020 American Community Survey five-year ZIP Code Tabulation Area (ZCTA)-level estimates of household digital access to telecommunication technology and broadband. We calculated driving times of the fastest route from population-weighted ZCTA centroids to the nearest hospital maternity unit. Rural-urban stratified generalized median regression models were conducted to examine differences in ZCTA-level proportions of household lacking digital access equipment (any digital device, smartphones, tablet), and lacking broadband subscriptions by spatial accessibility to maternity units. In 2020, 2,905 (16.6%) urban and 3,394 (39.5%) rural ZCTAs in the United States were located >30 minutes from the nearest hospital maternity units. Regardless of rurality, these communities farther away from a maternity unit had disproportionally lower broadband and device accessibility. Although urban communities have greater digital access to technology and broadband subscriptions compared to rural communities, disparities in the percentage of households with access to digital devices were more pronounced within urban areas, particularly between those with and without close proximity to a hospital maternity unit. Communities where nearest hospital maternity units were >30 minutes away had higher poverty and uninsurance rates than those with <15-minute access. Socioeconomically disadvantaged communities face significant barriers to maternity care access, both with substantial travel burdens and inadequate digital access. To optimize maternity care access, ongoing efforts (e.g., Affordable Connectivity Program introduced in the 2021 Infrastructure Act), should bridge the gaps in digital access and target communities with substantial travel burdens to care and limited digital access.


Isolation of Japanese encephalitis virus from mosquitoes (Diptera: Culicidae) collected in the Western Province of Papua New Guinea, 1997-1998.
After Japanese encephalitis (JE) virus emerged in the Torres Strait in Australia in 1995, investigations were initiated into the origin of the incursion. New Guinea was considered the most likely source, given its proximity to islands of the Torres Strait. Almost 400,000 adult mosquitoes were processed for virus isolation from 26 locations in the Western Province of Papua New Guinea (PNG) between February 1996 and February 1998, yielding three isolates of JE virus. Two isolates of Murray Valley encephalitis, 17 isolates of Sindbis, and 1 each of Sepik and Ross River viruses were also obtained. Nucleic acid sequences of the PNG JE isolates were determined in the prM region, and in a region overlapping a part of the fifth nonstructural protein and the 3' untranslated region. The PNG isolates belonged to genotype II, and shared > 99.2% identity with isolates from humans and mosquitoes from the Torres Strait, suggesting that PNG is the source of incursions of JE virus into Australia.


Tying the knot: linking cytokinesis to the nuclear cycle.
For the survival of both the parent and the progeny, it is imperative that the process of their physical division (cytokinesis) be precisely coordinated with progression through the mitotic cell cycle. Recent studies in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are beginning to unravel the nature of the links between cytokinesis and the nuclear division cycle. The cyclin-dependent kinases and a novel surveillance mechanism that monitors cytokinesis and/or morphogenesis appear to play important regulatory roles in forging these links. It is becoming increasingly clear that the inactivation of the mitosis-promoting cyclin-dependent kinase, which marks the completion of the nuclear division cycle, is essential for actomyosin ring constriction and division septum assembly in both yeasts. Additionally, the spindle pole bodies are emerging as important transient locale for proteins that might play a key role in coupling the completion of mitosis to the onset of cytokinesis.


