'Most rare workmen': optical practitioners in early seventeenth-century Delft.
A special interest in optics among various seventeenth-century painters living in the Dutch city of Delft has intrigued historians, including art historians, for a long time. Equally, the impressive career of the Delft microscopist Antoni van Leeuwenhoek has been studied by many historians of science. However, it has never been investigated who, at that time, had access to the mathematical and optical knowledge necessary for the impressive achievements of these Delft practitioners. We have tried to gain insight into Delft as a 'node' of optical knowledge by following the careers of three minor local figures in early seventeenth-century Delft. We argue that through their work, products, discussions in the vernacular and exchange of skills, rather than via learned publications, these practitioners constituted a foundation on which the later scientific and artistic achievements of other Delft citizens were built. Our Delft case demonstrates that these practitioners were not simple and isolated craftsmen; rather they were crucial components in a network of scholars, savants, painters and rich virtuosi. Decades before Vermeer made his masterworks, or Van Leeuwenhoek started his famous microscopic investigations, the intellectual atmosphere and artisanal knowledge in this city centred on optical topics.


Early lymph vessel development from embryonic stem cells.
The purpose of this study was to establish a model system for lymph vessel development based on directed differentiation of murine embryonic stem cells. Stem cells were aggregated to form embryoid bodies, and subsequently cultured in 3-dimensional collagen matrix for up to 18 days. Treatment with vascular endothelial growth factor (VEGF)-C and VEGF-A individually enhanced formation of lymphatic vessel structures, although combined treatment with VEGF-C and VEGF-A was most potent and gave rise to a network of LYVE-1, podoplanin, Prox1, and VEGF receptor-3 positive lymphatic vessel structures running parallel to and apparently emanating from, capillaries. In contrast, fibroblast growth factor-2, hepatocyte growth factor, or hypoxia had little or no effect on the development of the early lymphatics. Further, cells of hematopoietic origin were shown to express lymphatic markers. In summary, different subpopulations of lymphatic endothelial cells were identified on the basis of differential expression of several lymphatic and blood vessel markers, indicating vascular heterogeneity. We conclude that the present model closely mimics the early steps of lymph vessel development in mouse embryos.


Early intervention of patients at risk for acute respiratory failure and prolonged mechanical ventilation with a checklist aimed at the prevention of organ failure: protocol for a pragmatic stepped-wedged cluster trial of PROOFCheck.
Acute respiratory failure (ARF) often presents and progresses outside of the intensive care unit. However, recognition and treatment of acute critical illness is often delayed with inconsistent adherence to evidence-based care known to decrease the duration of mechanical ventilation (MV) and complications of critical illness. The goal of this trial is to determine whether the implementation of an electronic medical record-based early alert for progressive respiratory failure coupled with a checklist to promote early compliance to best practice in respiratory failure can improve the outcomes of patients at risk for prolonged respiratory failure and death. A pragmatic stepped-wedged cluster clinical trial involving 6 hospitals is planned. The study will include adult hospitalised patients identified as high risk for MV >48 hours or death because they were mechanically ventilated outside of the operating room or they were identified as high risk for ARF on the Accurate Prediction of PROlonged VEntilation (APPROVE) score. Patients with advanced directives limiting intubation will be excluded. The intervention will consist of (1) automated identification and notification of clinician of high-risk patients by APPROVE or by invasive MV and (2) checklist of evidence-based practices in ARF (Prevention of Organ Failure Checklist-PROOFCheck). APPROVE and PROOFCheck will be developed in the pretrial period. Primary outcome is hospital mortality. Secondary outcomes include length of stay, ventilator and organ failure-free days and 6-month and 12-month mortality. Predefined subgroup analysis of patients with limitation of aggressive care after study entry is planned. Generalised estimating equations will be used to compare patients in the intervention phase with the control phase, adjusting for clustering within hospitals and time. The study was approved by the institutional review boards. Results will be published in peer-reviewed journals and presented at international meetings. NCT02488174.


