Early colonization events in the mutualistic association between Steinernema carpocapsae nematodes and Xenorhabdus nematophila bacteria.
The bacterium Xenorhabdus nematophila is a mutualist of the entomopathogenic nematode Steinernema carpocapsae. During its life cycle, the bacterium exists both separately from the nematode and as an intestinal resident of a nonfeeding nematode form, the infective juvenile (IJ). The progression of X. nematophila from an ex vivo existence to a specific and persistent colonization of IJs is a model to understand the mechanisms mediating the initiation and maintenance of benign host-microbe interactions. To help characterize this process, we constructed an X. nematophila strain that constitutively expresses green fluorescent protein, which allowed its presence to be monitored within IJs. Using this strain, we showed that few bacterial cells initiate colonization of an individual IJ and that these grow inside the lumen of the IJ intestine in a reproducible polyphasic pattern during colonization. In accordance with these two observations, we demonstrated that the final population of bacteria in a nematode is of predominantly monoclonal origin, suggesting that only one or two bacterial clones initiate or persist during colonization of an individual nematode. These data suggest that X. nematophila initiates IJ colonization by competing for limited colonization sites or resources within the nematode intestine. This report represents the first description of the biological interactions occurring between X. nematophila and S. carpocapsae during the early stages of the colonization process, provides insights into the physiology of X. nematophila in its host niche, and will facilitate interpretation of future data regarding the molecular events mediating this process.


Adaptation of primate vestibuloocular reflex to altered peripheral vestibular inputs. II Spatiotemporal properties of the adapted slow-phase eye velocity.
1. The ability of the vestibuloocular reflex (VOR) to undergo adaptive modification after selective changes in the peripheral vestibular system was investigated in rhesus monkeys by recording three-dimensional eye movements before and after inactivation of selective semicircular canals. In the preceding paper we showed that the horizontal VOR gain evoked by passive yaw oscillations after lateral semicircular canal inactivation recovers gradually over time in a frequency-specific manner. Here we present the spatial tuning of the adapted slow-phase eye velocity and describe its spatiotemporal properties as a function of time after canal inactivation. 2. The spatial organization of the VOR was investigated during oscillations at different head positions in the pitch, roll, and yaw planes, as well as in the right anterior/left posterior and left anterior/right posterior canal planes. Acutely after bilateral inactivation of the lateral semicircular canals, a small horizontal response could still be elicited that peaked during rotations in pitched head positions that would maximally stimulate vertical semicircular canals. In addition, the phase of horizontal slow-phase velocity abruptly reversed through 180 degrees at positions close to upright, similarly to torsional slow-phase velocity. These spatial response properties suggest that the small, residual horizontal response components that are present acutely after plugging of both lateral canals originate from vertical semicircular canal signals. 3. As the horizontal response amplitude increased over time, consistent changes were also observed in the spatiotemporal tuning of horizontal slow-phase velocity. 1) The spatiotemporal response properties of horizontal slow-phase velocity acquired noncosine tuning characteristics, primarily in the pitch plane, in the right anterior/left posterior and left anterior/right posterior canal planes. Accordingly, horizontal response amplitude was nonzero during rotation in any head position in these planes and response phase varied significantly as a function of head orientation. 2) The peak horizontal response amplitude shifted spatially over time, such that 5-10 mo after plugging it was maximal during rotations at head positions close to upright. 4. In parallel to these unique spatiotemporal response properties characterizing the adapted horizontal VOR, torsional slow-phase velocity also exhibited small spatiotemporal changes after lateral canal inactivation that tended to precede in time the changes associated with the horizontal response components. In contrast, vertical slow-phase velocity in the plugged animals was unaltered and continued to be characterized by cosine-tuned spatial properties in three dimensions. 5. Recovery of the horizontal response gain during yaw oscillations in upright position, as well as the unique, noncosine spatiotemporal characteristics of the adapted horizontal VOR, were also observed in an animal with all but one vertical semicircular canals inactivated. There was, however, no sign of VOR gain recovery up to 2 mo after all semicircular canals were inactivated. These results suggest that the observed recovery of horizontal VOR is at least partly due to signals originating from the remaining intact vertical canal(s). Even in the presence of a single intact vertical canal, the improvement in horizontal gaze stability is at least partly restored through spatiotemporal changes in the processing of vestibuloocular signals that improve the gain and spatial tuning of horizontal VOR at the expense of temporal response properties.


Cognitive and emotional differences in young maltreated children: a translational application of dynamic skill theory.
Through a translational approach, dynamic skill theory enhances the understanding of the variation in the behavioral and cognitive presentations of a high-risk population-maltreated children. Two studies illustrate the application of normative developmental constructs from a dynamic skills perspective to samples of young maltreated and nonmaltreated children. Each study examines the emotional and cognitive development of maltreated children with attention to their developing world view or negativity bias and cognitive skills. Across both studies, maltreated children demonstrate negativity bias when compared to their nonmaltreated counterparts. Cognitive complexity demonstrated by the maltreated children is dependent upon a positive or negative context. Positive problem solving is more difficult for maltreated children when compared to their nonmaltreated counterparts. Differences by maltreatment type, severity, timing of the abuse, and identity of the perpetrator are also delineated, and variation in the resulting developmental trajectories in each case is explored. This translation of dynamic skill theory, as applied to maltreated children, enhances our basic understanding of their functioning, clarifies the nature of their developmental differences, and underscores the need for early intervention.


Novamin infusion: a new method to cure postoperative shivering with hypothermia.
Postoperative shivering is a frequent complication of surgery in developing countries and there is no satisfying method to treat it, let alone to cure it. We studied whether intravenous amino acid (AA) infusion can cure postoperative shivering in the postanesthesia care unit. Sixty postanesthesia care unit patients with shivering grade 2 or higher and tympanic temperature <36°C received randomly either infusion of Novamin 18 AAs (2 mL/kg/h), pethidine (0.5 mg/kg), or tramadol (1 mg/kg). Tympanic temperature, shivering grade, and thermal comfort were assessed every 5 min for 60 min. Blood glucose and lactic acid concentrations were measured before and after treatment. Postoperative nausea and vomiting were also recorded. Shivering stopped within 5 min in the pethidine and tramadol groups versus 90% stopped within 15 min in AA group. There were five cases of reshivering in the tramadol group versus none in the AA or pethidine groups. Tympanic temperature increased slowly in all patients but increased significantly faster in the AA group. Thermal comfort improved significantly faster in the AA group versus the other two groups, thermal comfort was significantly higher in the tramadol versus the pethidine group ≥35 min. Blood glucose concentration in AA group increased to 135.18 ± 9.18 mg/dL. There were some cases of nausea and vomiting in pethidine and tramadol groups but none in the AA group. Novamin infusion can stop postoperative shivering and alleviates hypothermia and improves thermal comfort more effectively than tramadol and pethidine with less nausea and vomiting and causes a clinically acceptable blood glucose increase with no reshivering episodes.


Variability in estimation of self-reported dietary intake data from elite athletes resulting from coding by different sports dietitians.
A routine activity for a sports dietitian is to estimate energy and nutrient intake from an athlete's self-reported food intake. Decisions made by the dietitian when coding a food record are a source of variability in the data. The aim of the present study was to determine the variability in estimation of the daily energy and key nutrient intakes of elite athletes, when experienced coders analyzed the same food record using the same database and software package. Seven-day food records from a dietary survey of athletes in the 1996 Australian Olympic team were randomly selected to provide 13 sets of records, each set representing the self-reported food intake of an endurance, team, weight restricted, and sprint/power athlete. Each set was coded by 3-5 members of Sports Dietitians Australia, making a total of 52 athletes, 53 dietitians, and 1456 athlete-days of data. We estimated within- and between- athlete and dietitian variances for each dietary nutrient using mixed modeling, and we combined the variances to express variability as a coefficient of variation (typical variation as a percent of the mean). Variability in the mean of 7-day estimates of a nutrient was 2- to 3-fold less than that of a single day. The variability contributed by the coder was less than the true athlete variability for a 1-day record but was of similar magnitude for a 7-day record. The most variable nutrients (e.g., vitamin C, vitamin A, cholesterol) had approximately 3-fold more variability than least variable nutrients (e.g., energy, carbohydrate, magnesium). These athlete and coder variabilities need to be taken into account in dietary assessment of athletes for counseling and research.


Modification of ocular defects in mouse developmental glaucoma models by tyrosinase.
Mutations in the cytochrome P450 family 1, subfamily B, polypeptide 1 (CYP1B1) gene are a common cause of human primary congenital glaucoma (PCG). Here we show that Cyp1b1-/- mice have ocular drainage structure abnormalities resembling those reported in human PCG patients. Using Cyp1b1-/- mice, we identified the tyrosinase gene (Tyr) as a modifier of the drainage structure phenotype, with Tyr deficiency increasing the magnitude of dysgenesis. The severe dysgenesis in eyes lacking both CYP1B1 and TYR was alleviated by administration of the tyrosinase product dihydroxyphenylalanine (l-dopa). Tyr also modified the drainage structure dysgenesis in mice with a mutant Foxc1 gene, which is also involved in PCG. These experiments raise the possibility that a tyrosinase/l-dopa pathway modifies human PCG, which could open new therapeutic avenues.


APOE4 Induces Site-Specific Tau Phosphorylation Through Calpain-CDK5 Signaling Pathway in EFAD-Tg Mice.
APOE4 is the greatest genetic risk factor for Alzheimer's disease (AD), particularly associated with increased levels of amyloid-β (Aβ) and amyloid deposition. However, it remains unclear whether APOE4 is associated with greater tau phosphorylation and neurofibrillary tangle formation, a hallmark of AD leading to structural disruption of the neuronal cytoskeleton. The current study used 3 and 7 month old EFAD mice, which express human APOE and over-express specifically human Aβ42 via 5 familial-AD (FAD) mutations, to investigate APOE genotype-specific effects on site-specific tau phosphorylation. The results reveal that AD-like site-specific tau phosphorylation was increased in E4FAD mice, accompanied by disrupted cortical neuronal morphology, compared to E3FAD mice. Further analysis demonstrated that the levels of CDK5, its regulatory subunits (p35 and p25) and calpain (including calpain1 and calpain2), but not GSK3β, were significantly increased in E4FAD mice compared to E3FAD mice. These results suggest that the APOE4 genotype contributes to increased site-specific tau phosphorylation via activation of the calpain-CDK5 signaling pathway.


Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal.
One of the most dominant influences in the patterning of multicellular embryos is exerted by the Hedgehog (Hh) family of secreted signaling proteins. Here, we identify a segment polarity gene in Drosophila melanogaster, skinny hedgehog (ski), and show that its product is required in Hh-expressing cells for production of appropriate signaling activity in embryos and in the imaginal precursors of adult tissues. The ski gene encodes an apparent acyltransferase, and we provide genetic and biochemical evidence that Hh proteins from ski mutant cells retain carboxyl-terminal cholesterol modification but lack amino-terminal palmitate modification. Our results suggest that ski encodes an enzyme that acts within the secretory pathway to catalyze amino-terminal palmitoylation of Hh, and further demonstrate that this lipid modification is required for the embryonic and larval patterning activities of the Hh signal.


Evaluating the efficacy of beef slaughter line interventions by quantifying the six major non-O157 Shiga toxin producing Escherichia coli serogroups using real-time multiplex PCR.
Six major Shiga toxin producing Escherichia coli (STEC) serogroups: O26, O103, O145, O111, O121, and O45 have been declared as adulterants in federally inspected raw beef in the USA effective June 4th, 2012 in addition to the routinely tested STEC O157: H7. This study tests a real-time multiplex PCR assay and pooling of the samples to optimize the detection and quantification (prevalence and contamination) of six major non-O157 STEC, regardless of possessing Shiga toxins. To demonstrate the practicality, one large-scale slaughter plant (Plant LS) and one small-scale slaughter plant (Plant SS) located in the Mid-Western USA were sampled, in 2011, before the establishment of 2013 USDA laboratory protocols. Carcasses were sampled at consecutive intervention stations and beef trimmings were collected at the end of the fabrication process. Plant SS had marginally more contaminated samples than Plant LS (p-value 0.08). The post-hide removal wash, steam pasteurization, and lactic acid (≤5%) spray used in Plant LS seemed to reduce the six serogroups effectively, compared to the hot-water wash and 7-day chilling at Plant SS. Compared to the culture isolation methods, quantification of the non-O157 STEC using real-time PCR may be an efficient way to monitor the efficacy of slaughter line interventions.


Adaptation by alternative RNA splicing of slow troponin T isoforms in type 1 but not type 2 Charcot-Marie-Tooth disease.
Slow troponin T (TnT) plays an indispensable role in skeletal muscle function. Alternative RNA splicing in the NH(2)-terminal region produces high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms of slow TnT. Normal adult slow muscle fibers express mainly HMW slow TnT. Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral polyneuropathies caused by various neuronal defects. We found in the present study that LMW slow TnT was significantly upregulated in demyelination form type 1 CMT (CMT1) but not axonal form type 2 CMT (CMT2) muscles. Contractility analysis showed an increased specific force in single fibers isolated from CMT1 but not CMT2 muscles compared with control muscles. However, an in vitro motility assay showed normal velocity of the myosin motor isolated from CMT1 and CMT2 muscle biopsies, consistent with their unchanged myosin isoform contents. Supporting a role of slow TnT isoform regulation in contractility change, LMW and HMW slow TnT isoforms showed differences in the molecular conformation in conserved central and COOH-terminal regions with changed binding affinity for troponin I and tropomyosin. In addition to providing a biochemical marker for the differential diagnosis of CMT, the upregulation of LMW slow TnT isoforms under the distinct pathophysiology of CMT1 demonstrates an adaptation of muscle function to neurological disorders by alternative splicing modification of myofilament proteins.


Fluoride exposure alters the ultra-structure of sperm flagellum via reducing key protein expressions in testis.
Excessive fluoride (F) ingestion via drinking water interfered with spermatogenesis and lowered sperm quality of human and animals. However, it is still unclear why the effects of fluoride on sperm quality focus on mostly sperm motility rather than sperm count. The objective of this study is to investigate the potential relationship between alteration in the structure and function of sperm flagellum and fluoride exposure in the environment. 40 male mice were allocated to four groups which were treated with 0, 25, 50, 100 mg/L NaF deionized water, respectively, for 8 weeks continuously. The testicular morphology, ultra-structure of fibrous sheath and axoneme of sperm flagellum, and eleven key function genes Akap3, Akap4, Dnah1, Eno4, Cfap43, Cfap44, Hydin, Spef2, Spag6, Spag16, and Cfap69 were examined by histology, transmission electron microscopy, and real-time PCR methods respectively. The results displayed that fluoride damaged the typical "9 + 2″ microtubule structure including fibrous sheathes and axoneme of sperm flagellum in testes of mice. Furthermore, the mRNA and protein expression levels of AKAP3 and AKAP4 related to fibrous sheathes formation, and CFAP43, CFAP44 and HYDIN in axoneme were down-regulated by fluoride exposure. Taken together, we revealed that fluoride altered the structures of the fibrous sheathes and axonemal in sperm flagellum via down-regulating the mRNA and protein expression levels of AKAP3, AKAP4, CFAP43, CFAP44, and HYDIN, which may be one of the reasons that fluoride lowered sperm quality and male reproductive function.


Cerebral blood flow velocity underestimates cerebral blood flow during modest hypercapnia and hypocapnia.
To establish the accuracy of transcranial Doppler ultrasound (TCD) measures of middle cerebral artery (MCA) cerebral blood flow velocity (CBFV) as a surrogate of cerebral blood flow (CBF) during hypercapnia (HC) and hypocapnia (HO), we examined whether the cross-sectional area (CSA) of the MCA changed during HC or HO and whether TCD-based estimates of CBFV were equivalent to estimates from phase contrast (PC) magnetic resonance imaging. MCA CSA was measured from 3T magnetic resonance images during baseline, HO (hyperventilation at 30 breaths/min), and HC (6% carbon dioxide). PC and TCD measures of CBFV were measured during these protocols on separate days. CSA and TCD CBFV were used to calculate CBF. During HC, CSA increased from 5.6 ± 0.8 to 6.5 ± 1.0 mm(2) (P < 0.001, n = 13), while end-tidal carbon dioxide partial pressure (PETCO2) increased from 37 ± 3 to 46 ± 5 Torr (P < 0.001). During HO, CSA decreased from 5.8 ± 0.9 to 5.3 ± 0.9 mm(2) (P < 0.001, n = 15), while PetCO2 decreased from 36 ± 4 to 23 ± 3 Torr (P < 0.001). CBFVs during baseline, HO, and HC were compared between PC and TCD, and the intraclass correlation coefficient was 0.83 (P < 0.001). The relative increase from baseline was 18 ± 8% greater (P < 0.001) for CBF than TCD CBFV during HC, and the relative decrease of CBF during HO was 7 ± 4% greater than the change in TCD CBFV (P < 0.001). These findings challenge the assumption that the CSA of the MCA does not change over modest changes in PETCO2.


Effect of the coil-globule transition on the free-energy barrier for intrachain crystal nucleation.
The rate of crystal nucleation of colloids and globular proteins can be enhanced by critical density fluctuations. It has been argued that a closely related phenomenon influences the rate of intramolecular "crystallization" of single-chain polymers. We report Monte Carlo simulations of the free-energy barrier that separates the crystalline state of a homopolymer chain from the disordered state. The simulations show that the barrier for nucleation of the ordered state is drastically lowered as the disordered state changes from a coil to a globule. We discuss the relation of the present findings to intramolecular nucleation of heteropolymers, such as proteins.


The effects of minoxidil, 1% pyrithione zinc and a combination of both on hair density: a randomized controlled trial.
Recent studies of antidandruff shampoos or tonics containing antifungal or antibacterial agents produced effects suggestive of a potential hair growth benefit. The purpose of this 6-month, 200-patient, randomized, investigator-blinded, parallel-group clinical study was to assess the hair growth benefits of a 1% pyrithione zinc shampoo. The efficacy of a 1% pyrithione zinc shampoo (used daily), was compared with that of a 5% minoxidil topical solution (applied twice daily), a placebo shampoo and a combination of the 1% pyrithione zinc shampoo and the 5% minoxidil topical solution. Two hundred healthy men between the ages of 18 and 49 years (inclusive) exhibiting Hamilton-Norwood type III vertex or type IV baldness were enrolled. Total hair counts, the primary efficacy measure, were obtained using fibre-optic microscopy and a computer-assisted, manual hair count method. Secondary measures of efficacy included assessments of hair diameter, as well as patient and investigator global assessments of improvement in hair growth. These were based on photographs of the scalp using both midline and vertex views. Hair count results showed a significant (P < 0.05) net increase in total visible hair counts for the 1% pyrithione zinc shampoo, the 5% minoxidil topical solution, and the combination treatment groups relative to the placebo shampoo after 9 weeks of treatment. The relative increase in hair count for the 1% pyrithione zinc shampoo was slightly less than half that for the minoxidil topical solution and was essentially maintained throughout the 26-week treatment period. No advantage was seen in using both the 5% minoxidil topical solution and the 1% pyrithione zinc shampoo. A small increase in hair diameter was observed for the minoxidil-containing treatment groups at week 17. Assessments of global improvements by the patients and investigator generally showed the benefit of 5% minoxidil. The benefit of the 1% pyrithione zinc shampoo used alone tended (P < 0.1) to be apparent only to the investigator. Hair count results show a modest and sustained improvement in hair growth with daily use of a 1% pyrithione zinc shampoo over a 26-week treatment period.


