Human vascular smooth muscle cells. Target for and source of tumor necrosis factor.
TNF-alpha (also known as cachectin) may produce many of its important effects in vivo by actions on blood vessels. Endothelial cells are well known to respond to TNF-alpha. We investigated whether vascular smooth muscle cells (SMC), the most abundant cell type in most vessels, also respond to TNF-alpha and the related cytokine lymphotoxin (TNF-beta). Both human rTNF-alpha and beta (0.1 to 100 ng/ml) induced transient accumulation of IL-1 mRNA by adult human vascular SMC that peaked between 1 and 4 h. The inhibitor of RNA synthesis actinomycin D (1 microgram/ml) blocked the induction of IL-1 mRNA, whereas inhibition of protein synthesis with cycloheximide (1 microgram/ml) resulted in a marked "superinduction" of both IL-1 alpha and IL-1 beta mRNA species. TNF-alpha treatment also increased intracellular biologically active IL-1 and subsequent release of IL-1 activity from SMC. Metabolic labeling and immunoprecipitation with specific antibodies demonstrated de novo synthesis of IL-1 alpha and IL-1 beta precursors in TNF-treated or lymphotoxin-treated SMC. TNF-alpha also activated other SMC functions including the concentration-dependent release of PGE2 from SMC, and time-dependent induction of the gene for (2'-5')-oligoadenylate synthetase, an enzyme thought to mediate the anti-viral and anti-proliferative actions of IFN. We also explored whether SMC, which both produce and respond to IL-1, might also express either of the TNF genes. Bacterial LPS (10 micrograms/ml) caused slight accumulation of TNF-alpha transcripts. Incubation of SMC for 4 h with inhibitors of protein synthesis alone caused little or no elevation of TNF-alpha mRNA, but simultaneous addition of LPS ("superinduction" conditions) induced large amounts of TNF-alpha (but not TNF-beta) mRNA. Cells treated with anisomycin (1 microgram/ml) and LPS, then washed to remove this reversible inhibitor of protein synthesis, released TNF-alpha into the medium, as assessed by the L929 cytotoxicity assay and by metabolic labeling and immunoprecipitation. Thus, SMC both respond to both TNF and lymphotoxin and can produce TNF-alpha, a cytokine with numerous effects on vascular cells of potential significance in the pathophysiology of septic shock and other inflammatory conditions.


White matter correlates of different aspects of facial affect recognition impairment following traumatic brain injury.
Although facial affect recognition deficits are well documented in individuals with moderate-to-severe traumatic brain injury (TBI), little research has examined the neural mechanisms underlying these impairments. Here, we use diffusion tensor imaging (DTI), specifically the scalars fractional anisotropy (FA), mean diffusivity (MD), and radial diffusivity (RD), to examine relationships between regional white-matter integrity and two facial affect sub-skills: perceptual affect recognition abilities (measured by an affect matching task) and verbal categorization of facial affect (measured by an affect labeling task). Our results showed that, within the TBI group, higher levels of white-matter integrity in tracts involved in affect recognition (inferior fronto-occipital, inferior longitudinal, and uncinate fasciculi) were associated with better performance on both tasks. Verbal categorization skills were specifically and positively correlated with integrity of the left uncinate fasciculus. Moreover, we observed a striking lateralization effect, with perceptual abilities having an almost exclusive relationship with integrity of right hemisphere tracts, while verbal abilities were associated with both left and right hemisphere integrity. The findings advance our understanding of the neurobiological mechanisms that underlie subcomponents of facial affect recognition and lead to different patterns of facial affect recognition impairment in adults with TBI.


The SH2B1 obesity locus and abnormal glucose homeostasis: lack of evidence for association from a meta-analysis in individuals of European ancestry.
The development of type 2 diabetes (T2D) is influenced both by environmental and by genetic determinants. Obesity is an important risk factor for T2D, mostly mediated by obesity-related insulin resistance. Obesity and insulin resistance are also modulated by the genetic milieu; thus, genes affecting risk of obesity and insulin resistance might also modulate risk of T2D. Recently, 32 loci have been associated with body mass index (BMI) by genome-wide studies, including one locus on chromosome 16p11 containing the SH2B1 gene. Animal studies have suggested that SH2B1 is a physiological enhancer of the insulin receptor and humans with rare deletions or mutations at SH2B1 are obese with a disproportionately high insulin resistance. Thus, the role of SH2B1 in both obesity and insulin resistance makes it a strong candidate for T2D. However, published data on the role of SH2B1 variability on the risk for T2D are conflicting, ranging from no effect at all to a robust association. The SH2B1 tag SNP rs4788102 (SNP, single nucleotide polymorphism) was genotyped in 6978 individuals from six studies for abnormal glucose homeostasis (AGH), including impaired fasting glucose, impaired glucose tolerance or T2D, from the GENetics of Type 2 Diabetes in Italy and the United States (GENIUS T2D) consortium. Data from these studies were then meta-analyzed, in a Bayesian fashion, with those from DIAGRAM+ (n = 47,117) and four other published studies (n = 39,448). Variability at the SH2B1 obesity locus was not associated with AGH either in the GENIUS consortium (overall odds ratio (OR) = 0.96; 0.89-1.04) or in the meta-analysis (OR = 1.01; 0.98-1.05). Our data exclude a role for the SH2B1 obesity locus in the modulation of AGH.


Analysis of the neuronal selectivity underlying low fMRI signals.
A prevailing assumption in neuroimaging studies is that relatively low fMRI signals are due to weak neuronal activation, and, therefore, they are commonly ignored. However, lower fMRI signals may also result from intense activation by highly selective, albeit small, subsets of neurons in the imaged voxel. We report on an approach that could form a basis for resolving this ambiguity imposed by the low (mm range) spatial resolution of fMRI. Our approach employs fMR-adaptation as an indicator for highly active neuronal populations even when the measured fMRI signal is low. In this study, we first showed that fMRI-adaptation is diminished when overall neuronal activity is lowered substantially by reducing image contrast. We then applied the same adaptation paradigm, but this time we lowered the fMRI signal by changing object shape. While the overall fMRI signal in category-related regions such as the face-related pFs was drastically reduced for non-face stimuli, the adaptation level obtained for these stimuli remained high. We hypothesize that the relatively greater adaptation level following exposure to "nonoptimal" object shapes is indicative of small subsets of neurons responding vigorously to these "nonoptimal" objects even when the overall fMRI activity is low. Our results show that fMR-adaptation can be used to differentiate between neuronal activation patterns that appear similar in the overall fMRI signal. The results suggest that it may be possible to employ fMR-adaptation to reveal functionally heterogeneous islands of activity, which are too small to image using conventional imaging methods.


Fibroblast growth factor 23 and risks of mortality and end-stage renal disease in patients with chronic kidney disease.
A high level of the phosphate-regulating hormone fibroblast growth factor 23 (FGF-23) is associated with mortality in patients with end-stage renal disease, but little is known about its relationship with adverse outcomes in the much larger population of patients with earlier stages of chronic kidney disease. To evaluate FGF-23 as a risk factor for adverse outcomes in patients with chronic kidney disease. A prospective study of 3879 participants with chronic kidney disease stages 2 through 4 who enrolled in the Chronic Renal Insufficiency Cohort between June 2003 and September 2008. All-cause mortality and end-stage renal disease. At study enrollment, the mean (SD) estimated glomerular filtration rate (GFR) was 42.8 (13.5) mL/min/1.73 m(2), and the median FGF-23 level was 145.5 RU/mL (interquartile range [IQR], 96-239 reference unit [RU]/mL). During a median follow-up of 3.5 years (IQR, 2.5-4.4 years), 266 participants died (20.3/1000 person-years) and 410 reached end-stage renal disease (33.0/1000 person-years). In adjusted analyses, higher levels of FGF-23 were independently associated with a greater risk of death (hazard ratio [HR], per SD of natural log-transformed FGF-23, 1.5; 95% confidence interval [CI], 1.3-1.7). Mortality risk increased by quartile of FGF-23: the HR was 1.3 (95% CI, 0.8-2.2) for the second quartile, 2.0 (95% CI, 1.2-3.3) for the third quartile, and 3.0 (95% CI, 1.8-5.1) for the fourth quartile. Elevated fibroblast growth factor 23 was independently associated with significantly higher risk of end-stage renal disease among participants with an estimated GFR between 30 and 44 mL/min/1.73 m(2) (HR, 1.3 per SD of FGF-23 natural log-transformed FGF-23; 95% CI, 1.04-1.6) and 45 mL/min/1.73 m(2) or higher (HR, 1.7; 95% CI, 1.1-2.4), but not less than 30 mL/min/1.73 m(2). Elevated FGF-23 is an independent risk factor for end-stage renal disease in patients with relatively preserved kidney function and for mortality across the spectrum of chronic kidney disease.


Gender Differentiated Preferences for a Community-Based Conservation Initiative.
Community-based conservation (CBC) aims to benefit local people as well as to achieve conservation goals, but has been criticised for taking a simplistic view of "community" and failing to recognise differences in the preferences and motivations of community members. We explore this heterogeneity in the context of Kenya's conservancies, focussing on the livelihood preferences of men and women living adjacent to the Maasai Mara National Reserve. Using a discrete choice experiment we quantify the preferences of local community members for key components of their livelihoods and conservancy design, differentiating between men and women and existing conservancy members and non-members. While Maasai preference for pastoralism remains strong, non-livestock-based livelihood activities are also highly valued and there was substantial differentiation in preferences between individuals. Involvement with conservancies was generally perceived to be positive, but only if households were able to retain some land for other purposes. Women placed greater value on conservancy membership, but substantially less value on wage income, while existing conservancy members valued both conservancy membership and livestock more highly than did non-members. Our findings suggest that conservancies can make a positive contribution to livelihoods, but care must be taken to ensure that they do not unintentionally disadvantage any groups. We argue that conservation should pay greater attention to individual-level differences in preferences when designing interventions in order to achieve fairer and more sustainable outcomes for members of local communities.