Validity of self-measured waist and hip circumferences: results from a community study in Malaysia.
Measures of central adiposity are better predictors of adverse health events than BMI. Nonetheless, BMI is more widely used in health research. One reason for this may be the limited research supporting the self-measurement of waist and hip circumference. The lack of validity studies is particularly acute in Asia. The main objective was to establish the validity of self-measurement of waist and hip circumference in a community setting and the correlation of those measures with BMI, blood pressure, and blood glucose levels. A community based, cross-sectional survey. A "healthy living expo" at a shopping mall in a rural town on peninsular Malaysia One hundred and thirty six (136) individuals volunteered to participate in the study, 125 of whom met the inclusion criteria. The ethnic distribution of the participants was 80% Chinese, 17% Malay and 3% Indian. Most participants were female (60%), with participants' ages ranging from 18 to 78 years (mean, 47.2). Self and assisted measurements of waist and hip were taken. Blood pressure, non-fasting blood glucose, height, and weight were also measured. Bland Altman plots and Lin's concordance coefficient were used to measure agreement between self and assisted measures. Pearson's correlation was used to examine the association of self and assisted measures with blood pressure, blood glucose, and BMI. There was a downwards bias in self measured waist (-0.81 cm) and hip (-1 cm) circumferences compared with assisted measures. The concordance for the self and assisted measures of waist, hip and the ratio of the two were, respectively, .96, .93 , and .84. The correlation between measures of central adiposity and BMI, blood pressure and blood glucose were similar for self and assisted measures. The results provide additional support for the use of self-measurement of waist and hip circumference studies of central adiposity, but is limited by the specificity of the setting.


High expression of human uroplakin Ia in urinary bladder transitional cell carcinoma.
Uroplakins (UPs) Ia, Ib, II, and III are tissue-specific and differentiation-dependent transmembrane proteins of the urothelium. We assessed the usefulness of human UP Ia as a histological marker by examining its expression in urinary bladder transitional cell carcinoma (TCC). A polyclonal antibody against human UP Ia was raised using a synthesized polypeptide. We applied our antibody to various organ tissues, including urothelium, and observed no crossreactivity. Analysis by RT-PCR of normal urothelium, TCC and other organ tissues indicated that the human UP Ia gene expression is highly specialized to urothelium, and is conserved in TCC. Using immunohistochemistry, we investigated the expression of UP Ia in TCC from patients who had undergone radical cystectomy and from autopsy cases. Positive staining (10% or more positive cancer cells) was noted in primary lesions from 61 of 63 (96.8%) cystectomy patients. Depending on pathological grade, high expression (50% or more positive cancer cells) was observed in 17 of 18 (94.4%) moderately- to well-differentiated TCC and in 36 of 45 (80.0%) poorly differentiated TCC. With regard to tumor invasion, high expression was noted in 20 of 22 (90.9%) superficial and 33 of 41 (80.5%) muscle-invasive TCC. Cause-specific survival rates were 68.6% and 75.0% in high- and low-expression patients, respectively (log-rank test, P = 0.855, mean follow-up; 65.0 months). In metastases, positive reactions were observed in 13 of 18 (72.2%) lesions. UP Ia may represent a specific histological marker judging from the stable expression, although its value as a prognostic factor remains undetermined.


Longitudinal Clival Fracture in a Child: Case Report and Review of the Literature.
Clival fracture is a rare and serious injury, which mostly results in the death of the victim before reaching the hospital. The entity is hardly diagnosed in the early phase because of high mortality rates and deficiencies in completion of radiological imaging. The incidence of diagnoses increased year by year after the usage of computed tomography gained widespread availability. In this article, we aimed to present radiological and clinical characteristics of a 5-year-old patient brought to the emergency department after a motor vehicle accident. The most current recommendations with regard to the management of clival fracture of a pediatric patient are also discussed.


