The targeted podocyte.
The podocyte plays a key role both in maintenance of the glomerular filtration barrier and in glomerular structural integrity. Podocyte injury and loss contribute to proteinuria and progressive sclerosis. Inhibitors of mammalian target of rapamycin (mTOR) have variably decreased or caused proteinuria and sclerosis in human disease and experimental settings. In this issue of the JCI, two interesting studies of podocyte-specific manipulation of the mTOR system shed light on the complexity of this pathway in the podocyte.


Community-level changes in condom use and uptake of HIV pre-exposure prophylaxis by gay and bisexual men in Melbourne and Sydney, Australia: results of repeated behavioural surveillance in 2013-17.
Pre-exposure prophylaxis (PrEP) has been rapidly rolled out in large, publicly funded implementation projects in Victoria and New South Wales, Australia. Using behavioural surveillance of gay and bisexual men, we analysed the uptake and effect of PrEP, particularly on condom use by gay and bisexual men not using PrEP. We collected data from the Melbourne and Sydney Gay Community Periodic Surveys (GCPS), cross-sectional surveys of adult gay and bisexual men in Melbourne, VIC, and Sydney, NSW. Recruitment occurred at gay venues or events and online. Eligible participants were 18 years or older (face-to-face recruitment) or 16 years or older (online recruitment), identified as male (including transgender participants who identified as male); and having had sex with a man in the past 5 years or identified as gay or bisexual, or both. Using multivariate logistic regression, we assessed trends in condom use, condomless anal intercourse with casual partners (CAIC), and PrEP use by gay and bisexual men, controlling for sample variation over time. Between Jan 1, 2013, and March 31, 2017, 27 011 participants completed questionnaires in the Melbourne (n=13 051) and Sydney (n=13 960) GCPS. 16 827 reported sex with casual male partners in the 6 months before survey and were included in these analyses. In 2013, 26 (1%) of 2692 men reported CAIC and were HIV-negative and using PrEP, compared with 167 (5%) of 3660 men in 2016 and 652 (16%) of 4018 men in 2017 (p<0·0001). Consistent condom use was reported by 1360 (46%) of 2692 men in 2013, 1523 (42%) of 3660 men in 2016, and 1229 (31%) of 4018 men in 2017 (p<0·0001). In 2013, 800 (30%) of 2692 men who were HIV-negative or untested and not on PrEP reported CAIC, compared with 1118 (31%) of 3660 men in 2016, and 1166 (29%) of 4018 in 2017 (non-significant trend). A rapid increase in PrEP use by gay and bisexual men in Melbourne and Sydney was accompanied by an equally rapid decrease in consistent condom use. Other jurisdictions should consider the potential for community-level increases in CAIC when modelling the introduction of PrEP and in monitoring its effect. Australian Government Department of Health, Victorian Department of Health and Human Services, and New South Wales Ministry of Health.


Adjuvant antibiotic-loaded bone cement: Concerns with current use and research to make it work.
Antibiotic-loaded bone cement (ALBC) is broadly used to treat orthopaedic infections based on the rationale that high-dose local delivery is essential to eradicate biofilm-associated bacteria. However, ALBC formulations are empirically based on drug susceptibility from routine laboratory testing, which is known to have limited clinical relevance for biofilms. There are also dosing concerns with nonstandardized, surgeon-directed, hand-mixed formulations, which have unknown release kinetics. On the basis of our knowledge of in vivo biofilms, pathogen virulence, safety issues with nonstandardized ALBC formulations, and questions about the cost-effectiveness of ALBC, there is a need to evaluate the evidence for this clinical practice. To this end, thought leaders in the field of musculoskeletal infection (MSKI) met on 1 August 2019 to review and debate published and anecdotal information, which highlighted four major concerns about current ALBC use: (a) substantial lack of level 1 evidence to demonstrate efficacy; (b) ALBC formulations become subtherapeutic following early release, which risks induction of antibiotic resistance, and exacerbated infection from microbial colonization of the carrier; (c) the absence of standardized formulation protocols, and Food and Drug Administration-approved high-dose ALBC products to use following resection in MSKI treatment; and (d) absence of a validated assay to determine the minimum biofilm eradication concentration to predict ALBC efficacy against patient specific micro-organisms. Here, we describe these concerns in detail, and propose areas in need of research.


Quantification of intrinsic water use efficiency along a moisture gradient in Northeastern China.
Water use efficiency (WUE) is an important ecophysiological characteristic of plants, especially in semiarid and arid regions. At the scale of community or ecosystem, WUE is difficult to quantify because the amount of water used per unit dry mass production is a function of microclimatic variables and species composition. In this study, we analyzed corrected intrinsic water use efficiency (IWUE(s)) of grass and shrub species along the western segment of the Northeast China Transect (NECT) and the relationship between IWUE(s) and mean annual rainfall, habitat degradation status, vegetation type, and plant functional type (C3 versus C4) at 22 survey sites. Site intrinsic water use efficiency (IWUE(v)) and its relationship with the aforementioned site variables were analyzed based on species frequencies at each site. First, it was concluded that photosynthetic pathway played a very important role in determining species IWUE(s). Mean IWUE(s) for C4 species was approximately double that of C3 species. Second, mean annual rainfall, vegetation type, and site degradation status significantly affected IWUE(s) (p < 0.01). Mean IWUE(s) at degraded sites was twice as high as that at nondegraded sites. The mean IWUE(s) for meadows was significantly higher than those for other vegetation types (p < 0.05). Third, the frequency of occurrence of C4 plants explained 36% of the variance in IWUE(v) across the survey sites. The mean frequency of C4 occurrence at degraded sites was more than double that at nondegraded sites. Consequently, mean IWUE(v) at degraded sites was more than double that at nondegraded sites. Dominant C4 species in saline-alkaline areas tended to have higher intrinsic WUE than dominant C4 species in sandy shrub communities.


High-dose isotretinoin in acne vulgaris: improved treatment outcomes and quality of life.
Isotretinoin, for acne treatment, is associated with high rates of permanent remission. However, at recommended doses of 0.5-1.0 mg/kg/day for 5-6 months [average cumulative dose: 120-150 mg/kg], more than 20% of patients experience a relapse within two years that requires further medical management. To examine outcomes of high-dose isotretinoin in a cohort with cystic acne, as well as measuring its impact on quality of life (QOL). A single dermatologist, single institution investigation within an academic tertiary care center in Bronx, NY. Eighty patients with nodulocystic acne, maintained on oral isotretinoin at a dose of 1.3 mg/kg/day or greater, were studied from 2006-2009 while additionally participating in a QOL survey. Main outcome measures included documented events, acne clearance, presence of relapse, and quality of life parameters. The mean daily dose of isotretinoin was 1.6 mg/kg/day for an average time course of 178 days [cumulative dose: 290 mg/kg]. No side effects or laboratory abnormalities led to discontinuation of treatment. There were no psychiatric symptoms. One-hundred percent (100%) of patients were disease-free upon completion of treatment. During the three-year study period, 10 patients (12.5%) developed a relapse that required an additional course of isotretinoin. Analysis of QOL domains (self-perception, role-social, symptoms) revealed significant improvement following isotretinoin therapy (p = 0.0124, p = 0.0066, p = 0.0265, respectively). Isotretinoin prescribed at 1.5 mg/kg/day or greater for 5-6 months [cumulative total dose of 290 mg/kg] is safe and effective compared to current standard dosing practices. We propose the use of high-dose isotretinoin (>1.3 mg/kg/day) as a treatment option in severe nodulocystic acne and encourage larger, prospective, multicenter studies into this therapeutic approach.


Long-term outcome of patients undergoing liver transplantation for mixed hepatocellular carcinoma and cholangiocarcinoma: an analysis of the UNOS database.
Mixed hepatocellular and cholangiocarcinoma (HCC-CC) have been associated with a poor prognosis after liver transplantation (LT). We aimed to evaluate long-term outcomes in patients undergoing LT for HCC-CC versus patients with hepatocellular carcinoma (HCC) or cholangiocarcinoma (CC). Retrospective analysis of the United Network for Organ Sharing (UNOS) database from 1994-2013. Overall survival (OS) in patients with HCC-CC, HCC, and CC, were compared. We identified 4049 patients transplanted for primary malignancy (94 HCC-CC; 3515 HCC; 440 CC). Mean age of patients with HCC-CC was 57 ± 10 years, and 77% were male. MELD score did not differ among the groups (p = 0.637). Hepatitis C virus was the most common secondary diagnosis within the HCC-CC (44%) and HCC (36%) cohorts, with primary sclerosing cholangitis in the CC (16%) cohort. OS rates at 1, 3 and 5 years for HCC-CC (82%, 47%, 40%) were similar to CC (79%, 58%, 47%), but significantly worse than HCC (86%, 72%, and 62% p = 0.002). Patients undergoing LT for HCC had significantly better survival compared to those transplanted for HCC-CC and CC. LT for mixed HCC-CC confers a survival rate similar to selected patients with CC. Efforts should be made to identify HCC-CC patients preoperatively.


Suppression of homologous recombination sensitizes human tumor cells to IGF-1R inhibition.
Inhibition of type 1 IGF receptor (IGF-1R) sensitizes to DNA-damaging cancer treatments, and delays repair of DNA double strand breaks (DSBs) by non-homologous end-joining and homologous recombination (HR). In a recent screen for mediators of resistance to IGF-1R inhibitor AZ12253801, we identified RAD51, required for the strand invasion step of HR. These findings prompted us to test the hypothesis that IGF-1R-inhibited cells accumulate DSBs formed at endogenous DNA lesions, and depend on residual HR for their repair. Indeed, initial experiments showed time-dependent accumulation of γH2AX foci in IGF-1R -inhibited or -depleted prostate cancer cells. We then tested effects of suppressing HR, and found that RAD51 depletion enhanced AZ12253801 sensitivity in PTEN wild-type prostate cancer cells but not in cells lacking functional PTEN. Similar sensitization was induced in prostate cancer cells by depletion of BRCA2, required for RAD51 loading onto DNA, and in BRCA2(-/-) colorectal cancer cells, compared with isogenic BRCA2(+/-) cells. We also assessed chemical HR inhibitors, finding that RAD51 inhibitor BO2 blocked RAD51 focus formation and sensitized to AZ12253801. Finally, we tested CDK1 inhibitor RO-3306, which impairs HR by inhibiting CDK1-mediated BRCA1 phosphorylation. R0-3306 suppressed RAD51 focus formation consistent with HR attenuation, and sensitized prostate cancer cells to IGF-1R inhibition, with 2.4-fold reduction in AZ12253801 GI50 and 13-fold reduction in GI80. These data suggest that responses to IGF-1R inhibition are enhanced by genetic and chemical approaches to suppress HR, defining a population of cancers (PTEN wild-type, BRCA mutant) that may be intrinsically sensitive to IGF-1R inhibitory drugs.