Isoliquiritigenin isolated from licorice Glycyrrhiza uralensis prevents 6-hydroxydopamine-induced apoptosis in dopaminergic neurons.
Licorice (Glycyrrhiza uralensis) is a medicinal herb containing various bioactive components implicated in antioxidative, anti-inflammatory, antiviral, and neuroprotective effects, but the effects of licorice against Parkinson's disease (PD)-related dopaminergic cell death have not been studied. In this study, we investigated the protective effects of isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in a dopaminergic cell line, SN4741. ISL (1 µM) significantly attenuated 6-OHDA (50 µM)-induced reactive oxygen species (ROS) and nitric oxide (NO) generation and apoptotic cell death. ISL pretreatment effectively suppressed 6-OHDA-mediated upregulation of Bax, p-c-Jun N-terminal kinase (JNK), p-p38 mitogen-activated protein (MAP) kinase, cytochrome c release, and caspase 3 activation. In addition, ISL significantly attenuated 6-OHDA-induced Bcl-2, brain-derived neurotrophic factor (BDNF), and mitochondrial membrane potential (MMP) reduction. Pharmacological inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway reversed ISL-mediated neuroprotection against 6-OHDA toxicity in SN4741 cells. These results provide the first evidence that ISL can protect dopaminergic cells under oxidative stress conditions by regulating the apoptotic process.


Ethanol-based proliposome delivery systems of paclitaxel for in vitro application against brain cancer cells.
In this study the anticancer activity of paclitaxel-loaded nano-liposomes on glioma cell lines was investigated. Soya phosphatidylcholine:cholesterol (SPC:Chol), hydrogenated soya phosphatidylcholine:cholesterol (HSPC:Chol) or dipalmitoylphosphatidylcholine:cholesterol (DPPC:Chol) in 1:1 mole ratio were used to prepare ethanol-based proliposomes. Following hydration of proliposomes, the size of resulting vesicles was subsequently reduced to nanometer scale via probe-sonication. The resulting formulations were characterized in terms of size, zeta potential and morphology of the vesicles, and entrapment efficiency of paclitaxel (PX) as well as the final pH of the preparations. DPPC-liposomes entrapped 35-92% of PX compared to 27-74% and 25-60% entrapped by liposomes made from SPC and HSPC formulations respectively, depending on drug concentration. The entrapment efficiency of liposomes was dependent on the lipid bilayer properties and ability of PX to modify surface charge of the vesicles. In vitro cytotoxicity studies revealed that PX-liposome formulations were more selective at inhibiting the malignant cells. The cytotoxicity of PX-liposomes was dependent on their drug-entrapment efficiency. This study has shown PX-liposomes generated from proliposomes have selective activity against glioma cell lines, and the synthetic DPPC phospholipid was most suitable for maximized drug entrapment and highest activity against the malignant cells in vitro.


Sodium/calcium exchange modulates intracellular calcium overload during posthypoxic reoxygenation in mammalian working myocardium. Evidence from aequorin-loaded ferret ventricular muscles.
We tested the hypothesis that the intracellular Ca2+ overload of ventricular myocardium during the period of posthypoxic reoxygenation is mediated by transsarcolemmal Ca2+ influx via Na+/Ca2+ exchange. In aequorin-loaded, ferret right ventricular papillary muscles, blockers of the sarcolemmal and the sarcoplasmic reticulum Ca2+ channels, slowed the Cai2+ transient, producing a convex ascent during membrane depolarization, followed by a concave descent during repolarization. The magnitude of the Cai2+ transient was affected by changes in the membrane potential, Nai+, Nao+, and Cao2+, and was blocked by Ni2+, or dichlorbenzamil. The calculated Na+/Ca2+ exchange current was in the reverse mode (Ca2+ influx) during the ascending phase of the Cai2+ transient, and was abruptly switched to the forward mode (Ca2+ efflux) at repolarization, matching the time course of the Cai2+ transient. During hypoxic superfusion, the Cai2+ transient was abbreviated, which was associated with a shorter action potential duration. In contrast, immediately after reoxygenation, the Cai2+ transient increased to a level greater than that of the control, even though the action potential remained abbreviated. This is the first demonstration on a beat-to-beat basis that, during reoxygenation, Ca2+ influx via Na+/Ca2+ exchange is augmented and transports a significant amount of Ca2+ into the ventricular myocardial cell. The activation of the exchanger at the time of reoxygenation appears to be mediated by Nai+ accumulation, which occurs during hypoxia.


Impact of Sarcopenia on One-Year Mortality among Older Hospitalized Patients with Impaired Mobility.
However, the information regarding the impact of sarcopenia on mortality in older individuals is rising, there is a lack of knowledge concerning this issue among geriatric hospitalized patients. Therefore, aim of the present study was to investigate the associations between sarcopenia and 1-year mortality in a prospectively recruited sample of geriatric inpatients with different mobility and dependency status. Sarcopenia was diagnosed using the criteria of the European Working Group on Sarcopenia in Older People (EWGSOP). Hand grip strength and skeletal muscle mass were measured using Jamar dynamometer and bioelectrical impedance analysis, respectively. Physical function was assessed with the Short Physical Performance Battery. Dependency status was defined by Barthel-Index (BI). Mobility limitation was defined according to walking ability as described in BI. The survival status was ascertained by telephone interview. The recruited population comprised 198 patients from a geriatric acute ward with a mean age of 82.8 ± 5.9 (70.2% females). 50 (25.3%) patients had sarcopenia, while 148 (74.7%) had no sarcopenia. 14 (28%) patients died among sarcopenic subjects compared with 28 (19%) non-sarcopenic subjects (P=0.229). After adjustment for potential confounders, sarcopenia was associated with increased mortality among patients with limited mobility prior to admission (n=138, hazard ratio, HR: 2.52, 95% CI: 1.17-5.44) and at time of discharge (n=162, HR: 1.93, 95% CI: 0.67-3.22). In a sub-group of patients with pre-admission BI<60 (n=45), <70 (n=73) and <80 (n=108), the risk of death was 3.63, 2.80 and 2.55 times higher in sarcopenic patients, respectively. In contrast, no significant relationships were observed between sarcopenia and mortality across the different scores of BI during admission and at time of discharge. Sarcopenia is significantly associated with higher risk of mortality among sub-groups of older patients with limited mobility and impaired functional status, independently of age and other clinical variables.


Comparisons of cardiovascular dysautonomia and cognitive impairment between de novo Parkinson's disease and de novo dementia with Lewy bodies.
Cognitive impairment may be correlated with cardiovascular dysautonomia, including blood pressure (BP) dysregulation, in Parkinson's disease (PD), but the association between these factors in dementia with Lewy bodies (DLB) is uncertain. This study aimed to clarify whether cardiovascular dysautonomia had an influence on cognitive function in Lewy body disease or not. Ninty-nine patients with de novo PD (n = 75) and DLB (n = 24) were evaluated using the Mini-Mental State Examination (MMSE) and Frontal Assessment Battery (FAB). Cardiac 123I-metaiodobenzylguanidine (MIBG) scintigraphy, orthostatic hypotension (OH), supine hypertension (SH), postprandial hypotension (PPH), nocturnal BP fall in 24-h ambulatory blood pressure monitoring (ABPM) and constipation were estimated. Associations of these factors with cognitive and executive dysfunction were examined. In DLB, MIBG uptake was reduced and OH, PPH and SH were severely disturbed, compared to PD. The nocturnal BP fall in ABPM was lower in DLB, and the failure of nocturnal BP fall in PD was associated with MMSE, after adjustment for other clinical features. FAB was significantly associated nocturnal BP fall, age and SH in PD, but no significant correlations among factors were found for DLB. The significant association between nocturnal BP dysregulation and cognitive or executive decline in PD might be due to impaired microvascular circulation or invasion of α-synuclein in the CNS. The lack of a correlation of BP insufficiency with cognitive impairment in DLB suggests initial involvement of Lewy body pathology in the neocortex, regardless of Lewy body invasion of the autonomic nervous system.


A Randomized, Controlled Trial of Liraglutide for Adolescents with Obesity.
Obesity is a chronic disease with limited treatment options in pediatric patients. Liraglutide may be useful for weight management in adolescents with obesity. In this randomized, double-blind trial, which consisted of a 56-week treatment period and a 26-week follow-up period, we enrolled adolescents (12 to <18 years of age) with obesity and a poor response to lifestyle therapy alone. Participants were randomly assigned (1:1) to receive either liraglutide (3.0 mg) or placebo subcutaneously once daily, in addition to lifestyle therapy. The primary end point was the change from baseline in the body-mass index (BMI; the weight in kilograms divided by the square of the height in meters) standard-deviation score at week 56. A total of 125 participants were assigned to the liraglutide group and 126 to the placebo group. Liraglutide was superior to placebo with regard to the change from baseline in the BMI standard-deviation score at week 56 (estimated difference, -0.22; 95% confidence interval [CI], -0.37 to -0.08; P = 0.002). A reduction in BMI of at least 5% was observed in 51 of 113 participants in the liraglutide group and in 20 of 105 participants in the placebo group (estimated percentage, 43.3% vs. 18.7%), and a reduction in BMI of at least 10% was observed in 33 and 9, respectively (estimated percentage, 26.1% vs. 8.1%). A greater reduction was observed with liraglutide than with placebo for BMI (estimated difference, -4.64 percentage points) and for body weight (estimated difference, -4.50 kg [for absolute change] and -5.01 percentage points [for relative change]). After discontinuation, a greater increase in the BMI standard-deviation score was observed with liraglutide than with placebo (estimated difference, 0.15; 95% CI, 0.07 to 0.23). More participants in the liraglutide group than in the placebo group had gastrointestinal adverse events (81 of 125 [64.8%] vs. 46 of 126 [36.5%]) and adverse events that led to discontinuation of the trial treatment (13 [10.4%] vs. 0). Few participants in either group had serious adverse events (3 [2.4%] vs. 5 [4.0%]). One suicide, which occurred in the liraglutide group, was assessed by the investigator as unlikely to be related to the trial treatment. In adolescents with obesity, the use of liraglutide (3.0 mg) plus lifestyle therapy led to a significantly greater reduction in the BMI standard-deviation score than placebo plus lifestyle therapy. (Funded by Novo Nordisk; NN8022-4180 ClinicalTrials.gov number, NCT02918279.).