A biomaterial-assisted mesenchymal stromal cell therapy alleviates colonic radiation-induced damage.
Healthy tissues surrounding abdomino-pelvic tumours can be impaired by radiotherapy, leading to chronic gastrointestinal complications with substantial mortality. Adipose-derived Mesenchymal Stromal Cells (Ad-MSCs) represent a promising strategy to reduce intestinal lesions. However, systemic administration of Ad-MSCs results in low cell engraftment within the injured tissue. Biomaterials, able to encapsulate and withstand Ad-MSCs, can overcome these limitations. A silanized hydroxypropylmethyl cellulose (Si-HPMC) hydrogel has been designed and characterized for injectable cell delivery using the operative catheter of a colonoscope. We demonstrated that hydrogel loaded-Ad-MSCs were viable, able to secrete trophic factors and responsive to the inflammatory environment. In a rat model of radiation-induced severe colonic damage, Ad-MSC + Si-HPMC improve colonic epithelial structure and hyperpermeability compared with Ad-MSCs injected intravenously or locally. This therapeutic benefit is associated with greater engraftment of Si-HPMC-embedded Ad-MSCs in the irradiated colonic mucosa. Moreover, macrophage infiltration near the injection site was less pronounced when Ad-MSCs were embedded in the hydrogel. Si-HPMC induces modulation of chemoattractant secretion by Ad-MSCs that could contribute to the decrease in macrophage infiltrate. Si-HPMC is suitable for cell delivery by colonoscopy and induces protection of Ad-MSCs in the tissue potentiating their therapeutic effect and could be proposed to patients suffering from colon diseases.


Theoretical models of catalytic domains of protein phosphatases 1 and 2A with Zn2+ and Mn2+ metal dications and putative bioligands in their catalytic centers.
The oligomeric metalloenzymes protein phosphatases dephosphorylate OH groups of Ser/Thr or Tyr residues of proteins whose actions depend on the phosphorus signal. The catalytic units of Ser/Thr protein phosphatases 1, 2A and 2B (PP1c, PP2Ac and PP2Bc, respectively), which exhibit about 45% sequence similarity, have their active centers practically identical. This feature strongly suggests that the unknown structure of PP2Ac could be successfully homology-modeled from the known structures of PP1c and/or PP2Bc. Initially, a theoretical model of PP1c was built, including a phosphate and a metal dication in its catalytic site. The latter was modeled, together with a structural hydroxyl anion, as a triangular pseudo-molecule (Zno or Mno), composed of two metal cations (double Zn2+ or Mn2+, respectively) and the OH- group. To the free PP1c two inhibitor sequences R29RRRPpTPAMLFR40 of DARPP-32 and R30RRRPpTPATLVLT42 of Inhibitor-1, and two putative substrate sequences LRRApSVA and QRRQRKpRRTI were subsequently docked. In the next step, a free PP2Ac model was built via homology re-modeling of the PP1c template and the same four sequences were docked to it. Thus, together, 20 starting model complexes were built, allowing for combination of the Zno and Mno pseudo-molecules, free enzymes and the peptide ligands docked in the catalytic sites of PP1c and PP2Ac. All models were subsequently subjected to 250-300 ps molecular dynamics using the AMBER 5.0 program. The equilibrated trajectories of the final 50 ps were taken for further analyses. The theoretical models of PP1c complexes, irrespective of the dication type, exhibited increased mobilities in the following residue ranges: 195-200, 273-278, 287-209 for the inhibitor sequences and 21-25, 194-200, 222-227, 261, 299-302 for the substrate sequences. Paradoxically, the analogous PP2Ac models appeared much more stable in similar simulations, since only their "prosegment" residues 6-10 and 14-18 exhibited an increased mobility in the inhibitor complexes while no areas of increased mobility were found in the substrate complexes. Another general observation was that the complexes with Mn dications were more stable than those with Zn dications for both PP1c and PP2Ac units.


