Measurement of protein turnover in rat liver. Analysis of the complex curve for decay of label in a mixture of proteins.
The curve for decay of 14C in rat liver protein labelled by injection of NaH14CO3 was analysed to obtain the average turnover rate of mixed liver protein. Three different methods of analysis were used. (1) Unlike decay curves from homogeneous proteins, the curve did not fit a single exponential, but a good fit was obtained with three exponentials. By assuming that the mixture contained three major components with different turnover rates, the calculated value for the average turnover rate (k) was close to 40% per day. (2) k was also calculated from the area under the decay curve, a method which makes no assumptions about the number of proteins in the mixture. This method also gave a value close to 40% per day. (3) It was shown empirically, both by simulation of decay of label in model mixtures of protein and with the decay curve measured in vivo, that k can be calculated from the time taken for the specific radioactivity to fall to 10% of its maximum value. This is an advantage, since the other two methods require the decay curve to be measured over a much longer period of time.


The effects of active and passive smoking on selected trace element levels in human milk.
Our study aimed to compare levels of six micro-elements and six potentially toxic elements in the breast milk of non-smoking women compared to those found in women who smoke tobacco and women exposed to second-hand smoke during pregnancy and lactation. This was a cross-sectional study conducted on 100 lactating women in western Iran. The studied subjects were in three groups: passive smokers, active smokers, and a control group. Concentrations of selected trace elements in breast milk (essential and non-essential metals) were determined using ICP-MS. Our results indicated that the parameters of education, fruit consumption, and cosmetics usage had a significant difference among the groups (p < 0.05). Moreover, for trace elements, the Kruskal-Wallis test was statistically significant for arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) (p < 0.05). The post hoc Dunn test revealed a significant difference in the levels of As, Cd, Hg, and Pb between non-smoker and passive/active smoker groups (p < 0.05). Our findings illustrate that exposure to cigarette smoke can cause an increase in the level of potentially toxic elements in human milk, which is dangerous for the consumption of premature newborns, but more research is needed to evaluate the potential toxic mechanisms of toxic metals.


N-cadherin dependent collective cell invasion of prostate cancer cells is regulated by the N-terminus of α-catenin.
Cancer cell invasion is the critical first step of metastasis, yet, little is known about how cancer cells invade and initiate metastasis in a complex extracellular matrix. Using a cell line from bone metastasis of prostate cancer (PC3), we analyzed how prostate cancer cells migrate in a physiologically relevant 3D Matrigel. We found that PC3 cells migrated more efficiently as multi-cellular clusters than isolated single cells, suggesting that the presence of cell-cell adhesion improves 3D cell migration. Perturbation of N-cadherin function by transfection of either the N-cadherin cytoplasmic domain or shRNA specific to N-cadherin abolished collective cell migration. Interestingly, PC3 cells do not express α-catenin, an actin binding protein in the cadherin complex. When the full-length α-catenin was re-introduced, the phenotype of PC3 cells reverted back to a more epithelial phenotype with a decreased cell migration rate in 3D Matrigel. Interestingly, we found that the N-terminal half of α-catenin was sufficient to suppress invasive phenotype. Taken together, these data suggest that the formation of N-cadherin junctions promotes 3D cell migration of prostate cancer cells, and this is partly due to an aberrant regulation of the N-cadherin complex in the absence of α-catenin.


Bacterial Community Assembly in a Typical Estuarine Marsh with Multiple Environmental Gradients.
Bacterial communities play essential roles in estuarine marsh ecosystems, but the interplay of ecological processes underlying their community assembly is poorly understood. Here, we studied the sediment bacterial communities along a linear gradient extending from the water-land junction toward a high marsh, using 16S rRNA gene amplicon sequencing. Bacterial community compositions differed significantly between sediment transects. Physicochemical properties, particularly sediment nutrient levels (i.e., total nitrogen [TN] and available phosphorus [AP]), as well as sediment physical structure and pH (P < 0.05), were strongly associated with the overall community variations. In addition, the topological properties of bacterial cooccurrence networks varied with distance to the water-land junction. Both node- and network-level topological features revealed that the bacterial network of sediments farthest from the junction was less intense in complexity and interactions than other sediments. Phylogenetic null modeling analysis showed a progressive transition from stochastic to deterministic community assembly for the water-land junction sites toward the emerging terrestrial system. Taken together, data from this study provide a detailed outline of the distribution pattern of the sediment bacterial community across an estuarine marsh and inform the mechanisms and processes mediating bacterial community assembly in marsh soils.IMPORTANCE Salt marshes represent highly dynamic ecosystems where the atmosphere, continents, and the ocean interact. The bacterial distribution in this ecosystem is of great ecological concern, as it provides essential functions acting on ecosystem services. However, ecological processes mediating bacterial assembly are poorly understood for salt marshes, especially the ones located in estuaries. In this study, the distribution and assembly of bacterial communities in an estuarine marsh located in south Hangzhou Bay were investigated. The results revealed an intricate interplay between stochastic and deterministic processes mediating the assembly of bacterial communities in the studied gradient system. Collectively, our findings illustrate the main drivers of community assembly, taking into consideration changes in sediment abiotic variables and potential biotic interactions. Thus, we offer new insights into estuarine bacterial communities and illustrate the interplay of ecological processes shaping the assembly of bacterial communities in estuarine marsh ecosystems.


The chemokine GRO-alpha (CXCL1) confers increased tumorigenicity to glioma cells.
The chemokine GRO-alpha (CXCL1) has been found to mediate the proliferation of glia progenitor cells during neural development. As malignant gliomas are thought to arise from glia progenitors or their differentiated counterparts, astrocytes or oligodendrocytes, we have investigated whether GRO-alpha regulates the tumor characteristics of glioma cells. We found first that resected glioma specimens were strongly immunoreactive for GRO-alpha expression in cells with the morphology of tumor cells. In culture, the U251 glioma line transfected to overexpress GRO-alpha had elevated levels of motility and invasiveness. GRO-alpha transfectants increased their expression of several proteins associated with migratory behavior, including matrix metalloproteinase-2, beta1-integrin and SPARC. The implantation of GRO-alpha glioma clones into the brain of nude mice caused the early demise of mice and this was associated with the formation of larger intracerebral tumors when compared with mice implanted with vector control lines. These results implicate GRO-alpha in gliomas and suggest that the dysregulation of a glia proliferative factor contributes to tumorigenesis. Targeting GRO-alpha may be a useful therapeutic tool to control brain tumor biology.


Is there a role for consolidative radiotherapy in the treatment of aggressive and localized non-Hodgkin lymphoma? A systematic review with meta-analysis.
Chemotherapy is the mainstay of non-Hodgkin lymphoma (NHL) treatment. Based on expert opinion, the use of radiotherapy (RT) is currently preferred in some institutions as consolidative treatment for patients with localized disease. The lack of conclusive data coming from conflicting studies about the impact of treatment demands a systematic review, which could provide the most reliable assessment for clinical decision-making. We evaluate the addition of RT post-CT, for aggressive and localized NHL (ALNHL). Randomized controlled trials (RCT) that evaluated chemotherapy alone versus chemotherapy plus RT were searched in databases. The outcomes were overall survival (OS), progression-free survival (PFS), overall response rate (ORR) and toxicity. Risk ratio (RR) and hazard ratio (HR) with their respective 95% confidence intervals (CI) were calculated using a fized-effect model. Four trials (1,796 patients) met the inclusion criteria. All trials tested the use of RT after systemic therapy comprising anthracycline-based chemotherapy. This systematic review showed that RT enhances PFS after chemotherapy (hazard ratio [HR] 0.81; 95% CI 0.67-0.98; p = 0.03), with no impact on ORR and OS. Some heterogeneity between trials could limit the conclusions about OS. Toxicity data could not be pooled due to differences in reporting adverse events. This systematic review with meta-analysis shows no improvement in survival when adding RT to systemic therapy for ALNHL. Our conclusions are limited by the available data. Further evaluations of new RT technologies and its association with biologic agents are needed.


Noise-robust speech recognition through auditory feature detection and spike sequence decoding.
Speech recognition in noisy conditions is a major challenge for computer systems, but the human brain performs it routinely and accurately. Automatic speech recognition (ASR) systems that are inspired by neuroscience can potentially bridge the performance gap between humans and machines. We present a system for noise-robust isolated word recognition that works by decoding sequences of spikes from a population of simulated auditory feature-detecting neurons. Each neuron is trained to respond selectively to a brief spectrotemporal pattern, or feature, drawn from the simulated auditory nerve response to speech. The neural population conveys the time-dependent structure of a sound by its sequence of spikes. We compare two methods for decoding the spike sequences--one using a hidden Markov model-based recognizer, the other using a novel template-based recognition scheme. In the latter case, words are recognized by comparing their spike sequences to template sequences obtained from clean training data, using a similarity measure based on the length of the longest common sub-sequence. Using isolated spoken digits from the AURORA-2 database, we show that our combined system outperforms a state-of-the-art robust speech recognizer at low signal-to-noise ratios. Both the spike-based encoding scheme and the template-based decoding offer gains in noise robustness over traditional speech recognition methods. Our system highlights potential advantages of spike-based acoustic coding and provides a biologically motivated framework for robust ASR development.


Nanoparticles binding to lipid membranes: from vesicle-based gels to vesicle tubulation and destruction.
While cells offer numerous inspiring examples in which membrane morphology and function are controlled by interactions with viruses or proteins, we still lack design principles for controlling membrane morphology in synthetic systems. With experiments and simulations, we show that spherical nanoparticles binding to lipid-bilayer membrane vesicles results in a remarkably rich set of collective morphologies that are controllable via the particle binding energy. We separately study cationic and anionic particles, where the adhesion is tuned by addition of oppositely charged lipids to the vesicles. When the binding energy is weak relative to a characteristic membrane-bending energy, vesicles adhere to one another and form a soft solid gel, a novel and useful platform for controlled release. With larger binding energy, a transition from partial to complete wrapping of the nanoparticles causes a remarkable vesicle destruction process culminating in rupture, nanoparticle-membrane tubules, and an apparent inversion of the vesicles. These findings help unify the diverse phenomena observed previously. They also open the door to a new class of vesicle-based, closed-cell gels that are more than 99% water and can encapsulate and release on demand, and show how to drive intentional membrane remodeling for shape-responsive systems.