Future perspectives in melanoma research "Melanoma Bridge", Napoli, November 30th-3rd December 2016.
Major advances have been made in the treatment of cancer with targeted therapy and immunotherapy; several FDA-approved agents with associated improvement of 1-year survival rates became available for stage IV melanoma patients. Before 2010, the 1-year survival were quite low, at 30%; in 2011, the rise to nearly 50% in the setting of treatment with Ipilimumab, and rise to 70% with BRAF inhibitor monotherapy in 2013 was observed. Even more impressive are 1-year survival rates considering combination strategies with both targeted therapy and immunotherapy, now exceeding 80%. Can we improve response rates even further, and bring these therapies to more patients? In fact, despite these advances, responses are heterogeneous and are not always durable. There is a critical need to better understand who will benefit from therapy, as well as proper timing, sequence and combination of different therapeutic agents. How can we better understand responses to therapy and optimize treatment regimens? The key to better understanding therapy and to optimizing responses is with insights gained from responses to targeted therapy and immunotherapy through translational research in human samples. Combination therapies including chemotherapy, radiotherapy, targeted therapy, electrochemotherapy with immunotherapy agents such as Immune Checkpoint Blockers are under investigation but there is much room for improvement. Adoptive T cell therapy including tumor infiltrating lymphocytes and chimeric antigen receptor modified T cells therapy is also efficacious in metastatic melanoma and outcome enhancement seem likely by improved homing capacity of chemokine receptor transduced T cells. Tumor infiltrating lymphocytes therapy is also efficacious in metastatic melanoma and outcome enhancement seem likely by improved homing capacity of chemokine receptor transduced T cells. Understanding the mechanisms behind the development of acquired resistance and tests for biomarkers for treatment decisions are also under study and will offer new opportunities for more efficient combination therapies. Knowledge of immunologic features of the tumor microenvironment associated with response and resistance will improve the identification of patients who will derive the most benefit from monotherapy and might reveal additional immunologic determinants that could be targeted in combination with checkpoint blockade. The future of advanced melanoma needs to involve education and trials, biobanks with a focus on primary tumors, bioinformatics and empowerment of patients and clinicians.


Leukotriene B4 enhances innate immune defense against the puerperal sepsis agent Streptococcus pyogenes.
Puerperal sepsis is a leading cause of maternal mortality worldwide. Streptococcus pyogenes [group A Streptococcus; (GAS)] is a major etiologic agent of severe postpartum sepsis, yet little is known regarding the pathogenesis of these infections. Tissue macrophages provide innate defense against GAS, and their actions are highly regulated. The intracellular second messenger cAMP can negatively regulate macrophage actions against GAS. Because leukotriene (LT) B(4) has been shown to suppress intracellular cAMP in macrophages, we hypothesized that it could enhance innate defenses against GAS. We assessed the capacity of LTB(4) to modulate antistreptococcal actions of human macrophages, including placental and decidual macrophages and used a novel intrauterine infection model of GAS in mice lacking the 5-lipoxygenase enzyme to determine the role of endogenous LTs in host defense against this pathogen. Animals lacking 5-lipoxygenase were significantly more vulnerable to intrauterine GAS infection than were wild-type mice and showed enhanced dissemination of bacteria out of the uterus and a more robust inflammatory response than did wild-type mice. In addition, LTB(4) reduced intracellular cAMP levels via the BLT1 receptor and was a potent stimulant of macrophage phagocytosis and NADPH oxidase-dependent intracellular killing of GAS. Importantly, interference was observed between the macrophage immunomodulatory actions of LTB(4) and the cAMP-inducing lipid PGE(2), suggesting that interplay between pro- and anti-inflammatory compounds may be important in vivo. This work underscores the potential for pharmacological targeting of lipid mediator signaling cascades in the treatment of invasive GAS infections.


Interaction of AP-1 with a cluster of NF-kappa B binding elements in the human TNF promoter region.
Transcriptional activation of the human TNF gene involves multiple regulatory elements whose functional properties vary between stimuli and cell types. Here we have used a COS-7 expression system to dissect the transactivating potential of NF-kappa B binding sites in the human TNF promoter region from other regulatory influences. In this model, NF-kappa B acts largely through a dense cluster of three binding sites located 600 nt upstream of the transcription start site. We show that the transcriptional activity of this complex is highly sensitive to the p65:p50 ratio that is expressed. We demonstrate that the AP-1 complex c-Jun/Fra2 is capable of binding to this region and that this inhibits the transactivating effects of NF-kappa B. These results are suggestive of a complex regulatory element that mediates fine control rather than acting as a simple on-off switch for TNF gene expression.