Spatial acuity in 2-to-3-year-old children with normal acoustic hearing, unilateral cochlear implants, and bilateral cochlear implants.
: To measure spatial acuity on a right-left discrimination task in 2-to-3-year-old children who use a unilateral cochlear implant (UCI) or bilateral cochlear implants (BICIs); to test the hypothesis that BICI users perform significantly better when they use two CIs than when using a single CI, and that they perform better than the children in the UCI group; to determine how well children with CIs perform compared with children who have normal acoustic hearing (NH); to determine the effect of intensity roving on spatial acuity. : Three groups of children between 26 and 36 months of age participated in this study: 8 children with NH (mean age: 30.9 months), 12 children who use a UCI (mean age: 31.9 months), and 27 children who use BICIs (mean age: 30.7 months). Testing was conducted in a large sound-treated booth with loudspeakers positioned in a horizontal arc with a radius of 1.2 m. The observer-based psychophysical procedure was used to measure the children's ability to identify the hemifield containing the sound source (right versus left). Two methods were used for quantifying spatial acuity, an adaptive-tracking method and a fixed-angle method. In Experiment 1 an adaptive tracking algorithm was used to vary source angle, and the minimum audible angle (MAA), the smallest angle at which right-left discrimination performance is better than chance, was estimated. All three groups participated in Experiment 1. In Experiment 2 source angles were fixed at ±50 degrees, and performance was evaluated by computing the number of SDs above chance. Children in the UCI and BICI groups participated in Experiment 2. : In Experiment 1, when stimulus intensity was roved by 8 dB, MAA thresholds were 3.3 degrees to 30.2 degrees (mean = 14.5 degrees) and 5.7 degrees to 69.6 degrees (mean = 30.9 degrees) in the NH group and in the BICI group, respectively. When the intensity level was fixed for the BICI group, performance did not improve. Within the BICI group, 5 out of 27 children obtained MAA thresholds within one SD of their peers who have NH; all five had >12 months of bilateral listening experience. In Experiment 2, BICIs provided some advantages when the intensity level was fixed. First, the BICI group outperformed the UCI group. Second, children in the BICI group who repeated the task with their 1st CI alone had statistically significantly better performance when using both devices. In addition, when intensity roving was introduced, a larger percentage of children who had 12 or more months of BICI experience continued to perform above chance than children who had <12 months of BICI experience. Taken together, the results suggest that children with BICIs have spatial acuity that is better than when using their first CI alone and than that of their peers who use UCIs. In addition, longer durations of BICI use tend to result in better performance, although this cannot be generalized to all participants. : This report is consistent with a growing body of evidence that spatial-hearing skills can emerge in young children who use BICIs. The observation that these skills are not concomitantly emerging in age- and experience-matched children who use UCIs suggests that BICIs provide cues that are necessary for these spatial-hearing skills that UCIs do not provide.


The Cost of Improving the Precision of the Variational Quantum Eigensolver for Quantum Chemistry.
New approaches into computational quantum chemistry can be developed through the use of quantum computing. While universal, fault-tolerant quantum computers are still not available, and we want to utilize today's noisy quantum processors. One of their flagship applications is the variational quantum eigensolver (VQE)-an algorithm for calculating the minimum energy of a physical Hamiltonian. In this study, we investigate how various types of errors affect the VQE and how to efficiently use the available resources to produce precise computational results. We utilize a simulator of a noisy quantum device, an exact statevector simulator, and physical quantum hardware to study the VQE algorithm for molecular hydrogen. We find that the optimal method of running the hybrid classical-quantum optimization is to: (i) allow some noise in intermediate energy evaluations, using fewer shots per step and fewer optimization iterations, but ensure a high final readout precision; (ii) emphasize efficient problem encoding and ansatz parametrization; and (iii) run all experiments within a short time-frame, avoiding parameter drift with time. Nevertheless, current publicly available quantum resources are still very noisy and scarce/expensive, and even when using them efficiently, it is quite difficult to perform trustworthy calculations of molecular energies.


Chromated copper arsenate-treated fence posts in the agronomic landscape: soil properties controlling arsenic speciation and spatial distribution.
Soils adjacent to chromated copper arsenate (CCA)-treated fence posts along a fence line transecting different soil series, parent material, drainage classes, and slope were used to determine which soil properties had the most influence on As spatial distribution and speciation. Metal distribution was evaluated at macroscopic (total metal concentration contour maps) and microscopic scales (micro-synchrotron X-ray fluorescence maps), As speciation was determined using extended X-ray absorption fine structure spectroscopy, and redox status and a myriad of other basic soil properties were elucidated. All geochemical parameters measured point to a condition in which the mobilization of As becomes more favorable moving down the topographic gradient, likely resulting through competition (Meh-P, SOM), neutral or slightly basic pH, and redox conditions that are favorable for As mobilization (higher Fe(II) and total-Fe concentrations in water extracts). On the landscape scale, with hundreds of kilometers of fence, the arsenic loading into the soil can be substantial (∼8-12 kg km). Although a significant amount of the As is stable, extended use of CCA-treated wood has resulted in elevated As concentrations in the local environment, increasing the risk of exposure and ecosystem perturbation. Therefore, a move toward arsenic-free alternatives in agricultural applications for which it is currently permitted should be considered.


Synthesis and antimycobacterial evaluation of benzofurobenzopyran analogues.
We recently reported that 3,3-dimethyl-3H-benzofuro[3,2,f][1]-benzopyran and its hydrogenated analogue are selective in vitro inhibitors of mycobacterial growth. However, their lack of in vivo activity on a murine model of Mycobacterium tuberculosis infection due to their poor bioavailability led to a structure-activity relationship investigation. We wish to report here the preparation of some structural analogues along with their biological effect on the growth of Mycobacterium smegmatis, M. tuberculosis, as well as on VERO cells for the most active compound.


Structural and mutational analysis of E2 trans-activating proteins of papillomaviruses reveals three distinct functional domains.
The E2 proteins of papillomaviruses are able to transactivate the viral enhancers by interacting with the sequence ACCGN4CGGT found in all papillomavirus long control regions. Analysis of the alignment of the amino acid sequences of 10 E2 proteins reveals three distinct regions: two partially conserved domains at the N and C termini of the proteins and a region variable in size and sequence in the middle. A computer prediction of the secondary structure of the 10 sequences outlines interesting conserved features, including two long amphiphilic alpha helices at the N terminus. To analyse the respective roles of the different segments of these proteins, we constructed a set of in-frame deletion and insertion mutations in the E2 coding sequences of the bovine papillomavirus type 1 (BPV1) and cottontail rabbit papillomavirus (CRPV). The test of their capacity to trans-activate or repress different viral constructs shows that the C-terminal domain of the E2 proteins is involved exclusively in DNA binding whereas the N-terminal domain is probably required for interaction with other components of the transcriptional machinery. The inner variable domain may confer flexibility to the protein such that it will facilitate contacts of the two others with their respective targets.


Hepatoprotective effects of soluble rice protein in primary hepatocytes and in mice.
The production of rice-derived by-products has increased owing to the growing use of processed rice products. The objective of this study was to isolate highly purified proteins from a rice by-product, rice syrup meal, and to examine their hepatoprotective effects in vitro and in vivo. Soluble rice protein (SRP70) was obtained via enzymatic processing of rice syrup meal using Termamyl SC and Alcalase. Mass spectrometry analysis showed that SRP70 contained low-molecular-weight (<600 Da) peptides. SRP70 did not affect the viability of rat primary hepatocytes and ameliorated tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity. t-BHP-induced elevations in hepatocyte alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities were reduced by SRP70 in a dose-dependent manner. In addition, t-BHP exposure increased the level of malondialdehyde, a toxic reactive aldehyde, which was dose-dependently decreased by SRP70 treatment. These SRP70-induced decreases in biochemical parameters were also observed in vivo in mice. In particular, SRP70 increased the activities of liver antioxidant enzymes in t-BHP-treated mice, including catalase and glutathione peroxidase, as well as increasing the level of glutathione, an antioxidant peptide. SRP70-mediated activation of antioxidant enzymes was shown to be due to the up-regulation in their gene expressions, while nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 4 (NOX4), a pro-oxidant enzyme, was down-regulated by SRP70. Hematoxylin and eosin staining also showed that SRP70 protected the liver from histopathological changes induced by t-BHP. Taken together, these data showed that SRP70, which is derived from a rice-processing by-product, had hepatoprotective effects in vitro and in vivo.


1999 National Hospital Discharge Survey: annual summary with detailed diagnosis and procedure data.
This report presents 1999 national estimates and selected trend data on the use of non-Federal short-stay hospitals in the United States. Estimates are provided by demographic characteristics of patients discharged, geographic region, ownership, and bed size of hospitals, principal expected source of payment, conditions diagnosed, and surgical and nonsurgical procedures performed. Measurements of hospital use include number and rate of discharges and days of care, and the average length of stay. Estimates of first-listed diagnoses, days of care, all-listed diagnoses, and all-listed procedures are presented according to their code number in the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM). The estimates are based on data collected through the National Hospital Discharge Survey. The survey has been conducted annually by the National Center for Health Statistics since 1965. In 1999 data were collected for approximately 300,000 discharges. Of the 487 eligible non-Federal short-stay hospitals in the sample, 458 (94 percent) responded to the survey. An estimated 32.1 million inpatients were discharged from non-Federal short-stay hospitals in 1999. These patients used an estimated 160.1 million days of care. Patients 65 years of age and over accounted for 40 percent of discharges and used 48 percent of days of care. More than half of all patients under age 65 in 1999 had a principal expected source of payment from a private source. Patients discharged from nonprofit hospitals accounted for nearly 78 percent of all discharges. Heart disease accounted for 4.5 million discharges, and deliveries made up 3.8 million discharges. An estimated 3.7 million newborn infants were discharged from short-stay hospitals after average stays of 3.2 days.