The COMBAT project: controlling and progressively minimizing the burden of vector-borne animal trypanosomosis in Africa.
Vector-borne diseases affecting livestock have serious impacts in Africa. Trypanosomosis is caused by parasites transmitted by tsetse flies and other blood-sucking Diptera. The animal form of the disease is a scourge for African livestock keepers, is already present in Latin America and Asia, and has the potential to spread further. A human form of the disease also exists, known as human African trypanosomosis or sleeping sickness. Controlling and progressively minimizing the burden of animal trypanosomosis (COMBAT) is a four-year research and innovation project funded by the European Commission, whose ultimate goal is to reduce the burden of animal trypanosomosis (AT) in Africa. The project builds on the progressive control pathway (PCP), a risk-based, step-wise approach to disease reduction or elimination. COMBAT will strengthen AT control and prevention by improving basic knowledge of AT, developing innovative control tools, reinforcing surveillance, rationalizing control strategies, building capacity, and raising awareness. Knowledge gaps on disease epidemiology, vector ecology and competence, and biological aspects of trypanotolerant livestock will be addressed. Environmentally friendly vector control technologies and more effective and adapted diagnostic tools will be developed. Surveillance will be enhanced by developing information systems, strengthening reporting, and mapping and modelling disease risk in Africa and beyond. The socio-economic burden of AT will be assessed at a range of geographical scales. Guidelines for the PCP and harmonized national control strategies and roadmaps will be developed. Gender equality and ethics will be pivotal in all project activities. The COMBAT project benefits from the expertise of African and European research institutions, national veterinary authorities, and international organizations. The project consortium comprises 21 participants, including a geographically balanced representation from 13 African countries, and it will engage a larger number of AT-affected countries through regional initiatives.


Selective autophagy and viruses.
In recent years, the process of selective autophagy has received much attention with respect to the clearance of protein aggregates, damaged mitochondria and bacteria. However, until recently, there have been virtually no studies on the selective autophagy of viruses, although they are perhaps one of the most ubiquitous unwanted constituents in human cells. Recently, we have shown that the ability of neuronal Atg5 to protect against lethal Sindbis virus central nervous system (CNS) infection in mice is associated with impaired viral capsid clearance, increased p62 accumulation and increased neuronal cell death. In vitro, we showed that p62 interacts with the Sindbis capsid protein and targets it for degradation in autophagosomes. Herein, we review these findings and broadly speculate about potential roles of selective viral autophagy in the regulation of host immunity and viral pathogenesis.


Differential responses of hypothalamus-pituitary-adrenal axis immediate early genes to corticosterone and circadian drive.
The hypothalamus-pituitary-adrenal (HPA) axis diurnal cycle of activity is manifest in circadian rhythms of ACTH and corticosterone secretion, which in the rat peak around the onset of the dark period. This cycle is thought to be driven by daily fluctuations in activity of CRH neurons within the paraventricular nucleus of the hypothalamus (PVN), controlled by suprachiasmatic nucleus inputs. In this study we examined whether the circadian drive that regulates ACTH and corticosterone basal secretion in the rat is reflected in PVN immediate early gene expression and, if so, whether different genes respond uniformly or uniquely to circadian stimulatory input. In addition, we examined how circadian drive and acute stress, two categories of stimuli that induce HPA axis activation, comparatively affect gene expression within different components of the HPA axis (c-fos mRNA, CRH heteronuclear RNA, and zif268 mRNA in PVN; c-fos mRNA, proopiomelanocortin heteronuclear RNA, and zinc finger 268 mRNA in anterior pituitary; c-fos mRNA and nerve growth factor I-B mRNA in adrenal cortex). Finally, we examined whether circadian differences in gene expression depend on endogenous glucocorticoids and, if so, whether the dependence is on an acute or permissive influence of the hormone. We found that a circadian drive that regulates HPA axis basal hormone secretion is also manifest on basal c-fos gene expression in the PVN. Moreover, we show that different immediate early genes within the HPA axis anatomical components display different diurnal patterns of gene expression. These differential patterns result, in part, from gene-specific responses to circadian signals and acute and/or permissive glucocorticoid actions.


Use-dependent hemispheric balance.
In the human brain, homologous regions of the primary motor cortices (M1s) are connected through transcallosal fibers. Interhemispheric communication between the two M1s plays a major role in the control of unimanual hand movements, and the strength of this connection seems to be dependent on arm activity. For instance, a lesion in the M1 can induce an increase in the excitability of the intact M1 and an abnormal high inhibitory influence onto the damaged M1. This can be attributable to either the disuse of the affected limb or the overuse of the unaffected one. Here, to directly investigate cortical modifications induced by an abnormal asymmetric use of the two limbs, we studied both the excitability of the two M1s and transcallosal interaction between them in healthy subjects whose right hand was immobilized for 10 h. The left "not-immobilized" arm was completely free to move in one group of participants (G1) and limited in the other one (G2). We found that the non-use reduced the excitability of the left M1 and decreased the inhibitory influence onto the right hemisphere in the two groups. However, an increase in the excitability of right M1 and a deeper inhibitory interaction onto the left hemisphere were evident only in G1. Thus, modifications in the right M1 were not directly produced by the non-use but would depend on the overuse of the "not-immobilized" arm. Our findings suggest that the balance between the two M1s is strongly use dependent.


Maintaining control: metabolism of molting Arctic seals in water and when hauled out.
Seals haul out of water for extended periods during the annual molt, when they shed and regrow their pelage. This behavior is believed to limit heat loss to the environment given increased peripheral blood flow to support tissue regeneration. The degree to which time in water, particularly during the molt, may affect thermoregulatory costs is poorly understood. We measured the resting metabolism of three spotted seals (Phoca largha), one ringed seal (Pusa hispida) and one bearded seal (Erignathus barbatus) during and outside the molting period, while resting in water and when hauled out. Metabolic rates were elevated in spotted and ringed seals during molt, but comparable in water and air for individuals of all species, regardless of molt status. Our data indicate that elevated metabolism during molt primarily reflects the cost of tissue regeneration, while increased haul out behavior is driven by the need to maintain elevated skin temperatures to support tissue regeneration.


Interleukin-22 (IL-22) production by pulmonary Natural Killer cells and the potential role of IL-22 during primary influenza virus infection.
We set out to test the hypothesis that interleukin-22 (IL-22), a cytokine crucial for epithelial cell homeostasis and recovery from tissue injury, would be protective during influenza virus infection. Recent studies have identified phenotypically and functionally unique intestinal NK cells capable of producing the cytokine IL-22. Unlike gut NK cells that produce IL-22, the surface phenotypes of lung NK cells were similar to those of spleen NK cells and were characteristically mature. With mitogen stimulation, both single and double IL-22- and gamma interferon (IFN-gamma)-producing lung NK cells were detected. However, only the IL-22(+) IFN-gamma(-) lung NK subset was observed after stimulation with IL-23. IL-23 receptor (IL-23R) blocking dramatically inhibited IL-22 production, but not IFN-gamma production. Furthermore, we found that NK1.1(+) or CD27(-) lung NK cells were the primary sources of IL-22. After influenza virus infection, lung NK cells were quickly activated to produce both IFN-gamma and IL-22 and had increased cytotoxic potential. The level of IL-22 in the lung tissue declined shortly after infection, gradually returning to the baseline after virus clearance, although the IL-22 gene expression was maintained. Furthermore, depletion of NK cells with or without influenza virus infection reduced the protein level of IL-22 in the lung. Anti-IL-22 neutralization in vivo did not dramatically affect weight loss and survival after virus clearance. Unexpectedly, anti-IL-22-treated mice had reduced virus titers. Our data suggest that during primary respiratory viral infection, IL-22 seems to a play a marginal role for protection, indicating a differential requirement of this cytokine for bacterial and viral infections.


Over-expression of nicotinamide phosphoribosyltransferase in ovarian cancers.
Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD(+)) synthesis and is required for cell growth, survival, DNA replication and repair, and angiogenesis. Nampt expression increases gene expression which promotes cell survival and increases SirT1 activity, promoting angiogenesis, and it is increased in several human malignancies. Recently, others have shown that ovarian serous adenocarcinomas (OSAs) express high levels of activated Stat3. Since Nampt expression is increased by Stat3, we hypothesized that Nampt protein might be highly expressed in OSAs. Using tissue microarray (TMA) and the avidin-biotin complex immunohistochemical technique we examined Nampt expression in 47 samples of benign ovarian tissue and 49 samples of ovarian serous adenoacarcinomas. Our data show that Nampt protein expression is significantly increased in OSAs as compared to benign ovarian tissue (0.49+/-0.12 benign vs. 4.78+/-0.46 malignant; +/-standard error of the mean). This is the first report demonstrating Nampt overexpression in OSA, which may shed light on the pathogenesis of OSA. Further studies of the role of Nampt overexpresion in OSA may shed light on the prognosis and clinical course of OSA. Last, since an effective pharmacologic Nampt inhibitor is currently in clinical use, further studies of Nampt overexpression in OSA may be used in selecting patients for Nampt inhibitor therapy.