No transcription-translation feedback in circadian rhythm of KaiC phosphorylation.
An autoregulatory transcription-translation feedback loop is thought to be essential in generating circadian rhythms in any model organism. In the cyanobacterium Synechococcus elongatus, the essential clock protein KaiC is proposed to form this type of transcriptional negative feedback. Nevertheless, we demonstrate here temperature-compensated, robust circadian cycling of KaiC phosphorylation even without kaiBC messenger RNA accumulation under continuous dark conditions. This rhythm persisted in the presence of a transcription or translation inhibitor. Moreover, kinetic profiles in the ratio of KaiC autophosphorylation-dephosphorylation were also temperature compensated in vitro. Thus, the cyanobacterial clock can keep time independent of de novo transcription and translation processes.


Integrated biclustering of heterogeneous genome-wide datasets for the inference of global regulatory networks.
The learning of global genetic regulatory networks from expression data is a severely under-constrained problem that is aided by reducing the dimensionality of the search space by means of clustering genes into putatively co-regulated groups, as opposed to those that are simply co-expressed. Be cause genes may be co-regulated only across a subset of all observed experimental conditions, biclustering (clustering of genes and conditions) is more appropriate than standard clustering. Co-regulated genes are also often functionally (physically, spatially, genetically, and/or evolutionarily) associated, and such a priori known or pre-computed associations can provide support for appropriately grouping genes. One important association is the presence of one or more common cis-regulatory motifs. In organisms where these motifs are not known, their de novo detection, integrated into the clustering algorithm, can help to guide the process towards more biologically parsimonious solutions. We have developed an algorithm, cMonkey, that detects putative co-regulated gene groupings by integrating the biclustering of gene expression data and various functional associations with the de novo detection of sequence motifs. We have applied this procedure to the archaeon Halobacterium NRC-1, as part of our efforts to decipher its regulatory network. In addition, we used cMonkey on public data for three organisms in the other two domains of life: Helicobacter pylori, Saccharomyces cerevisiae, and Escherichia coli. The biclusters detected by cMonkey both recapitulated known biology and enabled novel predictions (some for Halobacterium were subsequently confirmed in the laboratory). For example, it identified the bacteriorhodopsin regulon, assigned additional genes to this regulon with apparently unrelated function, and detected its known promoter motif. We have performed a thorough comparison of cMonkey results against other clustering methods, and find that cMonkey biclusters are more parsimonious with all available evidence for co-regulation.


A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment.
Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross-reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin-fixed paraffin-embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (rs > 0.9, P-value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.


Identification of risk groups for development of central nervous system leukemia in adults with acute lymphocytic leukemia.
The risk of development of CNS leukemia was investigated in 153 adults with acute lymphocytic leukemia (ALL) who received systemic combination chemotherapy without CNS prophylaxis. Overall, 31 patients (20%) developed CNS leukemia after a median of 6 months of therapy; the estimated 1-year incidence of CNS leukemia was 21% (SE, 3.9%). Characteristics significantly associated with CNS involvement included the presence of elevated hemoglobin creatinine, alkaline phosphatase, fibrinogen, and lactic dehydrogenase levels; B-cell leukemia; and high leukemic cell proliferative activity. Multivariate analysis identified lactic dehydrogenase levels of greater than or equal to 600 U/L and greater than or equal to 14% of cells in the S + G2M compartment to have independent additive poor prognostic significance. Patients were categorized into different risk groups for CNS leukemia with 1-year incidences ranging from 4% to 55%. While related to a high occurrence of CNS leukemia at diagnosis (33%) and subsequently (100%), the low incidence of B-cell disease excluded it from the multivariate analysis. The use of systemic chemotherapy containing multiple agents with good CNS penetration and in high doses (VAD regimen) in 90 patients was associated with a trend for lower CNS leukemia at 1 year (15% v 31%), especially in the low-risk category. We propose to develop future therapies for adults with ALL that include risk-oriented CNS prophylactic approaches.