Determinants of survival in adult HIV patients on antiretroviral therapy in Oromiyaa, Ethiopia.
The antiretroviral treatment (ART) scale-up service has been a recent development in Ethiopia, but its impact on mortality has not been well investigated. The aim of this study was to assess the early survival outcome of the scale-up service by utilizing routine hospital data. All adult HIV/AIDS patients who started on antiretroviral treatment in Shashemene and Assela hospitals from January 1, 2006 to May 31, 2006 were included and followed up for 2 years. Data were extracted from standard patient medical registrations. Kaplan-Meier curves were used to estimate survival probability and the Cox proportional hazard model was applied to determine predictors of mortality. Two alterative assumptions (real case and worst case) were made in determining predictors of mortality. The median age of patients was 33 years and 57% were female. Eighty-five percent had CD4 <200 cells/µL with a median CD4 count of 103 cells/µL. The median survival time was 104.4 weeks. A total of 28 (10.3%) deaths were observed during the 2-year period and 48 patients (18%) were lost to follow up. The majority of deaths occurred in the first 4 months of treatment. In multivariate analysis, 2-year survival was significantly associated with the clinical stage of the disease, baseline hemoglobin, and cotrimoxazole prophylaxis therapy (CPT) at or before ART initiation in both assumptions. The median CD4 count and body weight showed a marked improvement during the first 6 months of treatment, followed by stagnation thereafter. The study has shown an overall low mortality but a high loss to follow-up rate of the cohort. Advanced clinical stage, anemia, low body weight, and lack of CPT initiation were independent predictors of mortality - but not gender. CPT initiation should be encouraged in routine HIV care services, and patient retention mechanisms have to be strengthened. Stagnation in immunological and weight recovery after the first 6 months should be further investigated. The utilization of routine data should be encouraged in order to facilitate appropriate decision making.


Alteration of ambient GABA by phasic and tonic neuronal activation.
Neurons of primary auditory cortex (AI) emit spikes (action potentials) in two distinct manners, responding to sounds in an onset or a sustained manner. The former AI neurons are called phasic cells and the latter tonic cells. The phasic cells generate spikes for a brief time period (less than hundreds of milliseconds) at the onset of an auditory stimulus (e.g., a tone frequency sound), and the tonic cells continuously generate spikes throughout the stimulation period. Simulating a neural network model of AI, we investigated whether and how the onset discharges influence the sustained discharges that are believed to play a central role in encoding auditory information. Onset discharges, triggered by a phasic input, briefly excited GABAergic interneurons and transiently increased the level of ambient GABA, which was immediately recognized by extrasynaptic GABA(a) receptors and provided inhibitory currents into neurons. The transient alteration of ambient GABA allowed tonic cells to respond selectively to a tonic input. The timing of phasic input relative to a tonic one had a great impact on the responsiveness of tonic cells. We found optimal timing for the best selective responsiveness: phasic input preceding tonic input by several tens of milliseconds. Offset discharges induced by a secondary input to phasic cells, applied at the end of the tonic input period, suddenly terminated the sustained discharges and allowed the network to return rapidly to the ongoing-spontaneous neuronal state. We suggest that the transporter-mediated alteration of ambient GABA, triggered by onset discharges, may improve the response property of AI neurons. Offset discharges may have a role in resetting AI neurons so that they can prepare for the next auditory input.


Ultrasound-mediated delivery of siESE complexed with microbubbles attenuates HER2+/- cell line proliferation and tumor growth in rodent models of breast cancer.
The highly tunable, noninvasive and spatially targeted nature of microbubble-enhanced, ultrasound-guided (MB+US) drug delivery makes it desirable for a wide variety of therapies. In breast cancer, both HER2+ and HER2- type neoplasms pose significant challenges to conventional therapeutics in greater than 40% of breast cancer patients, even with the widespread application of biologics such as trastuzumab. To address this therapeutic challenge, we examined the novel combination of tumor-injected microbubble-bound siRNA complexes and monodisperse size-isolated microbubbles (4-µm diameter) to attenuate tumor growth in vivo, as well as MB+US-facilitated shRNA and siRNA knockdown of ESE-1, an effector linked to dysregulated HER2 expression in HER2+/- cell line propagation. We first screened six variants of siESE and shESE for efficient knockdown of ESE in breast cancer cell lines. We demonstrated efficient reduction of BT-474 (PR+, ER+, HER2+; luminal B) and MDA-MB-468 (PR-, ER-, HER2-; triple-negative) clonogenicity and non-adherent growth after knockdown of ESE-1. A significant reduction in proliferative potential was seen for both cell lines using MB+US to deliver shESE and siESE. We then demonstrated significant attenuation of BT-474 xenograft tumor growth in Nod/SCID female mice using direct injection of microbubble-adsorbed siESE to the tumor and subsequent sonication. Our results suggest a positive effect on drug delivery from MB+US, and highlights the feasibility of using RNAi and MB+US for breast cancer pathologies. RNAi coupled with MB+US may also be an effective theranostic approach to treat other acoustically accessible tumors, such as melanoma, thyroid, parotid and skin cancer.


Neutrophil extracellular traps in COVID-19.
In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have the potential to propagate inflammation and microvascular thrombosis - including in the lungs of patients with acute respiratory distress syndrome. We now report that sera from patients with COVID-19 have elevated levels of cell-free DNA, myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3); the latter 2 are specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute-phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.


Complete genome of the cellulolytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evolutionary adaptations.
We present here the complete 2.4-Mb genome of the cellulolytic actinobacterial thermophile Acidothermus cellulolyticus 11B. New secreted glycoside hydrolases and carbohydrate esterases were identified in the genome, revealing a diverse biomass-degrading enzyme repertoire far greater than previously characterized and elevating the industrial value of this organism. A sizable fraction of these hydrolytic enzymes break down plant cell walls, and the remaining either degrade components in fungal cell walls or metabolize storage carbohydrates such as glycogen and trehalose, implicating the relative importance of these different carbon sources. Several of the A. cellulolyticus secreted cellulolytic and xylanolytic enzymes are fused to multiple tandemly arranged carbohydrate binding modules (CBM), from families 2 and 3. For the most part, thermophilic patterns in the genome and proteome of A. cellulolyticus were weak, which may be reflective of the recent evolutionary history of A. cellulolyticus since its divergence from its closest phylogenetic neighbor Frankia, a mesophilic plant endosymbiont and soil dweller. However, ribosomal proteins and noncoding RNAs (rRNA and tRNAs) in A. cellulolyticus showed thermophilic traits suggesting the importance of adaptation of cellular translational machinery to environmental temperature. Elevated occurrence of IVYWREL amino acids in A. cellulolyticus orthologs compared to mesophiles and inverse preferences for G and A at the first and third codon positions also point to its ongoing thermoadaptation. Additional interesting features in the genome of this cellulolytic, hot-springs-dwelling prokaryote include a low occurrence of pseudogenes or mobile genetic elements, an unexpected complement of flagellar genes, and the presence of three laterally acquired genomic islands of likely ecophysiological value.


Protein phosphorylation in isolated human adipocytes-adrenergic control of the phosphorylation of hormone-sensitive lipase.
The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32PO4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the beta-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (Mr 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the alpha-2 agonist clonidine. Epinephrine, a combined alpha and beta agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the alpha-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.


An antigenic peptide produced by reverse splicing and double asparagine deamidation.
A variety of unconventional translational and posttranslational mechanisms contribute to the production of antigenic peptides, thereby increasing the diversity of the peptide repertoire presented by MHC class I molecules. Here, we describe a class I-restricted peptide that combines several posttranslational modifications. It is derived from tyrosinase and recognized by tumor-infiltrating lymphocytes isolated from a melanoma patient. This unusual antigenic peptide is made of two noncontiguous tyrosinase fragments that are spliced together in the reverse order. In addition, it contains two aspartate residues that replace the asparagines encoded in the tyrosinase sequence. We confirmed that this peptide is naturally presented at the surface of melanoma cells, and we showed that its processing sequentially requires translation of tyrosinase into the endoplasmic reticulum and its retrotranslocation into the cytosol, where deglycosylation of the two asparagines by peptide-N-glycanase turns them into aspartates by deamidation. This process is followed by cleavage and splicing of the appropriate fragments by the standard proteasome and additional transport of the resulting peptide into the endoplasmic reticulum through the transporter associated with antigen processing (TAP).