Patient-days: a better measure of incidence of occupational bloodborne exposures.
There is currently no accepted standard denominator to calculate and to report the incidence of occupational exposures to bloodborne pathogens (OEBBPs) in health care. We performed a multicenter study of OEBBP injuries reported from 31 community hospitals in the southeastern United States from January 2003 to December 2006. A qualitative design was used to assess 4 commonly used denominators to calculate the incidence of OEBBP: patient-days; staffed beds; occupied beds and full-time employee equivalents (FTEs). Six criteria were used to assess the quality and suitability of each denominator as a standard method to calculate incidence of OEBBP. We also analyzed the correlation of hospital rankings produced by these 4 denominators. During 4 years of study, a total of 3375 occupational exposures were reported. Patient-days outperformed others as a denominator to calculate rates of OEBBP when judged by 6 predefined criteria. Data for staffed beds, occupied beds, and FTE were manually collected, infrequently reported, and often subject to missing data. Furthermore, FTE and staffed beds data also captured unoccupied beds and non-clinical employee data that were not associated with risk of OEBBP. Patient-days may be the most suitable and readily available denominator for standard reporting and benchmarking of incidence of OEBBP. Patient-days may be used as a standard method for comparing rates of OEBBP.


Loss of LGR4/GPR48 causes severe neonatal salt wasting due to disrupted WNT signaling altering adrenal zonation.
Disorders of isolated mineralocorticoid deficiency, which cause potentially life-threatening salt-wasting crisis early in life, have been associated with gene variants of aldosterone biosynthesis or resistance; however, in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone-producing adrenal zona glomerulosa (zG). Herein, we describe a highly consanguineous family with multiple perinatal deaths and infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex-specific ablation of Lgr4, using Lgr4fl/fl mice mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG, and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling, which results in abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.


miRNA Biomarkers in Breast Cancer Detection and Management.
Breast cancer is considered as a heterogeneous disease comprising various types of neoplasms, which involves different profile changes in both mRNA and micro-RNA (miRNA) expression. Extensive studies on mRNA expression in breast tumor have yielded some very interesting findings, some of which have been validated and used in clinic. Recent miRNA research advances showed great potential for the development of novel biomarkers and therapeutic targets. miRNAs are a new class of small non-coding regulatory RNAs that are involved in regulating gene expression at the posttranscriptional level. It has been demonstrated that miRNA expression is frequently deregulated in breast cancer, which warrants further in-depth investigation to decipher their precise regulatory role in tumorigenesis. We address briefly the regulatory mechanism of miRNA, the expression of miRNAs in tumorigenesis, and their potential use as breast cancer biomarkers for early disease diagnosis and prognosis. In addition, we discuss the use of the Formalin-Fixed, Paraffin-Embedded (FFPE) tissue as an invaluable source for breast cancer biomarker discovery and validation, and the potential use of circulating miRNAs in blood for early breast cancer detection. We envision the potential use of miRNAs in breast cancer management in the near future, particularly in improving the early diagnosis, prognosis and treatment.


Mass Spectrometric Quantitation of Apurinic/Apyrimidinic Sites in Tissue DNA of Rats Exposed to Tobacco-Specific Nitrosamines and in Lung and Leukocyte DNA of Cigarette Smokers and Nonsmokers.
Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N7POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 108 nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and nonsmokers. Rats treated with 5 or 21 mg/kg bw NNK for 4 days by s.c. injection had 2-6 and 2-17 times more AP sites than controls in liver and lung DNA (p < 0.05). Increases in AP sites were also found in liver DNA of rats exposed for 10 and 30 weeks (p < 0.05) but not for 50 and 70 weeks to 5 ppm of NNK in their drinking water. Levels of N7POBG were significantly correlated with AP sites in rats treated with NNK. In rats treated with 14 ppm (S)-NNN in their drinking water for 10 weeks, increased AP site formation compared to controls was observed in oral and nasal respiratory mucosa DNA (p < 0.05). No significant increase in AP sites was found in human lung and leukocyte DNA of cigarette smokers compared to nonsmokers, although AP sites in leukocyte DNA were significantly correlated with urinary levels of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). This is the first study to use mass spectrometry based methods to examine AP site formation by carcinogenic tobacco-specific nitrosamines in laboratory animals and to evaluate AP sites in DNA of smokers and nonsmokers.


The proteome of lentil (Lens culinaris Medik.) seeds: discriminating between landraces.
Lentil (Lens culinaris Medik.) is one of the most ancient crops of the Mediterranean region used for human nutrition; an extensive differentiation of L. culinaris over millennia has resulted in a number of different landraces. As a consequence of environmental and socio-economic issues, the disappearance of many of them occurred in more recent times. To investigate the potential of proteomics as a tool in phylogenetic studies, testing the possibility to identify specific markers of different plant landraces, 2-D gel electrophoretic maps of mature seeds were obtained from seven lentil populations belonging to a local ecotype (Capracotta) and five commercial varieties (Turca Rossa, Canadese, Castelluccio di Norcia, Rascino and Colfiorito). 2-DE analysis resolved hundreds of protein species in each lentil sample, among which only 122 were further identified by MALDI-TOF PMF and/or nanoLC-ESI-LIT-MS/MS, probably as a result of the poor information available on L. culinaris genome. A comparison of these maps revealed that 103 protein spots were differentially expressed within and between populations. The multivariate statistical analyses carried out on these variably expressed spots showed that 24 protein species were essential for population discrimination, thus determining their proposition as landrace markers. Besides providing the first reference map of mature lentil seeds, our data confirm previous studies based on morphological/genetic observations and further support the valuable use of proteomic techniques as phylogenetic tool in plant studies.


Formation of aerobic granules for the treatment of real and low-strength municipal wastewater using a sequencing batch reactor operated at constant volume.
This study aimed at evaluating the formation of aerobic granular sludge (AGS) for the treatment of real and low-strength municipal wastewater using a column sequencing batch reactor (SBR) operated in fill-draw mode (constant volume). The focus was on understanding how the wastewater upflow velocity (VWW) applied during the anaerobic feeding influenced the sludge properties and in turn the substrate conversion. Two different strategies were tested: (1) washing-out the flocs by imposing high wastewater upflow velocities (between 5.9 and 16 m h-1) during the anaerobic feeding (Approach #1) and (2) selective utilization of organic carbon during the anaerobic feeding (1 m h-1) combined with a selective sludge withdrawal (Approach #2). A column SBR of 190 L was operated in constant volume during 1500 days and fed with real and low-strength municipal wastewater. The formation of AGS with SVI30 of around 80 mL gTSS-1 was observed either at very low (1 m h-1) or at high VWW (16 m h-1). At 16 m h-1 the AGS was mainly composed of large and round granules (d > 0.63 mm) with a fluffy surface, while at 1 m h-1 the sludge was dominated by small granules (0.25 < d < 0.63 mm). The AGS contained a significant fraction of flocs during the whole operational period. A considerable and continuous washout of biomass occurred at VWW higher than 5.9 m h-1 (Approach #1) due to the lower settling velocity of the AGS fed with municipal wastewater. The low sludge retention observed at VWW higher than 5.9 m h-1 deteriorated the substrate conversion and in turn the effluent quality. High solid concentrations (and thus solid retention time) were maintained during Approach #2 (VWW of 1 m h-1), which resulted in an excellent effluent quality. The study demonstrated that the formation of AGS is possible during the treatment of real and low-strength municipal wastewater in a SBR operated at constant volume. Low wastewater upflow velocities should be applied during the anaerobic feeding phase in order to ensure enough biomass retention and efficient substrate removal.


Insulin resistance in NASH.
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease observed in the clinical practice of hepatology. The coexistence of metabolic syndrome in this cohort of patients has made insulin resistance central to the pathogenesis of these disorders. The metabolic consequence of insulin resistance is impaired hepatic glucose output and abnormal lipid handling. In the face of continued metabolic insults the normal hepatic regulatory mechanism gets overwhelmed and fat accumulates in the hepatocytes. The subsequent fate of steatotic hepatocytes depends on the capacity of additional factors such as adipocytokines and toxicity induced by the free fatty acids themselves to induce inflammatory response. This latter process is responsible for the producing the phenotype of non-alcoholic steatohepatitis (NASH). Irrespective of the process by which these phenotypic response occurs, it is now universally accepted that in the absence of insulin resistance the spectrum of changes one associates with NAFLD does not develop. In this review we will discuss the various processes that are involved in the pathogenesis of NAFLD.


Antigen affinity discrimination is an intrinsic function of the B cell receptor.
Antibody affinity maturation, a hallmark of adaptive immune responses, results from the selection of B cells expressing somatically hypermutated B cell receptors (BCRs) with increased affinity for antigens. Despite the central role of affinity maturation in antibody responses, the molecular mechanisms by which the increased affinity of a B cell for antigen is translated into a selective advantage for that B cell in immune responses is incompletely understood. We use high resolution live-cell imaging to provide evidence that the earliest BCR-intrinsic events that follow within seconds of BCR-antigen binding are highly sensitive to the affinity of the BCR for antigen. High affinity BCRs readily form oligomers and the resulting microclusters grow rapidly, resulting in enhanced recruitment of Syk kinase and calcium fluxes. Thus, B cells are able to read the affinity of antigen by BCR-intrinsic mechanisms during the earliest phases of BCR clustering, leading to the initiation of B cell responses.


Effect of oxybuprocaine 0.4% in preventing surgically induced miosis.
In a group of 68 patients a double-blind study was performed to assess the effect of preoperative oxybuprocaine 0.4% (Dorsacaine, Novesin) eye drops in comparison with a placebo in preventing surgically induced miosis during extracapsular cataract extraction. One drop of oxybuprocaine 0.4% or placebo was instilled 10 and 5 minutes preoperatively. The pupil diameter was recorded at different stages of the operation. It was found that oxybuprocaine reduced the amount of pupil constriction during the operation significantly as compared with the placebo group, facilitating the removal of lens material and the implantation of an intraocular lens. The effect of oxybuprocaine is considered to be due to anaesthetic action on sensory nerves in the eye, which may inhibit the release of a miotic substance.