Incidence of cervical cytological abnormalities with aging in the women's health initiative: a randomized controlled trial.
To estimate the incidence of cytological abnormalities and cervical cancer and to determine the effect of oral estrogen and progestin on cervical cytology among postmenopausal women participating in a multi-institution clinical trial. The study was a longitudinal analysis of a prospective cohort of 16,608 postmenopausal women (aged 50-79 years) participating in the Women's Health Initiative (WHI) clinical trial of estrogen plus progestin. Eligible participants had a cervical smear within 1 year before randomization and at 3- and 6-year follow-ups. Outcomes measured were low-grade and high-grade squamous intraepithelial lesions (LSIL, HSIL) and cervical cancer at follow-up years 3 and 6. Of 15,733 eligible participants with a uterus, 7,663 were assigned to placebo and 8,070 to estrogen plus progestin. At baseline, 318 women (2%) had low-grade abnormalities on cervical cytology. The annual incidence rate of any new cytological abnormality in the estrogen plus progestin group was significantly higher than that in the placebo group (hazard ratio 1.4, 95% confidence interval [CI] 1.2-1.6). Independent risk factors for HSIL and cervical cancer over a 6-year follow-up (after stratifying for baseline cytologic abnormalities) included sexual activity in the past year while not being married or living as married (hazard ratio 3.5, 95% CI 1.5-8.3). Risk factors did not include age or use of estrogen plus progestin. Use of estrogen plus progestin was associated with increased incidence of any cytologic abnormality, although it had no impact on the incidence of HSIL or cervical cancer. Sexually active older women who are not married or living as married may benefit from continued cervical cancer screening. Clinicaltrials.gov, www.clinicaltrials.gov, NCT00000611.


Acute heroin overdose.
Acute heroin overdose is a common daily experience in the urban and suburban United States and accounts for many preventable deaths. Heroin acts as a pro-drug that allows rapid and complete central nervous system absorption; this accounts for the drug's euphoric and toxic effects. The heroin overdose syndrome (sensitivity for diagnosing heroin overdose, 92%; specificity, 76%) consists of abnormal mental status, substantially decreased respiration, and miotic pupils. The response of naloxone does not improve the sensitivity of this diagnosis. Most overdoses occur at home in the company of others and are more common in the setting of other drugs. Heroin-related deaths are strongly associated with use of alcohol or other drugs. Patients with clinically significant respiratory compromise need treatment, which includes airway management and intravenous or subcutaneous naloxone. Hospital observation for several hours is necessary for recurrence of hypoventilation or other complications. About 3% to 7% of treated patients require hospital admission for pneumonia, noncardiogenic pulmonary edema, or other complications. Methadone maintenance is an effective preventive measure, and others strategies should be studied.


New targets for therapy in prostate cancer: modulation of stromal-epithelial interactions.
Changes in genomic and phenotypic expression of progressing prostate tumors and their stroma occur in a dynamic fashion based on bidirectional signaling from stromal-epithelial interactions. These interactions may underlie the ability of prostate cancer cells to survive and proliferate in the prostate and bone. By investigating the phenotypic and genotypic changes of stromal cells adjacent to cancer cells and the reciprocal changes of cancer cells, novel molecular markers may be developed to diagnose cancer earlier before pathologic appearance of cancer cells at the primary site. Attacking epithelial and stromal elements together is a unique approach to both localized and metastatic prostate cancer therapy. Co-targeting both tumor cells and stroma requires identifying a reliable tumor and tissue-specific cis-DNA element, such as osteocalcin (OC) promoter. OC expression is elevated in prostate tumor cells and in prostate and bone stromal cells interdigitating with both localized and metastatic prostate epithelium. We have previously designed an adenovirus-based therapeutic gene vehicle and demonstrated that a replication-competent adenoviral vector (Ad vector) is highly efficient in blocking the growth of cancer cells in culture, including cells without androgen receptor as well as cells that do or do not make prostate-specific antigen. In vivo, intravenous administration of an Ad-OC vector was effective against preexisting human prostate cancer subcutaneous and bone xenografts. The addition of vitamin D(3) enhanced further viral replication at target sites. Co-targeting tumor cells and stroma using systemic Ad vector is a viable and promising option for treatment of both localized and metastatic prostate cancer.


No resistance to tenofovir disoproxil fumarate detected after up to 144 weeks of therapy in patients monoinfected with chronic hepatitis B virus.
Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue with potent activity against human immunodeficiency virus type 1 and hepatitis B virus (HBV). To date, no reports of HBV clinical resistance to TDF have been confirmed. In two phase 3 studies (GS-US-174-0102 and GS-US-174-0103), 375 hepatitis B e antigen-negative (HBeAg(-) ) patients and 266 HBeAg(+) patients with chronic hepatitis B (some nucleoside-naive and some lamivudine-experienced) were randomized 2:1 to receive TDF (n = 426) or adefovir dipivoxil (ADV; n = 215) for 48 weeks. After week 48, eligible patients received open-label TDF with no interruption. The studies are being continued through week 384/year 8; week 144 data are presented here. Per protocol, viremic patients (HBV DNA level ≥ 400 copies/mL or 69 IU/mL) had the option of adding emtricitabine (FTC) at or after week 72. Resistance analyses of HBV polymerase/reverse transcriptase (pol/RT) were based on population dideoxy sequencing. Phenotypic analyses were conducted in HepG2 cells with recombinant HBV derived from patient serum. Most patients maintained TDF monotherapy treatment across both studies (607/641, 95%). A resistance analysis of HBV pol/RT was performed at the baseline for all patients, for viremic patients at week 144 or at the last time when they were on TDF monotherapy (34 on TDF and 19 on ADV-TDF), and for patients who remained viremic after the addition of FTC (7/20 on TDF and 5/14 on ADV-TDF). No patient developed amino acid substitutions associated with resistance to TDF. Virological breakthrough on TDF monotherapy was infrequent over 144 weeks (13/426, 3%) and was attributed to documented nonadherence in most cases (11/13, 85%). Persistent viremia (≥400 copies/mL) through week 144 was rare (5/641, 0.8%) and was not associated with virological resistance to TDF by population or clonal analyses. No nucleoside-naive or nucleoside-experienced patient developed HBV pol/RT mutations associated with TDF resistance after up to 144 weeks of exposure to TDF monotherapy.


Cyclic tensile stress during physiological occlusal force enhances osteogenic differentiation of human periodontal ligament cells via ERK1/2-Elk1 MAPK pathway.
Physiological occlusal force constitutively exists in the oral environment and is important for periodontal homeostasis and remodeling. Cyclic tensile stress (CTS) triggers the biological response of periodontal ligament (PDL). However, a few reports have studied the correlation between CTS during physiological occlusal force and PDL cell activities such as osteogenic differentiation. In the present study, human PDL cells (hPDLCs) were subjected to 10% elongation CTS loading at 0.5 Hz for 24 h, which represents the physiological conditions of occlusal force. Gene expression microarray was used to investigate the mechano-induced differential gene profile and pathway analysis in vitro. The osteogenic relative factors, that is, SPP1, RUNX2, and SP7, were assessed by real-time PCR and Western blot. The involvement of mitogen-activated protein kinase (MAPK) signaling pathways was investigated by Western blot with a specific inhibitor. The expressions of SPP1, RUNX2, SP7, p-ERK1/2, and p-Elk1 were up-regulated after 10% CTS exposure. However, these up-regulated expressions were prevented by ERK1/2 inhibitor U0126 in the physiological occlusal force-applied hPDLCs. These results showed that 10% CTS could enhance osteogenic differentiation of hPDLCs via ERK1/2-Elk1 MAPK pathway, indicating that CTS during physiological occlusal force is a potent agent for PDL remodeling.


Hydrazone Linker as a Useful Tool for Preparing Chimeric Peptide/Nonpeptide Bifunctional Compounds.
The area of multitarget compounds, joining two pharmacophores within one molecule, is a vivid field of research in medicinal chemistry. Not only pharmacophoric elements are essential for the design and activity of such compounds, but the type and length of linkers used to connect them are also crucial. In the present contribution, we describe compound 1 in which a typical opioid peptide sequence is combined with a fragment characteristic for neurokinin-1 receptor (NK1R) antagonists through a hydrazone bridge. The compound has a high affinity for μ- and δ-opioid receptors (IC50= 12.7 and 74.0 nM, respectively) and a weak affinity for the NK1R. Molecular modeling and structural considerations explain the observed activities. In in vivo test, intrathecal and intravenous administrations of 1 exhibited a strong analgesic effect, which indicates potential BBB penetration. This letter brings an exemplary application of the hydrazone linker for fast, facile, and successful preparation of chimeric compounds.


Deletion of an additional domain located between SXXK and SXN active-site fingerprints in penicillin-binding protein 4 from Escherichia coli.
It was suggested previously that the primary structure of penicillin-binding protein 4 (PBP4) is new and unique among proteins that interact with penicillin. Our proposal that PBP4 carries an additional domain, located between the active-site fingerprints SXXK and SXN, was investigated by mutational deletion analysis. A clustered set of internal deletions was created in this region by exonuclease treatment of the dacB coding DNA, starting from two internal restriction sites. PBP4 mutants carrying internal deletions were selected by screening for immunoreactive forms of PBP4 with reduced molecular weight that were still active with respect to penicillin binding. DNA sequencing revealed 24 distinct PBP4 mutants with internal deletions ranging from 37 to 113 amino acids. The amino- and carboxy-terminal end points of the deletions were not randomly distributed but tended to cluster in certain areas. Overproduction of the individual mutated forms of PBP4 resulted in accumulation of the major portion of the proteins in the particulate cell fraction. The yield of soluble and active mutated forms of the protein was reduced from below 1% to 79% of the level obtained for the native protein. The deletions that were introduced had minor effects on the deacylation rate of bound benzylpenicillin. Two pairs of cysteine residues (Cys-139-Cys-153 and Cys-197-Cys-214) that are located in the deletable region may form disulfide bridges.