Characterization of Salmonella enterica isolates from turkeys in commercial processing plants for resistance to antibiotics, disinfectants, and a growth promoter.
Salmonella enterica isolates from turkeys in two commercial processing plants (1 and 2) were characterized for susceptibility to antibiotics, disinfectants, and the organoarsenical growth promoter, 4-hydroxy-3-nitrophenylarsonic acid (3-NHPAA, roxarsone), and it's metabolites, NaAsO(2) (As(III)) and Na(2)HAsO(4) • 7H(2)O (As(V)). The 130 Salmonella serovars tested demonstrated a low incidence of resistance to the antibiotics gentamicin (GEN), kanamycin (KAN), sulfamethoxazole (SMX), streptomycin (STR), and tetracycline (TET). Isolates resistant to antibiotics were most often multidrug resistant. Serovars Hadar and Typhimurium were resistant to KAN, STR, and TET and GEN, SMX, and STR, respectively. All isolated Salmonella serovars were resistant to the disinfectant chlorhexidine with minimum inhibitory concentrations (MICs; 1-8 μg/mL), and they were susceptible to triclosan and benzalkonium chloride. The didecyldimethylammonium chloride component was the most active ammonium chloride tested. No cross-resistance was observed between antibiotics and disinfectants. The MICs for 3-NHPAA (4096 μg/mL) were consistent between processing Plant 1 and Plant 2, but MICs for the 3-NHPAA metabolites (As(III) and As(V)) were higher in Plant 1 than in Plant 2. In Plant 1, 76% of the isolates had MICs >256 μg/mL for As(III) and 92% of the isolates had MICs >1024 μg/mL for As(V). In Plant 2, all of the isolates had MICs ≤256 μg/mL for As(III) and 90% of the isolates had MICs ≤1024 μg/mL for As(V). Only 4 Salmonella serovars were isolated from Plant 1, but 10 serovars were isolated from Plant 2. S. enterica serovar Derby from Plant 1 was highly resistant to As(III) and As(V) with MICs >1024 and >8192 μg/mL, respectively, suggesting previous exposure to high arsenic metabolite concentrations. These levels may have been high enough to kill other Salmonella serovars, thus possibly explaining the lack of serovar diversity observed in Plant 1. The application of a growth promoter may affect the serovar diversity in treated birds.


Status of infection surveillance and control programs in the United States, 1992-1996. Association for Professionals in Infection Control and Epidemiology, Inc.
Nosocomial infections have been recognized as a source of morbidity and mortality throughout the world for several decades. In the United States, an estimated 2.1 million nosocomial infections occur annually in acute care hospitals alone. Infection surveillance and control programs (ISCPs) play a vital role in addressing this problem, but no national studies have described the status and composition of these programs since the 1970s. In January 1997, a voluntary survey was sent by mail to members of the Association for Professionals in Infection Control and Epidemiology, Inc. Only one response per facility was requested. The survey asked for information for the years 1992 to 1996 (study period), and questions pertained to characteristics of the health care facility in which the respondent worked, characteristics of the ISCP and its personnel, and the overall level of administration support for infection control activities. Completed questionnaires were received from personnel at 187 health care facilities located in 40 states and the District of Columbia. The majority (76.5%) of responding facilities were nongovernment owned, and 57.2% were classified as general acute care facilities. The number of licensed beds at these facilities remained stable throughout the study period, but all other measures of facility size and activity (eg, number of patient days and number of nurses) decreased by as much as 28.9%. In 1992, ISCPs were most likely to be organizationally located in the Nursing Department, but by 1996, many had been transferred to departments of Medical Records, Quality Assurance, or Risk Management. Throughout the course of the study period, the number of facilities performing surveillance for health care-associated infections in outpatient settings increased by 44.0%, from 100 to 144. In 1996, only 47.6% of facilities had a hospital epidemiologist (HE), and HEs devoted a median of 15% or less of their time to infection control activities. For the most part, HEs were trained in infectious diseases, and few had certification in infection control. Infection control professionals (ICPs) were much more common than were HEs (ICPs were reported at 97.9% of respondents' facilities in 1996), and they spent the majority (80% in 1996) of their time on infection control activities. During the course of the study period, increasing numbers of facilities had ICPs who had certification in infection control. Furthermore, most respondents did not report a change over time in the level of administration support for infection control activities. Health care delivery has changed dramatically during the past 20 years. This study presents an updated description of ISCPs in the United States. Our results illustrate several changing parameters, such as departmental shifts and increased outpatient surveillance, that reflect adjustments in health care priorities during the study period. As the transformation of the health care system continues, continued evaluation of the status of ISCPs on a national level will be necessary. Diligent monitoring, proactive measures, and collaboration between infection control organizations and government agencies will be vital for the prevention and control of health care-associated infections in the future.


PharmEcology: A pharmacological approach to understanding plant-herbivore interactions: an introduction to the symposium.
A central goal in understanding the ecology and evolution of animals is to identify factors that constrain or expand breadth of diet. Selection of diet in many animals is often constrained by chemical deterrents (i.e., secondary metabolites) in available food items. The integration of chemistry and ecology has led to a significant understanding of the chemical complexity of prey (e.g., animals, plants, and algae) and the resultant foraging behavior of consumers. However, most of the literature on chemical defenses of marine and terrestrial prey lacks a mechanistic understanding of how consumers tolerate, or avoid, chemically-defended foods. In order to understand ecological patterns of foraging and co-evolutionary relationships between prey and consumers, we must advance our understanding of the physiological mechanisms responsible for chemical interactions. Such mechanistic studies require the integration of the discipline of pharmacology with ecology, which we call "PharmEcology." Pharmacology provides the tools and insight to investigate the fate (what the body does to a chemical) and action (what a chemical does to the body) of chemicals in living organisms, whereas ecology provides the insight into the interactions between organisms (e.g., herbivores) and their environment (e.g., plants). Although, the general concepts of pharmacology were introduced to ecologists studying plant-herbivore interactions over 30 years ago, the empirical use of pharmacology to understand mechanisms of chemical interactions has remained limited. Moreover, many of the recent biochemical, molecular and technical advances in pharmacology have yet to be utilized by ecologists. The PharmEcology symposium held at a meeting of the Society for Integrative and Comparative Biology in January of 2009 was developed to define novel research directions at the interface of pharmacology and ecology.


Structuring a written examination to assess ASBH health care ethics consultation core knowledge competencies.
As clinical ethics consultants move toward professionalization, the process of certifying individual consultants or accrediting programs will be discussed and debated. With certification, some entity must be established or ordained to oversee the standards and procedures. If the process evolves like other professions, it seems plausible that it will eventually include a written examination to evaluate the core knowledge competencies that individual practitioners should possess to meet peer practice standards. The American Society for Bioethics and Humanities (ASBH) has published core knowledge competencies for many years that are accepted by experts as the prevailing standard. Probably any written examination will be based upon the ASBH core knowledge competencies. However, much remains to be done before any examination may be offered. In particular, it seems likely that a recognized examining board must create and validate examination questions and structure the examination so as to establish meaningful, defensible parameters after dealing with such challenging questions as: Should the certifying examination be multiple choice or short-answer essay? How should the test be graded? What should the pass rate be? How may the examination be best administered? To advance the field of health care ethics consultation, thought leaders should start to focus on the written examination possibilities, to date unaddressed carefully in the literature. Examination models-both objective and written-must be explored as a viable strategy about how the field of health care ethics consultations can grow toward professionalization.


Perceived sleep quality predicts cognitive function in adults with major depressive disorder independent of depression severity.
The aim of this study was to examine the role of perceived sleep quality in predicting subjective as well as objective cognitive function in adults with major depressive disorder (MDD). Adults with recurrent MDD (n = 100) experiencing a major depressive episode of at least moderate severity and age-, sex-, and education-matched healthy controls (HC) (n = 100) were recruited to participate in a clinical trial validating the THINC-integrated tool (THINC-it; NCT02508493) for cognitive function. The THINC-it includes subjective and objective measures of cognitive function. Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI). Compared with HC, individuals with MDD reported significantly poorer sleep quality, as assessed by domain and global PSQI scores (all P values <.05). Both perceived sleep quality (P < .001) and depression severity (P = .002) were found to independently predict impairments in subjective cognitive performance. Only perceived sleep quality predicted objective cognitive impairments (P = .017). Exploratory mediation analysis revealed depression severity to be a partial mediator of the relationship between perceived sleep quality and subjective cognitive performance (95% confidence interval [CI]: -0.56, -0.33). The results indicate that the subjective and objective cognitive impairments are differentially related to perceived sleep quality and depression severity and emphasize the importance of treating sleep disturbances in MDD.


Measuring Family Members' Satisfaction with End-of-Life Care in Long-Term Care: Adaptation of the CANHELP Lite Questionnaire.
Improving end-of-life care (EOLC) in long-term care (LTC) homes requires quality measurement tools that assess families' satisfaction with care. This research adapted and pilot-tested an EOLC satisfaction measure (Canadian Health Care Evaluation Project (CANHELP) Lite Questionnaire) for use in LTC to measure families' perceptions of the EOLC experience and to be self-administered. Phase 1. A literature review identified key domains of satisfaction with EOLC in LTC, and original survey items were assessed for inclusiveness and relevance. Items were modified, and one item was added. The revised questionnaire was administered to 118 LTC family members and cognitive interviews were conducted. Further modifications were made including reformatting to be self-administered. The new instrument was pilot-tested with 134 family members. Importance ratings indicated good content and face validity. Cronbach's alpha coefficients (range: .88-.94) indicated internal consistency. This research adapted and pilot-tested the CANHELP for use in LTC. This paper introduces the new, valid, internally consistent, self-administered tool (CANHELP Lite Family Caregiver LTC) that can be used to measure families' perceptions of and satisfaction with EOLC. Future research should further validate the instrument and test its usefulness for quality improvement and care planning.


Impact of endocrine disruptor chemicals in gynaecology.
The potential hazardous effects that estrogen- and androgen-like chemicals may have both on wildlife and human health have attracted much attention from the scientific community. Endocrine disruptors (EDCs) are chemicals that have the capacity to interfere with normal signalling systems. EDCs may mimic, block or modulate the synthesis, release, transport, metabolism and binding or elimination of natural hormones. Even though potential EDCs may be present in the environment at only very low levels, they may still cause harmful effects, especially when several different compounds act on one target. EDCs include persistent pollutants, agrochemicals and widespread industrial compounds. Not all EDCs are man-made compounds; many plants produce substances (phytoestrogens) that can have different endocrine effects either adverse or beneficial in certain circumstances. Natural substances such as sex hormones from urban or farm wastes can become concentrated in industrial, agricultural and urban areas; thus, such wastes may be considered potential 'EDCs' for humans and/or wildlife. Much attention has focussed on changing trends in male reproductive parameters in relation to EDC exposure; however, studies on the female reproductive system have been less comprehensive. We have focussed this article on four major aspects of female reproductive health: fertility and fecundability, endometriosis, precocious puberty and breast and endometrial cancer.