Increased leptin levels in serum and peritoneal fluid of patients with pelvic endometriosis.
Pelvic endometriosis is an immune-related chronic inflammatory disease, characterized by ectopic implants of endometrium in the peritoneal cavity and associated with increased secretion of proinflammatory cytokines and neoangiogenesis. Leptin, the adipocyte-derived hormone, has been shown to have a role in food intake, basal metabolism, and reproductive function. Leptin levels are dynamically regulated, being elevated by inflammatory mediators and reduced by starvation. Leptin itself can influence the proinflammatory immune responses of CD4+ T lymphocytes, and reports have also shown this hormone to be an angiogenic factor in vitro and in vivo. We investigated whether leptin concentrations in serum and peritoneal fluid (PF) differed between 13 patients with different stages of endometriosis and 15 age- and body mass index-matched controls. We found a statistically significant (P < 0.05) increase in leptin levels in serum (30.3 +/- 14.8 ng/mL) and PF (35.9 +/- 17.4 ng/mL) of patients with endometriosis, compared with our control population (serum, 15.6 +/- 8.4; PF, 17.5 +/- 7.2 ng/mL). Regression equations, relating leptin to body mass index, were also significantly different in endometriosis patients, compared with controls. Higher levels of leptin were observed in the earlier stages of endometriosis than advanced-stage disease. These data suggest that the proinflammatory and neoangiogenic actions of leptin may contribute to the pathogenesis of endometriosis.


Skin-specific antibodies neutralizing mycolactone toxin during the spontaneous healing of Mycobacterium ulcerans infection.
Buruli ulcer, a neglected tropical infectious disease, is caused by Mycobacterium ulcerans. Without treatment, its lesions can progress to chronic skin ulcers, but spontaneous healing is observed in 5% of cases, suggesting the possible establishment of a host strategy counteracting the effects of M. ulcerans. We reveal here a skin-specific local humoral signature of the spontaneous healing process, associated with a rise in antibody-producing cells and specific recognition of mycolactone by the mouse IgG2a immunoglobulin subclass. We demonstrate the production of skin-specific antibodies neutralizing the immunomodulatory activity of the mycolactone toxin, and confirm the role of human host machinery in triggering effective local immune responses by the detection of anti-mycolactone antibodies in patients with Buruli ulcer. Our findings pave the way for substantial advances in both the diagnosis and treatment of Buruli ulcer in accordance with the most recent challenges issued by the World Health Organization.


Management of lumbar spinal stenosis.
Lumbar spinal stenosis (LSS) affects more than 200,000 adults in the United States, resulting in substantial pain and disability. It is the most common reason for spinal surgery in patients over 65 years. Lumbar spinal stenosis is a clinical syndrome of pain in the buttocks or lower extremities, with or without back pain. It is associated with reduced space available for the neural and vascular elements of the lumbar spine. The condition is often exacerbated by standing, walking, or lumbar extension and relieved by forward flexion, sitting, or recumbency. Clinical care and research into lumbar spinal stenosis is complicated by the heterogeneity of the condition, the lack of standard criteria for diagnosis and inclusion in studies, and high rates of anatomic stenosis on imaging studies in older people who are completely asymptomatic. The options for non-surgical management include drugs, physiotherapy, spinal injections, lifestyle modification, and multidisciplinary rehabilitation. However, few high quality randomized trials have looked at conservative management. A systematic review concluded that there is insufficient evidence to recommend any specific type of non-surgical treatment. Several different surgical procedures are used to treat patients who do not improve with non-operative therapies. Given that rapid deterioration is rare and that symptoms often wax and wane or gradually improve, surgery is almost always elective and considered only if sufficiently bothersome symptoms persist despite trials of less invasive interventions. Outcomes (leg pain and disability) seem to be better for surgery than for non-operative treatment, but the evidence is heterogeneous and often of limited quality.


Epidemiological aspects of human rotavirus infection in children hospitalized with acute gastroenteritis in an area of northern Italy.
Human rotavirus (HRV) is recognized as the most common cause of severe gastroenteritis in children under 5 years of age. Due to the lack of recent reports about the surveillance of HRV infection in Italy, in this study we assessed the prevalence rate of HRV infection on 1,340 stool samples belonging to 1,264 pediatric patients hospitalized with acute gastroenteritis in the period January 2000--December 2002. The stool samples were submitted to virological investigations by electron microscopy (EM) and conventional cell culture, as well as from January 2002 by RT-PCR for norovirus detection. Reovirus-like particles observed by EM were identified by electropherotyping. Single HRV infections were detected in 302 cases (23.9%, ranging from 19.1% in 2000 to 30.2% in 2001). Mixed infections were observed in 28 cases in which HRV was found to be associated with adenovirus in 16 cases (1.3%), with picornavirus in 4 (0.3%), and with norovirus in 8 (2.1% of the 388 cases examined in 2002). The 3 major epidemic periods of HRV infections were March--May 2000 (66 cases), December 2000--May 2001 (128 cases) and September 2001--April 2002 (105 cases) with peaks in March, January and March, and January, respectively. In the periods of major incidence, single HRV infection accounted even for 52.5% of the gastroenteritis cases monthly examined. According to age distribution, 68.9% (208 cases) of HRV infected children was under 4 years (69.6%: 230/330 cases, including mixed infections) and 16.9% (51 cases) was in the 5-12-year age-group. The epidemiological aspects of HRV infection, also compared to other enteric virus infections, will contribute to assess the magnitude of the problem of HRV in different settings and to devise strategies for intervention.


[Virion polypeptides of the nuclear polyhedrosis virus of the silkworm].
The nuclear polyhedrosis virus of the silkworm B. mori was obtained by dissolution of polyhedra at pH 10,5. It was shown that the virus particles contain a protease, which cleaves a few virion polypeptides during the dissolution of polyhedra under alkaline conditions. The proteolytic activity is inactivated by polyhedra treatment with phenylmethylsulfonylfluoride, diisopropylfluorophosphate, HgCl2 and at 80 degrees. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed 18 virion polypeptides with molecular weights ranging between 10000-110000. It was assumed that the virion includes the polyhedral protein and its fragments. A possible role of the polyhedral protein and its fragments in the virion is discussed.


A phylogenomic resolution of the sea urchin tree of life.
Echinoidea is a clade of marine animals including sea urchins, heart urchins, sand dollars and sea biscuits. Found in benthic habitats across all latitudes, echinoids are key components of marine communities such as coral reefs and kelp forests. A little over 1000 species inhabit the oceans today, a diversity that traces its roots back at least to the Permian. Although much effort has been devoted to elucidating the echinoid tree of life using a variety of morphological data, molecular attempts have relied on only a handful of genes. Both of these approaches have had limited success at resolving the deepest nodes of the tree, and their disagreement over the positions of a number of clades remains unresolved. We performed de novo sequencing and assembly of 17 transcriptomes to complement available genomic resources of sea urchins and produce the first phylogenomic analysis of the clade. Multiple methods of probabilistic inference recovered identical topologies, with virtually all nodes showing maximum support. In contrast, the coalescent-based method ASTRAL-II resolved one node differently, a result apparently driven by gene tree error induced by evolutionary rate heterogeneity. Regardless of the method employed, our phylogenetic structure deviates from the currently accepted classification of echinoids, with neither Acroechinoidea (all euechinoids except echinothurioids), nor Clypeasteroida (sand dollars and sea biscuits) being monophyletic as currently defined. We show that phylogenetic signal for novel resolutions of these lineages is strong and distributed throughout the genome, and fail to recover systematic biases as drivers of our results. Our investigation substantially augments the molecular resources available for sea urchins, providing the first transcriptomes for many of its main lineages. Using this expanded genomic dataset, we resolve the position of several clades in agreement with early molecular analyses but in disagreement with morphological data. Our efforts settle multiple phylogenetic uncertainties, including the position of the enigmatic deep-sea echinothurioids and the identity of the sister clade to sand dollars. We offer a detailed assessment of evolutionary scenarios that could reconcile our findings with morphological evidence, opening up new lines of research into the development and evolutionary history of this ancient clade.


Imp3p and Imp4p mediate formation of essential U3-precursor rRNA (pre-rRNA) duplexes, possibly to recruit the small subunit processome to the pre-rRNA.
In eukaryotes, formation of short duplexes between the U3 small nucleolar RNA (snoRNA) and the precursor rRNA (pre-rRNA) at multiple sites is a prerequisite for three endonucleolytic cleavages that initiate small subunit biogenesis by releasing the 18S rRNA precursor from the pre-rRNA. The most likely role of these RNA duplexes is to guide the U3 snoRNA and its associated proteins, designated the small subunit processome, to the target cleavage sites on the pre-rRNA. Studies by others in Saccharomyces cerevisiae have identified the proteins Mpp10p, Imp3p, and Imp4p as candidates to mediate U3-pre-rRNA interactions. We report here that Imp3p and Imp4p appear to stabilize an otherwise unstable duplex between the U3 snoRNA hinge region and complementary bases in the external transcribed spacer of the pre-rRNA. In addition, Imp4p, but not Imp3p, seems to rearrange the U3 box A stem structure to expose the site that base-pairs with the 5' end of the 18S rRNA, thereby mediating duplex formation at a second site. By mediating formation of both essential U3-pre-rRNA duplexes, Imp3p and Imp4p may help the small subunit processome to dock onto the pre-rRNA, an event indispensable for ribosome biogenesis and hence for cell growth.


Transanal minimally invasive surgery for benign and malignant rectal lesions: midterm outcomes from a tertiary center.
Although transanal minimally invasive surgery (TAMIS) for rectal neoplasia has gained wide acceptance, the mid-term and long-term outcomes are not widely reported in the literature. Describe the mid-term outcomes of patients who underwent TAMIS for benign and malignant rectal lesions in a single center. Retrospective cohort study. Tertiary referral center. Demographic, clinical, and oncological outcomes of patients who underwent TAMIS between January 2015 and December 2022 were prospectively collected. The indication for TAMIS was based on the National Comprehensive Cancer Network guidelines. The follow up for the cancer patients included clinical examination, tumor markers every 6 months and MRI rectum at the end of one year. In addition, colonoscopy and CT scan at years one and three and a final CT scan and colonoscopy at year five. Mid-term oncological and clinical outcome. Thirty elective TAMIS procedures included adenocarcinoma for 33.3% (n=10) of the patients, 20% (n=6) neuroendocrine tumor and the 40% (n=12) were adenomatous lesions. Negative resection margins were achieved in all malignant lesions. Perioperative complications occurred in 2 patients (6.6%), one patient had breaching into the peritoneal cavity, and postoperative hypotension occurred in another patient. The median follow-up time was 23 months (range: 5-72 months). Two patients with adenoma and positive margins developed recurrent adenoma (6.6%) and one patient with initial polypectomy biopsy of adenocarcinoma, had TAMIS with histopathology of adenoma and distant metastasis had developed. TAMIS for local excision of rectal neoplasia is a valid option with favorable mid-term outcomes provided there is adherence to careful selection criteria. Retrospective nature and small number of the patients.