Gaps in Public Awareness About BRCA and Genetic Testing in Prostate Cancer: Social Media Landscape Analysis.
Genetic testing, particularly for BRCA1/2, is increasingly important in prostate cancer (PCa) care, with impact on PCa management and hereditary cancer risk. However, the extent of public awareness and online discourse on social media is unknown, and presents opportunities to identify gaps and enhance population awareness and uptake of advances in PCa precision medicine. The objective of this study was to characterize activity and engagement across multiple social media platforms (Twitter, Facebook, and YouTube) regarding BRCA and genetic testing for PCa compared with breast cancer, which has a long history of public awareness, advocacy, and prominent social media presence. The Symplur Signals online analytics platform was used to obtain metrics for tweets about (1) #BRCA and #breastcancer, (2) #BRCA and #prostatecancer, (3) #genetictesting and #breastcancer, and (4) #genetictesting and #prostatecancer from 2016 to 2020. We examined the total number of tweets, users, and reach for each hashtag, and performed content analysis for a subset of tweets. Facebook and YouTube were queried using analogous search terms, and engagement metrics were calculated. During a 5-year period, there were 10,005 tweets for #BRCA and #breastcancer, versus 1008 tweets about #BRCA and #prostatecancer. There were also more tweets about #genetictesting and #breastcancer (n=1748), compared with #genetic testing and #prostatecancer (n=328). Tweets about genetic testing (12,921,954) and BRCA (75,724,795) in breast cancer also had substantially greater reach than those about PCa (1,463,777 and 4,849,905, respectively). Facebook groups and pages regarding PCa and BRCA/genetic testing had fewer average members, new members, and new posts, as well as fewer likes and followers, compared with breast cancer. Facebook videos had more engagement than YouTube videos across both PCa and breast cancer content. There is substantially less social media engagement about BRCA and genetic testing in PCa compared with breast cancer. This landscape analysis provides insights into strategies for leveraging social media platforms to increase public awareness about PCa germline testing, including use of Facebook to share video content and Twitter for discussions with health professionals.


Prospective and retrospective ECG-gating for CT coronary angiography perform similarly accurate at low heart rates.
To compare, in patients with suspicion of coronary artery disease (CAD) and low heart rates, image quality, diagnostic performance, and radiation dose values of prospectively and retrospectively electrocardiography (ECG)-gated dual-source computed tomography coronary angiography (CTCA) for the diagnosis of significant coronary stenoses. Two-hundred consecutive patients with heart rates ≤70 bpm were retrospectively enrolled; 100 patients undergoing prospectively ECG-gated CTCA (group 1) and 100 patients undergoing retrospectively-gated CTCA (group 2). Coronary artery segments were assessed for image quality and significant luminal diameter narrowing. Sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and accuracy of both CTCA groups were determined using conventional catheter angiography (CCA) as reference standard. Radiation dose values were calculated. Both groups were comparable regarding gender, body weight, cardiovascular risk profile, severity of CAD, mean heart rate, heart rate variability, and Agatston score (all p>0.05). There was no significant difference in the rate of non-assessable coronary segments between group 1 (1.6%, 24/1404) and group 2 (1.4%, 19/1385; p=0.77); non-diagnostic image quality was significantly (p<0.001) more often attributed to stair step artifacts in group 1. Segment-based sensitivity, specificity, PPV, NPV, and accuracy were 98%, 98%, 88%, 100%, and 100% among group 1; 96%, 99%, 90%, 100%, and 98% among group 2, respectively. Parameters of diagnostic performance were similar (all p>0.05). Mean effective radiation dose of prospectively ECG-gated CTCA (2.2±0.4 mSv) was significantly (p<0.0001) smaller than that of retrospectively ECG-gated CTCA (8.1±0.6 mSv). Prospectively ECG-gated CTCA yields similar image quality, performs as accurately as retrospectively ECG-gated CTCA in patients having heart rates ≤70 bpm while being associated with a lower mean effective radiation dose.


Prevalence and psychosocial risk factors associated with internet addiction in a nationally representative sample of college students in Taiwan.
The aim of this study was to examine the prevalence of Internet addiction in a nationally representative sample of college students and to identify any associated psychosocial risk factors. The present study was constructed using a cross-sectional design with 3,616 participants. Participants were surveyed during the middle of the spring and fall semesters and recruited from colleges around Taiwan using stratified and cluster random sampling methods. Associations between Internet addiction and psychosocial risk factors were examined using stepwise logistic regression analysis. The prevalence of Internet addiction was found to be 15.3 percent (95 percent confidence interval, 14.1 percent to 16.5 percent). More depressive symptoms, higher positive outcome expectancy of Internet use, higher Internet usage time, lower refusal self-efficacy of Internet use, higher impulsivity, lower satisfaction with academic performance, being male, and insecure attachment style were positively correlated with Internet addiction. The prevalence of Internet addiction among college students in Taiwan was high, and the variables mentioned were independently predictive in the logistic regression analysis. This study can be used as a reference for policy making regarding the design of Internet addiction prevention programs and can also aid in the development of strategies designed to help Internet-addicted college students.