A PKC-beta inhibitor protects against cardiac microvascular ischemia reperfusion injury in diabetic rats.
PKC-beta inhibitor Ruboxistaurin (RBX or LY333531) can be used to reverse diabetic microvascular complication. However, it has not been previously established whether RBX can protect against ischemia/reperfusion (I/R) injury of cardiac microvessels in diabetic rats. STZ-induced diabetic rats were randomized into four groups and underwent I/R procedures. Cardiac barrier function and the region of cardiac microvascular lesion were examined. Cell monolayer barrier function was detected in cultured cardiac microvascular endothelial cells (CMECs) subjected to simulated I/R (SI/R). PKC-beta siRNA was transfected into CMECs to silence PKC-beta. Apoptosis Index of CMECs was detected by TUNEL assay and phosphor-LIMK2 protein expression was examined by Western blot analysis. RBX and insulin administration significantly reduced the cardiac microvascular lesion region and Apoptosis Index of endothelial cells (all P < 0.05 vs. no-treatment group). RBX decreased phosphor-LIMK2 expression (P < 0.05 vs. no-treatment group). RBX pretreatment and transfection with PKC-beta siRNA induced a rapid barrier enhancement in CMECs monolayer as detected by increased transendothelial electrical resistance (TER) and decreased FITC-dextran clearance (all P < 0.05 vs. no-treatment group). Meanwhile, RBX pretreatment and transfection with PKC-beta siRNA significantly decreased TUNEL positive CMECs and phosphor-LIMK2 expression in cultured CMECs (all P < 0.05 vs. no-treatment group). RBX pretreatment reduced F-actin/G-actin in cultured CMECs, reproducing the same effect as PKC-beta siRNA. These data indicate that PKC-beta inhibitor (RBX) may be helpful in attenuating the risk of severe cardiac microvascular I/R injury in diabetic rats partly due to its maintenance of endothelial barrier function and anti-apoptotic effect.


Dietary impact on esophageal cancer in humans: a review.
Cancer is the abnormal growth of cells that tend to proliferate in an uncontrolled way. About 32.6 million people in the world are affected by different types of cancer; 456 000 have esophageal cancer, which is the eighth most common cancer and the sixth most common cause of cancer-related deaths with a toll of 400 000 in 2012. About 80% of cases were reported from the less developed regions of the world. An incidence ratio of 2.4 : 1 for men and women reveals the gender disparity. Esophageal cancer is mainly of two types, namely, squamous cell carcinoma and adenocarcinoma. Most of the studies show a positive correlation of esophageal cancer with alcohol and tobacco consumption, but a negative correlation with fruit and vegetable intake. The type and amount of cereal, fat, meat and beverages consumed are also under study. Significant statistical synergistic interaction has been registered in studies considering the joint effect of the amount of beverage swallowed and its temperature, especially among heavy consumers. The role of non-nutritional components is gaining increasing attention because of their antioxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic properties. Socioeconomic status is another factor that cannot be ignored. Nevertheless, the predominant cause appears to be different in different regions, and in spite of the great advancements in the medical field, this still remains an enigma for researchers.


On the mechanism of CFTR inhibition by a thiazolidinone derivative.
The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.


Validation of a screening protocol for identifying low-risk candidates with type 1 diabetes mellitus for kidney with or without pancreas transplantation.
Certain clinical risk factors are associated with significant coronary artery disease in kidney transplant candidates with diabetes mellitus. We sought to validate the use of a clinical algorithm in predicting post-transplantation mortality in patients with type 1 diabetes. We also examined the prevalence of significant coronary lesions in high-risk transplant candidates. All patients with type 1 diabetes evaluated between 1991 and 2001 for kidney with/without pancreas transplantation were classified as high-risk based on the presence of any of the following risk factors: age >or=45 yr, smoking history >or=5 pack years, diabetes duration >or=25 yr or any ST-T segment abnormalities on electrocardiogram. Remaining patients were considered low risk. All high-risk candidates were advised to undergo coronary angiography. The primary outcome of interest was all-cause mortality post-transplantation. Eighty-four high-risk and 42 low-risk patients were identified. Significant coronary artery stenosis was detected in 31 high-risk candidates. Mean arterial pressure was a significant predictor of coronary stenosis (odds ratio 1.68; 95% confidence interval 1.14-2.46), adjusted for age, sex and duration of diabetes. In 75 candidates who underwent transplantation with median follow-up of 47 months, the use of clinical risk factors predicted all eight deaths. No deaths occurred in low-risk patients. A significant mortality difference was noted between the two risk groups (p = 0.03). This clinical algorithm can identify patients with type 1 diabetes at risk for mortality after kidney with/without pancreas transplant. Patients without clinical risk factors can safely undergo transplantation without further cardiac evaluation.


Towards a statistical mechanical theory of active fluids.
We present a stochastic description of a model of N mutually interacting active particles in the presence of external fields and characterize its steady state behavior in the absence of currents. To reproduce the effects of the experimentally observed persistence of the trajectories of the active particles we consider a Gaussian force having a non-vanishing correlation time τ, whose finiteness is a measure of the activity of the system. With these ingredients we show that it is possible to develop a statistical mechanical approach similar to the one employed in the study of equilibrium liquids and to obtain the explicit form of the many-particle distribution function by means of the multidimensional unified colored noise approximation. Such a distribution plays a role analogous to the Gibbs distribution in equilibrium statistical mechanics and provides complete information about the microscopic state of the system. From here we develop a method to determine the one- and two-particle distribution functions in the spirit of the Born-Green-Yvon (BGY) equations of equilibrium statistical mechanics. The resulting equations which contain extra-correlations induced by the activity allow us to determine the stationary density profiles in the presence of external fields, the pair correlations and the pressure of active fluids. In the low density regime we obtained the effective pair potential ϕ(r) acting between two isolated particles separated by a distance, r, showing the existence of an effective attraction between them induced by activity. Based on these results, in the second half of the paper we propose a mean field theory as an approach simpler than the BGY hierarchy and use it to derive a van der Waals expression of the equation of state.


Urinary Podocalyxin as a Biomarker to Diagnose Membranous Nephropathy.
A non-invasive diagnostic marker of membranous nephropathy (MN) is desirable. The urinary level of podocalyxin (PCX) is higher in various glomerular diseases, including MN. The aim of this study was to construct a diagnostic model of MN with the combination of urinary PCX and clinical parameters. We performed this cross-sectional study to construct the diagnostic models for MN by using data and samples from the multicenter kidney biopsy registry of Nagoya University and its affiliated hospitals. Urinary (u-) PCX was measured by sandwich ELISA. We constructed 3 types of diagnostic models in 105 training samples: u-PCX univariate model, the combined model of clinical parameters other than u-PCX (clinical model), and the combined model of both u-PCX and clinical parameters (combined model). We assessed the clinical usefulness of the diagnostic models through the comparison of c-statistics and decision curve analysis (DCA) in 209 validation samples. The clinical model consisted of age, glomerular filtration rate, and diabetes mellitus. In the training cohort, the c-statistics were 0.868 [95% CI, 0.799-0.937] in the combined model. In the validation cohort, sensitivity was 80.5% and specificity was 73.5% on the cut-off value. The net benefit of the combined model was better between threshold probabilities of 40-80% in DCA. In this study, we demonstrated the utility of u-PCX as a diagnostic marker for MN and the clinical usefulness of the diagnostic models, through the combination of u-PCX and clinical parameters including age, glomerular filtration rate, and diabetes mellitus.


Carney triad, SDH-deficient tumors, and Sdhb+/- mice share abnormal mitochondria.
Carney triad (CTr) describes the association of paragangliomas (PGL), pulmonary chondromas, and gastrointestinal (GI) stromal tumors (GISTs) with a variety of other lesions, including pheochromocytomas and adrenocortical tumors. The gene(s) that cause CTr remain(s) unknown. PGL and GISTs may be caused by loss-of-function mutations in succinate dehydrogenase (SDH) (a condition known as Carney-Stratakis syndrome (CSS)). Mitochondrial structure and function are abnormal in tissues that carry SDH defects, but they have not been studied in CTr. For the present study, we examined mitochondrial structure in human tumors and GI tissue (GIT) of mice with SDH deficiency. Tissues from 16 CTr tumors (n=12), those with isolated GIST (n=1), and those with CSS caused by SDHC (n=1) and SDHD (n=2) mutations were studied by electron microscopy (EM). Samples of GIT from mice with a heterozygous deletion in Sdhb (Sdhb(+) (/-), n=4) were also studied by EM. CTr patients presented with mostly epithelioid GISTs that were characterized by plump cells containing a centrally located, round nucleus and prominent nucleoli; these changes were almost identical to those seen in the GISTs of patients with SDH. In tumor cells from patients, regardless of diagnosis or tumor type, cytoplasm contained an increased number of mitochondria with a 'hypoxic' phenotype: mitochondria were devoid of cristae, exhibited structural abnormalities, and were of variable size. Occasionally, mitochondria were small and round; rarely, they were thin and elongated with tubular cristae. Many mitochondria exhibited amorphous fluffy material with membranous whorls or cystic structures. A similar mitochondrial hypoxic phenotype was seen in Sdhb(+) (/-) mice. We concluded that tissues from SDH-deficient tumors, those from mouse GIT, and those from CTr tumors shared identical abnormalities in mitochondrial structure and other features. Thus, the still-elusive CTr defect(s) is(are) likely to affect mitochondrial function, just like germline SDH-deficiency does.