Botulinum neurotoxin A activity is dependent upon the presence of specific gangliosides in neuroblastoma cells expressing synaptotagmin I.
Botulinum neurotoxin A (BoNT/A) is the deadliest of all known biological substances. Although its toxicity makes BoNT/A a biological warfare threat, its biologic activity makes it an increasingly useful therapeutic agent for the treatment of muscular disorders. However, almost 200 years after its discovery, the neuronal cell components required for the activity of this deadly toxin have not been unequivocally identified. In this work, neuroblastoma cells expressing synaptotagmin I, a protein shown to be bound by BoNT/A, were used to determine whether specific gangliosides were necessary for BoNT/A activity as measured by synaptosomal-associated protein of 25 kDa (SNAP-25) cleavage. Ganglioside GT1b was found to support BoNT/A activity significantly more effectively than GD1a, which was far more effective than GM1 when added to ganglioside-deficient murine cholinergic Neuro 2a or to human adrenergic SK-N-SH neuroblastoma cells. Whereas both cell lines expressed synaptotagmin I, SNAP-25 cleavage was not observed in the absence of complex gangliosides. These results indicate that 1) gangliosides are required for BoNT/A activity, 2) synaptotagmin I in the absence of gangliosides does not support BoNT/A activity, and 3) Neuro 2a cells are an efficient model system for studying the biological activity of BoNT/A.


Opportunities and challenges of hydrogel microspheres for tendon-bone healing after anterior cruciate ligament reconstruction.
Poor angiogenesis and bony ingrowth are the major factors causing unsatisfactory healing between the tendon graft and the bone tunnel surface. Exogenous biological factors, biomaterials, and cells have been considered as new strategies to promote healing quality in recent years. However, it remains challenging for their clinical use because of insufficient in-situ retention time and release efficiency. Increasing attention has been paid to the hydrogel microspheres (HMPs) as potential drug-loading deliveries in biomedicine due to their minimally invasive manner, extended drug retention time, and high loading efficiency. In this review, the healing mechanism between the tendon graft and the bone tunnel is introduced, which is followed by a brief summarization of current methods applied for enhancement of the healing quality. Then, the preclinical studies focusing on HMPs as novel drug carriers are summarized to address the aforementioned concerns in the treatment of tendon-bone healing. Of note, the challenges and perspectives of HMPs in clinical conversion are also outlooked. Collectively, this review may inspire researchers and clinicians to develop clinical available HMPs in orthopedics such as sports medicine from both material and biomedical aspects.


Glucocorticoid supplementation during ovarian stimulation for IVF or ICSI.
Ovarian response to stimulation during in-vitro fertilisation (IVF) and intra-cytoplasmic sperm injection (ICSI) plays an important role in determining live birth rates. Adjuvant treatments during ovarian stimulation that have different modes of action have been used to improve ovarian response to stimulation and outcome of IVF. Glucocorticoids (GCs) are a class of steroid hormones that have been used either alone or in combination with other stimulatory regimens in order to improve folliculogenesis and pregnancy rates. However, considerable uncertainty remains over whether administration of glucocorticoid during ovarian stimulation until oocyte recovery is superior to no glucocorticoid in improving live birth rates in women undergoing IVF/ICSI. To determine the safety and effectiveness of systemic glucocorticoids during ovarian stimulation for IVF and ICSI cycles. We searched the Cochrane Gynaecology and Fertility Group Specialised Register, the Cochrane Central Register of Studies Online (CRSO), MEDLINE, Embase, CINAHL and PsycINFO from inception to 10 October 2016. We handsearched reference lists of articles, trial registers and relevant conference proceedings and contacted researchers in the field. We included randomised controlled trials (RCTs) comparing adjuvant treatment with systemic glucocorticoids during ovarian stimulation for IVF or ICSI cycles versus no adjuvant treatment. Two review authors independently selected studies, assessed risk of bias and extracted the data. Our primary outcome was live birth. Secondary outcomes included clinical pregnancy, multiple pregnancy, miscarriage, ovarian hyperstimulation syndrome (OHSS) and side-effects. We calculated odds ratios (ORs) with 95% confidence intervals (CIs) and pooled the data using a fixed-effect model. The quality of the evidence was assessed using GRADE methods. Four RCTs were included in the review (416 women). The trials compared glucocorticoid supplementation during IVF stimulation versus placebo. Two of the studies had data in a form that we could not enter into analysis, so results include data from only two trials (310) women. For the outcome of live birth, data were available for only 212 women, as the larger study had data available from only one study centre.One of the studies gave inadequate description of randomisation methods, but the other was at low risk of bias in all domains. The evidence was rated as low or very low quality for all outcomes, mainly due to imprecision, with low sample sizes and few events.There was insufficient evidence to determine whether there was any difference between the groups in live birth rate (OR 1.08, 95% CI 0.45 to 2.58; 2 RCTs, n = 212, I2 = 0%, low-quality evidence). Our findings suggest that if the chance of live birth with placebo is assumed to be 15%, the chance following supplementation would be between 7% and 31%. There was no conclusive evidence of a difference in the clinical pregnancy rate (OR 1.69, 95% CI 0.98 to 2.90; 2 RCTs, n = 310, I2 = 0%, low-quality evidence).The evidence suggests that if the chance of clinical pregnancy with placebo is assumed to be 24%, the chance following treatment with glucocorticoid supplementation would be between 23% and 47%. There was also insufficient evidence to determine whether there was any difference between the groups in multiple-pregnancy rate (OR 3.32 , 95% CI 0.12 to 91.60; 1 RCT , n = 20, very low-quality evidence) or miscarriage rate (OR 1.00, 95% CI 0.05 to 18.57; 1 RCT, n = 20, very low-quality evidence). Neither of the studies reported OHSS or side-effects. The safety and effectiveness of glucocorticoid administration in women undergoing controlled ovarian hyperstimulation for IVF/ICSI cycles (until the day of oocyte retrieval) is unclear due to the small number of studies and low event rates. Whilst glucocorticoids possible increase the clinical pregnancy rate, there may be little or no impact on live birth rate. More research is needed.


Secondary adrenal insufficiency after intrathecal steroid administration.
A 38-year-old man developed secondary adrenal insufficiency as a consequence of intrathecal methylprednisolone administration. Evidence in support of this diagnosis included an absent plasma cortisol response to insulin-induced hypoglycemia, an inadequate adrenal response to exogenous corticotropin stimulation, a typical delayed response to prolonged corticotropin infusion over 3 days, and the findings of an elevated level of prednisolone in the cerebrospinal fluid a full 2 months after its administration. It is therefore recommended that patients receiving intrathecal steroids be carefully observed for the possible development of secondary adrenal insufficiency.


Efavirenz therapy in rhesus macaques infected with a chimera of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type 1.
The specificity of nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for the RT of human immunodeficiency virus type 1 (HIV-1) has prevented the use of simian immunodeficiency virus (SIV) in the study of NNRTIs and NNRTI-based highly active antiretroviral therapy. However, a SIV-HIV-1 chimera (RT-SHIV), in which the RT from SIVmac239 was replaced with the RT-encoding region from HIV-1, is susceptible to NNRTIs and is infectious to rhesus macaques. We have evaluated the antiviral activity of efavirenz against RT-SHIV and the emergence of efavirenz-resistant mutants in vitro and in vivo. RT-SHIV was susceptible to efavirenz with a mean effective concentration of 5.9 +/- 4.5 nM, and RT-SHIV variants selected with efavirenz in cell culture displayed 600-fold-reduced susceptibility. The efavirenz-resistant mutants of RT-SHIV had mutations in RT similar to those of HIV-1 variants that were selected under similar conditions. Efavirenz monotherapy of RT-SHIV-infected macaques produced a 1.82-log-unit decrease in plasma viral-RNA levels after 1 week. The virus load rebounded within 3 weeks in one treated animal and more slowly in a second animal. Virus isolated from these two animals contained the K103N and Y188C or Y188L mutations. The RT-SHIV-rhesus macaque model may prove useful for studies of antiretroviral drug combinations that include efavirenz.


Hard Decisions Shape the Neural Coding of Preferences.
Hard decisions between equally valued alternatives can result in preference changes, meaning that subsequent valuations for chosen items increase and decrease for rejected items. Previous research suggests that this phenomenon is a consequence of cognitive dissonance reduction after the decision, induced by the mismatch between initial preferences and decision outcomes. In contrast, this functional magnetic resonance imaging and eye-tracking study with male and female human participants found that preferences are already updated online during the process of decision-making. Preference changes were predicted from activity in left dorsolateral prefrontal cortex and precuneus while making hard decisions. Fixation durations during this phase predicted both choice outcomes and subsequent preference changes. These preference adjustments became behaviorally relevant only for choices that were remembered and were in turn associated with hippocampus activity. Our results suggest that preferences evolve dynamically as decisions arise, potentially as a mechanism to prevent stalemate situations in underdetermined decision scenarios.SIGNIFICANCE STATEMENT Most theories of decision-making assume that we always choose the best option available, based on a set of stable preferences. However, what happens for hard decisions when the available options are preferred equally? We show that in such stalemate situations, decision-makers adjust their preferences dynamically during the process of decision-making, and these preference adjustments are predicted by a left prefrontal-parietal network. We also show that eye movements during decision-making are predictive of the magnitude of the upcoming value change. Our results suggest that preferences are dynamic, adjusted every time a hard decision is made, prompting a re-evaluation of existing frameworks of decision-making.