Epstein-Barr virus-induced gene 3-deficiency leads to impaired antitumor T-cell responses and accelerated tumor growth.
Epstein-Barr virus-induced gene 3 (EBI3) encoded protein can form heterodimers with IL-27P28, and IL-12P35 to form IL-27, and IL-35. However, IL-27 stimulates, whereas IL-35 inhibits antitumor T-cell responses. IL-27 also limits the Foxp3+ regulatory T cell (Treg) population, whereas IL-35 has been shown to expand Tregs and foster Treg suppressive functions. It remains unclear which group of forces are dominant during antitumor T-cell responses. In this study, we evaluated the tumor growth and antitumor T-cell responses in EBI3-deficient mice that lack both IL-27 and IL-35. We found that injecting B16 melanoma cells into EBI3-deficient C57BL/6 mice, or J558 plasmacytoma cells into EBI3-deficient BALB/c mice resulted in significantly increased tumor growth relative to those implanted in wild-type control mice. Tumors from EBI3-deficient mice contained significantly decreased proportions of CD8+ T cells and increased proportions of CD4+FoxP3+ Treg cells as compared to those from EBI3-intact mice. Tumor-infiltrating T cells from EBI3-deficient mice were impaired in their capacity to produce IFNγ. Phenotypically, Tregs from EBI3-deficient mice were highly suppressive and produced IL-10 in the tumor microenvironment. Depletion of Tregs or inactivation of the IL-10 pathway significantly abrogated tumor growth enhancement in Ebi3-/- mice. Finally, we showed that Ebi3-/- mice administered a melanoma vaccine failed to mount a CD8+ T-cell response and the vaccine failed to confer tumor rejection in EBI3-deficient mice. Taken together, these results suggest that Ebi3-/- mice show a phenotype of IL-27-deficiency rather than IL-35-deficiency during anti-tumor T-cell responses. Thus, our results suggest that endogenous IL-27 is critical for both spontaneous and vaccine-induced antitumor T-cell responses.


Bactericidal activity of ethanolic extracts of propolis against Staphylococcus aureus isolated from mastitic cows.
Staphylococcus aureus is an important pathogen for both humans and animals, and it has been an ubiquitous etiological agent of bovine mastitis in dairy farms worldwide. Elimination of S. aureus with classic antibiotics is difficult, and the current study aimed to evaluate the efficacy of ethanolic extracts of propolis (EEP) against S. aureus cultivated in complex media or milk. EEP (0-0.5 mg ml(-1)) decreased growth of S. aureus in BHI media and 1 mg ml(-1) was bactericidal against washed cell suspensions (10(7) CFU ml(-1)). Propolis extracts also killed S. aureus cells resuspended in milk, but the bactericidal dose was at least 20-fold greater. Cultures that were transferred for at least 60 generations with sub-lethal doses of propolis did not change much their sensibility to EEP. Atomic force microscopy images revealed changes in morphology and cell size of S. aureus cells exposed to EEP (0.5 mg ml(-1)). Our results indicate that propolis extracts might be effective against mastitis-causing S. aureus strains in vivo, but milk constituents affect the inhibitory activity of propolis. Considering that propolis-resistance appears to be a phenotype not easily selected, the use of EEP combined or not with other antimicrobial agents might be useful for mastitis control in vivo.


Decoding the spectra of low-finesse extrinsic optical fiber Fabry-Perot interferometers.
A theoretical model is developed to address the fringe visibility and additional phase in the interference spectra of low-finesse extrinsic optical fiber excited Fabry-Pérot interferometers. The model described in the paper applies to both single-mode and multimode fiber excitations; according to the theory, the fringe visibility and additional phase term are primarily determined by the working wavelength and angular power density distribution outputting from the excitation fiber, rather than based on spatial and temporal degree of coherence. Under certain approximations, the output interference intensity and the spatial power density distribution projected onto the fiber axis form a Fourier-transform pair, which potentially provides a tool for spatial density distribution analysis of fiber output. With excellent agreement with experiments, the theory presented in this paper leads to design guidelines for Fabry-Pérot interferometric sensors and insightful physical understanding of such devices.