Denervation augments alpha-2 but not alpha-1 adrenergic responses in canine saphenous veins.
Experiments were performed in order to determine the influence of sympathetic denervation on alpha-1 and alpha-2 adrenergic responses in canine saphenous veins. In female dogs anesthetized with sodium pentobarbital, the left lumbar sympathetic chain was excised from L1 to L7. After a 3- to 5-week period, the left (denervated) and right (innervated) saphenous veins were removed, cut into rings and suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution. Denervation reduced significantly the norepinephrine content of the venous rings and the contractile responses evoked by the indirect sympathomimetic amine, tyramine. The contractile responses evoked by exogenous norepinephrine were augmented by denervation under control conditions (16.7-fold shift in concentration-effect curve) and also after inhibition of neuronal and extraneuronal uptake and beta adrenoceptors (3.8-fold shift in curve). Denervation increased the contractile responses evoked by the alpha-2 adrenergic agonist, UK 14,304 (5-fold shift in concentration-effect curve), but not those produced by the alpha-1 adrenergic agonist, phenylephrine. The selective augmentation of alpha-2 adrenergic responses by denervation may reflect the preferential innervation of alpha-2 adrenoceptors in the canine saphenous vein.


Korean Red Ginseng Extract Enhances the Anticancer Effects of Imatinib Mesylate Through Abrogation p38 and STAT5 Activation in KBM-5 Cells.
Although imatinib mesylate (IM) in the treatment of chronic myelogenous leukemia (CML) remains the best example of successful targeted therapy, majority of patients with CML suffer its toxicity profile and develop chemoresistance to existing therapeutic agents. Thus, there is a need to develop novel alternative therapies for the treatment of CML. Here, we investigated whether Korean red ginseng extract (KRGE) could suppress the proliferation and induce chemosensitization in human CML cells. Also, we used a human phospho-antibody array containing 46 antibodies against signaling molecules to examine a subset of phosphorylation events after treatment. Korean red ginseng extract broadly suppressed the proliferation of five different cell lines, but KRGE was found to be the most potent inducer of apoptosis against KBM-5 cells. It also abrogated the expression of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), survivin, inhibitors of apoptosis protein 1/2, COX-2 (Cyclooxygenase-2), cyclin D1, matrix metalloproteinase-9, and VEGF (Vascular endothelial growth factor), as well as upregulated the expression of pro-apoptotic gene products. Interestingly, KRGE also enhanced the cytotoxic and apoptotic effect of IM in KBM-5 cells. The combination treatment of KRGE and IM caused pronounced suppression of p38 and signal transducer and activator of transcription 5 phosphorylation and induced phosphorylation of p53 compared with the individual treatment. Our results demonstrate that KRGE can enhance the anticancer activity of IM and may have a substantial potential in the treatment of CML.


Prevalence of Albuminuria in a General Population Cohort of Patients With Established Chronic Heart Failure.
Numerous studies have reported an association between albuminuria and adverse outcomes in adults with chronic heart failure (CHF). However, the prevalence of albuminuria in adults with established CHF remains unclear. This study was a cross-sectional analysis of the National Health and Nutrition Examination Survey (NHANES) 1999-2012. Adults aged ≥18 years were included, and diagnosis of CHF was based on participant self-report. The primary outcome was the prevalence of microalbuminuria (albumin-to-creatinine ratio 30-300 mg/g) and macroalbuminuria (albumin-to-creatinine ratio >300 mg/g) in adults with CHF. The secondary outcome was the adjusted odds ratio of any albuminuria in adults with and without CHF. During the study period, 37,961 adults did not have CHF and 1,214 adults had CHF. In adults with CHF, 22.1% (95% confidence interval [CI] 19.6%-24.7%) had microalbuminuria and 10.4% (95% CI 8.1%-12.7%) macroalbuminuria. In adjusted analyses, the odds of albuminuria in adults with CHF was 1.89-fold higher (95% CI 1.59-2.26; P < .001) than in adults without CHF. Taken together, albuminuria is more common in adults with CHF than in those without CHF, even after adjustment for important demographic and clinical confounders.


Lack of correlation of Burkholderia pseudomallei quantities in blood, urine, sputum and pus.
We evaluated the correlation of Burkholderia pseudomallei quantities in blood versus urine, sputum or pus. Correlations between bacterial counts in blood and other samples were not found. It is likely that an initial seeding event to extracellular organs is followed by independent growth of B. pseudomallei, and that bacteria in the urine were not passively filtered from the bloodstream.


SH2 and PTB domains in tyrosine kinase signaling.
Intracellular signaling pathways that involve protein tyrosine kinases (PTKs) are critical for the control of most cellular processes. Dysfunctions in PTKs, or in the signaling pathways that they regulate, result in a variety of diseases such as cancer, diabetes, immune deficiency, and many others. SH2 (Src homology region 2) and PTB (phosphotyrosine-binding) domains are small protein modules that mediate protein-protein interactions involved in many signal transduction pathways. Both domains were initially identified as modules that recognize phosphorylated tyrosines in receptor tyrosine kinases and other signaling proteins. Subsequent studies have shown that, while binding of SH2 domains to their target proteins is strictly regulated by tyrosine phosphorylation, most PTB domains actually bind to their (nonphosphorylated) targets constitutively. The functions of SH2 and PTB domains include targeting of their host proteins to different cellular compartments, assembly of key components of signaling pathways in response to extracellular signals, and the control of autoinhibition, activation and dimerization of their host proteins. The information flow from the cell surface to different cellular compartments to regulate the cell cycle, cell shape and movement, cell proliferation, differentiation and cell survival are all controlled in part by SH2 and PTB domains that can recognize phosphotyrosine or particular amino acid sequence motifs in a wide variety of target molecules.


Identification of photofragmentation patterns in trihalide anions by global analysis of vibrational wavepacket dynamics in broadband transient absorption data.
Trihalide anions are linear molecules that can be photodissociated with ultraviolet (UV) light. Whereas deep-UV excitation leads to three-body dissociation, for near-UV excitation just one molecular bond is cleaved, which notionally opens up the possibility for different fragmentation patterns. Here, we explore whether the dihalide fragment is formed as an anionic or neutral species and whether heteronuclear trihalides can lead to two different dihalides. The analysis is based on pronounced wavepacket dynamics induced by femtosecond UV pulses and associated both with the initial trihalide and the nascent dihalide species. For the trihalide anions I3-, Br3-, IBr2-, and ICl2- (point group D∞h), as well as for I2Br- and I2Cl- (point group C∞v) in dichloromethane solution, we identify dihalide fragments by their characteristic vibrational wavenumbers, which we achieve from globally fitting the vibrational wavepacket oscillations, considering a wavelength-dependent phase. No signature from neutral species is found right after excitation, hence there is only one diatomic product in D∞h trihalides. For the investigated C∞v trihalides, which could allow a homonuclear and a heteronuclear product, only the homonuclear one is observed. Since dihalide anions are unstable intermediates, their absorption and the ground-state bleach of the trihalide anion show a biexponential recovery for all samples due to recombining fragment pairs. The rate of the electron transfer yielding a neutral dihalogen and an atomic anion, a prerequisite for the recombination, gives rise to the biexponential behavior; fast recombination is mediated by vibrational excess energy, while slow recombination occurs for cooled-down dihalogens. These data reveal the fragmentation and recombination dynamics from a time-domain approach rather than frequency-domain vibrational spectroscopy and contribute to the in-depth comprehension of these versatile model molecules.


The effect of Massachusetts health reform on 30 day hospital readmissions: retrospective analysis of hospital episode statistics.
To analyse changes in overall readmission rates and disparities in such rates, among patients aged 18-64 (those most likely to have been affected by reform), using all payer inpatient discharge databases (hospital episode statistics) from Massachusetts and two control states (New York and New Jersey). Difference in differences analysis to identify the post-reform change, adjusted for secular changes unrelated to reform. US hospitals in Massachusetts, New York, and New Jersey. Adults aged 18-64 admitted for any cause, excluding obstetrical. Readmissions at 30 days after an index admission. After adjustment for known confounders, including age, sex, comorbidity, hospital ownership, teaching hospital status, and nurse to census ratio, the odds of all cause readmission in Massachusetts was slightly increased compared with control states post-reform (odds ratio 1.02, 95% confidence interval 1.01 to 1.04, P<0.05). Racial and ethnic disparities in all cause readmission rates did not change in Massachusetts compared with control states. In analyses limited to Massachusetts only, there were minimal overall differences in changes in readmission rates between counties with differing baseline uninsurance rates, but black people in counties with the highest uninsurance rates had decreased odds of readmission (0.91, 0.84 to 1.00) compared with black people in counties with lower uninsurance rates. Similarly, white people in counties with the highest uninsurance rates had decreased odds of readmission (0.96, 0.94 to 0.99) compared with white people in counties with lower uninsurance rates. In the United States, and in Massachusetts in particular, extending health insurance coverage alone seems insufficient to improve readmission rates. Additional efforts are needed to reduce hospital readmissions and disparities in this outcome.