Changes in gene expression profiles of bovine embryos produced in vitro, by natural ovulation, or hormonal superstimulation.
Embryos produced by hormonal superstimulation have been used as an in vivo control in most published research on embryo gene expression. However, it is not known if this is the most appropriate control for gene expression profile studies. We compared the expression of GRB-10, IGF-II, IGF-IIR, MnSOD, GPX-4, catalase, BAX, and interferon-tau genes, in embryos produced in vivo by hormonal superovulation (SOV), by in vitro fertilization (IVF) or in vivo without any hormonal stimulus (NOV). GRB-10 was less expressed in NOV than IVF embryos, whereas no differences were found for the other genes. The genes related to stress response were then grouped and compared; the sum of expression of MnSOD, GPX-4, and catalase genes tended to be greater in IVF than NOV embryos. A correlation analysis was performed; we found a distinct behavior for NOV embryos when compared with SOV and IVF in the expression of GRB-10, IGF-II and IGF-IIR genes. However, the behavior of these genes was similar in SOV and IVF embryos. We conclude that ovarian hormonal stimulation can affect embryos by altering gene expression. Although this conclusion was based on investigation of only a few genes, we suggest that SOV embryos should be used with caution as a control in gene expression studies.


Processing of hematopoietic stem cells from peripheral blood before cryopreservation: use of a closed automated system.
Hematopoietic stem cell transplantation is commonly used to treat several oncohematologic diseases. The autologous hematopoietic progenitor cells collected through apheresis (HPC-A) must be cryopreserved and stored before use in vivo. Cell processing that precedes cryopreservation of HPC-A includes volume reduction aimed at reducing the amount of dimethyl sulfoxide used, as well as storage space. The aim of our study was to assess the effectiveness of volume reduction performed with an automated closed system, namely, the Sepax S100 cell separation device (Biosafe SA). A total of 165 procedures were carried out on concentrates collected from 104 adult and pediatric patients. As a control group, 30 HPC-A units processed according to the standard method (i.e., centrifugation at a speed of 850 × g for 10 minutes, followed by manual plasma reduction) were evaluated. The volume reduction obtained was 59% (range, 20.54%-84.21%; standard deviation [SD], ± 12.19%), going from 236 mL (range, 100-443 mL; SD, ± 80.41 mL) to 97 mL (range, 33.00-263.00 mL; SD, ± 47.41 mL); recovery of nucleated cells was 90% (range, 64.84%-105.93%; SD, ± 8.76%), while that of CD34+ cells was 91% (range, 59.30%-119.37%; SD, ± 13.30%). These values did not differ from those obtained using the standard method. Automated processing required 20 minutes versus 40 minutes of manual processing. Our data demonstrate that volume reduction carried out with the Sepax S100 automated system was particularly effective; cell recovery was excellent and the time spent was short. Moreover, the closed system allows cell processing to be carried out in a contamination-controlled environment, in accordance with good manufacturing practice guidelines.


Evaluation and treatment of cryptorchidism: AUA guideline.
Cryptorchidism is one of the most common pediatric disorders of the male endocrine glands and the most common genital disorder identified at birth. This guideline is intended to provide physicians and non-physician providers (primary care and specialists) with a consensus of principles and treatment plans for the management of cryptorchidism (typically isolated non-syndromic). A systematic review and meta-analysis of the published literature was conducted using controlled vocabulary supplemented with key words relating to the relevant concepts of cryptorchidism. The search strategy was developed and executed by reference librarians and methodologists to create an evidence report limited to English-language, published peer-reviewed literature. This review yielded 704 articles published from 1980 through 2013 that were used to form a majority of the guideline statements. Clinical Principles and Expert Opinions were used for guideline statements lacking sufficient evidence-based data. Guideline statements were created to inform clinicians on the proper methods of history-taking, physical exam, and evaluation of the boy with cryptorchidism, as well as the various hormonal and surgical treatment options. Imaging for cryptorchidism is not recommended prior to referral, which should occur by 6 months of age. Orchidopexy (orchiopexy is the preferred term) is the most successful therapy to relocate the testis into the scrotum, while hormonal therapy is not recommended. Successful scrotal repositioning of the testis may reduce but does not prevent the potential long-term issues of infertility and testis cancer. Appropriate counseling and follow-up of the patient is essential.


Measuring the attitudes and impact of the eighty-hour workweek rules on orthopaedic surgery residents.
The literature on graduate medical education contains anecdotal reports of some effects of the new eighty-hour workweek on the attitudes and performance of residents. However, there are relatively few studies detailing the attitudes of large numbers of residents in a particular surgical specialty toward the new requirements. Between July and November 2004, a survey created by the Academic Advocacy Committee of the American Academy of Orthopaedic Surgeons was distributed by mail, fax, and e-mail to a total of 4207 orthopaedic residents at the postgraduate year-1 through year-6 levels of training. The survey responses were tabulated electronically, and the results were recorded. The survey response rate was 13.2% (554 residents). Sixty-eight percent (337) of the 495 respondents whose postgraduate-year level was known were at the postgraduate year-4 level or higher. Attitudes concerning the duty rules were mixed. Twenty-three percent of the 554 respondents thought that eighty hours constituted an appropriate number of duty hours per week; 41% believed that eighty hours were too many, and 34% thought that eighty hours were not sufficient. Thirty-three percent of the respondents had worked greater than eighty hours during at least a single one-week period since the new rules were implemented; this occurred more commonly among the postgraduate year-3 and more junior residents. Orthopaedic trauma residents had the most difficulty adhering to the new duty-hour restrictions. Eighty-two percent of the respondents indicated that their residency programs have been forced to make changes to their call schedules or to hire ancillary staff to address the rules. The use of physician assistants, night-float systems, and so-called home-call assignments were the most common strategies used to achieve compliance. Resident attitudes toward the work rules are mixed. The rules have forced residency programs to restructure. Junior residents have more favorable attitudes toward the new standards than do senior residents. Self-reporting of duty hours is the most common method of monitoring in orthopaedic training programs. Such systems allow ample opportunity for inaccuracies in the measurement of hours worked. Although residents report an improved quality of life as a result of these new rules, the attitude that the quality of training is diminished persists.


The Weather Research and Forecasting Model with Aerosol-Cloud Interactions (WRF-ACI): Development, Evaluation, and Initial Application.
The Weather Research and Forecasting Model with Aerosol-Cloud Interactions (WRF-ACI) is developed for studying aerosol effects on gridscale and subgrid-scale clouds using common aerosol activation and ice nucleation formulations and double-moment cloud microphysics in a scale-aware subgrid-scale parameterization scheme. Comparisons of both the standard WRF and WRF-ACI models' results for a summer season against satellite and reanalysis estimates show that the WRF-ACI system improves the simulation of cloud liquid and ice water paths. Correlation coefficients for nearly all evaluated parameters are improved, while other variables show slight degradation. Results indicate a strong cloud lifetime effect from current climatological aerosols increasing domain average cloud liquid water path and reducing domain average precipitation as compared to a simulation with aerosols reduced by 90%. Increased cloud-top heights indicate a thermodynamic invigoration effect, but the impact of thermodynamic invigoration on precipitation is overwhelmed by the cloud lifetime effect. A combination of cloud lifetime and cloud albedo effects increases domain average shortwave cloud forcing by ~3.0 W m-2. Subgrid-scale clouds experience a stronger response to aerosol levels, while gridscale clouds are subject to thermodynamic feedbacks because of the design of the WRF modeling framework. The magnitude of aerosol indirect effects is shown to be sensitive to the choice of autoconversion parameterization used in both the gridscale and subgrid-scale cloud microphysics, but spatial patterns remain qualitatively similar. These results indicate that the WRF-ACI model provides the community with a computationally efficient tool for exploring aerosol-cloud interactions.


Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis.
The effects of cryptococcal polysaccharide and selected serum factors on (i) the attachment of Cryptococcus neoformans to macrophages and (ii) the subsequent ingestion of yeast cells by the macrophages were investigated. Percent attachment was measured after incubation of yeast cells with macrophages at 4 C. Percent engulfment was determined after incubation of yeast cells and macrophages at 37 C. Nonencapsulated yeast cells readily attached to macrophages at the low temperature and were engulfed at a high rate at 37 C, whereas encapsulated yeast cells attached to macrophages at low rates and were engulfed at low rates. Addition of varying doses of purified cryptococcal polysaccharide to nonencapsulated yeast cells inhibited attachment at approximately the same concentration of polysaccharide required for inhibition of engulfment. Nonencapsulated yeast cells that attached to macrophages at 4 C were eluted from the macrophages by addition of purified cryptococcal polysaccharide to the incubation medium. Heat-labile opsonins were not required for attachment of yeast cells to macrophages, but they were necessary for maximal initial rates of phagocytosis. Heat-stable components of serum facilitated attachment of cryptococci, but their most important function appeared to be triggering the ingestion of attached yeast. Specific antiserum had no effect on the attachment and engulfment of nonencapsulated cryptococci, and the antiserum produced only a small enhancement of the engulfment of encapsulated cryptococci.