Brain surface contraction mapped in first-episode schizophrenia: a longitudinal magnetic resonance imaging study.
Schizophrenia is associated with structural brain abnormalities, but the timing of onset and course of these changes remains unclear. Longitudinal magnetic resonance imaging (MRI) studies have demonstrated progressive brain volume decreases in patients around and after the onset of illness, although considerable discrepancies exist regarding which brain regions are affected. The anatomical pattern of these progressive changes in schizophrenia is largely unknown. In this study, MRI scans were acquired repeatedly from 16 schizophrenia patients approximately 2 years apart following their first episode of illness, and also from 14 age-matched healthy subjects. Cortical Pattern Matching, in combination with Structural Image Evaluation, using Normalisation, of Atrophy, was applied to compare the rates of cortical surface contraction between patients and controls. Surface contraction in the dorsal surfaces of the frontal lobe was significantly greater in patients with first-episode schizophrenia (FESZ) compared with healthy controls. Overall, brain surface contraction in patients and healthy controls showed similar anatomical patterns, with that of the former group exaggerated in magnitude across the entire brain surface. That the pattern of structural change in the early course of schizophrenia corresponds so closely to that associated with normal development is consistent with the hypothesis that a schizophrenia-related factor interacts with normal adolescent brain developmental processes in the pathophysiology of schizophrenia. The exaggerated progressive changes seen in patients with schizophrenia may reflect an increased rate of synaptic pruning, resulting in excessive loss of neuronal connectivity, as predicted by the late neurodevelopmental hypothesis of the illness.


W-tree indexing for fast visual word generation.
The bag-of-visual-words representation has been widely used in image retrieval and visual recognition. The most time-consuming step in obtaining this representation is the visual word generation, i.e., assigning visual words to the corresponding local features in a high-dimensional space. Recently, structures based on multibranch trees and forests have been adopted to reduce the time cost. However, these approaches cannot perform well without a large number of backtrackings. In this paper, by considering the spatial correlation of local features, we can significantly speed up the time consuming visual word generation process while maintaining accuracy. In particular, visual words associated with certain structures frequently co-occur; hence, we can build a co-occurrence table for each visual word for a large-scale data set. By associating each visual word with a probability according to the corresponding co-occurrence table, we can assign a probabilistic weight to each node of a certain index structure (e.g., a KD-tree and a K-means tree), in order to re-direct the searching path to be close to its global optimum within a small number of backtrackings. We carefully study the proposed scheme by comparing it with the fast library for approximate nearest neighbors and the random KD-trees on the Oxford data set. Thorough experimental results suggest the efficiency and effectiveness of the new scheme.


A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach.
The presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.


Long-Term Follow-Up with Video of a Patient with Deafness-Dystonia Syndrome Treated with DBS-GPi.
The prevalence of deafness-dystonia syndrome (DDS) is relatively low. To our knowledge, only 2 cases of this syndrome treated with deep brain stimulation (DBS) have been reported. We present a patient with DDS of unknown cause, refractory to medical treatment, who has been successfully treated with DBS of the internal globus pallidus (DBS-GPi) and followed up for 4 years. A 21-year-old male, with progressive bilateral sensorineural hearing loss since the age of 3, developed dystonic movements at the age of 12. The patient presented with progressive segmental craniocervical dystonia with jaw-opening, tongue protrusion, retrocollis and gradual overflow including upper limb dystonia. Pharmacological therapy was ineffective. At the age of 17, the patient's condition deteriorated with the risk of developing a dystonic state. DBS-GPi implantation resulted in a striking improvement. The Burke-Marsden-Fahn Dystonia Rating Scale (BMFDRS) score improved from 75 points before the surgery to 10 points at 3 months after DBS-GPi implantation. Neurological examination at the age of 21 showed mild dystonic movements, mainly oromandibular dystonia (BMFDRS: 15 points). The clinical phenotype of our patient was consistent with Mohr-Tranebjaerg syndrome (MTS). We performed genetic analysis of the TIMM8A gene (the only gene in which mutations are known to cause MTS), but the result was negative; however, other potentially new mutations have to be considered. Based on our case with the longest reported follow-up of 4 years and 2 earlier reports, we advise to consider DBS-GPi in patients with DDS with unsatisfactory effect of pharmacological treatment.


Ten-year outcome of enzyme replacement therapy with agalsidase beta in patients with Fabry disease.
Fabry disease results from deficient α-galactosidase A activity and globotriaosylceramide accumulation causing renal insufficiency, strokes, hypertrophic cardiomyopathy and early demise. We assessed the 10-year outcome of recombinant α-galactosidase A therapy. The outcomes (severe clinical events, renal function, cardiac structure) of 52/58 patients with classic Fabry disease from the phase 3 clinical trial and extension study, and the Fabry Registry were evaluated. Disease progression rates for patients with low renal involvement (LRI, n=32) or high renal involvement (HRI, n=20) at baseline were assessed. 81% of patients (42/52) did not experience any severe clinical event during the treatment interval and 94% (49/52) were alive at the end of the study period. Ten patients reported a total of 16 events. Patients classified as LRI started therapy 13 years younger than HRI (mean 25 years vs 38 years). Mean slopes for estimated glomerular filtration rate for LRI and HRI were -1.89 mL/min/1.73 m(2)/year and -6.82 mL/min/1.73 m(2)/year, respectively. Overall, the mean left ventricular posterior wall thickness and interventricular septum thickness remained unchanged and normal. Patients who initiated treatment at age ≥ 40 years exhibited significant increase in left ventricular posterior wall thickness and interventricular septum thickness. Mean plasma globotriaosylceramide normalised within 6 months. This 10-year study documents the effectiveness of agalsidase beta (1 mg/kg/2 weeks) in patients with Fabry disease. Most patients remained alive and event-free. Patients who initiated treatment at a younger age and with less kidney involvement benefited the most from therapy. Patients who initiated treatment at older ages and/or had advanced renal disease experienced disease progression.


Effects of vestibular rehabilitation on gait performance in poststroke patients: a pilot randomized controlled trial.
The effects of vestibular rehabilitation on poststroke patients are unknown. This study aimed to investigate whether or not vestibular rehabilitation would improve both the vestibulo-ocular reflex and gait performance of patients with poststroke hemiparesis. Twenty-eight patients with stroke were assigned randomly to either an experimental group (N=14) or a control group (N=14). The experimental group performed the conventional physical therapy for 40 min and vestibular rehabilitation for 20 min, as a 60 min session, during the first 3 weeks and then completed only the conventional intervention for 60 min for the following 3 weeks. The control group performed only the 60 min conventional physical therapy for 6 weeks. Both groups were measured using the gaze stabilization test, the 10 m walking test, the timed up and go test, and the dynamic gait index. Patients were assessed at baseline, and at 3 and 6 weeks. Although the control group showed no significant difference in any outcome measures, the experimental group showed an improvement in gaze stabilization test scoring, which increased significantly after 3 weeks compared with the baseline (P=0.030). The dynamic gait index was also significantly increased after 3 and 6 weeks compared with the baseline (P=0.049 and 0.024, respectively). This study indicated that vestibular rehabilitation might improve poststroke patients' vestibulo-ocular reflex. Moreover, patients might show improved gait performance at least up to 3 weeks after the vestibular intervention by the sensory reweight to coordinate vestibular input.


Distinct actions of the fermented beverage kefir on host behaviour, immunity and microbiome gut-brain modules in the mouse.
Mounting evidence suggests a role for the gut microbiota in modulating brain physiology and behaviour, through bi-directional communication, along the gut-brain axis. As such, the gut microbiota represents a potential therapeutic target for influencing centrally mediated events and host behaviour. It is thus notable that the fermented milk beverage kefir has recently been shown to modulate the composition of the gut microbiota in mice. It is unclear whether kefirs have differential effects on microbiota-gut-brain axis and whether they can modulate host behaviour per se. To address this, two distinct kefirs (Fr1 and UK4), or unfermented milk control, were administered to mice that underwent a battery of tests to characterise their behavioural phenotype. In addition, shotgun metagenomic sequencing of ileal, caecal and faecal matter was performed, as was faecal metabolome analysis. Finally, systemic immunity measures and gut serotonin levels were assessed. Statistical analyses were performed by ANOVA followed by Dunnett's post hoc test or Kruskal-Wallis test followed by Mann-Whitney U test. Fr1 ameliorated the stress-induced decrease in serotonergic signalling in the colon and reward-seeking behaviour in the saccharin preference test. On the other hand, UK4 decreased repetitive behaviour and ameliorated stress-induced deficits in reward-seeking behaviour. Furthermore, UK4 increased fear-dependent contextual memory, yet decreased milk gavage-induced improvements in long-term spatial learning. In the peripheral immune system, UK4 increased the prevalence of Treg cells and interleukin 10 levels, whereas Fr1 ameliorated the milk gavage stress-induced elevation in neutrophil levels and CXCL1 levels. Analysis of the gut microbiota revealed that both kefirs significantly changed the composition and functional capacity of the host microbiota, where specific bacterial species were changed in a kefir-dependent manner. Furthermore, both kefirs increased the capacity of the gut microbiota to produce GABA, which was linked to an increased prevalence in Lactobacillus reuteri. Altogether, these data show that kefir can signal through the microbiota-gut-immune-brain axis and modulate host behaviour. In addition, different kefirs may direct the microbiota toward distinct immunological and behavioural modulatory effects. These results indicate that kefir can positively modulate specific aspects of the microbiota-gut-brain axis and support the broadening of the definition of psychobiotic to include kefir fermented foods. Video abstract.