Conformationally gated metal uptake by apomanganese superoxide dismutase.
Metal uptake by apomanganese superoxide dismutase in vitro is a complex process exhibiting multiphase "gated" reaction kinetics and a striking sigmoidal temperature profile that has led to a model of conformationally gated metal binding, requiring conversion between "closed" and "open" forms. This work systematically explores the structural determinants of metal binding in both wild-type (WT) apoprotein and mutational variants as a test of mechanistic models. The pH dependence of metalation under physiological conditions (37 degrees C) shows it is linked to ionization of a single proton with a p K a of 7.7. Size exclusion chromatography demonstrates that the apoprotein is dimeric even when it is fully converted to the open form. The role of molecular motions in metal binding has been probed by using disulfide engineering to introduce covalent constraints into the protein. While restricting motion at domain interfaces has no effect, constraining the subunit interface significantly perturbs metal uptake but does not prevent the process. Mutagenesis of residues in the active site environment results in a dramatic shift in the transition temperature by as much as 20 degrees C or a loss of pH sensitivity. On the basis of these results, a mechanism for metal uptake by manganese superoxide dismutase involving reorientation of active site residues to form a metal entry channel is proposed.


Single administration of 5-HT1A agonists decreases 5-HT1A presynaptic, but not postsynaptic receptor-mediated responses: relationship to antidepressant-like action.
The 5-HT1A agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), buspirone or TVXQ 7821 (ipsapirone) but not the 5-HT1B agonist RU 24969, attenuated the hyperphagic response to 8-OH-DPAT administered on the next day. Attenuation was still apparent on the fifth day after either 8-OH-DPAT or buspirone but not on the tenth day after 8-OH-DPAT administration. The ability of 8-OH-DPAT to reduce raphe 5-HIAA levels was also impaired by previous 8-OH-DPAT treatment. However, the 8-OH-DPAT or 5-methoxy-N,N-dimethyltryptamine-induced 5-HT syndromes were unaltered. The results indicate that a single pretreatment with 5-HT1A agonists rapidly desensitises 5-HT1A presynaptic receptor-mediated responses. This effect may mediate the antidepressant-like action of the drugs in an animal model of depression.


Response rate of bone mineral density to teriparatide in postmenopausal women with osteoporosis.
It is desirable for clinicians to know what bone mineral density (BMD) response they can expect in women treated with osteoporosis therapies. The focus of this analysis was to determine what percentage of women attained a lumbar spine BMD response to teriparatide that equaled or exceeded the least significant change (LSC) value of 3%. Data from three clinical trials involving postmenopausal women with osteoporosis were examined. The Fracture Prevention Trial was a double-blinded, placebo-controlled clinical trial examining the safety and efficacy of teriparatide 20 and 40 microg/day. The other two trials were double-blinded, head-to-head comparisons of alendronate 10 mg/day and teriparatide 20 or 40 microg/day, respectively. Only treatment-compliant women who had lumbar spine BMD measurements at all specified time points in these trials were included. For reference, we also examined the percentage of women with lumbar spine BMD responses to alendronate. Hip BMD responses that equaled or exceeded 3% were also examined. According to the LSC criteria, 91% of the teriparatide 20 microg/day group and 94% of the teriparatide 40 microg/day group were lumbar spine BMD responders at 18 months in the Fracture Prevention Trial. In the teriparatide 20 microg/alendronate head-to-head trial, 94% of women receiving teriparatide had a lumbar spine BMD response that equaled or exceeded the 3% criterion at 18 months compared to 75% of those receiving alendronate 10 mg/day (p < 0.01). In the teriparatide 40 microg/day group of the other head-to-head trial, 92% of women achieved the 3% criterion for the lumbar spine at 12 months compared to 69% of those receiving alendronate 10 mg/day (p < 0.01). The median 3-month change in amino-terminal extension peptide of procollagen type 1 [PINP] in women who had a lumbar spine BMD response to teriparatide at 18 months was larger than in women who did not have a lumbar spine BMD response. However, the median 3-month PINP change in lumbar spine BMD nonresponders still exceeded the LSC value of 10 microg/L. Although the percentage of teriparatide-treated women with a hip BMD response that met the 3% criterion was significantly greater than for placebo, there was no significant difference between the percentage of teriparatide 20 microg/day and alendronate 10 mg/day responders in the comparison trial. The baseline characteristics of teriparatide lumbar spine responders and nonresponders were similar. This analysis demonstrates that the vast majority of treatment-compliant postmenopausal women with osteoporosis and minimal prior bisphosphonate exposure have a lumbar spine BMD response to teriparatide that meets or exceeds the LSC. The characteristics of teriparatide responders and nonresponders were not significantly different; thus, we were unable to discern any characteristics that could be used to identify potential nonresponders.


Macrolide resistance among invasive Streptococcus pneumoniae isolates.
Macrolide antibiotics, including erythromycin, clarithromycin, and azithromycin, are the mainstays of empirical pneumonia therapy. Macrolide resistance among Streptococcus pneumoniae, the most common cause of community-acquired pneumonia, is increasing in the United States. Whether resistance is a significant problem or whether macrolides remain useful for treatment of most resistant strains is unknown. To examine the epidemiology of macrolide-resistant pneumococci in the United States. Analysis of 15 481 invasive isolates from 1995 to 1999 collected by the Centers for Disease Control and Prevention's Active Bacterial Core surveillance system in 8 states. Trends in macrolide use (1993-1999) and resistance and factors associated with resistance, including examination of 2 subtypes, the M phenotype, associated with moderate minimum inhibitory concentrations (MICs), and the MLS(B) phenotype, associated with high MICs and clindamycin resistance. From 1993 to 1999, macrolide use increased 13%; macrolide use increased 320% among children younger than 5 years. Macrolide resistance increased from 10.6% in 1995 to 20.4% in 1999. M phenotype isolates increased from 7.4% to 16.5% (P<.001), while the proportion with the MLS(B) phenotype was stable (3%-4%). The median erythromycin MIC (MIC(50)) of M phenotype isolates increased from 4 microg/mL to 8 microg/mL. In 1999, M phenotype strains were more often from children than persons 5 years or older (25.2% vs 12.6%; P<.001) and from whites than blacks (19.3% vs 11.2%; P<.001). In the setting of increasing macrolide use, pneumococcal resistance has become common. Most resistant strains have MICs in the range in which treatment failures have been reported. Further study and surveillance are critical to understanding the clinical implications of our findings.


[Hygiene problems associated with characteristics of the sludge from purification plants in Liguria].
Sludge samples from five municipal sewage treatment plants in Liguria (Italy) were examined periodically during a whole year. Four of them are located in Genova and receive the sewage corresponding respectively to 20,000-53,000-125,000 and 285,000 equivalent inhabitants. The last one is in Chiavari and receives the sewage corresponding to 40,000 equivalent inhabitants. All plants included a preliminary screening treatment, primary sedimentation and a secondary activated sludge process. Sludge was thickened by centrifuge. The bacteriological and chemical characteristics of the sludge after centrifuging (detection and estimation of total and fecal coliforms, fecal streptococci and enumeration and isolation of Salmonella, pH, moisture, total and volatile residue, nitrogen, phosphorus, heavy metals) were measured in order to establish the possibility of land disposal. Dry solids contained on average 34 g/kg total nitrogen and 1,4 g/kg phosphorus. Among heavy metals copper and zinc reached high levels: 544 and 1587 mg/kg of dry solids. Other metals were present at lower concentrations (As 6, Cd 2, Cr 70, Hg 2, Ni 53, pb 416 mg/kg). Comparing these results with those of a research carried out contemporarily in another region (Emilia Romagna) it was observed that in Ligury there is a higher content of copper and bacteria (with an average of 69 Salmonella in 1 g of dry solids). The results showed that sludge produced in the five treatment plants in Ligury (Italy) was apt to be used as fertilizer in soil, but that care has to be taken for what concerns microbial contamination. For what regards the differences among the five sewage treatment plants under observation some heavy metals (arsenic and chromium) reached very high average levels (10-14 mg/kg of arsenic and 70-118 mg/kg of chromium) in the sludge of two of them, which treat the sewage from highly industrialized areas. On the contrary lead and cadmium were present at the same levels in all plants (416 mg/kg of lead and 2 mg/kg of cadmium) with the exception of one (Ge-Quinto) where cadmium was practically absent (0,2 mg/kg). The overall average concentrations of heavy metals resulted definitively lower than maximum concentration proposed by CEE (1986) and by some italian region as Lombardia. Some differences among plants for what regards microbiological parameters were noted only for total coliforms.


Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain.
The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell defense mechanisms. These results suggest the mechanisms of tolerance for the Populus hydrolysate-tolerant mutant strain of C. thermocellum are based on increased cellular efficiency caused apparently by downregulation of non-critical genes and increasing the expression of genes in energy production and conversion rather than tolerance to specific hydrolysate components. The wild type, conversely, responds to hydrolysate media by down-regulating growth genes and up-regulating stress response genes.


Postural stability is altered by the stimulation of pain but not warm receptors in humans.
It is now recognized that large diameter myelinated afferents provide the primary source of lower limb proprioceptive information for maintaining an upright standing position. Small diameter afferents transmitting noxious stimuli, however, can also influence motor behaviors. Despite the possible influence of pain on motor behaviors, the effects of pain on the postural control system have not been well documented. Two cutaneous heat stimulations (experiment 1: non-noxious 40 degrees C; experiment 2: noxious 45 degrees C) were applied bilaterally on the calves of the subject with two thermal grills to stimulate A delta and C warm receptors and nociceptors in order to examine their effects on postural stability. The non-noxious stimulation induced a gentle sensation of warmth and the noxious stimulation induced a perception of heat pain (visual analogue scores of 0 and 46 mm, respectively). For both experiments, ten healthy young adults were tested with and without heat stimulations of the lower limbs while standing upright on a force platform with eyes open, eyes closed and eyes closed with tendon co-vibration of tibialis anterior and triceps surae muscles. The center of pressure displacements were analyzed to examine how both stimulations affected the regulation of quiet standing and if the effects were exacerbated when vision was removed or ankle proprioception perturbed. The stimulation of the warm receptors (40 degrees C) did not induce any postural deterioration. With pain (45 degrees C), subjects showed a significant increase in standard deviation, range and mean velocity of postural oscillations as well as standard deviation of the center of pressure velocity. The effects of heat pain were exacerbated when subjects had both their eyes closed and ankle tendons vibrated (increased standard deviation of the center of pressure velocity and mean velocity of the center of pressure). A non-noxious stimulation (40 degrees C) of the small diameter afferents is not a sufficiently intense sensory stimulation to alter the control of posture. A painful stimulation (45 degrees C) of the skin thermoreceptors, however, yielded a deterioration of the postural control system. The observed deteriorating effects of the combined stimulation of nociceptors and Ia afferents (when ankle tendons were vibrated) could result from the convergence of these afferents at the spinal level. This could certainly lead to the hypothesis that individuals suffering from lower limb pain present alterations of the postural control mechanisms; especially populations already at risk of falling (for example, frail elderly) or populations suffering from concomitant lower limb pain and sensory deficits (for example, diabetic polyneuropathy).