Assessing callous-unemotional traits: development of a brief, reliable measure in a large and diverse sample of preadolescent youth.
Callous-unemotional (CU) traits are critical to developmental, diagnostic, and clinical models of antisocial behaviors (AB). However, assessments of CU traits within large-scale longitudinal and neurobiologically focused investigations remain remarkably sparse. We sought to develop a brief measure of CU traits using items from widely administered instruments that could be linked to neuroimaging, genetic, and environmental data within already existing datasets and future studies. Data came from a large and diverse sample (n = 4525) of youth (ages~9-11) taking part in the Adolescent Brain and Cognitive Development (ABCD) Study. Moderated nonlinear factor analysis was used to assess measurement invariance across sex, race, and age. We explored whether CU traits were distinct from other indicators of AB, investigated unique links with theoretically-relevant outcomes, and replicated findings in an independent sample. The brief CU traits measure demonstrated strong psychometric properties and evidence of measurement invariance across sex, race, and age. On average, boys endorsed higher levels of CU traits than girls and CU traits were related to, yet distinguishable from other indicators of AB. The CU traits construct also exhibited expected associations with theoretically important outcomes. Study findings were also replicated across an independent sample of youth. In a large, multi-site study, a brief measure of CU traits can be measured distinctly from other dimensions of AB. This measure provides the scientific community with a method to assess CU traits in the ABCD sample, as well as in other studies that may benefit from a brief assessment of CU.


Comparison of generalized estimating equations and quadratic inference functions using data from the National Longitudinal Survey of Children and Youth (NLSCY) database.
The generalized estimating equations (GEE) technique is often used in longitudinal data modeling, where investigators are interested in population-averaged effects of covariates on responses of interest. GEE involves specifying a model relating covariates to outcomes and a plausible correlation structure between responses at different time periods. While GEE parameter estimates are consistent irrespective of the true underlying correlation structure, the method has some limitations that include challenges with model selection due to lack of absolute goodness-of-fit tests to aid comparisons among several plausible models. The quadratic inference functions (QIF) method extends the capabilities of GEE, while also addressing some GEE limitations. We conducted a comparative study between GEE and QIF via an illustrative example, using data from the "National Longitudinal Survey of Children and Youth (NLSCY)" database. The NLSCY dataset consists of long-term, population based survey data collected since 1994, and is designed to evaluate the determinants of developmental outcomes in Canadian children. We modeled the relationship between hyperactivity-inattention and gender, age, family functioning, maternal depression symptoms, household income adequacy, maternal immigration status and maternal educational level using GEE and QIF. Basis for comparison include: (1) ease of model selection; (2) sensitivity of results to different working correlation matrices; and (3) efficiency of parameter estimates. The sample included 795, 858 respondents (50.3% male; 12% immigrant; 6% from dysfunctional families). QIF analysis reveals that gender (male) (odds ratio [OR] = 1.73; 95% confidence interval [CI] = 1.10 to 2.71), family dysfunctional (OR = 2.84, 95% CI of 1.58 to 5.11), and maternal depression (OR = 2.49, 95% CI of 1.60 to 2.60) are significantly associated with higher odds of hyperactivity-inattention. The results remained robust under GEE modeling. Model selection was facilitated in QIF using a goodness-of-fit statistic. Overall, estimates from QIF were more efficient than those from GEE using AR (1) and Exchangeable working correlation matrices (Relative efficiency = 1.1117; 1.3082 respectively). QIF is useful for model selection and provides more efficient parameter estimates than GEE. QIF can help investigators obtain more reliable results when used in conjunction with GEE.