Are polymorphic markers within the alpha-1-antitrypsin gene associated with risk of human immunodeficiency virus disease?
We sought to determine whether variants of the human alpha-1-antitrypsin (AAT) gene, also known as "PI," or "SERPINA1," are associated with human immunodeficiency virus (HIV) infection in 2 African-based populations from HIV-pandemic sub-Saharan Africa. Eleven commonly occurring African-associated polymorphic markers in the coding and intronic regions of the AAT gene were analyzed via denaturing gradient gel electrophoresis. A significant association between HIV-1 infection and the presence of an allelic variant was observed in the case of the M2 and A332A haplotypes, thus presenting AAT as a potentially novel HIV-1 susceptibility locus.


Extracranial carotid artery stenting: the Puerto Rico medical center endovascular neurosurgery service experience.
Extracranial carotid artery stenting (CAS) represents a viable alternative for high-risk surgical patients. The aim of this study was to determine the clinical features and outcome of 25 patients that underwent CAS at the Puerto Rico Medical Center. A retrospective review of a series of 25 high-risk surgical patients that underwent CAS from June 2005 to January 2010 was performed. Patients were followed-up at clinics with computed tomography angiography and/or digital subtraction angiography. Patient ages ranged from 52 to 88 years. Twenty-one of the patients had severe cervical carotid stenosis (more than 80%). Those with moderate stenosis (from 50% to 80%) were treated when they were symptomatic or when stenosis recurred after carotid endarterectomy. Among the 25 patients, only 2 presented with restenosis (more than 50% luminal diameter). Both had a history of radiation-induced disease, but neither required retreatment. Five patients required post-stenting angioplasty due to a less than 50% improvement in luminal diameter. There was 1 death, and 1 patient presented delayed neurocognitive deterioration. The combined long-term morbidity and mortality in the subgroup with at least two years of follow-up was 8.3%. There were no intracerebral hemorrhages or recurrent strokes/transient ischemic attacks. The restriction of post-stenting angioplasty to only those cases without significant revascularization appears to help reduce restenosis rates while ensuring a gradual increase in intracranial blood flow. The latter may not apply to patients with a prior history of radiotherapy.


GENES AS INSTRUMENTS FOR STUDYING RISK BEHAVIOR EFFECTS: AN APPLICATION TO MATERNAL SMOKING AND OROFACIAL CLEFTS.
This study uses instrumental variable (IV) models with genetic instruments to assess the effects of maternal smoking on the child's risk of orofacial clefts (OFC), a common birth defect. The study uses genotypic variants in neurotransmitter and detoxification genes relateded to smoking as instruments for cigarette smoking before and during pregnancy. Conditional maximum likelihood and two-stage IV probit models are used to estimate the IV model. The data are from a population-level sample of affected and unaffected children in Norway. The selected genetic instruments generally fit the IV assumptions but may be considered "weak" in predicting cigarette smoking. We find that smoking before and during pregnancy increases OFC risk substantially under the IV model (by about 4-5 times at the sample average smoking rate). This effect is greater than that found with classical analytic models. This may be because the usual models are not able to consider self-selection into smoking based on unobserved confounders, or it may to some degree reflect limitations of the instruments. Inference based on weak-instrument robust confidence bounds is consistent with standard inference. Genetic instruments may provide a valuable approach to estimate the "causal" effects of risk behaviors with genetic-predisposing factors (such as smoking) on health and socioeconomic outcomes.


Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons.
Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.


X-inactivation patch size in human female tissue confounds the assessment of tumor clonality.
Most models of tumorigenesis assume that tumors are monoclonal in origin. This conclusion is based largely on studies using X chromosome-linked markers in females. One important factor, often ignored in such studies, is the distribution of X-inactivated cells in tissues. Because lyonization occurs early in development, many of the progeny of a single embryonic stem cell are grouped together in the adult, forming patches. As polyclonality can be demonstrated only at the borders of X-inactivation patches, the patch size is crucial in determining the chance of demonstrating polyclonality and hence the number of tumors that need to be examined to exclude polyclonality. Previously studies using X-linked genes such as glucose-6-phosphate dehydrogenase have been handicapped by the need to destroy the tissues to study the haplotypes of glucose-6-phosphate dehydrogenase [Fialkow, P.-J. (1976) Biochim. Biophys. Acta 458, 283-321] or to determine the restriction fragment length polymorphisms of X chromosome-linked genes [Vogelstein, B., Fearon, E. R., Hamilton, S. R. & Feinberg, A. P. (1985) Science 227, 642-645]. Here we visualize X-inactivation patches in human females directly. Results show that the patch size is relatively large in both the human colon and breast, confounding assessment of tumor clonality with traditional X-inactivation studies.


The development and validation of the Bronchiectasis Health Questionnaire.
Health-related quality of life or health status is significantly impaired in bronchiectasis. There is a paucity of brief, simple-to-use, disease-specific health status measures. The aim of this study was to develop and validate the Bronchiectasis Health Questionnaire (BHQ), a new health status measure that is brief and generates a single overall score.Patients with bronchiectasis were recruited from two outpatient clinics, during a clinically stable stage. The development of the questionnaire followed three phases: item generation and item reduction using Rasch analysis, validation, and repeatability testing. The BHQ was translated into 11 languages using standardised methodology.206 patients with bronchiectasis completed a preliminary 65-item questionnaire. 55 items were removed due to redundancy or poor fit to the Rasch model. The final version of the BHQ consisted of 10 items. Internal consistency was good (Cronbach's α=0.85). Convergent validity of the BHQ with the St George's Respiratory Questionnaire was high (r= -0.82; p<0.001) and moderate with lung function (forced expiratory volume in 1 s % predicted r= -0.27; p=0.001). There was a significant association between BHQ scores and number of exacerbations of bronchiectasis in the last 12 months (p<0.001), hospital admissions (p=0.001) and computed tomography scan bronchiectasis pulmonary lobe counts (p<0.001). BHQ scores were significantly worse in patients with sputum bacterial colonisation versus no colonisation (p=0.048). The BHQ was highly repeatable after 2 weeks (intraclass correlation coefficient 0.89).The BHQ is a brief, valid and repeatable, self-completed health status questionnaire for bronchiectasis that generates a single total score. It can be used in the clinic to assess bronchiectasis from the patient's perspective.


Multivariate Analysis of Anthropometric Traits Using Summary Statistics of Genome-Wide Association Studies from GIANT Consortium.
Meta-analysis of single trait for multiple cohorts has been used for increasing statistical power in genome-wide association studies (GWASs). Although hundreds of variants have been identified by GWAS, these variants only explain a small fraction of phenotypic variation. Cross-phenotype association analysis (CPASSOC) can further improve statistical power by searching for variants that contribute to multiple traits, which is often relevant to pleiotropy. In this study, we performed CPASSOC analysis on the summary statistics from the Genetic Investigation of ANthropometric Traits (GIANT) consortium using a novel method recently developed by our group. Sex-specific meta-analysis data for height, body mass index (BMI), and waist-to-hip ratio adjusted for BMI (WHRadjBMI) from discovery phase of the GIANT consortium study were combined using CPASSOC for each trait as well as 3 traits together. The conventional meta-analysis results from the discovery phase data of GIANT consortium studies were used to compare with that from CPASSOC analysis. The CPASSOC analysis was able to identify 17 loci associated with anthropometric traits that were missed by conventional meta-analysis. Among these loci, 16 have been reported in literature by including additional samples and 1 is novel. We also demonstrated that CPASSOC is able to detect pleiotropic effects when analyzing multiple traits.


Fas splicing regulation during early apoptosis is linked to caspase-mediated cleavage of U2AF65.
U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa (U2AF65) is an essential splicing factor in the recognition of the pre-mRNA 3' splice sites during the assembly of the splicing commitment complex. We report here that U2AF65 is proteolyzed during apoptosis. This cleavage is group I or III caspase dependent in a noncanonical single site localized around the aspartic acid(128) residue and leads to the separation of the N- and C-terminal parts of U2AF65. The U2AF65 N-terminal fragment mainly accumulates in the nucleus within nuclear bodies (nucleoli-like pattern) and to a much lesser extent in the cytoplasm, whereas the C-terminal fragment is found in the cytoplasm, even in localization studies on apoptosis induction. From a functional viewpoint, the N-terminal fragment promotes Fas exon 6 skipping from a reporter minigene, by acting as a dominant-negative version of U2AF65, whereas the C-terminal fragment has no significant effect. The dominant-negative behavior of the U2AF65 N-terminal fragment can be reverted by U2AF35 overexpression. Interestingly, U2AF65 proteolysis in Jurkat cells on induction of early apoptosis correlates with the down-regulation of endogenous Fas exon 6 inclusion. Thus, these results support a functional link among apoptosis induction, U2AF65 cleavage, and the regulation of Fas alternative splicing.


[Apitherapy--the role of honey in the chronic wound healing process].
With increasing frequency, modern medicine directs attention to natural products and biological therapy methods and their use in clinical practice. Bee honey as well as other bee products (propolis, royal jelly and venom) have already found use in medicine not only as immune system stimulants but also as chronic wound healing promoters. The major arguments for implementing apitherapy are the low cost in comparison with conventional therapies and the fact that honey has proved effective against resistant hospital pathogens. Besides antimicrobial characteristics associated with no antimicrobial resistance risk, honey has anti-inflammatory effects. Controlled clinical trials have shown honey to be effective in chronic wound healing and gave rise to medihoney. Nevertheless, further study is needed to confirm the efficacy and safety of honey in clinical practice.