Untreated Stroke as Collateral Damage of COVID-19: "Time Is Brain" Versus "Stay at Home".
For decades, neurologists have been advocating that anyone with acute focal deficits report immediately to the closest hospital's emergency room. Major advancements in the hyperacute diagnosis and treatment of stroke have justified our call-to-action slogan of "Time is Brain"-faster therapy leads to superior outcomes. However, this mantra has been recently usurped by the catchphrase "Stay at Home" during the coronavirus disease 2019 (COVID-19) pandemic. Fewer patients are presenting to hospitals with acute stroke; our census is down. Presumably the etiology of this phenomenon is either strict "social distancing" that some people may misperceive to exclude even emergent situations, or fears of contracting the virus while hospitalized. In this Short Report, we describe the year-over-year drop in stroke volume (ischemic and hemorrhagic both) coinciding with a paradoxical rise in acute reperfusion therapies at our university hospital. These data imply that stroke patients with mild/moderate symptoms are most likely staying home, and not receiving urgent therapies, and correspondingly, only the most severely disabled stroke patients are ultimately seeking and receiving help. We must remind our patients and the general public that our services are essential and available, as stroke still remains a medical emergency, and carries a likely higher overall mortality risk than COVID-19. As neurologists, we also must be vigilant for the atypical presentations and varied etiologies of stroke associated with COVID-19 as well.


Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes.
Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.


Effects of short-term and long-term treatment with inhaled corticosteroids on bone metabolism in patients with airways obstruction. Dutch CNSLD Study Group.
Recent reports have suggested short-term changes in serum parameters of bone metabolism with inhaled corticosteroids. The relevance of these findings to the balance between bone formation and resorption during years of corticosteroid treatment remains uncertain. Two novel markers of bone turnover were first compared with conventional markers in a pilot study and subsequently measured in a long-term double blind study of inhaled corticosteroids. In study I 15 patients were newly started on at least 800 micrograms inhaled corticosteroids daily. At entry and after four weeks serum levels of alkaline phosphatase, osteocalcin, and PICP (procollagen type I carboxy terminal propeptide; a procollagen splice product) were measured as markers of bone formation, as well as the urinary hydroxyproline/creatinine ratio and serum levels of ICTP (type I collagen carboxy terminal telopeptide; a collagen degradation product) as markers of bone resorption. In study II 70 patients with airways obstruction received 800 micrograms beclomethasone daily in addition to terbutaline and 85 received bronchodilators only in a double blind fashion. Serum levels of PICP and ICTP were measured before and after 2.5 years of treatment. In study I a decrease in osteocalcin levels was accompanied by an increase in levels of PICP and a small and non-significant rise in alkaline phosphatase. There were no changes in hydroxyproline or ICTP. In study II no differences were found in serum levels of PICP between the treatment groups; an increase in serum ICTP was found in the group treated without inhaled corticosteroids compared with the group treated with inhaled corticosteroids. No detrimental long-term effect of inhaled corticosteroids was found with three conventional and two novel parameters of bone metabolism. The results indicate that long-term changes in bone turnover during treatment with inhaled corticosteroids should not be deduced from short-term studies with single serum parameters of bone metabolism, but well designed long-term studies of, for example, bone densitometry should be awaited before quoting detrimental effects of inhaled corticosteroids on bone metabolism.


Cerebrospinal fluid immunoglobulins and complement in meningococcal meningitis.
Cerebrospinal fluid (CSF) immunoglobulins (IgA, IgG, IgM) were measured in 107 patients with meningococcal meningitis. Levels were correlated significantly with CSF total protein, and both CSF immunoglobulin and protein increased with age. C3 was measured in the CSF of 38 patients and was also closely correlated with the CSF protein level. C3 breakdown products were found in all six CSFs tested. The CSF immunoglobulin and complement were thought to have leaked from the plasma due to inflammation of the meninges and had little value in diagnosis or prognosis.


Community member perspectives from transgender women and men who have sex with men on pre-exposure prophylaxis as an HIV prevention strategy: implications for implementation.
An international randomized clinical trial (RCT) on pre-exposure prophylaxis (PrEP) as an human immunodeficiency virus (HIV)-prevention intervention found that taken on a daily basis, PrEP was safe and effective among men who have sex with men (MSM) and male-to-female transgender women. Within the context of the HIV epidemic in the United States (US), MSM and transgender women are the most appropriate groups to target for PrEP implementation at the population level; however, their perspectives on evidenced-based biomedical research and the results of this large trial remain virtually unknown. In this study, we examined the acceptability of individual daily use of PrEP and assessed potential barriers to community uptake. We conducted semi-structured interviews with an ethnoracially diverse sample of thirty HIV-negative and unknown status MSM (n = 24) and transgender women (n = 6) in three California metropolitan areas. Given the burden of disease among ethnoracial minorities in the US, we purposefully oversampled for these groups. Thematic coding and analysis of data was conducted utilizing an approach rooted in grounded theory. While participants expressed general interest in PrEP availability, results demonstrate: a lack of community awareness and confusion about PrEP; reservations about PrEP utilization, even when informed of efficacious RCT results; and concerns regarding equity and the manner in which a PrEP intervention could be packaged and marketed in their communities. In order to effectively reduce HIV health disparities at the population level, PrEP implementation must take into account the uptake concerns of those groups who would actually access and use this biomedical intervention as a prevention strategy. Recommendations addressing these concerns are provided.


Inhibition of lindane-induced toxicity using alpha-lipoic acid and vitamin E in the brain of Mus musculus.
In the present investigation, we have used adenosine triphosphatase (ATPase) activity as biochemical test of toxic action of lindane that was explained by lipid peroxidation model. Study was also undertaken to ascertain the potential protective role of alpha-lipoic acid (ALA) and vitamin E on the same parameters. Highly acute dose of lindane, i.e., 40 mg/kg bw for 18 h exposure, was used for creating lesions in brain. Lipid peroxidation was measured in terms of glutathione peroxidase and thio barbituric acid-reacting substances (TBARS). Various brain regions under investigation were cerebellum and pons-medulla oblongata. Healthy, male, Swiss mice (7-8 weeks old) were allocated into four groups. First group was control, second group was treated with lindane, third group was treated purely with antioxidants, and fourth group received both antioxidants and lindane treatment. Results revealed the significant difference (at 1% and 5% in all groups) in all studied parameters from control. Increased TBARS level in second group suggests that lindane enhances the production of free radicals in studied brain regions. Antioxidants under test are efficient remedy for neurotoxicity caused by lindane. We conclude that lindane manifests toxic effects on brain ATPase and enhances lipid peroxidation. ALA and vitamin E in combination may provide protection against lindane-induced acute toxicity.


Impairment of interferon-induced IRF-7 gene expression due to inhibition of ISGF3 formation by trichostatin A.
Two members of the signal transducer and activator of transcription family, STAT1 and STAT2, form, together with interferon regulatory factor 9 (IRF-9), the ISGF3 complex that activates the expression of the interferon-stimulated genes (ISG). The ISGF3 complex also participates in the virus-induced alpha/beta interferon (IFN-alpha/beta) gene amplification cascade by up-regulating IRF-7 gene expression. Here, we show that treatment of cells with trichostatin A (TSA), a deacetylase inhibitor, inhibits the virus-induced activation of IFN-alpha/beta promoters and dramatically reduces the ability of different ISG promoters to respond to IFN stimulation. Impairment of IFN-alpha/beta and ISG expression by TSA in infected cells is due to the blockage of interferon-stimulated ISGF3 complex formation, which leads to the abolition of IRF-7 gene expression. We also show that the TSA-dependent inhibition of ISGF3 is related to impaired nuclear accumulation of STAT2. Our data suggest that an acetylation/deacetylation mechanism participates in the regulation of cellular distribution and function of STAT2 in IFN-alpha/beta signaling.


Evidence for genes controlling resistance to Heligmosomoides bakeri on mouse chromosome 1.
Resistance to infections with Heligmosomoides bakeri is associated with a significant quantitative trait locus (QTL-Hbnr1) on mouse chromosome 1 (MMU1). We exploited recombinant mice, with a segment of MMU1 from susceptible C57Bl/10 mice introgressed onto MMU1 in intermediate responder NOD mice (strains 1094 and 6109). BALB/c (intermediate responder) and C57Bl/6 mice (poor responder) were included as control strains and strain 1098 (B10 alleles on MMU3) as NOD controls. BALB/c mice resisted infection rapidly and C57Bl/6 accumulated heavy worm burdens. Fecal egg counts dropped by weeks 10-11 in strain 1098, but strains 1094 and 6109 continued to produce eggs, harbouring more worms when autopsied (day 77). PubMed search identified 3 genes (Ctla4, Cd28, Icos) as associated with 'Heligmosomoides' in the B10 insert. Single nucleotide polymorphism (SNP) differences in Ctla4 could be responsible for regulatory changes in gene function, and a SNP within a splice site in Cd28 could have an impact on function, but no polymorphisms with predicted effects on function were found in Icos. Therefore, one or more genes encoded in the B10 insert into NOD mice contribute to the response phenotype, narrowing down the search for genes underlying the H. bakeri resistance QTL, and suggest Cd28 and Ctla4 as candidate genes.