Back to the Wild: On a Quest for Donors Toward Salinity Tolerant Rice.
Salinity stress affects global food producing areas by limiting both crop growth and yield. Attempts to develop salinity-tolerant rice varieties have had limited success due to the complexity of the salinity tolerance trait, high variation in the stress response and a lack of available donors for candidate genes for cultivated rice. As a result, finding suitable donors of genes and traits for salinity tolerance has become a major bottleneck in breeding for salinity tolerant crops. Twenty-two wild Oryza relatives have been recognized as important genetic resources for quantitatively inherited traits such as resistance and/or tolerance to abiotic and biotic stresses. In this review, we discuss the challenges and opportunities of such an approach by critically analyzing evolutionary, ecological, genetic, and physiological aspects of Oryza species. We argue that the strategy of rice breeding for better Na+ exclusion employed for the last few decades has reached a plateau and cannot deliver any further improvement in salinity tolerance in this species. This calls for a paradigm shift in rice breeding and more efforts toward targeting mechanisms of the tissue tolerance and a better utilization of the potential of wild rice where such traits are already present. We summarize the differences in salinity stress adaptation amongst cultivated and wild Oryza relatives and identify several key traits that should be targeted in future breeding programs. This includes: (1) efficient sequestration of Na+ in mesophyll cell vacuoles, with a strong emphasis on control of tonoplast leak channels; (2) more efficient control of xylem ion loading; (3) efficient cytosolic K+ retention in both root and leaf mesophyll cells; and (4) incorporating Na+ sequestration in trichrome. We conclude that while amongst all wild relatives, O. rufipogon is arguably a best source of germplasm at the moment, genes and traits from the wild relatives, O. coarctata, O. latifolia, and O. alta, should be targeted in future genetic programs to develop salt tolerant cultivated rice.


[The relationship between polipharmacy, chronic complications and depression in individuals with type 2 diabetes mellitus].
The objectives of this study were: to characterize the polipharmacy in subjects with type 2 diabetes mellitus (DM2) and to verify the correlation between polipharmacy and number of medications for DM2 complications with depression indicators (Beck Depression inventory (BDI) and urinary cortisol (CORT) levels). A sample composed of 40 patients with DM2 from the Diabetes League of HCFM-USP was analyzed for depression indicators (CORT and BDI) in addition to evaluation for polipharmacy and number of DM2 complications. The results showed oral hypoglycemic agents, insulins, antihypertensives, diuretics, lipid-lowering drugs and thrombolytics are the most frequent medications used. In this sample, 75% used from 5 to 8 medicines daily and 12.5% used more than eight medicines/day; all of them used to take each medication at least 3 times daily. Between 1 and 3 DM2 complications were observed in 60% of the individuals and 22.5% showed more than 3 DM2 complications. No significant correlations were observed between depression indicators (BDI and CORT), number of medications and DM2 complications. However, positive correlation was observed between CORT and daily frequency of medication (Spearman, r = 0.319, p = 0.019).


Nano-organized shells and their application in controlled release.
The release characteristics of hollow-shell drug-delivery carriers are strongly dependent on the properties of the capsule shell, in particular its thickness and porous structure. The aim of this investigation was to conduct a detailed study of the relationship between capsule processing parameters, the resulting shell characteristics and subsequent release of an encapsulated liquid. Hollow spherical polymer capsules of constant outer diameter were prepared using electrohydrodynamic processing and the shell thickness of the capsules varied between 100-150 nm. For each type of capsule, the size and structure of channels present in the shell were extensively studied using electron microscopy. To investigate the effect upon the release characteristics the capsules were loaded with a water-soluble dye of molecular weight approximately 961 and release profiles determined using ultraviolet spectroscopy. The channel diameter was found to be similar for all shell thicknesses (-5 nm). The majority of the channels were radially aligned and through the full thickness of the shell. It was found that the rate of release decreased with increasing shell thickness and it became increasingly linear with respect to time; modeling confirmed that the release was diffusion dominated. The results of the study show that by controlling the structural characteristics of the shell of the hollow drug-carrier particles at the nanoscale through their forming methodology, the release profile can in turn be tailored according to the application requirements.


Deathbed visions from India: a study of family observations in northern Kerala.
This is an interview-based study of 104 families and their observations of the last weeks and days of a dying family member. Forty families reported "unusual experiences and behaviors" from the dying person in their last period of life. Thirty of these dying persons displayed behavior consistent with deathbed visions-interacting or speaking with deceased relatives, mostly their dead parents. There were six cases of reported premonitions of death and five possible confusional states with one patient reported to have had both a deathbed vision and confusional experiences. Socio-demographic factors such as gender, age, occupation, or cause and place of death were not found to be significant. Hindu patients appeared to be more likely than Muslim patients to report these experiences. Use of opiates (or not) did not appear to influence reports. The findings are discussed with reference to past studies of deathbed visions as well as their implications for the future pastoral care of dying people and their families.


Ferritin and serum iron levels in adult patients with sickle cell anaemia at Ibadan, Nigeria.
The ferritin and serum iron levels in 100 adult patients with sickle cell anaemia (SCA) and in ten pregnant (SCA) females were measured. All the adult sickle cell anaemia patients had serum ferritin levels ranging between 325 ng/dl and > 1500 ng/dl. These levels are within and above the normal range. The same trend was observed in the pregnant adult females with SCA. However, in the pregnant women, significantly lower ferritin levels than in the non-pregnant women were recorded. There is, therefore, a reduction in ferritin levels in pregnancy. This could be due to the increased iron requirements and demand by the developing foetus. Serum iron was below normal in 30% of the pregnant women, only 6% of all the adult patients had serum iron levels below the normal levels. Given the adequacy of ferritin levels in the patients studied and the very low incidence of below normal serum iron levels, it is concluded that the sickle cell anaemia patients in the area of study have adequate levels of iron and ferritin in their serum. Iron should therefore be given only in proven cases of iron deficiency anaemia.


Sclerosing Polycystic "Adenosis" of Salivary Glands: A Neoplasm Characterized by PI3K Pathway Alterations More Correctly Named Sclerosing Polycystic Adenoma.
Sclerosing polycystic adenosis (SPA) is a rare benign salivary gland lesion that usually arises from the parotid gland. SPA was originally interpreted to be a non-neoplastic alteration analogous to fibrocystic changes of the breast, but now there is uncertainty about whether it may represent a neoplasm. SPA often contains intraductal proliferations with an appearance similar to ductal neoplasia of the breast, and one study reported X-chromosome inactivation using polymorphisms of the human androgen receptor (Skalova et al., in AJSP 30:939-944, 2006). We investigated the genetics of SPA through targeted next generation sequencing (NGS). Four cases of SPA were retrieved from the authors' consultation files. A custom, targeted NGS panel including 1425 cancer-related genes was performed on all cases, followed by immunohistochemistry for PTEN. All four cases developed in females, ranging from 40 to 69 years (mean 52.5 years), affecting the parotid (n = 3) and submandibular glands (n = 1). All cases exhibited characteristic histologic features of SPA: well-circumscribed lesions with fibrosis and an admixture of ducts, myoepithelial cells and acinar cells, the latter containing brightly eosinophilic intracytoplasmic granules. Two cases had intraductal apocrine epithelial proliferations. By targeted NGS, loss-of-function mutations in PTEN were revealed in all 4 cases. In addition, 2 of 4 cases harbored PIK3CA mutations and 2 of 4 possessed PIK3R1 alterations; one case lacked both PIK3CA and PIK3R1 mutations. PTEN expression by immunohistochemistry was lost in the ductal and acinar elements but not the myoepithelial cells in all cases. SPA is characterized by genetic alterations in the PI3K pathway, with PTEN mutations seen most frequently. This molecular profile is similar to salivary duct carcinoma and the apocrine variant of intraductal carcinoma (i.e., salivary duct carcinoma-in situ). PI3K pathway alterations were found in cases both with and without intraductal apocrine proliferations, and PTEN immunohistochemistry suggested that the ductal and acinar cells, but not myoepithelial cells, were affected. Taken together, these findings strongly support that SPA is a neoplasm, more correctly named "sclerosing polycystic adenoma." The salivary duct carcinoma-like genetic alterations, coupled with the fact that the surrounding myoepithelial cells appear to be non-neoplastic, suggest a close relationship between SPA and apocrine intraductal carcinoma.


Dental plaque development on a hydroxyapatite disk in young adults observed by using a barcoded pyrosequencing approach.
Dental plaque is a dynamic microbial biofilm ecosystem that comprises hundreds of species including difficult-to-cultivate bacteria. We observed the assembly of a plaque bacterial community through 16S rRNA gene analysis. Plaque samples that accumulated on a hydroxyapatite disk for 1, 2, 3, 4, 5, and 7 days with saliva on day 0 were collected from 19 young adults using a removable resin splint. Quantitative PCR analysis showed that the total bacterial amount gradually increased and reached a plateau on day 4. Barcoded pyrosequencing analysis revealed that the microbial richness and diversity particularly increased between days 5 and 7. A principal coordinate analysis plot based on unweighted UniFrac showed the community assembly in a time-related manner, which became increasingly similar to the salivary microbiota. Facultative anaerobic bacteria such as Streptococcus, Neisseria, Abiotrophia, Gemella, and Rothia were predominant in the plaque bacterial community in the earlier days, whereas obligate anaerobes, such as Porphyromonas, Fusobacterium, Prevotella, and Capnocytophaga showed increased dominance on later days. UniFrac analysis also demonstrated that dental caries experience had a significant effect on the assembly process. Our results reveal the development pattern of the plaque bacterial community as well as the inter-individual differences associated with dental caries experience.


Skin Colour, Perception of Racism and Depression among Adolescents in Urban Brazil.
The relation between race, ethnicity and health has been recognised as having an important role in the understanding of social inequalities in health. In Brazil, miscegenation (the mixing of different ethnicities or races, especially in marriage) is recognised as a sign of racial tolerance, but individuals with black skin colour have poorer social and health indicators than whites. The hypothesis that perceived racial discrimination is associated with depression and depressive symptoms among adolescents living in a Brazilian urban area is analysed, taking into consideration sociodemographic variables, socioeconomic status and skin colour. This paper presents results from a cross-sectional study carried out with a random one-stage cluster sampling of households in the city of Salvador, Bahia, Brazil. The study population comprises 973 individuals from 10 to 21 years of age who answered questionnaires about racial discrimination, socio-demographic and health-related variables. Major depression according to DSM-IV criteria was assessed using a validated Portuguese version of the Patient Health Questionnaire (PHQ). The overall prevalence of major depression was estimated as 10.4%. No statistically significant differences were found for prevalence of major depression or any depressive symptom according to skin colour (Black versus non-Black). However, adolescents who reported racial discrimination were more likely to have major depression, even after adjustment for age, sex, socioeconomic status, skin colour and self-esteem (Adjusted Prevalence ratio = 2.00, 95% Confidence Interval: 1.37-2.97). Low self-esteem was not associated with racial discrimination. Depression among adolescents is a common disorder. Skin colour, a biological component of ethnic differences, was not a factor associated with depression or depressive symptoms. Instead, the perception of racial discrimination was a strong potential risk factor for major depression in this population group, which needs to be addressed in further studies and considered in preventive and mental health care programs.