GATA-4 and FOG-2 expression in pediatric ovarian sex cord-stromal tumors replicates embryonal gonadal phenotype: results from the TREP project.
GATA proteins are a family of zinc finger transcription factors regulating gene expression, differentiation and proliferation in various tissues. The expression of GATA-4 and FOG-2, one of its modulators, was studied in pediatric Sex Cord-Stromal tumors of the ovary, in order to evaluate their potential role as diagnostic markers and prognostic factors. Clinical and histological data of 15 patients, enrolled into the TREP Project since 2000 were evaluated. When available, immunostaines for FOG-2, GATA-4, α-Inhibin, Vimentin and Pancytokeratin were also analyzed. In our series there were 6 Juvenile Granulosa Cell Tumors (JGCT), 6 Sertoli-Leydig Cell Tumors (SLCT), 1 Cellular Fibroma, 1 Theca Cell Tumor and 1 Stromal Sclerosing Tumor (SST). Thirteen patients obtained a complete remission (CR), 1 reached a second CR after the removal of a metachronous tumor and 1 died of disease. Inhibin was detectable in 11/15, Vimentin in 13/15, Pancytokeratin in 6/15, GATA-4 in 5/13 and FOG-2 in 11/15. FOG-2 was highly expressed in 5/6 JGCT, while GATA-4 was weakly detectable only in 1 of the cases. SLCT expressed diffusely FOG-2 (4/6) and GATA-4 (3/5). GATA-4 and FOG-2 were detected in fibroma and thecoma but not in the SST. Pediatric granulosa tumors appear to express a FOG-2/GATA-4 phenotype in keeping with primordial ovarian follicles. High expression of GATA-4 does not correlate with aggressive behaviour as seen in adults, but it is probably involved in cell proliferation its absence can be associated with the better outcome of JGCT. SLCTs replicate the phenotype of Sertoli cells during embryogenesis in normal testis. In this group, the lack of expression of FOG-2 in tumors in advanced stages might reveal a hypothetical role in inhibiting GATA-4 cell proliferation pathway. In fibroma/thecoma group GATA-4 and FOG-2 point out the abnormal activation of GATA pathway and might be involved in the onset of these tumors.


Double network hydrogels based on semi-rigid polyelectrolyte physical networks.
Applying the double network principle to develop tough hydrogels with different polymer chemistries is important for the potential application of hydrogel materials. Synthesis of the two interpenetrated networks with contrasting structure and properties required for double networks usually involves a two-step polymerization process. In this work, we present a new method to synthesize tough double network hydrogels by post-physical crosslinking of linear semi-rigid polyelectrolytes entrapped in a chemically crosslinked neutral network. Owing to their semi-rigid structure, the linear polyelectrolytes form a brittle physical network above their overlap concentration in multi-valent ZrCl2O ion solutions without macroscopic phase separation within the flexible neutral network. The double network hydrogels thus prepared exhibit high modulus (∼1.7 MPa), strength (∼1.3 MPa), fracture strain (∼7.3), and strain energy density (∼5.9 MJ m-3), while containing over 80% water. These materials also exhibit modest self-healing ability (∼51% after 30 minutes), demonstrating an additional benefit of a physical sacrificial network. This method is simpler than the conventional two-step polymerization and could be applied to develop tough hydrogels from rigid polyelectrolytes, including biopolymers such as DNA, HA, and chondroitin sulfate.


Social semantics: altruism, cooperation, mutualism, strong reciprocity and group selection.
From an evolutionary perspective, social behaviours are those which have fitness consequences for both the individual that performs the behaviour, and another individual. Over the last 43 years, a huge theoretical and empirical literature has developed on this topic. However, progress is often hindered by poor communication between scientists, with different people using the same term to mean different things, or different terms to mean the same thing. This can obscure what is biologically important, and what is not. The potential for such semantic confusion is greatest with interdisciplinary research. Our aim here is to address issues of semantic confusion that have arisen with research on the problem of cooperation. In particular, we: (i) discuss confusion over the terms kin selection, mutualism, mutual benefit, cooperation, altruism, reciprocal altruism, weak altruism, altruistic punishment, strong reciprocity, group selection and direct fitness; (ii) emphasize the need to distinguish between proximate (mechanism) and ultimate (survival value) explanations of behaviours. We draw examples from all areas, but especially recent work on humans and microbes.