Online survey of current autopsy practice.
The Royal College of Pathologists (RCPath) has for several years published guidance on good autopsy practice. However, pressures such as time, cost and the introduction of the Human Tissue Act have generated suggestions that there is a discrepancy between the published guidelines and what can realistically be achieved in daily practice. The aims of this study were to determine the extent to which practising pathologists agree with this complaint, and what suggestions they might have for its resolution. All histopathologists in the UK on the RCPath database (n = 1213) were sent an email invitation to participate in an online questionnaire. 406 pathologists completed the survey, providing numerical data and free-text responses. Results concerning pressures of time, resources and limitations on examination and sampling were in keeping with those expected from recent issues raised. The view that RCPath guidelines are higher than can be achieved in routine coronial autopsy practice was widely supported, but only 45% stated that the RCPath should publish separate guidelines to differentiate between hospital ("consent") autopsies and medico-legal cases. The circumstances under which coronial autopsies are conducted in many parts of the UK make it difficult or impossible to comply with current RCPath guidance. Pathologists disagree on whether this situation demands a reduction of RCPath standards, an improvement in autopsy practice in medico-legal cases to current RCPath standards, or the implementation of "double standards". Resolution of this dilemma requires clarification of exactly what a coronial autopsy is trying to achieve.


Regulation of Kv1 channel trafficking by the mamba snake neurotoxin dendrotoxin K.
Modulation of voltage-gated potassium (Kv) channel surface expression can profoundly affect neuronal excitability. Some, but not all, mammalian Shaker or Kv1 alpha subunits contain a dominant endoplasmic reticulum (ER) retention signal in their pore region, preventing surface expression of Kv1.1 homotetrameric channels and of heteromeric Kv1 channels containing more than one Kv1.1 subunit. The critical amino acid residues within this ER pore-region retention signal are also critical for high-affinity binding of snake dendrotoxins (DTX). This suggests that ER retention may be mediated by an ER protein with a domain structurally similar to that of DTX. One facet of such a model is that expression of soluble DTX in the ER lumen should compete for binding to the retention protein and allow for surface expression of retained Kv1.1. Here, we show that luminal DTX expression dramatically increased both the level of cell surface Kv1.1 immunofluorescence staining and the proportion of Kv1.1 with processed N-linked oligosaccharides. Electrophysiological analyses showed that luminal DTX expression led to significant increases in Kv1.1 currents. Together, these data showed that luminal DTX expression increases surface expression of functional Kv1.1 homotetrameric channels and support a model whereby a DTX-like ER protein regulates abundance of cell surface Kv1 channels.


Dispersal and selection mediate hybridization between a native and invasive species.
Hybridization between native and non-native species has serious biological consequences, but our understanding of how dispersal and selection interact to influence invasive hybridization is limited. Here, we document the spread of genetic introgression between a native (Oncorhynchus clarkii) and invasive (Oncorhynchus mykiss) trout, and identify the mechanisms influencing genetic admixture. In two populations inhabiting contrasting environments, non-native admixture increased rapidly from 1984 to 2007 and was driven by surprisingly consistent processes. Individual admixture was related to two phenotypic traits associated with fitness: size at spawning and age of juvenile emigration. Fish with higher non-native admixture were larger and tended to emigrate at a younger age--relationships that are expected to confer fitness advantages to hybrid individuals. However, strong selection against non-native admixture was evident across streams and cohorts (mean selection coefficient against genotypes with non-native alleles (s) = 0.60; s.e. = 0.10). Nevertheless, hybridization was promoted in both streams by the continuous immigration of individuals with high levels of non-native admixture from other hybrid source populations. Thus, antagonistic relationships between dispersal and selection are mediating invasive hybridization between these fish, emphasizing that data on dispersal and natural selection are needed to fully understand the dynamics of introgression between native and non-native species.


Structured illumination microscopy combined with machine learning enables the high throughput analysis and classification of virus structure.
Optical super-resolution microscopy techniques enable high molecular specificity with high spatial resolution and constitute a set of powerful tools in the investigation of the structure of supramolecular assemblies such as viruses. Here, we report on a new methodology which combines Structured Illumination Microscopy (SIM) with machine learning algorithms to image and classify the structure of large populations of biopharmaceutical viruses with high resolution. The method offers information on virus morphology that can ultimately be linked with functional performance. We demonstrate the approach on viruses produced for oncolytic viriotherapy (Newcastle Disease Virus) and vaccine development (Influenza). This unique tool enables the rapid assessment of the quality of viral production with high throughput obviating the need for traditional batch testing methods which are complex and time consuming. We show that our method also works on non-purified samples from pooled harvest fluids directly from the production line.


Validation and genotyping of multiple human polymorphic inversions mediated by inverted repeats reveals a high degree of recurrence.
In recent years different types of structural variants (SVs) have been discovered in the human genome and their functional impact has become increasingly clear. Inversions, however, are poorly characterized and more difficult to study, especially those mediated by inverted repeats or segmental duplications. Here, we describe the results of a simple and fast inverse PCR (iPCR) protocol for high-throughput genotyping of a wide variety of inversions using a small amount of DNA. In particular, we analyzed 22 inversions predicted in humans ranging from 5.1 kb to 226 kb and mediated by inverted repeat sequences of 1.6-24 kb. First, we validated 17 of the 22 inversions in a panel of nine HapMap individuals from different populations, and we genotyped them in 68 additional individuals of European origin, with correct genetic transmission in ∼ 12 mother-father-child trios. Global inversion minor allele frequency varied between 1% and 49% and inversion genotypes were consistent with Hardy-Weinberg equilibrium. By analyzing the nucleotide variation and the haplotypes in these regions, we found that only four inversions have linked tag-SNPs and that in many cases there are multiple shared SNPs between standard and inverted chromosomes, suggesting an unexpected high degree of inversion recurrence during human evolution. iPCR was also used to check 16 of these inversions in four chimpanzees and two gorillas, and 10 showed both orientations either within or between species, providing additional support for their multiple origin. Finally, we have identified several inversions that include genes in the inverted or breakpoint regions, and at least one disrupts a potential coding gene. Thus, these results represent a significant advance in our understanding of inversion polymorphism in human populations and challenge the common view of a single origin of inversions, with important implications for inversion analysis in SNP-based studies.


The magnitude of the electromotive force of canine ventricular myocardium.
The isolated and perfused dog heart was placed in a cubic container filled with Tyrode's solution. Ventricular ectopic beats were produced by electrical stimulation of the left ventricular wall, and initial QRS vectors of these beats were determined with orthogonal leads from the surface of the container. At the same instants, the activated area on the epicardial surface was measured by means of a large number of contiguous bipolar leads from the epicardial surface. The QRS vector and the activated epicardial area were found to be nearly porportional. By use of these results and a calibration system with artificial dipoles, the double layer moment of the ventricular activation wave was calculated as 0.13 mA.cm per unit area. This value corresponds to 60% of the maximal possible strength of the tissue electromotive force. Lowering the conductivity of the surrounding solution increased the QRS voltage but not as much as the potential caused by a constant-current dipole within the solution. The relationship between the QRS voltage and the conductivity of the medium was analyzed by a simplified model of the system and was found to correspond approximately to that of a constant-current source within a spherical heart with a resistivity 2 to 3 times that of Tyrode's solution.


Spatial Release From Informational Masking: Evidence From Functional Near Infrared Spectroscopy.
Informational masking (IM) can greatly reduce speech intelligibility, but the neural mechanisms underlying IM are not understood. Binaural differences between target and masker can improve speech perception. In general, improvement in masked speech intelligibility due to provision of spatial cues is called spatial release from masking. Here, we focused on an aspect of spatial release from masking, specifically, the role of spatial attention. We hypothesized that in a situation with IM background sound (a) attention to speech recruits lateral frontal cortex (LFCx) and (b) LFCx activity varies with direction of spatial attention. Using functional near infrared spectroscopy, we assessed LFCx activity bilaterally in normal-hearing listeners. In Experiment 1, two talkers were simultaneously presented. Listeners either attended to the target talker (speech task) or they listened passively to an unintelligible, scrambled version of the acoustic mixture (control task). Target and masker differed in pitch and interaural time difference (ITD). Relative to the passive control, LFCx activity increased during attentive listening. Experiment 2 measured how LFCx activity varied with ITD, by testing listeners on the speech task in Experiment 1, except that talkers either were spatially separated by ITD or colocated. Results show that directing of auditory attention activates LFCx bilaterally. Moreover, right LFCx is recruited more strongly in the spatially separated as compared with colocated configurations. Findings hint that LFCx function contributes to spatial release from masking in situations with IM.


Mesoscale substrate curvature overrules nanoscale contact guidance to direct bone marrow stromal cell migration.
The intrinsic architecture of biological tissues and of implanted biomaterials provides cells with large-scale geometrical cues. To understand how cells are able to sense and respond to complex structural environments, a deeper insight into the cellular response to multi-scale and conflicting geometrical cues is needed. In this study, we subjected human bone marrow stromal cells (hBMSCs) to mesoscale cylindrical surfaces (diameter 250-5000 µm) and nanoscale collagen fibrils (diameter 100-200 nm) that were aligned perpendicular to the cylinder axis. On flat surfaces and at low substrate curvatures (cylinder diameter d > 1000 µm), cell alignment and migration were governed by the nanoscale collagen fibrils, consistent with the contact guidance effect. With increasing surface curvature (decreasing cylinder diameter, d < 1000 µm), cells increasingly aligned and migrated along the cylinder axis, i.e. the direction of zero curvature. An increase in phosphorylated myosin light chain levels was observed with increasing substrate curvature, suggesting a link between substrate-induced cell bending and the F-actin-myosin machinery. Taken together, this work demonstrates that geometrical cues of up to 10× cell size can play a dominant role in directing hBMSC alignment and migration and that the effect of nanoscale contact guidance can even be overruled by mesoscale curvature guidance.