Wolbachia interferes with ferritin expression and iron metabolism in insects.
Wolbachia is an intracellular bacterium generally described as being a facultative reproductive parasite. However, Wolbachia is necessary for oogenesis completion in the wasp Asobara tabida. This dependence has evolved recently as a result of interference with apoptosis during oogenesis. Through comparative transcriptomics between symbiotic and aposymbiotic individuals, we observed a differential expression of ferritin, which forms a complex involved in iron storage. Iron is an essential element that is in limited supply in the cell. However, it is also a highly toxic precursor of Reactive Oxygen Species (ROS). Ferritin has also been shown to play a key role in host-pathogen interactions. Measuring ferritin by quantitative RT-PCR, we confirmed that ferritin was upregulated in aposymbiotic compared to symbiotic individuals. Manipulating the iron content in the diet, we showed that iron overload markedly affected wasp development and induced apoptotic processes during oogenesis in A. tabida, suggesting that the regulation of iron homeostasis may also be related to the obligate dependence of the wasp. Finally, we demonstrated that iron metabolism is influenced by the presence of Wolbachia not only in the obligate mutualism with A. tabida, but also in facultative parasitism involving Drosophila simulans and in Aedes aegypti cells. In these latter cases, the expression of Wolbachia bacterioferritin was also increased in the presence of iron, showing that Wolbachia responds to the concentration of iron. Our results indicate that Wolbachia may generally interfere with iron metabolism. The high affinity of Wolbachia for iron might be due to physiological requirement of the bacterium, but it could also be what allows the symbiont to persist in the organism by reducing the labile iron concentration, thus protecting the cell from oxidative stress and apoptosis. These findings also reinforce the idea that pathogenic, parasitic and mutualistic intracellular bacteria all use the same molecular mechanisms to survive and replicate within host cells. By impacting the general physiology of the host, the presence of a symbiont may select for host compensatory mechanisms, which extends the possible consequences of persistent endosymbiont on the evolution of their hosts.


Human leukocyte antigen and clinical and demographic characteristics in psoriatic arthritis and psoriasis in Chinese patients.
Psoriasis and psoriatic arthritis (PsA) are interrelated disorders. To date, no study has compared the differences of genes between patients with PsA and psoriasis and healthy controls in a Chinese population. We conducted a retrospective study to determine the human leukocyte antigen (HLA) -A, -B, -Cw, -DR, and -DQ alleles in Chinese patients with PsA and psoriasis. HLA studies were performed using polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) genotyping methods in 91 patients with PsA and 80 with psoriasis and 75 controls. Age at disease onset, sex, disease duration, enthesitis, and uveitis were also analyzed. Among the patients with PsA and psoriasis, the frequency of HLA-B27 was significantly higher in PsA and HLA-A*30, -Cw*06, -DR*07 in psoriasis compared with controls. In contrast, HLA-B*58 was more common in controls than in PsA and psoriasis groups, and the prevalence of HLA-DR*17 was significantly higher in controls than in those with psoriasis. Comparing PsA and psoriasis, the prevalence of HLA-B*27 and HLA-Cw*12 were more common in PsA patients, while the prevalence of HLA-DR*07 was higher in those with psoriasis (p < 0.05). Among PsA patients, the association between HLA-B*27 and axial joint involvement and uveitis was significant (p < 0.05). Certain HLA alleles are found in Chinese patients with psoriasis (HLA-A*30, -Cw*06, -DR*07) and PsA (HLA-B*27). Psoriasis patients with the HLA-B*27 and/or -Cw*12 may have higher risk of developing PsA. Ours is the first study to assess the genetic role of HLA in patients with psoriasis and PsA in a Chinese population.


Detection and phylogenetic analysis of infectious pancreatic necrosis virus in Chile.
Infectious pancreatic necrosis virus (IPNV) is the etiological agent of a highly contagious disease that is endemic to salmon farming in Chile and causes great economic losses to the industry. Here we compared different diagnostic methods to detect IPNV in field samples, including 3 real-time reverse transcription PCR (qRT-PCR) assays, cell culture isolation, and indirect fluorescent antibody test (IFAT). Additionally, we performed a phylogenetic analysis to investigate the genogroups prevailing in Chile, as well as their geographic distribution and virulence. The 3 qRT-PCR assays used primers that targeted regions of the VP2 and VP1 genes of the virus and were tested in 46 samples, presenting a fair agreement within their results. All samples were positive for at least 2 of the qRT-PCR assays, 29 were positive for cell culture, and 23 for IFAT, showing less sensitivity for these latter 2 methods. For the phylogenetic analysis, portions of 1180 and 523 bp of the VP2 region of segment A were amplified by RT-PCR, sequenced and compared with sequences from reference strains and from isolates reported by previous studies carried out in Chile. Most of the sequenced isolates belonged to genogroup 5 (European origin), and 5 were classified within genogroup 1 (American origin). Chilean isolates formed clusters within each of the genogroups found, evidencing a clear differentiation from the reference strains. To our knowledge, this is the most extensive study completed for IPNV in Chile, covering isolates from sea- and freshwater salmon farms and showing a high prevalence of this virus in the country.


Loop diuretics increase bone turnover and decrease BMD in osteopenic postmenopausal women: results from a randomized controlled study with bumetanide.
To study effects of loop diuretics on bone, 87 women were randomized to 1 year of treatment with bumetanide or placebo. Compared with placebo, bumetanide decreased BMD by 2% at the total hip and by 1.4% at the whole body. Levels of biochemical bone markers were lower in the placebo than in the bumetanide group. Thus, treatment with loop diuretics affects bone metabolism. Loop diuretics may affect bone because they increase the renal calcium excretion and alters the diurnal rhythm of plasma PTH levels. We studied the effects of 1 year of treatment with the loop diuretic bumetanide on bone metabolism. In a double-blinded design, 87 healthy postmenopausal women with osteopenia were randomized to 1-year bumetanide treatment 2 mg/day or placebo. BMD, plasma levels of calcitropic hormones, and biochemical bone markers were measured at baseline, after 1 year of treatment (week 52), and 6 months after withdrawal of treatment (week 78). Calcium (800 mg/day) and vitamin D (10 microg/day) were administered to all participants during the entire 1.5-year study period. Compared with placebo, urinary calcium (+17%) and plasma PTH levels (+9%) increased significantly in response to bumetanide. After 1 year of treatment, BMD in the bumetanide compared with the placebo group was significantly decreased by 2% at the total hip and ultradistal forearm and by 1.4% at the whole body. In addition, levels of biochemical markers of bone turnover differed significantly (approximately 20%) between groups, with lower levels in the placebo than in the bumetanide group. Six months after the end of treatment, the effects of bumetanide were weakening. Treatment with loop diuretics affects bone turnover and decreases BMD. Further studies may reveal whether loop diuretics should be considered as a risk factor for fracture.


Polyphasic characterization reveals the presence of novel fish-associated Chryseobacterium spp. in the Great Lakes of North America.
Recent reports suggest an emergence of novel Chryseobacterium spp. associated with aquaculture-reared fish worldwide. Herein, we report on multiple Chryseobacterium spp. infecting Great Lakes fishes that are highly similar to previously detected isolates from Europe, Africa, and Asia but have never before been reported in North America. Polyphasic characterization, which included extensive physiological, morphological, and biochemical analyses, fatty acid profiling, and phylogenetic analyses based upon partial 16S rRNA gene sequences, highlighted the diversity of Great Lakes' fish-associated chryseobacteria and also suggested that at least 2 taxa represent potentially novel Chryseobacterium spp. Screening for the ability of representative chryseobacteria to elicit lesions in experimentally challenged fish showed that they induced varying degrees of pathology, some of which were severe and resulted in host death. Median lethal dose (LD50) experiments for the isolate that elicited the most extensive pathology (Chryseobacterium sp. T28) demonstrated that the LD50 exceeded 4.5 × 108 cfu, thereby suggesting its role as a facultative fish-pathogenic bacterium. Histopathological changes in T28-infected fish included epithelial hyperplasia of the secondary lamellae and interlamellar space that resulted in secondary lamellar fusion, monocytic infiltrate, and mucus cell hyperplasia, all of which are consistent with branchitis, along with monocytic myositis, hemorrhage within the muscle, liver, adipose tissue, and ovaries, spongiosis of white matter of the brain, multifocal edema within the granular cell layer of the cerebellar cortex, and renal tubular degeneration and necrosis. The findings of this study underscore the widespread presence of chryseobacteria infecting Great Lakes fish.