Characterization of Enterotoxigenic Bacillus cereus sensu lato and Staphylococcus aureus Isolates and Associated Enterotoxin Production Dynamics in Milk or Meat-Based Broth.
Bacillus cereus sensu lato species, as well as Staphylococcus aureus, are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins. This study characterised enterotoxin genes of these species and examined growth and enterotoxin production dynamics of isolates when grown in milk or meat-based broth. All B. cereus s. l. isolates harboured nheA, hblA and entFM toxin genes, with lower prevalence of bceT and hlyII. When grown at 16 °C, toxin production by individual B. cereus s. l. isolates varied depending on the food matrix; toxin was detected at cell densities below 5 log10(CFU/mL). At 16 °C no staphylococcal enterotoxin C (SEC) production was detected by S. aureus isolates, although low levels of SED production was noted. At 30 °C all S. aureus isolates produced detectable enterotoxin in the simulated meat matrix, whereas SEC production was significantly reduced in milk. Relative to B. cereus s. l. toxin production, S. aureus typically required reaching higher cell numbers to produce detectable levels of enterotoxin. Phylogenetic analysis of the sec and sel genes suggested population evolution which correlated with animal host adaptation, with subgroups of bovine isolates or caprine/ovine isolates noted, which were distinct from human isolates. Taken together, this study highlights the marked differences in the production of enterotoxins both associated with different growth matrices themselves, but also in the behaviour of individual strains when exposed to different food matrices.


Differences in estimates of size distribution of beryllium powder materials using phase contrast microscopy, scanning electron microscopy, and liquid suspension counter techniques.
Accurate characterization of the physicochemical properties of aerosols generated for inhalation toxicology studies is essential for obtaining meaningful results. Great emphasis must also be placed on characterizing particle properties of materials as administered in inhalation studies. Thus, research is needed to identify a suite of techniques capable of characterizing the multiple particle properties (i.e., size, mass, surface area, number) of a material that may influence toxicity. The purpose of this study was to characterize the morphology and investigate the size distribution of a model toxicant, beryllium. Beryllium metal, oxides, and alloy particles were aerodynamically size-separated using an aerosol cyclone, imaged dry using scanning electron microscopy (SEM), then characterized using phase contrast microscopy (PCM), a liquid suspension particle counter (LPC), and computer-controlled SEM (CCSEM). Beryllium metal powder was compact with smaller sub-micrometer size particles attached to the surface of larger particles, whereas the beryllium oxides and alloy particles were clusters of primary particles. As expected, the geometric mean (GM) diameter of metal powder determined using PCM decreased with aerodynamic size, but when suspended in liquid for LPC or CCSEM analysis, the GM diameter decreased by a factor of two (p < 0.001). This observation suggested that the smaller submicrometer size particles attached to the surface of larger particles and/or particle agglomerates detach in liquid, thereby shifting the particle size distribution downward. The GM diameters of the oxide materials were similar regardless of sizing technique, but observed differences were generally significant (p < 0.001). For oxides, aerodynamic cluster size will dictate deposition in the lung, but primary particle size may influence biological activity. The GM diameter of alloy particles determined using PCM became smaller with decreasing aerodynamic size fraction; however, when suspended in liquid for CCSEM and LPC analyses, GM particle size decreased by a factor of two (p < 0.001) suggesting that alloy particles detach in liquid. Detachment of particles in liquid could have significance for the expected versus actual size (and number) distribution of aerosol delivered to an exposure subject. Thus, a suite of complimentary analytical techniques may be necessary for estimating size distribution. Consideration should be given to thoroughly understanding the influence of any liquid vehicle which may alter the expected aerosol size distribution.