Inhibition of tumor growth and vasculogenic mimicry by curcumin through down-regulation of the EphA2/PI3K/MMP pathway in a murine choroidal melanoma model.
This study aims to investigate the underlying mechanism by which curcumin inhibits tumor growth and reduces vasculogenic mimicry (VM) in a murine choroidal melanoma model. Sixty mice were given subretinal injection with B16F10 cells and divided into a treatment and a control group. Curcumin was administered to the treatment group once a day at a dose of 100 mg/kg for 18 days starting at d3 (the day of inoculation is designated as d0); an equivalent volume of poloxamer-F68 was administered to the control group. Immunohistochemical and histochemical double staining were ued to detect the different blood supply patterns. The amounts of epithelial cell kinase (EphA2), phosphatidylinositol-3-kinase (PI3K), and matrixmetalloproteinase-2 and -9 (MMP-2, MMP-9) proteins expressed in the tumor tissue were analyzed using immunohistochemical staining; mRNA levels were measured using real-time PCR analysis. Results indicate that the tumor volume is reduced (P=0.000) and that the numbers of VM (P=0.000), mosaic vessels (P=0.031), and endothelium-dependent vessels (P=0.000) are significantly decreased by curcumin (P=0.001). The expression levels of EphA2, PI3K, MMP-2, and -9 are also lower in the treatment group than in the control group (P=0.001); similarly, mRNA levels in the treatment group are lower than those in the control group (P=0.000). In conclusion, curcumin has the ability to inhibit the growth of engrafted melanoma VM channels through the regulation of vasculogenic factors that could be related to the down-regulation of the EphA2/PI3K/MMPs signaling pathway. Thus, curcumin has the potential of being a clinical inhibitor of VM of choroidal melanoma.


Short- and Longer-Term Association Between Body Mass Index and Health Status in Cardiac Rehabilitation Patients.
The association between body mass index (BMI) and subjective health status before and after cardiac rehabilitation (CR) and 1 year later was compared in patients undergoing primary percutaneous coronary intervention (pPCI) who did (CR group) and did not receive CR (no-CR group). The aim was to investigate the association between BMI and subjective health status based on the Short Form-12 questionnaire. Between 2009 and 2011, 242 patients with pPCI with an acute myocardial infarction completed a CR program and were compared with 115 patients in the no-CR group. All patients completed the Short Form-12 questionnaire at baseline, at 12 weeks, and at 1-year followup. The CR program consisted of a 2 sessions per week for 1.5 hours each for 12 weeks. Patients were categorized into 3 groups based on BMI: normal weight, overweight, and obese. Compared with patients in the no-CR group, CR group patients in the overweight group significantly improved their subjective health status after CR and these improvements were sustained at 1-year followup. CR patients in the normal weight and obese groups did not significantly improve subjective health status. The overweight patients had the highest improvement in subjective health status (OR = 3.4 post-CR and 5.1 at 1 year of followup). After CR, overweight patients showed the best improvement in subjective health status. CR did not significantly improve subjective health status in normal-weight and obese patients.


Is There a Role for Informal Caregivers in the Management of Diabetic Foot Ulcers? A Narrative Review.
Successful management of diabetic foot ulceration (DFU) is crucial for preventing long-term morbidity and lowering risk of amputations. This can be achieved with a multifaceted approach involving a multidisciplinary team, with the patient at the centre. However, not all healthcare setups enable this, and the rate of lower limb amputations continues to rise. It is therefore time to consider new approaches to diabetic foot care, capitalising on engagement from patients in self-management while supported by their informal caregivers (ICGs) to help improve outcome. The role of ICGs in DFU care has the potential to make a significant difference in outcome, yet this resource remains, in most cases, underutilised. Limited research has been conducted in this area to reveal the true impact on patient outcomes and the caregivers themselves. This narrative review aims to explore how ICGs can benefit DFU management with applicability to different healthcare setups while benefiting from established experience in the care of other chronic health conditions.


Caregiver Asthma Knowledge, Aptitude, and Practice in High Healthcare Utilizing Children: Effect of an Educational Intervention.
Factors underlying high healthcare utilization among Hispanic and African American (AA) children with asthma are not well known. We hypothesized that low parental knowledge and suboptimal practices are associated with high healthcare utlization and sought to elucidate these factors and identify ethnicity-specific differences. We also hypothesized that a targeted educational intervention will decrease emergency department (ED) visits and hospitalizations. A 57-item questionnaire investigating asthma knowledge, aptitude, and practice was administered during a hospitalization to 268 caregivers (158 Hispanic and 110 AA) of high healthcare utilizer children. Responses were compared between ethnicities. Participants were randomized into an education group and a control group to investigate the impact of an in-hospital educational intervention on future ED visits and hospitalizations. More than 80% of caregivers knew that asthma is associated with muscle constriction and mucus production. Overall, 66.7%-86.9% of caregivers found preventive steps including allergen avoidance, regular primary care physician (PCP) follow-up, and medication adherence helpful, but only 45.2% reported adherence to controller medications. Similarly, caregivers found management steps, including albuterol use, avoidance of ineffective medications, and need to contact PCP helpful but 33% Hispanic caregivers contacted their child's PCP at the time of the exacerbation leading the current hospitalization, compared with 17% AA caregivers (P=0.006). Moreover, 40% and 30% Hispanic and 27% and 18% AA caregivers felt stressed and helpless, respectively, about their child's asthma. Despite high baseline levels of knowledge, there were fewer ED visits in the education group (1.56±1.94) compared with the control group (2.05±2.32) (P=0.02) 2 years after the intervention. Although Hispanic and AA caregivers of children with high healthcare utilization were knowledgeable of asthma pathophysiology, and preventive and management steps, they reported being stressed and helpless and were unable to implement the steps at the time of an exacerbation, seeking care at the ED rather than contacting their PCP. The high health utilizers who underwent a targeted educational intervention had fewer ED visits 2 years following the intervention.


Ultrasound imaging of the distal radioulnar joint: a new method to assess ulnar radial translation in forearm rotation.
A cross-sectional reliability study was conducted with 23 normal participants to establish normal values, and the repeatability and validity of distal radioulnar joint translation measurements using ultrasound imaging. Static transverse images of maximal supination, neutral and maximal pronation were examined to assess translation, using a method consistent with the rheumatoid arthritis subluxation ratio. Translation while gripping a 1 kg weight in supinated and pronated positions was then compared with non-gripping translation. There was significantly more ulnar radial translation found with pronation than supination, when compared with neutral. Gripping in pronation did not produce statistically significant changes in translation, whereas the changes produced by gripping in supination were significant. Internal consistency was deemed very high and the rheumatoid arthritis subluxation ratio values measured using ultrasound imaging were consistent with previously documented values measured by computerized tomography. This study demonstrated that translational movement of the distal radioulnar joint can be reliably detected in healthy participants using ultrasound imaging. This may reduce dependency on other imaging modalities to diagnose distal radioulnar joint instability. 2.


Follicular dendritic cells: origin, phenotype, and function in health and disease.
Follicular dendritic cells (FDCs) were originally identified by their specific morphology and by their ability to trap immune-complexed antigen in B cell follicles. By virtue of the latter as well as the provision of chemokines, adhesion molecules, and trophic factors, FDCs participate in the shaping of B cell responses. Importantly, FDCs also supply tingible body macrophages (TBMs) with the eat-me-signaling molecule milk fat globule-EGF factor 8 (Mfge8), thereby enabling the disposal of apoptotic B cells. Recent studies have provided fundamental insights into the multiple functions of FDCs in both physiological and pathophysiological contexts and into their origin. Here we review these findings, and discuss current concepts related to FDC histogenesis both in lymphoid organs and in inflammatory lymphoneogenesis.


Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2.
Ascorbate (vitamin C) is a vital antioxidant molecule in the brain. However, it also has a number of other important functions, participating as a cofactor in several enzyme reactions, including catecholamine synthesis, collagen production, and regulation of HIF-1 alpha. Ascorbate is transported into the brain and neurons via the sodium-dependent vitamin C transporter 2 (SVCT2), which causes accumulation of ascorbate within cells against a concentration gradient. Dehydroascorbic acid, the oxidized form of ascorbate, is transported via glucose transporters of the GLUT family. Once in cells, it is rapidly reduced to ascorbate. The highest concentrations of ascorbate in the body are found in the brain and in neuroendocrine tissues such as adrenal, although the brain is the most difficult organ to deplete of ascorbate. Combined with regional asymmetry in ascorbate distribution within different brain areas, these facts suggest an important role for ascorbate in the brain. Ascorbate is proposed as a neuromodulator of glutamatergic, dopaminergic, cholinergic, and GABAergic transmission and related behaviors. Neurodegenerative diseases typically involve high levels of oxidative stress and thus ascorbate has been posited to have potential therapeutic roles against ischemic stroke, Alzheimer's disease, Parkinson's disease, and Huntington's disease.


Biological activities of anti-merozoite surface protein-1 antibodies induced by adjuvant-assisted immunizations in mice with different immune gene knockouts.
Immunizations with Plasmodium falciparum MSP1-42 or MSP1-19 induce antibodies that inhibit parasites in vitro, which correlates with in vivo protective immunity by vaccination. We previously showed that several adjuvant formulations can induce anti-MSP1-19 antibodies in interleukin-6, intercellular adhesion molecule 1, CD80, and CD86 knockout (KO) mice and at levels similar to those obtained in the healthy uninfected hosts. Here, we determine whether these immune gene KOs or the immunopotentiating activities of the adjuvants have a more important influence on the induction of parasite-inhibitory anti-MSP1-19 antibodies. Results showed that the biological activities of the anti-MSP1-19 antibodies induced by these adjuvants were not affected by the immune gene KOs. All adjuvant formulations that induced significant inhibitory antibody responses (i.e., >50% inhibition of parasite growth) contained monophosphoryl lipid A (MPL) in emulsion carriers, whereas MPL or emulsion carriers alone were ineffective. The ability to retain vaccine efficacy by the MSP1-19 and adjuvant formulations in the altered immunological background is a valuable and significant attribute in light of many instances of skewed immune status in the targeted vaccine populations.