Advance directives in skilled nursing facility residents transferred to emergency departments.
Ten years have passed since Congress enacted the Patient Self-Determination Act to promote the use of advance directives (ADs). This study was performed to determine the frequency, type, demographic distribution, and utility of ADs that accompany residents of skilled nursing facilities (SNFs) transferred to emergency departments (EDs). This was an observational, cross-sectional cohort of SNF residents, transferred to two urban, academic EDs. Chart review and physician interviews were conducted on consecutive patients arriving during 12-hour data collection shifts. Among 715 patients entered, 315 [44%, 95% confidence interval (95% CI) = 40% to 48%] had an AD. Advance directives were significantly more prevalent among white (50%) than African American (34%) or Hispanic (39%) patients (p < 0.001), and varied from 0% to 94% among SNFs. Of the 315 patients with ADs, do-not-resuscitate (DNR) orders were the most prevalent (65%, 95% CI = 58% to 69%). Although 75% (95% CI = 69% to 81%) of the DNR orders addressed cardiopulmonary resuscitation (CPR), only 12% (95% CI = 8% to 16%) addressed intubation. Among 39 patients who required intubation or CPR, 44% had ADs, 82% (95% CI = 57% to 96%) of which were deemed useful. Despite a decade of legislation promoting their use, ADs are lacking in most SNF residents transferred to EDs for evaluation and in most settings in which a clinical indication exists for intubation or CPR. Variation in their prevalence appears to be associated with both ethnicity and SNF origin. Although about three-fourths of DNR ADs addressed CPR, only about one in ten offered guidance regarding intubation. When available, ADs are used in most instances to guide emergency care.


Determination of the bulk scattering parameters of diffusing materials.
Diffusors are widely used optical components having numerous applications. They are commonly used to homogenize light beams and to create particular intensity distributions. The angular scattering profile of bulk scattering diffusing materials is determined by three bulk scattering parameters that are, however, not commonly available. This hampers an accurate implementation of bulk diffusors in ray tracing simulations. In this paper, the bulk scattering parameters of a concentration series of milk diluted with water were determined with the inverse adding-doubling method. Using these values as input, the macroscopic angular scattering profile was simulated using ray tracing software. The simulation results were compared to experimental data, and a good agreement between measured and simulated data was found. The method was also proven to be successful when applied to commercial diffusors.


Vestibular and oculomotor disturbances caused by industrial solvents.
Hydrocarbon solvents, xylene, styrene, methylchloroform, and trichlorethylene, given intravenously to rabbits produce a positional nystagmus. Due to their additional influence on rotatory nystagmus, one may conclude that their mode of action takes place in the central nervous system. Optokinetic (OKN) responses in rabbits, cats, and humans were also influenced by styrene. OKN responses have not yet been tested for the other solvents. A comparison is made with the effects of alpha-chloralose and the GABA-antagonists bicuculline and picrotoxin which produced similar disturbances. The GABA agonist, baclofen, prevents positional induced styrene nystagmus. It seems likely therefore that the solvents may act by blocking the cerebellar inhibition of vestibulo-oculomotor reflexes. Solvents given simultaneously in some combinations are either additive or synergistic in effect. The implications of these results for occupational medicine are self-evident.


Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions.
Despite the importance of membrane proteins in cellular processes, studies of these hydrophobic proteins present major technical challenges, including expression and purification for structural and biophysical studies. A modified strategy of that proposed previously by Saidijam et al. (2005) and others, for the routine expression of bacterial membrane proteins involved in environmental sensing and antimicrobial resistance (AMR), is proposed which results in purification of sufficient proteins for biophysical experiments. We report expression successes amongst a collection of enterococcal vancomycin resistance membrane proteins: VanTG, VanTG-M transporter domain, VanZ and the previously characterised VanS (A-type) histidine protein kinase (HPK). Using the same strategy, we report on the successful amplification and purification of intact BlpH and ComD2 HPKs of Streptococcus pneumoniae. Near-UV circular dichroism revealed both recombinant proteins bound their pheromone ligands BlpC and CSP2. Interestingly, CSP1 also interacted with ComD. Finally, we evaluate the alternative strategy for studying sensory HPKs involving isolated soluble sensory domain fragments, exemplified by successful production of VicKESD of Enterococcus faecalis VicK. Purified VicKESD possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicKESD denaturation occurs between 15 and 50 °C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicKESD. Our findings provide information on a modified strategy for the successful expression, production and/or storage of bacterial membrane HPKs, AMR proteins and sensory domains for their future crystallisation, and ligand binding studies.