Low-Load Resistance Training With Blood Flow Restriction Improves Clinical Outcomes in Musculoskeletal Rehabilitation: A Single-Blind Randomized Controlled Trial.
Background: There is growing evidence to support the use of low-load blood flow restriction (LL-BFR) exercise in musculoskeletal rehabilitation. Purpose: The purpose of this study was to evaluate the efficacy and feasibility of low-load blood flow restricted (LL-BFR) training versus conventional high mechanical load resistance training (RT) on the clinical outcomes of patient's undergoing inpatient multidisciplinary team (MDT) rehabilitation. Study design: A single-blind randomized controlled study. Methods: Twenty-eight lower-limb injured adults completed a 3-week intensive MDT rehabilitation program. Participants were randomly allocated into a conventional RT (3-days/week) or twice-daily LL-BFR training group. Outcome measurements were taken at baseline and 3-weeks and included quadriceps and total thigh muscle cross-sectional area (CSA) and volume, muscle strength [five repetition maximum (RM) leg press and knee extension test, isometric hip extension], pain and physical function measures (Y-balance test, multistage locomotion test-MSLT). Results: A two-way repeated measures analysis of variance revealed no significant differences between groups for any outcome measure post-intervention (p > 0.05). Both groups showed significant improvements in mean scores for muscle CSA/volume, 5-RM leg press, and 5-RM knee extension (p < 0.01) after treatment. LL-BFR group participants also demonstrated significant improvements in MSLT and Y-balance scores (p < 0.01). The Pain scores during training reduced significantly over time in the LL-BFR group (p = 0.024), with no adverse events reported during the study. Conclusion: Comparable improvements in muscle strength and hypertrophy were shown in LL-BFR and conventional training groups following in-patient rehabilitation. The LL-BFR group also achieved significant improvements in functional capacity. LL-BFR training is a rehabilitation tool that has the potential to induce positive adaptations in the absence of high mechanical loads and therefore could be considered a treatment option for patients suffering significant functional deficits for whom conventional loaded RT is contraindicated. Trial Registration: ISRCTN Reference: ISRCTN63585315, dated 25 April 2017.


Galectin-3 modulates Th17 responses by regulating dendritic cell cytokines.
Galectin-3 is a β-galactoside-binding animal lectin with diverse functions, including regulation of T helper (Th) 1 and Th2 responses. Current data indicate that galectin-3 expressed in dendritic cells (DCs) may be contributory. Th17 cells have emerged as critical inducers of tissue inflammation in autoimmune disease and important mediators of host defense against fungal pathogens, although little is known about galectin-3 involvement in Th17 development. We investigated the role of galectin-3 in the induction of Th17 immunity in galectin-3-deficient (gal3(-/-)) and gal3(+/+) mouse bone marrow-derived DCs. We demonstrate that intracellular galectin-3 negatively regulates Th17 polarization in response to the dectin-1 agonist curdlan (a β-glucan present on the cell wall of fungal species) and lipopolysaccharide, agents that prime DCs for Th17 differentiation. On activation of dectin-1, gal3(-/-) DCs secreted higher levels of the Th17-axis cytokine IL-23 compared with gal3(+/+) DCs and contained higher levels of activated c-Rel, an NF-κB subunit that promotes IL-23 expression. Levels of active Raf-1, a kinase that participates in downstream inhibition of c-Rel binding to the IL23A promoter, were impaired in gal3(-/-) DCs. Modulation of Th17 by galectin-3 in DCs also occurred in vivo because adoptive transfer of gal3(-/-) DCs exposed to Candida albicans conferred higher Th17 responses and protection against fungal infection. We conclude that galectin-3 suppresses Th17 responses by regulating DC cytokine production.