Multi-resolution simulation of DNA transport through large synthetic nanostructures.
Modeling and simulation has become an invaluable partner in development of nanopore sensing systems. The key advantage of the nanopore sensing method - the ability to rapidly detect individual biomolecules as a transient reduction of the ionic current flowing through the nanopore - is also its key deficiency, as the current signal itself rarely provides direct information about the chemical structure of the biomolecule. Complementing experimental calibration of the nanopore sensor readout, coarse-grained and all-atom molecular dynamics simulations have been used extensively to characterize the nanopore translocation process and to connect the microscopic events taking place inside the nanopore to the experimentally measured ionic current blockades. Traditional coarse-grained simulations, however, lack the precision needed to predict ionic current blockades with atomic resolution whereas traditional all-atom simulations are limited by the length and time scales amenable to the method. Here, we describe a multi-resolution framework for modeling electric field-driven passage of DNA molecules and nanostructures through to-scale models of synthetic nanopore systems. We illustrate the method by simulating translocation of double-stranded DNA through a solid-state nanopore and a micron-scale slit, capture and translocation of single-stranded DNA in a double nanopore system, and modeling ionic current readout from a DNA origami nanostructure passage through a nanocapillary. We expect our multi-resolution simulation framework to aid development of the nanopore field by providing accurate, to-scale modeling capability to research laboratories that do not have access to leadership supercomputer facilities.


Association of serotonin and dopamine gene pathways with behavioral subphenotypes in dementia.
Genetic association studies investigating the association between genes of serotonergic and dopaminergic systems and behavioral and psychological symptoms in dementia (BPSD) are contradictory. We have utilized 1008 probable Alzheimer's disease (AD) patients from the UK and used the 12-item Neuropsychiatric Inventory. We applied a multiple indicators-multiple causes (MIMIC) approach to investigate the effect of 11 polymorphisms on the 4 behavioral subphenotypes "psychosis", "moods", "agitation", and "behavioural dyscontrol". Significant associations were observed between the serotonin transporter gene (SERT) polymorphism STin2 and "psychosis"; the dopamine transporter gene (DAT) 3' variable number tandem repeats (VNTR) and "agitation"; and the dopamine receptor 4 (DRD4) VNTR and "moods" factors. Direct associations were identified between the dopamine receptor 3 (DRD3) BalI polymorphism and depression; the dopamine receptor 1 (DRD1) and dopamine transporter gene 3' VNTR polymorphisms and aberrant motor behavior; the DRD4 VNTR and sleep disturbances; and the SERT gene VNTR 5HTTLPR and apathy items. Significant interactions observed between polymorphisms suggested epistatic effects and interactions between polymorphisms and medications highlighted potential treatment response. This multiple indicators multiple causes (MIMIC) model efficiently captured the complexity of the interrelations between genetic variation, behavioral symptoms, and clinical variables.


The generation of oligodendroglial cells is preserved in the rostral migratory stream during aging.
The subventricular zone (SVZ) is the largest source of newly generated cells in the adult mammalian brain. SVZ-derived neuroblasts migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into mature neurons. Additionally, a small proportion of SVZ-derived cells contribute to the generation of myelinating oligodendrocytes. The production of new cells in the SVZ decreases during aging, affecting the incorporation of new neurons into the OB. However, the age-related changes that occur across the RMS are not fully understood. In this study we evaluate how aging affects the cellular organization of migrating neuroblast chains, the proliferation, and the fate of the newly generated cells in the SVZ-OB system. By using electron microscopy and immunostaining, we found that the RMS path becomes discontinuous and its cytoarchitecture is disorganized in aged mice (24-month-old mice). Subsequently, OB neurogenesis was impaired in the aged brain while the production of oligodendrocytes was not compromised. These findings provide new insight into oligodendrocyte preservation throughout life. Further exploration of this matter could help the development of new strategies to prevent neurological disorders associated with senescence.


Timing of Aspirin and Other Nonsteroidal Anti-Inflammatory Drug Use Among Patients With Colorectal Cancer in Relation to Tumor Markers and Survival.
Purpose Regular use of aspirin is associated with improved survival for patients with colorectal cancer (CRC). However, the timing of and the subtype of CRC that would benefit the most from using aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) in relation to survival is unclear. Patients and Methods In all, 2,419 patients age 18 to 74 years with incident invasive CRC who were diagnosed from 1997 to 2008 were identified from population-based cancer registries in the United States, Canada, and Australia. Detailed epidemiologic questionnaires were administered at study enrollment and at 5-year follow-up. Survival outcomes were completed through linkage to national death registries. BRAF- and KRAS-mutation status, microsatellite instability, and CpG island methylator phenotype were also evaluated. Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% CIs for overall survival (OS) and CRC-specific survival. Results After a median of 10.8 years of follow-up since diagnosis, 381 deaths (100 as a result of CRC) were observed. Compared with nonusers, postdiagnostic aspirin-only users had more favorable OS (HR, 0.75; 95% CI, 0.59 to 0.95) and CRC-specific survival (HR, 0.44; 95% CI, 0.25 to 0.71), especially among those who initiated aspirin use (OS: HR, 0.64; 95% CI, 0.47 to 0.86; CRC-specific survival: HR, 0.40; 95% CI, 0.20 to 0.80). The association between any NSAID use after diagnosis and OS differed significantly by KRAS-mutation status ( Pinteraction = .01). Use of any NSAID after diagnosis was associated with improved OS only among participants with KRAS wild-type tumors (HR, 0.60; 95% CI, 0.46 to 0.80) but not among those with KRAS-mutant tumors (HR, 1.24; 95% CI, 0.78 to 1.96). Conclusion Among long-term CRC survivors, regular use of NSAIDs after CRC diagnosis was significantly associated with improved survival in individuals with KRAS wild-type tumors.


Perceptions of parents and healthcare professionals regarding minimal invasive tissue sampling to identify the cause of death in stillbirths and neonates: a qualitative study protocol.
Globally, around 2.6 million neonatal deaths occur world-wide every year and the numbers of stillbirths is almost similar. Pakistan is ranked among the highest countries in the world for neonatal mortality. In 2016, for every 1000 babies born in Pakistan, 46 died before the end of the first month of life. Also, Pakistan had the highest rate of stillbirths (43.1/1000 births) in 2015. To meet sustainable development (SDG) targets of reducing neonatal mortality and stillbirths, it is essential to gain understanding about the causes of neonatal death and stillbirths. In Pakistan, full autopsies are conducted only in medico-legal cases and are very rarely performed to identify a definitive cause of death (CoD) and because of cost and insufficient staff are generally not feasible. Recently, minimally invasive tissue sampling (MITS) has been used to determine CoD in neonates and stillbirths as it addresses some of the socio-cultural and religious barriers to autopsy. However, it is not known how families and communities will perceive this procedure; therefore, exploring family and healthcare professionals' perceptions regarding MITS is essential in determining acceptable and feasible approaches for Pakistan. The study will employ an exploratory qualitative research design. The study will be conducted at the National Institute of Child Health (NICH) hospital of Karachi. The data collection method will consist of key-informant interviews (KIIs) and focus group discussions (FGDs). FGDs will be conducted with the families and relatives of newborns who are visiting the outpatient department (OPD) and well-baby clinics of NICH hospital. KIIs will be conducted with the NICH - medical director, healthcare providers, professionals involved in proceedings related to death and dying, religious leaders, health sector representatives from the government, public health experts, maternal and child health (MCH) specialists, obstetricians and neonatologists and experts from the bioethics committee. Study data will be analyzed using NVivo 10 software. The research will help explore specific cultural, religious and socio-behavioral factors that may increase or decrease the acceptability of MITS for identifying COD in neonates and stillbirths. The findings of the qualitative study will provide a better understanding of parents' and healthcare professionals' attitudes towards the use of MITS on neonatal deaths and stillborns.


A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication.
Attention-Deficit/Hyperactivity Disorder (ADHD) has a very high heritability (0.8), suggesting that about 80% of phenotypic variance is due to genetic factors. We used the integration of statistical and functional approaches to discover a novel gene that contributes to ADHD. For our statistical approach, we started with a linkage study based on large multigenerational families in a population isolate, followed by fine mapping of targeted regions using a family-based design. Family- and population-based association studies in five samples from disparate regions of the world were used for replication. Brain imaging studies were performed to evaluate gene function. The linkage study discovered a genome region harbored in the Latrophilin 3 gene (LPHN3). In the world-wide samples (total n=6360, with 2627 ADHD cases and 2531 controls) statistical association of LPHN3 and ADHD was confirmed. Functional studies revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication. Linkage and replicated association of ADHD with a novel non-candidate gene (LPHN3) provide new insights into the genetics, neurobiology, and treatment of ADHD.


Sex differences in the circadian regulation of sleep and waking cognition in humans.
The sleep-wake cycle and circadian rhythmicity both contribute to brain function, but whether this contribution differs between men and women and how it varies across cognitive domains and subjective dimensions has not been established. We examined the circadian and sleep-wake-dependent regulation of cognition in 16 men and 18 women in a forced desynchrony protocol and quantified the separate contributions of circadian phase, prior sleep, and elapsed time awake on cognition and sleep. The largest circadian effects were observed for reported sleepiness, mood, and reported effort; the effects on working memory and temporal processing were smaller. Although these effects were seen in both men and women, there were quantitative differences. The amplitude of the circadian modulation was larger in women in 11 of 39 performance measures so that their performance was more impaired in the early morning hours. Principal components analysis of the performance measures yielded three factors, accuracy, effort, and speed, which reflect core performance characteristics in a range of cognitive tasks and therefore are likely to be important for everyday performance. The largest circadian modulation was observed for effort, whereas accuracy exhibited the largest sex difference in circadian modulation. The sex differences in the circadian modulation of cognition could not be explained by sex differences in the circadian amplitude of plasma melatonin and electroencephalographic slow-wave activity. These data establish the impact of circadian rhythmicity and sex on waking cognition and have implications for understanding the regulation of brain function, cognition, and affect in shift-work, jetlag, and aging.