Prognostic value of magnetic resonance imaging findings in patients with sciatica.
OBJECTIVE This study aimed to determine the prognostic value of MRI variables to predict outcome in patients with herniated disc-related sciatica, and whether MRI could facilitate the decision making between early surgery and prolonged conservative care in these patients. METHODS A prospective observational evaluation of patients enrolled in a randomized trial with 1-year follow-up was completed. A total of 283 patients with sciatica who had a radiologically confirmed disc herniation were randomized either to surgery or to prolonged conservative care with surgery if needed. Outcome measures were recovery and leg pain severity. Recovery was registered on a 7-point Likert scale. Complete/near complete recovery was considered a satisfactory outcome. Leg pain severity was measured on a 0- to 100-mm visual analog scale. Multiple MRI characteristics of the degenerated disc herniation were independently scored by 3 spine experts. Cox models were used to study the influence of MRI variables on rate of recovery, and linear mixed models were used to determine the predictive value of MRI variables for leg pain severity during follow-up. The interaction of each MRI predictor with treatment allocation was tested. There were no study-specific conflicts of interest. RESULTS Baseline MRI variables associated with less leg pain severity were the reader's assessment of presence of nerve root compression (p < 0.001), and assessment of extrusion compared with protrusion of the disc herniation (p = 0.006). Both variables tended to be associated, but not significantly, with satisfactory outcome during follow-up (HR 1.45, 95% CI 0.93-2.24, and HR 1.24, 95% CI 0.96-1.61, respectively). The size of disc herniation at baseline was not associated with outcome. There was no significant change in the effects between treatment groups. CONCLUSIONS MRI assessment of the presence of nerve root compression and extrusion of a herniated disc at baseline was associated with less leg pain during 1-year follow-up, irrespective of a surgical or conservative treatment. MRI findings seem not to be helpful in determining which patients might fare better with early surgery compared with a strategy of prolonged conservative care. Clinical trial registration no.: ISRCTN26872154 ( controlled-trials.com ).


In vitro alteration of receptors for vasoactive intestinal peptide changes the in vivo localization of mouse T cells.
The capacity of T lymphocytes exposed in vitro to the neuropeptide vasoactive intestinal peptide (VIP) to bind VIP in vitro and to migrate to different tissues in vivo has been studied. VIP treatment of T cells resulted in a time- and dose-dependent loss of the ability of T cells to specifically bind radioiodinated VIP. Altered binding was due to a decrease in the expression of cellular receptors for VIP on the treated cells rather than an alteration in the affinity of the cells for the neuropeptide. Alteration of VIP receptor expression was not associated with a change in the expression of Thy-1, Lyt-1, or Lyt-2 surface markers by the treated cells. VIP treatment of T cells in vitro resulted, however, in a dose-dependent decrease in the ability of the treated cells to localize in mesenteric lymph nodes (MLN) and Peyer's patches of recipient animals at early times after cell transfer, and this was due to a selective decrease in the rate of accumulation of the treated cells in these tissues. There was no alteration in the distribution of VIP-treated cells in the blood, spleen, liver, or other major organs of the recipient animals. It is concluded that the presence of VIP receptors on T cells facilitates the entry of T cells into MLN and Peyer's patches in vivo, and it is proposed that this effect is mediated by T cell-VIP interactions in the vicinity of the specialized endothelium of those tissues.


Axonal pathfinding in the absence of normal pathways and impulse activity.
Retinal axons were challenged to grow to their targets both along abnormal pathways and in the absence of impulse activity. Eye primordia were first transplanted from normal to ectopic sites in axolotl embryos. Most of the hosts were genetically eyeless, others were enucleated normal embryos. These axolotl embryos were then parabiotically joined to California newt embryos. Both operations were completed by stage 28, which is before axons have left the eye. The result of the parabiosis was a paralysis of the "eyeless" axolotl twin due to the newt's tetrodotoxin (TTX), while the newt twin remained normally active. When the axolotl twin reached early larval stage, about 1 week later, the projection from the silent transplanted retina was assessed using horseradish peroxidase (HRP) injections into the retina, after which the animals were killed and prepared histologically to reveal the presence of HRP in neuronal processes. The results from 17 such cases show normal topographic retinotectal projections: the dorsal retina projecting to the ventrolateral tectum, and the ventral retina projecting to the dorsomedial tectum. Unusual pathways were often taken to achieve these destinations. Control animals, both normal axolotl larvae developing alone and normal axolotl larvae parabiosed to newts, also showed the normal retinotectal projection patterns. These results indicate that the retinal projections in the experimental group were basically normal. Thus, fibers need neither impulse activity nor a particular pathway to navigate to their correct targets during development. Both factors can be eliminated simultaneously, yet retinal axons still find their way to the tectum and make an ordered map. This indicates that other factors, such as the chemoaffinity mechanisms proposed by Sperry (Sperry, R.W. (1963) Proc. Natl. Acad. Sci. U.S.A. 50: 703-710), may play a more major role in axonal pathfinding in this system.


Homoclinic snaking near a codimension-two Turing-Hopf bifurcation point in the Brusselator model.
Spatiotemporal Turing-Hopf pinning solutions near the codimension-two Turing-Hopf point of the one-dimensional Brusselator model are studied. Both the Turing and Hopf bifurcations are supercritical and stable. The pinning solutions exhibit coexistence of stationary stripes of near critical wavelength and time-periodic oscillations near the characteristic Hopf frequency. Such solutions of this nonvariational problem are in contrast to the stationary pinning solutions found in the subcritical Turing regime for the variational Swift-Hohenberg equations, characterized by a spatially periodic pattern embedded in a spatially homogeneous background state. Numerical continuation was used to solve periodic boundary value problems in time for the Fourier amplitudes of the spatiotemporal Turing-Hopf pinning solutions. The solution branches are organized in a series of saddle-node bifurcations similar to the known snaking structures of stationary pinning solutions. We find two intertwined pairs of such branches, one with a defect in the middle of the striped region, and one without. Solutions on one branch of one pair differ from those on the other branch by a π phase shift in the spatially periodic region, i.e., locations of local minima of solutions on one branch correspond to locations of maxima of solutions on the other branch. These branches are connected to branches exhibiting collapsed snaking behavior, where the snaking region collapses to almost a single value in the bifurcation parameter. Solutions along various parts of the branches are described in detail. Time dependent depinning dynamics outside the saddle nodes are illustrated, and a time scale for the depinning transitions is numerically established. Wavelength variation within the snaking region is discussed, and reasons for the variation are given in the context of amplitude equations. Finally, we compare the pinning region to the Maxwell line found numerically by time evolving the amplitude equations.


Comparison of Administration Routes on the Protective Effects of Bee Venom Phospholipase A2 in a Mouse Model of Parkinson's Disease.
Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Progressive loss of dopaminergic neurons in the substantia nigra (SN) and their synaptic terminal connections in the striatum are main characterizations of PD. Although many efforts have been made to develop therapeutics, no treatment has been proven effective. We previously demonstrated that bvPLA2 can protect dopaminergic neurons by modulating neuroinflammatory responses in an MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced mouse model of PD. The cellular basis for the neuroprotective response of bvPLA2 was the induction of CD4+CD25+ regulatory T cells (Tregs), a population known to suppress immune activation and maintain homeostasis and tolerance to self-antigen. The aim of the present study was to investigate the effects of different routes of bvPLA2 administration in a PD mouse model. Neurobehavioral assessment revealed progressive deterioration in locomotor functions of the MPTP group compared with the control group. However, such functions were improved following subcutaneous (s.c.) bvPLA2 administration. The results showed that the s.c. route of bvPLA2 administration contributed to the induction of Treg cells and the reduction of Th1 and Th17 populations, demonstrating that the neuroprotective effects were associated with reduced tyrosine hydroxylase (TH)-positive dopaminergic neurons and microglia. These results suggested that the s.c. bvPLA2 injection could be beneficial for treating aspects of PD.


Automated force volume image processing for biological samples.
Atomic force microscopy (AFM) has now become a powerful technique for investigating on a molecular level, surface forces, nanomechanical properties of deformable particles, biomolecular interactions, kinetics, and dynamic processes. This paper specifically focuses on the analysis of AFM force curves collected on biological systems, in particular, bacteria. The goal is to provide fully automated tools to achieve theoretical interpretation of force curves on the basis of adequate, available physical models. In this respect, we propose two algorithms, one for the processing of approach force curves and another for the quantitative analysis of retraction force curves. In the former, electrostatic interactions prior to contact between AFM probe and bacterium are accounted for and mechanical interactions operating after contact are described in terms of Hertz-Hooke formalism. Retraction force curves are analyzed on the basis of the Freely Jointed Chain model. For both algorithms, the quantitative reconstruction of force curves is based on the robust detection of critical points (jumps, changes of slope or changes of curvature) which mark the transitions between the various relevant interactions taking place between the AFM tip and the studied sample during approach and retraction. Once the key regions of separation distance and indentation are detected, the physical parameters describing the relevant interactions operating in these regions are extracted making use of regression procedure for fitting experiments to theory. The flexibility, accuracy and strength of the algorithms are illustrated with the processing of two force-volume images, which collect a large set of approach and retraction curves measured on a single biological surface. For each force-volume image, several maps are generated, representing the spatial distribution of the searched physical parameters as estimated for each pixel of the force-volume image.