A laccase exclusively expressed by Metarhizium anisopliae during isotropic growth is involved in pigmentation, tolerance to abiotic stresses and virulence.
Insect pathogenic fungi including Metarhizium anisopliae offer an environmentally friendly alternative to chemical pesticides. However, their use has been limited by their relatively slow killing speed compared to chemicals and low tolerance to abiotic stresses. We report here on a class 1 laccase (MLAC1) that is involved in both virulence and tolerance to environmental stresses. Mlac1 is expressed during isotropic growth (swelling) but not during polarized growth (e.g., germ tubes and hyphae); Mlac1 is therefore expressed exclusively in the later stages of conidiation and in blastospores when M. anisopliae is living as a saprophyte. During infection processes, Mlac1 is also expressed by appressoria (infection structures) on the cuticle surface and hyphal bodies inside the insect haemocoel. Disrupting Mlac1 reduced virulence to caterpillars because of impaired appressoria and delayed post-infection events. It also produced a yellow-conidia phenotype with increased conidial susceptibility to heat shock (45 degrees C for 2h) and UV-B stress. The relationship between M. anisopliae's pigment-synthesis pathway and its adaptation to diverse natural habitats is discussed.


A NEW AND RAPID METHOD FOR THE ISOLATION AND CULTIVATION OF TUBERCLE BACILLI DIRECTLY FROM THE SPUTUM AND FECES.
Sixty-nine positive cultures were obtained from sixty-nine specimens of sputum from practically all stages of tuberculosis. Six of these specimens were negative by direct microscopic examination, but the cultures gave positive findings. These six specimens have been positive for tubercle bacilli at some time. Nineteen positive cultures were isolated from thirty-two specimens of feces. All these thirty-two specimens, upon direct microscopical examination, gave positive findings, some showing only a few tubercle bacilli. Six specimens were not free from contaminating organisms, and the remaining seven were negative. The method presented in this paper has proved very simple and accurate for the isolation of tubercle bacilli from sputum. The partial success in isolating tubercle bacilli from feces may be due to the fact that many of the bacilli may be dead.


Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications.
Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and straightforward approach for in vivo incorporation of a tag into proteins for relative quantitation by mass spectrometry. SILAC is a simple, yet powerful, method for investigating the dynamics of protein abundance and posttranslational modifications. Here, we provide detailed instructions for using this method to study protein complexes, protein-protein interactions, and the dynamics of protein abundance and posttranslational modifications. We expect that SILAC will become a routine technique because of its applicability to most areas of cell biology. We have also developed a Web site (http://www.silac.org) to provide researchers with updated information about this method and related resources.


Brillouin-based fiber-optic distributed temperature sensor with optical preamplification.
Optical preamplification has been used in a fiber-optic distributed temperature sensor based on spontaneous Brillouin scattering and the use of direct detection, resulting in improved signal-to-noise ratios. The fiber-based optical preamplifier system comprises a three-port circulator, an erbium-doped fiber amplifier with a small-signal gain of 27 dB, and a fiber Bragg grating with 47-GHz bandwidth. An improvement of 17 dB in the optical signal-to-noise ratio for the Brillouin signal is demonstrated in a 23-km sensor. The limit to the signal-to-noise ratio is attributed to spontaneous-spontaneous beat noise generated at the photodetector by amplified spontaneous emission from the optical amplifier.


Nontuberculous mycobacterial infections: a clinical review.
Nontuberculous mycobacteria (NTM) are important environmental pathogens that can cause a broad spectrum of diseases. The last few years brought several changes in this expanding field: The number of infections that can be associated with specific species as well as the number of new species as etiological agents has exploded due to the development of new diagnostic tools. The incidence of disseminated Mycobacterium avium complex (MAC) infections in HIV patients is decreasing with more potent anti-HIV treatments, while the rate of pulmonary NTM infection and disease as well as the prevalence of Buruli ulcer, a chronic, necrotizing, progressive ulcerous disease caused by Mycobacterium ulcerans, is increasing. The disease manifestations depend on the interaction between the specific mycobacterial pathogen and the host's immune system. This article presents an update of the epidemiology, diagnosis, and treatment of NTM-associated pulmonary disease, lymphadenitis, skin and soft tissue disease, skeletal infection and foreign body- and catheter-related NTM infections.


Identification of a 17-kDa protein associated with the peripheral-type benzodiazepine receptor in vascular and other smooth muscle types.
The existence of the peripheral-type benzodiazepine receptor (PBR) in vascular smooth muscle has been demonstrated in this laboratory. The present study utilized the photoaffinity ligand [3H]PK14105 to identify the protein subunit in rat aortic and other smooth muscle types to which high affinity ligands for the PBR bind. [3H]PK14105 bound to mitochondrial fractions isolated from rat aortic smooth muscle with high affinity (Kd = 7.0 +/- 0.5 nM) and high density (Bmax = 10.1 +/- 1.5 pmol/mg protein). The rank order of potency of a series of PBR ligands displacing the binding was PK11195 approximately equal to Ro5-4864 greater than protoporphyrin IX greater than flunitrazepam greater than diazepam much greater than clonazepam. [3H]PK14105 bound with comparable affinity and density to mitochondria isolated from rat myometrium and gastric smooth muscle as well. With ultraviolet irradiation, [3H]PK14105 specifically labeled a single protein of approximately 17 kDa in all three smooth muscle types examined. This protein was identical in size to that identified by [3H]PK14105 in rat adrenal gland. In adrenal gland an additional, minor protein of approximately 43 kDa was also specifically labeled by [3H]PK14105. Utilizing a probe designed from the known nucleotide sequence of the PBR in rat adrenal gland, an mRNA transcript of approximately 0.8 kilobases in size was identified in rat aortic smooth muscle by Northern blot analysis. These data indicate that a protein subunit of approximately 17 kDa comprises, at least in part, the PBR not only in vascular smooth muscle, but also in other smooth muscle types and adrenal gland as well.


ATF3-mediated epigenetic regulation protects against acute kidney injury.
A variety of stress stimuli, including ischemia-reperfusion (I/R) injury, induce the transcriptional repressor ATF3 in the kidney. The functional consequences of this upregulation in ATF3 after renal I/R injury are not well understood. Here, we found that ATF3-deficient mice had higher renal I/R-induced mortality, kidney dysfunction, inflammation (number of infiltrating neutrophils, myeloperoxidase activity, and induction of IL-6 and P-selectin), and apoptosis compared with wild-type mice. Furthermore, gene transfer of ATF3 to the kidney rescued the renal I/R-induced injuries in the ATF3-deficient mice. Molecular and biochemical analysis revealed that ATF3 interacted directly with histone deacetylase 1 (HDAC1) and recruited HDAC1 into the ATF/NF-kappaB sites in the IL-6 and IL-12b gene promoters. The ATF3-associated HDAC1 deacetylated histones, which resulted in the condensation of chromatin structure, interference of NF-kappaB binding, and inhibition of inflammatory gene transcription after I/R injury. Taken together, these data demonstrate epigenetic regulation mediated by the stress-inducible gene ATF3 after renal I/R injury and suggest potential targeted approaches for acute kidney injury.


Antenatal Three-Dimensional Printing of Aberrant Facial Anatomy.
Congenital airway obstruction poses a life-threatening challenge to the newborn. We present the first case of three-dimensional (3D) modeling and 3D printing of complex fetal maxillofacial anatomy after prenatal ultrasound indicated potential upper airway obstruction from a midline mass of the maxilla. Using fetal MRI and patient-specific computer-aided modeling, the craniofacial anatomy of the fetus was manufactured using a 3D printer. This model demonstrated the mass to be isolated to the upper lip and maxilla, suggesting the oral airway to be patent. The decision was made to deliver the infant without a planned ex utero intrapartum treatment procedure. The neonate was born with a protuberant cleft lip and palate deformity, without airway obstruction, as predicted by the patient-specific model. The delivery was uneventful, and the child was discharged without need for airway intervention. This case demonstrates that 3D modeling may improve prenatal evaluation of complex patient-specific fetal anatomy and facilitate the multidisciplinary approach to perinatal management of complex airway anomalies.


FSHD muscular dystrophy region gene 1 binds Suv4-20h1 histone methyltransferase and impairs myogenesis.
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with a strong epigenetic component. It is associated with deletion of a macrosatellite repeat leading to over-expression of the nearby genes. Among them, we focused on FSHD region gene 1 (FRG1) since its over-expression in mice, Xenopus laevis and Caenorhabditis elegans, leads to muscular dystrophy-like defects, suggesting that FRG1 plays a relevant role in muscle biology. Here we show that, when over-expressed, FRG1 binds and interferes with the activity of the histone methyltransferase Suv4-20h1 both in mammals and Drosophila. Accordingly, FRG1 over-expression or Suv4-20h1 knockdown inhibits myogenesis. Moreover, Suv4-20h KO mice develop muscular dystrophy signs. Finally, we identify the FRG1/Suv4-20h1 target Eid3 as a novel myogenic inhibitor that contributes to the muscle differentiation defects. Our study suggests a novel role of FRG1 as epigenetic regulator of muscle differentiation and indicates that Suv4-20h1 has a gene-specific function in myogenesis.


Mass-producible and efficient optical antennas with CMOS-fabricated nanometer-scale gap.
Optical antennas have been widely used for sensitive photodetection, efficient light emission, high resolution imaging, and biochemical sensing because of their ability to capture and focus light energy beyond the diffraction limit. However, widespread application of optical antennas has been limited due to lack of appropriate methods for uniform and large area fabrication of antennas as well as difficulty in achieving an efficient design with small mode volume (gap spacing < 10nm). Here, we present a novel optical antenna design, arch-dipole antenna, with optimal radiation efficiency and small mode volume, 5 nm gap spacing, fabricated by CMOS-compatible deep-UV spacer lithography. We demonstrate strong surface-enhanced Raman spectroscopy (SERS) signal with an enhancement factor exceeding 108 from the arch-dipole antenna array, which is two orders of magnitude stronger than that from the standard dipole antenna array fabricated by e-beam lithography. Since the antenna gap spacing, the critical dimension of the antenna, can be defined by deep-UV lithography, efficient optical antenna arrays with nanometer-scale gap can be mass-produced using current CMOS technology.