TMBETA-NET: discrimination and prediction of membrane spanning beta-strands in outer membrane proteins.
We have developed a web-server, TMBETA-NET for discriminating outer membrane proteins and predicting their membrane spanning beta-strand segments. The amino acid compositions of globular and outer membrane proteins have been systematically analyzed and a statistical method has been proposed for discriminating outer membrane proteins. The prediction of membrane spanning segments is mainly based on feed forward neural network and refined with beta-strand length. Our program takes the amino acid sequence as input and displays the type of the protein along with membrane-spanning beta-strand segments as a stretch of highlighted amino acid residues. Further, the probability of residues to be in transmembrane beta-strand has been provided with a coloring scheme. We observed that outer membrane proteins were discriminated with an accuracy of 89% and their membrane spanning beta-strand segments at an accuracy of 73% just from amino acid sequence information. The prediction server is available at http://psfs.cbrc.jp/tmbeta-net/.


Rabies virus nucleoprotein functions to evade activation of the RIG-I-mediated antiviral response.
The rabies virus Ni-CE strain causes nonlethal infection in adult mice after intracerebral inoculation, whereas the parental Nishigahara (Ni) strain kills mice. We previously reported that the chimeric CE(NiN) strain with the N gene from the Ni strain in the genetic background of the Ni-CE strain kills adult mice, indicating that the N gene is related to the different pathogenicities of Ni and Ni-CE strains. In the present study, to obtain an insight into the mechanism by which the N gene determines viral pathogenicity, we compared the effects of Ni, Ni-CE, and CE(NiN) infections on host gene expressions using a human neuroblastoma cell line. Microarray analysis of these infected cells revealed that the expression levels of particular genes in Ni- and CE(NiN)-infected cells, including beta interferon (IFN-beta) and chemokine genes (i.e., CXCL10 and CCL5) were lower than those in Ni-CE-infected cells. We also demonstrated that Ni-CE infection activated the interferon regulatory factor 3 (IRF-3)-dependent IFN-beta promoter and induced IRF-3 nuclear translocation more efficiently than did Ni or CE(NiN) infection. Furthermore, we showed that Ni-CE infection, but not Ni or CE(NiN) infection, strongly activates the IRF-3 pathway through activation of RIG-I, which is known as a cellular sensor of virus infection. These findings indicate that the N protein of rabies virus (Ni strain) has a function to evade the activation of RIG-I. To our knowledge, this is the first report that the Mononegavirales N protein functions to evade induction of host IFN and chemokines.


Cognitive and psychomotor performance tests and experiment design in multiple chemical sensitivity.
People suffering from multiple chemical sensitivity (MCS) complain of a variety of symptoms that could impair cognitive and psychomotor function either directly or indirectly. This paper discusses the use of cognitive and psychomotor performance tests together with some experiment designs that could be considered for use to assess fitness of MCS sufferers for work or the efficacy of diagnostic, preventative, or therapeutic measures. The tests could also contribute to the body of objective information on MCS and help sway the opinion of those who are dubious of its authenticity. The credentials of cognitive and psychomotor performance tests are derived from their successful use in studying the effects of drugs, and the types of tests are illustrated by describing those used by the United Kingdom Defence Evaluation and Research Agency Chemical and Biological Defence Human Studies Group, which has been involved in the assessment of drugs and chemicals on work performance for many years. The tests include mathematical, verbal and spatial processing, tracking, reaction time, attention and vigilance, and memory tests. The discussion of experiment designs includes both repeated measures and parallel groups designs together with their advantages and disadvantages and some suggested modifications to accommodate the particular problems posed by MCS.


Hard and soft tissue changes around implants installed in regular-sized and reduced alveolar bony ridges. An experimental study in dogs.
To study bony and soft tissue changes at implants installed in alveolar bony ridges of different widths. In 6 Labrador dogs, the mandibular premolars and first molars were extracted, and a buccal defect was created in the left side at the third and fourth premolars by removing the buccal bone and the inter-radicular and interdental septa. Three months after tooth extraction, full-thickness mucoperiosteal flaps were elevated, and implants were installed, two at the reduced (test) and two at the regular-sized ridges (control). Narrow or wide abutments were affixed to the implants. After 3 months, biopsies were harvested, and ground sections prepared for histological evaluation. A higher vertical buccal bony crest resorption was found at the test (1.5 ± 0.7 mm and 1.0 ± 0.7 mm) compared to the control implants (1.0 ± 0.5 mm and 0.7 ± 0.4 mm), for both wide and narrow abutment sites. A higher horizontal alveolar resorption was identified at the control compared to the test implants. The difference was significant for narrow abutment sites. The peri-implant mucosa was more coronally positioned at the narrow abutment, in the test sites, while for the control sites, the mucosal adaptation was more coronal at the wide abutment sites. These differences, however, did not reach statistical significance. Implants installed in regular-sized alveolar ridges had a higher horizontal, but a lower vertical buccal bony crest resorption compared to implants installed in reduced alveolar ridges. Narrow abutments in reduced ridges as well as wide abutments in regular-sized ridges yielded less soft tissue recession compared to their counterparts.


BANNER: an executable survey of advances in biomedical named entity recognition.
There has been an increasing amount of research on biomedical named entity recognition, the most basic text extraction problem, resulting in significant progress by different research teams around the world. This has created a need for a freely-available, open source system implementing the advances described in the literature. In this paper we present BANNER, an open-source, executable survey of advances in biomedical named entity recognition, intended to serve as a benchmark for the field. BANNER is implemented in Java as a machine-learning system based on conditional random fields and includes a wide survey of the best techniques recently described in the literature. It is designed to maximize domain independence by not employing brittle semantic features or rule-based processing steps, and achieves significantly better performance than existing baseline systems. It is therefore useful to developers as an extensible NER implementation, to researchers as a standard for comparing innovative techniques, and to biologists requiring the ability to find novel entities in large amounts of text.


Local remodeling of synthetic extracellular matrix microenvironments by co-cultured endometrial epithelial and stromal cells enables long-term dynamic physiological function.
Mucosal barrier tissues, comprising a layer of tightly-bonded epithelial cells in intimate molecular communication with an underlying matrix-rich stroma containing fibroblasts and immune cells, are prominent targets for drugs against infection, chronic inflammation, and other disease processes. Although human in vitro models of such barriers are needed for mechanistic studies and drug development, differences in extracellular matrix (ECM) needs of epithelial and stromal cells hinder efforts to create such models. Here, using the endometrium as an example mucosal barrier, we describe a synthetic, modular ECM hydrogel suitable for 3D functional co-culture, featuring components that can be remodeled by cells and that respond dynamically to sequester local cell-secreted ECM characteristic of each cell type. The synthetic hydrogel combines peptides with off-the-shelf reagents and is thus accessible to cell biology labs. Specifically, we first identified a single peptide as suitable for initial attachment of both endometrial epithelial and stromal cells using a 2D semi-empirical screen. Then, using a co-culture system of epithelial cells cultured on top of gel-encapsulated stromal cells, we show that inclusion of ECM-binding peptides in the hydrogel, along with the integrin-binding peptide, leads to enhanced accumulation of basement membrane beneath the epithelial layer and more fibrillar collagen matrix assembly by stromal cells over two weeks in culture. Importantly, endometrial co-cultures composed of either cell lines or primary cells displayed hormone-mediated differentiation as assessed by morphological changes and secretory protein production. A multiplex analysis of apical cytokine and growth factor secretion comparing cell lines and primary cells revealed strikingly different patterns, underscoring the importance of using primary cell models in analysis of cell-cell communication networks. In summary, we define a "one-size-fits-all" synthetic ECM that enables long-term, physiologically responsive co-cultures of epithelial and stromal cells in a mucosal barrier format.


Bacteriophage F336 recognizes the capsular phosphoramidate modification of Campylobacter jejuni NCTC11168.
Bacteriophages infecting the food-borne human pathogen Campylobacter jejuni could potentially be exploited to reduce bacterial counts in poultry prior to slaughter. This bacterium colonizes the intestinal tract of poultry in high numbers, and contaminated poultry meat is regarded as the major source of human campylobacteriosis. In this study, we used phage F336 belonging to the Myoviridae family to select a C. jejuni NCTC11168 phage-resistant strain, called 11168R, with the aim of investigating the mechanisms of phage resistance. We found that phage F336 has reduced adsorption to 11168R, thus indicating that the receptor is altered. While proteinase K-treated C. jejuni cells did not affect adsorption, periodate treatment resulted in reduced adsorption, suggesting that the phage binds to a carbohydrate moiety. Using high-resolution magic angle spinning nuclear magnetic resonance (NMR) spectroscopy, we found that 11168R lacks an O-methyl phosphoramidate (MeOPN) moiety attached to the GalfNAc on the capsular polysaccharide (CPS), which was further confirmed by mass spectroscopy. Sequence analysis of 11168R showed that the potentially hypervariable gene cj1421, which encodes the GalfNAc MeOPN transferase, contains a tract of 10 Gs, resulting in a nonfunctional gene product. However, when 11168R reverted back to phage sensitive, cj1421 contained 9 Gs, and the GalfNAc MeOPN was regained in this strain. In summary, we have identified the phase-variable MeOPN moiety, a common component of the diverse capsular polysaccharides of C. jejuni, as a novel receptor of phages infecting this bacterium.


The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy.
Skeletal muscle atrophy and the inhibition of muscle regeneration are known to occur as a natural consequence of aging, yet the underlying mechanisms that lead to these processes in atrophic myofibers remain largely unclear. Our research has revealed that the maintenance of proper mitochondrial-associated endoplasmic reticulum membranes (MAM) is vital for preventing skeletal muscle atrophy in microgravity environments. We discovered that the deletion of the mitochondrial fusion protein Mitofusin2 (MFN2), which serves as a tether for MAM, in human induced pluripotent stem (iPS) cells or the reduction of MAM in differentiated myotubes caused by microgravity interfered with myogenic differentiation process and an increased susceptibility to muscle atrophy, as well as the activation of the Notch signaling pathway. The atrophic phenotype of differentiated myotubes in microgravity and the regenerative capacity of Mfn2-deficient muscle stem cells in dystrophic mice were both ameliorated by treatment with the gamma-secretase inhibitor DAPT. Our findings demonstrate how the orchestration of mitochondrial morphology in differentiated myotubes and regenerating muscle stem cells plays a crucial role in regulating Notch signaling through the interaction of MAM.