Effect of maternal depression on premature infant health during initial hospitalization.
This study evaluates the effect of maternal depression on neonatal health status in premature infants during their initial hospitalization. Infants younger than 34 weeks' gestation born to nondrug abusing mothers were enrolled in the study. Thirty-one mother-infant pairs were identified. Maternal depression was evaluated by the Center for Epidemiologic Studies-Depression Scale (CES-D). Scores > or = 16 defined maternal depression. Initial infant physiologic health status was determined by the Score for Neonatal Acute Physiology (SNAP). In-hospital health status was assessed by the following variables: days receiving supplemental oxygen, days on mechanical ventilation (VENT), and days not on enteral feeding (NPO). Health status variables evaluated for long-term outcome included bronchopulmonary dysplasia at 28 days (BPD), BPD at 34 weeks' postmenstrual age (BPD-34), and intraventricular hemorrhage (IVH). Seventeen (55%) of 31 mothers manifested depression on the CES-D. No epidemiologic differences were found between this group and the nondepressed mothers. No differences in gestation or birth weight was detected between the preterm infants of depressed versus nondepressed mothers. The CES-D scores correlated significantly with SNAP (r = .36, P < .02). Infants of depressed mothers experienced significantly worse outcomes in the occurrence of BPD (P = .015), BPD-34 (P = .049), and IVH (P = .055). This study confirms that maternal depression occurs frequently in mothers of preterm infants and adversely affects the presenting neonatal health status of their babies during the initial hospitalization. Maternal depression was related to the severity of the initial neonatal illness and was significantly related to IVH and BPD. These factors may have long-term consequences for subsequent growth, neurodevelopment, and recurrence of related health problems.


AVJ-514 Trial - Baseline Characteristics and 30-Day Outcomes Following MitraClip® Treatment in a Japanese Cohort.
The MitraClip®system is a transcatheter-based therapeutic option for patients with chronic mitral regurgitation (MR) who are at high risk for surgery. A prospective, multicenter, single-arm study was initiated to confirm the transferability of this system to Japan.Methods and Results:Patients with symptomatic chronic moderate-to-severe (3+) or severe (4+) functional or degenerative MR with a Society of Thoracic Surgery (STS) score ≥8%, or the presence of 1 predefined risk factor were enrolled. Patients with left ventricular (LV) ejection fraction (EF) <30% were excluded. MR severity and LV function were assessed by an independent echocardiography core lab. Primary outcome included major adverse events (MAE) at 30 days and acute procedural success (APS). A total of 30 patients (age: 80±7 years; STS score: 10.3%±6.6%) were treated with the MitraClip®. At baseline, all patients had MR 3+/4+ with 53%/47% patients with degenerative/functional etiology with mean LVEF of 50.2±12.8%, and 37% of patients were NYHA class III/IV. APS was achieved in 86.7% with no occurrence of MAE. At 30 days, 86.7% of patients had MR ≤2+ and 96.7% were NYHA class I/II. The MitraClip®procedure resulted in clinically meaningful improvements in MR severity, function and quality of life measures, and low MAE rates. These early results suggest the transferability of this therapy to appropriately selected Japanese patients. (Trial Registration: clinicaltrials.gov Identifier NCT02520310.).


Effect of the Integrated Addition of a Red Tara Pods (Caesalpinia spinosa) Extract and NaCl over the Neo-Formed Contaminants Content and Sensory Properties of Crackers.
A 2k factorial design with three centrals points was considered to evaluate the effect of adding red Tara pods extract (Caesalpinia spinosa) (440-2560 µg/mL of dough water) and NaCl (0.3-1.7 g/100 g of flour) on the acrylamide (AA) and hydroxymethylfurfural (HMF) content and sensory attributes of crackers. Additionally, the best formulation, defined as that with the lowest AA and HMF content, was compared with a commercial formulation cracker. Red Tara pods extracts were obtained through conventional extraction using pure water (60 °C, 35 min). AA and HMF content were quantified by GC-MS and HPLC-DAD, respectively. The sensory evaluation was carried out using a descriptive analysis on a 10 cm non-structured linear scale. Red Tara pods extract significantly reduced (p < 0.05) the AA and HMF content, while NaCl only influenced the HMF formation. However, the sensory attributes did not significantly change (p > 0.05), excepting the violet-gray color and salty flavor, but at acceptable levels compared with the control sample. The higher the red Tara pods extract concentration (2560 µg/mL of dough water), the lower the neo-formed contaminants (NFCs) content of crackers (AA: 53 µg/kg and HMF: 1236 µg/kg) when salt level was below 3 g/100 g of flour. The action of the proanthocyanidins present in the extracts which trapped the carbonyl groups of sugars probably avoided the formation of both NFCs. Contrarily, NaCl addition (from 0.3 to 1.7 g/100 g of flour) significantly increased (p < 0.05) the HMF formation (from 1236 µg/kg to 4239 µg/kg, respectively), probably through the dehydration of carbohydrates during the Maillard reaction. When explored treatments were compared with a commercial formulated cracker, the highest mitigation effect (reductions of 40% and 32% AA and HMF, respectively) was reached with the addition of 2560 µg/mL of dough water of red Tara pods extract and 0.3 g/100 g of flour of NaCl. The addition of red Tara pods extracts integrated with the control of NaCl levels mitigated the NFCs in crackers, preserving their sensory properties. Future research should be focused on scaling this mitigation technology, considering a better chemical characterization of red Tara pods extracts as well as the validation of its use as functional food ingredient.


Simvastatin impairs ADP-stimulated respiration and increases mitochondrial oxidative stress in primary human skeletal myotubes.
Statins, the widely prescribed cholesterol-lowering drugs for the treatment of cardiovascular disease, cause adverse skeletal muscle side effects ranging from fatigue to fatal rhabdomyolysis. The purpose of this study was to determine the effects of simvastatin on mitochondrial respiration, oxidative stress, and cell death in differentiated primary human skeletal muscle cells (i.e., myotubes). Simvastatin induced a dose-dependent decrease in viability of proliferating and differentiating primary human muscle precursor cells, and a similar dose-dependent effect was noted in differentiated myoblasts and myotubes. Additionally, there were decreases in myotube number and size following 48 h of simvastatin treatment (5 μM). In permeabilized myotubes, maximal ADP-stimulated oxygen consumption, supported by palmitoylcarnitine+malate (PCM, complex I and II substrates) and glutamate+malate (GM, complex I substrates), was 32-37% lower (P<0.05) in simvastatin-treated (5 μM) vs control myotubes, providing evidence of impaired respiration at complex I. Mitochondrial superoxide and hydrogen peroxide generation were significantly greater in the simvastatin-treated human skeletal myotube cultures compared to control. In addition, simvastatin markedly increased protein levels of Bax (proapoptotic, +53%) and Bcl-2 (antiapoptotic, +100%, P<0.05), mitochondrial PTP opening (+44%, P<0.05), and TUNEL-positive nuclei in human skeletal myotubes, demonstrating up-regulation of mitochondrial-mediated myonuclear apoptotic mechanisms. These data demonstrate that simvastatin induces myotube atrophy and cell loss associated with impaired ADP-stimulated maximal mitochondrial respiratory capacity, mitochondrial oxidative stress, and apoptosis in primary human skeletal myotubes, suggesting that mitochondrial dysfunction may underlie human statin-induced myopathy.


Base excision repair deficiency caused by polymerase beta haploinsufficiency: accelerated DNA damage and increased mutational response to carcinogens.
The base excision repair pathway (BER) is believed to maintain genomic integrity by repairing DNA damage arising spontaneously or induced by oxidizing and alkylating agents. To establish the role of DNA polymerase beta (beta-pol) in BER and beta-pol-dependent BER in maintaining genomic stability, we have measured the impact of a gene-targeted disruption in the beta-pol gene on DNA repair capacity and on in vivo sensitivity to carcinogens. We have extensively phenotyped the DNA beta-pol heterozygous (beta-pol(+/-)) mouse as expressing approximately 50% less beta-pol mRNA and protein and as exhibiting an equivalent reduction in the specific activity of beta-pol. We measured BER activity by in vitro G:U mismatch and (8-OH)G:C repair and find that there is a significant reduction in the ability of extracts from beta-pol(+/-) mice to repair these types of DNA damage. In vivo, the beta-pol(+/-) mice sustain higher levels of DNA single-strand breaks as well as increased chromosomal aberrations as compared with beta-pol(+/+) littermates. Additionally, we show that reduction in beta-pol expression and BER activity results in increased mutagenicity of dimethyl sulfate as evidenced by a 2-fold increase in LacI mutation frequency. Importantly, the beta-pol(+/-) mice do not exhibit increased sensitivity to DNA damage induced by N-nitroso-N-ethylurea, ionizing radiation, or UV radiation, which induce damage processed by alternative repair pathways, demonstrating that this model is specifically a BER-deficient model.


A novel GFP nude rat model to investigate tumor-stroma interactions.
A key strategy for the study of the tumor microenvironment is to implant human tumor cells in an immunodeficient rodent strain ubiquitously expressing a fluorescent marker. Here, a novel nude rat expressing a green fluorescent protein (GFP) transgene was established and engrafted with primary human tumor tissue in order to separate tumor from stromal cell populations for subsequent molecular analysis. SD-TG (GFP) 2BalRrrc transgenic rats were crossed with HsdHan™: rnu/rnu Rowett nude rats to develop a GFP expressing immunocompromised rat. PCR and flow cytometry were used to follow the GFP genotype and phenotype in newborns. After three to four generations, animals were implanted with 4 T1 dsRed murine breast cancer cells or primary human glioblastoma (GBM) biopsies to generate xenografts for subsequent separation by fluorescence-activated cell sorting (FACS). Fluorecence microscopy and reverse transcription-PCR demonstrated that GFP, under the control of the human ubiquitin C promoter, was stably maintained and expressed in diverse organs over several generations. Immunophenotyping of blood samples by flow cytometry confirmed that the immunodeficient features of the parental rat strain, HsdHan™: rnu/rnu, were retained in the GFP nude rat. Both the murine cell line and human GBM biopsies engrafted, and stromal cell populations were isolated from dissociated xenografts by FACS to > 95% purity. A GFP transgene was stably introduced into a nude rat background in which human and murine cancer cells successfully engrafted. This animal strain provides a novel in vivo system for detailed cellular and molecular characterization of tumor-stroma interactions.


CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.
Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family.IMPORTANCEPlasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes-the intron-in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.