[An interstitial miniature antenna for localized in vivo P-31 MR spectroscopy].
Phosphorus spectroscopy can be used to assess response in tumor therapy and to monitor response. An additional response parameter would be useful for individualization in oncological therapy. Methodical problems of localisation and contamination make it more difficult to interpret and reproduce the spectra. Interstitial and endoluminal spectroscopy antennas placed directly within or close to the tumor could provide help in this problem. We developed an interstitial 31P MRS antenna together with a tuning network which can be used in thermometry catheters for hyperthermia within an internal lumen of 1.1 mm in diameter. A prototype of this type of miniature antenna suitable for use in Siemens MRI scanners at 1.5 T was described spectroscopically with regard to excitation profile, range and SNR. In terms of quality, the excitation profiles of the interstitial antennas in relation to orientation correspond to those of comparable but considerably larger endocavitary antennas and catheter coils for MR imaging and spectroscopy. Maximum sensitivity was achieved by aligning the coil normal perpendicular to the B0 field. Signal losses of up to 50% have to be reckoned with when using other orientations. The maximum range of the interstitial antenna was determined using spectroscopy and was found to be 5 mm, i.e. 9 times coil radius. The sensitivity (SNR, detection sensitivity) of the studied type of interstitial antenna allows in vivo 31P spectroscopy to be performed despite the unusually low axial dimension (coil radius r = 0.55 mm). The prototype of the described interstitial antenna was used to measure an in vivo spectrum from the back muscle of a rabbit in 10 min. Nevertheless, the detection volume of at least some ml necessary for 31P spectroscopy results mainly from the large antenna length. The sensitivity (SNR, detection sensitivity) of the interstitial antenna needs to be further improved in order to assess treatment response in patients. However, the construction principle is suitable for intracavitary 31P spectroscopy antenna with larger diameters, which can be used for advanced rectal, cervical and prostate carcinomas.


hipBA toxin-antitoxin systems mediate persistence in Caulobacter crescentus.
Antibiotic persistence is a transient phenotypic state during which a bacterium can withstand otherwise lethal antibiotic exposure or environmental stresses. In Escherichia coli, persistence is promoted by the HipBA toxin-antitoxin system. The HipA toxin functions as a serine/threonine kinase that inhibits cell growth, while the HipB antitoxin neutralizes the toxin. E. coli HipA inactivates the glutamyl-tRNA synthetase GltX, which inhibits translation and triggers the highly conserved stringent response. Although hipBA operons are widespread in bacterial genomes, it is unknown if this mechanism is conserved in other species. Here we describe the functions of three hipBA modules in the alpha-proteobacterium Caulobacter crescentus. The HipA toxins have different effects on growth and macromolecular syntheses, and they phosphorylate distinct substrates. HipA1 and HipA2 contribute to antibiotic persistence during stationary phase by phosphorylating the aminoacyl-tRNA synthetases GltX and TrpS. The stringent response regulator SpoT is required for HipA-mediated antibiotic persistence, but persister cells can form in the absence of all hipBA operons or spoT, indicating that multiple pathways lead to persister cell formation in C. crescentus.


Global convergence in the temperature sensitivity of respiration at ecosystem level.
The respiratory release of carbon dioxide (CO(2)) from the land surface is a major flux in the global carbon cycle, antipodal to photosynthetic CO(2) uptake. Understanding the sensitivity of respiratory processes to temperature is central for quantifying the climate-carbon cycle feedback. We approximated the sensitivity of terrestrial ecosystem respiration to air temperature (Q(10)) across 60 FLUXNET sites with the use of a methodology that circumvents confounding effects. Contrary to previous findings, our results suggest that Q(10) is independent of mean annual temperature, does not differ among biomes, and is confined to values around 1.4 +/- 0.1. The strong relation between photosynthesis and respiration, by contrast, is highly variable among sites. The results may partly explain a less pronounced climate-carbon cycle feedback than suggested by current carbon cycle climate models.


Retinal Blood Velocity Waveform Characteristics With Aging and Arterial Stiffening in Hypertensive and Normotensive Subjects.
We aimed to explore the velocity waveform characteristics of the retinal artery associated with age and the cardio-ankle vascular index (CAVI) as a conventional arterial stiffness marker by applying the Doppler optical coherence tomography (DOCT) flowmeter. In this cross-sectional study, DOCT flowmeter imaging was performed in 66 participants aged 21 to 83 years (17 men, 49 women) with no history of eye diseases and no systemic diseases, except for hypertension. Retinal blood velocity waveform was analyzed where several parameters in time (upstroke time, T1, T2, T3, and T4) and area under the waveform (area elevation, area declination, A1, A2, A3, and A4) were extracted. Systolic blood pressure-adjusted Pearson's coefficients were calculated to determine the correlations of each parameter with age or CAVI. Corrected upstroke time (UTc) was the waveform parameter most positively correlated with age (r = 0.497, P < 0.001). Area declination was the waveform parameter most negatively correlated with age (r = -0.682, P < 0.001) and CAVI (r = -0.601, P < 0.001). We extracted the waveform parameters associated with the risks of arterial stiffening. The velocity waveform analysis of the retinal artery with DOCT flowmeter potentially could become a new method for arterial stiffness identification. DOCT flowmeter could evaluate arterial stiffening in a different way from the conventional method of measuring arterial stiffening using pressure waveform. Because the DOCT flowmeter can easily, quickly, and noninvasively provide a retinal blood velocity waveform, this system could be useful as a routine medical examination for arterial stiffening.


Detection of soluble HLA-G molecules in plasma and amniotic fluid.
Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P<0.05) lower than in HLA-A11 negative ones (mean: 24.5+/-2.0 SEM ng/ml; n=80). sHLA-G levels in women at delivery (mean: 22.9+/-2.2 SEM ng/ml; n=28) were in the range of controls but were significantly (P<0.001) reduced in the corresponding cord bloods (mean: 13.8+/-1.5 SEM ng/ml; n=28). sHLA-G levels in amniotic fluids (mean: 15.5 + 1.0 SEM ng/ml; n=50) were significantly (P<0.001) lower than in plasma. sHLA-G levels were 5 and 11% of those of sHLA-I antigens in plasmas and amniotic fluids, respectively. Individual sHLA-G levels were not correlated with sHLA-I levels. SDS-PAGE analysis of plasma sHLA-G antigens revealed two molecular variants with a 35 kD and a 27 kD MW corresponding to the sizes of sHLA-G1 and -G2 isoforms. In conclusion, our study has shown that the two-step assay we have developed is reliable in measuring sHLA-G antigen levels. This assay will facilitate the analysis of the biological and clinical significance of sHLA-G antigens in plasma.


Chromosomal Instability-Driven Cancer Progression: Interplay with the Tumour Microenvironment and Therapeutic Strategies.
Chromosomal instability (CIN) is a prevalent characteristic of solid tumours and haematological malignancies. CIN results in an increased frequency of chromosome mis-segregation events, thus yielding numerical and structural copy number alterations, a state also known as aneuploidy. CIN is associated with increased chances of tumour recurrence, metastasis, and acquisition of resistance to therapeutic interventions, and this is a dismal prognosis. In this review, we delve into the interplay between CIN and cancer, with a focus on its impact on the tumour microenvironment-a driving force behind metastasis. We discuss the potential therapeutic avenues that have resulted from these insights and underscore their crucial role in shaping innovative strategies for cancer treatment.


Regulation of renal urea transporters.
Urea is important for the conservation of body water due to its role in the production of concentrated urine in the renal inner medulla. Physiologic data demonstrate that urea is transported by facilitated and by active urea transporter proteins. The facilitated urea transporter (UT-A) in the terminal inner medullary collecting duct (IMCD) permits very high rates of transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. Four isoforms of the UT-A urea transporter family have been cloned to date. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thereby preventing loss of urea from the medulla and decreasing urine-concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in Jk null individuals (who lack Kidd antigen). In addition to these facilitated urea transporters, three sodium-dependent, secondary active urea transport mechanisms have been characterized functionally in IMCD subsegments: (1) active urea reabsorption in the apical membrane of initial IMCD from low-protein fed or hypercalcemic rats; (2) active urea reabsorption in the basolateral membrane of initial IMCD from furosemide-treated rats; and (3) active urea secretion in the apical membrane of terminal IMCD from untreated rats. This review focuses on the physiologic, biophysical, and molecular evidence for facilitated and active urea transporters, and integrative studies of their acute and long-term regulation in rats with reduced urine-concentrating ability.


Functional dissection of a small anaerobically induced bZIP transcription factor from tomato.
A small anaerobically induced tomato transcription factor was isolated from a subtractive library. This factor, designated ABZ1 (anaerobic basic leucine zipper), is anaerobically induced in fruits, leaves and roots and encodes a nuclear localized protein. ABZ1 shares close structural and sequence homology with the S-family of small basic leucine zipper (bZIP) transcription factors that are implicated in stress response. Nuclear localization of ABZ1 is mediated by the basic region and occurs under normoxic conditions. ABZ1 binds to G-box-like target sites as a dimer. Binding can be abolished by heterodimerization with a truncated protein retaining the leucine zipper but lacking the DNA binding domain. The protein binds in a sequence specific manner to the CaMV 35S promoter which is down regulated when ABZ1 is coexpressed. This correlates with the anaerobic down regulation of the 35S promoter in tomato and tobacco. These results may suggest that small bZIP proteins are involved in the negative regulation of gene expression under anaerobic conditions.