Epidemiological processes involved in the emergence of vector-borne diseases: West Nile fever, Rift Valley fever, Japanese encephalitis and Crimean-Congo haemorrhagic fever.
Over the past few decades, the geographical distribution of arthropod-borne zoonoses has dramatically expanded. The influence of human-induced or ecological changes on the risk of disease outbreaks is undeniable. However, few hypotheses have been proposed which address the re-emergence of these diseases, the spread of these viruses to previously uninfected areas and their establishment therein. Host and vector movements play an important role in the dissemination of pathogens, and the ability of these diseases to colonise previously uninfected areas may be explained by the diversity of hosts and vectors, the presence of favourable ecological conditions, and the successful adaptations of vectors or pathogens to new ecosystems. The objective of this paper is to describe the epidemiological processes of the vector-borne diseases Rift Valley fever, West Nile fever, Japanese encephalitis and Crimean-Congo haemorrhagic fever.


Combined analgesia and local anesthesia to minimize pain during circumcision.
Pain of circumcision is only partially relieved by single modalities, such as penile nerve block, lidocaine-prilocaine cream, and sucrose pacifiers. To assess the effectiveness of a combination of interventions on the pain response of infants undergoing circumcision. Cohort study. Group 1 included infants circumcised using the Mogen clamp and combined analgesics (lidocaine dorsal penile nerve block, lidocaine-prilocaine, acetaminophen, and sugar-coated gauze dipped in grape juice). Group 2 included infants circumcised using the Gomco clamp and lidocaine-prilocaine. Infants were videotaped during circumcision, and pain was assessed using facial activity scores and percentage of time spent crying. There were 57 infants in group 1 and 29 infants in group 2. Birth characteristics did not differ between groups. Infants in group 1 were older than infants in group 2 (17 days vs 2 days) (P < .001). The mean duration of the procedure was 55 seconds and 577 seconds for infants in group 1 and 2, respectively (P < .001). Facial action scores and percentage of time spent crying were significantly lower during circumcision for infants in group 1 (P < .001). The percentage of time spent crying was 18% and 40% for infants in groups 1 and 2, respectively. No adverse effects were observed in infants in group 1; 1 infant in group 2 had a local skin infection. Infants circumcised with the Mogen clamp and combined analgesia have substantially less pain than those circumcised with the Gomco clamp and lidocaine-prilocaine cream. Because of the immense pain during circumcision, combined local anesthesia and analgesia using the Mogen clamp should be considered.


Regional and inter-regional economic rules and the enforcement of the right to health: The case of Colombia.
The regional policy level is often seen as a (potential) source of progressive policy-making in health (and in social policy more widely), complementing or substituting national policy levels, which are perceived as underperforming. While it can certainly be argued that there are important opportunities to adopt regional approaches to tackle border-crossing health issues, this article draws the attention to the fact that the linkage between (inter-)regional and national policy levels is not uni-directional. While in some instances the regional level may indeed take the lead in the promotion of (the right to) health, in other instances it may well be the other way round. This article focuses on the case of Colombia, where international economic rules have deeply permeated public policies in the health sector. On one hand, Colombia has been opening markets through the conclusion of regional integration arrangements (e.g. Andean Community and the Pacific Alliance) and the new generation of Free Trade Agreements. On the other hand, Colombia has been one of the most active emerging countries in promoting the right to health as a justiciable fundamental right, in line with the International Covenant on Economic, Social, and Cultural Rights of the United Nations mainly due to the judicial activism of the Colombian Constitutional Court with interesting implications for regional social governance. The article shows that national courts can play an important role in the protection of the right to health in a context of economic integration and the absence of regional balancing policies.


Early changes in the time course of myocardial contraction after correcting aortic regurgitation.
Correcting aortic regurgitation causes significant changes in left ventricular loading conditions, but few observations have been made intraoperatively of early effects on myocardial function. We studied 18 patients (mean age, 59+/-12 years; 14 men) in whom aortic regurgitation was corrected with a stentless biologic valve. Overall left ventricular function was studied by thermodilution cardiac output, ventricular filling pressure, and systemic arterial pressure. Regional myocardial function was assessed from intraoperative transesophageal M-mode echocardiography and high fidelity ventricular pressure recordings before cardiopulmonary bypass, and 0.5, 1, 3, 6, 12, and 20 hours after operation. Time course of contraction, and magnitude of left ventricular systolic wall stress, dimensional shortening, myocardial power, and stroke work were measured. Global hemodynamics: there was an immediate decrease in left ventricular stroke volume (58+/-31 mL versus 80+/-30 mL, p = 0.004) and stroke work index (250+/-86 mJ/m versus 401+/-198 mJ/m, p = 0.005), but systemic arterial pressure (79+/-11 mm Hg versus 65+/-10 mm Hg, p = 0.002), increased at constant heart rate and end-diastolic pressure. Regional myocardial function and timing: peak systolic wall stress, dimensional shortening rate, and myocardial power production were all unchanged with operation. However, myocardial stroke work decreased (3.0+/-1.3 mJ/cm versus 4.8+/-2.4 mJ/cm, p = 0.009), attributable to shortening of the duration of systole (475+/-91 ms versus 543+/-67 ms, p<0.001). Diastolic time increased from 34%+/-18% to 71%+/-33% of systolic pulse duration (p<0.001). Correcting aortic regurgitation causes an early decrease in regional and global stroke work and increases diastolic time, although systolic wall stress does not decrease immediately. These beneficial effects are achieved by reducing the duration rather than altering the peak intensity (power) of myocardial contraction.


Health impact metrics for air pollution management strategies.
Health impact assessments (HIAs) inform policy and decision making by providing information regarding future health concerns, and quantitative HIAs now are being used for local and urban-scale projects. HIA results can be expressed using a variety of metrics that differ in meaningful ways, and guidance is lacking with respect to best practices for the development and use of HIA metrics. This study reviews HIA metrics pertaining to air quality management and presents evaluative criteria for their selection and use. These are illustrated in a case study where PM2.5 concentrations are lowered from 10 to 8μg/m(3) in an urban area of 1.8 million people. Health impact functions are used to estimate the number of premature deaths, unscheduled hospitalizations and other morbidity outcomes. The most common metric in recent quantitative HIAs has been the number of cases of adverse outcomes avoided. Other metrics include time-based measures, e.g., disability-adjusted life years (DALYs), monetized impacts, functional-unit based measures, e.g., benefits per ton of emissions reduced, and other economic indicators, e.g., cost-benefit ratios. These metrics are evaluated by considering their comprehensiveness, the spatial and temporal resolution of the analysis, how equity considerations are facilitated, and the analysis and presentation of uncertainty. In the case study, the greatest number of avoided cases occurs for low severity morbidity outcomes, e.g., asthma exacerbations (n=28,000) and minor-restricted activity days (n=37,000); while DALYs and monetized impacts are driven by the severity, duration and value assigned to a relatively low number of premature deaths (n=190 to 230 per year). The selection of appropriate metrics depends on the problem context and boundaries, the severity of impacts, and community values regarding health. The number of avoided cases provides an estimate of the number of people affected, and monetized impacts facilitate additional economic analyses useful to policy analysis. DALYs are commonly used as an aggregate measure of health impacts and can be used to compare impacts across studies. Benefits per ton metrics may be appropriate when changes in emissions rates can be estimated. To address community concerns and HIA objectives, a combination of metrics is suggested.


Identification of Chemical Probes Targeting MBD2.
Epigenetics has received much attention in the past decade. Many insights on epigenetic (dys)regulation in diseases have been obtained, and clinical therapies targeting them are in place. However, the readers of the epigenetic marks are lacking enlightenment behind this revolution, and it is poorly understood how DNA methylation is being read and translated to chromatin function and cellular responses. Chemical probes targeting the methyl-CpG readers, such as the methyl-CpG binding domain proteins (MBDs), could be used to study this mechanism. We have designed analogues of 5-methylcytosine to probe the MBD domain of human MBD2. By setting up a protein thermal shift assay and an AlphaScreen-based test, we were able to identify three fragments that bind MBD2 alone and disrupt the MBD2-methylated DNA interactions. Two-dimensional NMR experiments and virtual docking gave valuable insights into the interaction of the ligands with the protein showing that the compounds interact with residues that are important for DNA recognition. These constitute the starting point for the design of potent chemical probes for MBD proteins.


Iatrogenic and sporadic Creutzfeldt-Jakob disease in 2 sisters without mutation in the prion protein gene.
Human genetic prion diseases have invariably been linked to alterations of the prion protein (PrP) gene PRNP. Two sisters died from probable Creutzfeldt-Jakob disease (CJD) in Switzerland within 14 y. At autopsy, both patients had typical spongiform change in their brains accompanied by punctuate deposits of PrP. Biochemical analyses demonstrated proteinase K-resistant PrP. Sequencing of PRNP showed 2 wild-type alleles in both siblings. Retrospectively, clinical data revealed a history of dural transplantation in the initially deceased sister, compatible with a diagnosis of iatrogenic CJD. Clinical and familial histories provided no evidence for potential horizontal transmission. This observation of 2 siblings suffering from CJD without mutations in the PRNP gene suggests potential involvement of non-PRNP genes in prion disease etiology.


Live-cell microscopy - tips and tools.
Imaging of living cells and tissue is now common in many fields of the life and physical sciences, and is instrumental in revealing a great deal about cellular dynamics and function. It is crucial when performing such experiments that cell viability is at the forefront of any measurement to ensure that the physiological and biological processes that are under investigation are not altered in any way. Many cells and tissues are not normally exposed to light during their life cycle, so it is important for microscopy applications to minimize light exposure, which can cause phototoxicity. To ensure minimal light exposure, it is crucial that microscope systems are optimized to collect as much light as possible. This can be achieved using superior-quality optical components and state-of-the-art detectors. This Commentary discusses how to set up a suitable environment on the microscope stage to maintain living cells. There is also a focus on general and imaging-platform-specific ways to optimize the efficiency of light throughput and detection. With an efficient optical microscope and a good detector, the light exposure can be minimized during live-cell imaging, thus minimizing phototoxicity and maintaining cell viability. Brief suggestions for useful microscope accessories as well as available fluorescence tools are also presented. Finally, a flow chart is provided to assist readers in choosing the appropriate imaging platform for their experimental systems.