Revisiting Polybia paulista wasp venom using shotgun proteomics - Insights into the N-linked glycosylated venom proteins.
The partial proteome of Polybia paulista wasp venom was previously reported elsewhere using a gel-dependent approach and resulted in the identification of a limited number of venom toxins. Here, we reinvestigated the P. paulista venom using a gel-free shotgun proteomic approach; the highly dynamic range of this approach facilitated the detection and identification of 1673 proteins, of which 23 venom proteins presented N-linked glycosylation as a posttranslational modification. Three different molecular forms of PLA1 were identified as allergenic proteins, and two of these forms were modified by N-linked glycosylation. This study reveals an extensive repertoire of hitherto undescribed proteins that were classified into the following six different functional groups: (i) typical venom proteins; (ii) proteins related to the folding/conformation and PTMs of toxins; (iii) proteins that protect toxins from oxidative stress; (iv) proteins involved in chemical communication; (v) housekeeping proteins; and (vi) uncharacterized proteins. It was possible to identify venom toxin-like proteins that are commonly reported in other animal venoms, including arthropods such as spiders and scorpions. Thus, the findings reported here may contribute to improving our understanding of the composition of P. paulista venom, its envenoming mechanism and the pathologies experienced by the victim after the wasp stinging accident. BIOLOGICAL SIGNIFICANCE: The present study significantly expanded the number of proteins identified in P. paulista venom, contributing to improvements in our understanding of the envenoming mechanism produced by sting accidents caused by this wasp. For example, novel wasp venom neurotoxins have been identified, but no studies have assessed the presence of this type of toxin in social wasp venoms. In addition, 23 N-linked glycosylated venom proteins were identified in the P. paulista venom proteome, and some of these proteins might be relevant allergens that are immunoreactive to human IgE.


Aplastic anemia: what have we learned from animal models and from the clinic.
Aplastic anemia (AA) is currently perceived as an immune-mediated disease in which aberrant effector cells recognize and destroy primitive marrow elements, resulting in pancytopenia. The immune hypothesis is based on clinical observations of responsiveness of AA to immunomodulatory agents such as anti-thymocyte globulin (ATG) and the requirement of cyclosporine to maintain response; evidence of an immune system in disarray provided by abnormal regulatory, TH1, TH17, and expanded CD8(+) T-cell populations, and animal models of immune-mediated marrow destruction, where many of the observed clinical and in vitro alterations can be confirmed and expanded. Murine models mimicking AA have used exposure to agents that result in marrow destruction through a direct toxic effect, but models that explore antigenic disparities between strains have resulted in immune-mediated destruction of the marrow, more closely modeling human AA. Many experiments in mice have helped confirm and elucidate specific mechanisms of marrow destruction. However, clinical development of regimens in AA has not relied on establishing their success in murine model. Instead, drugs and their combinations investigated in AA were those shown clinically to be active in AA, in other hematologic diseases, or in other specialties such as in rheumatology, and solid and bone marrow transplantation. In this review, the evolution of murine models and their clinical relevance in AA are discussed.


A homozygous nonsense mutation (428G-->A) in the human secretor (FUT2) gene provides resistance to symptomatic norovirus (GGII) infections.
Noroviruses (formerly Norwalk-like viruses) are a major cause of acute gastroenteritis worldwide and are associated with a significant number of nosocomial and food-borne outbreaks. In this study we show that the human secretor FUT2 gene, which codes for an alpha(1,2)-fucosyltransferase synthesizing the H-type 1 antigen in saliva and mucosa, is associated with susceptibility to norovirus infections. Allelic polymorphism characterization at nucleotide 428 for symptomatic (n = 53) and asymptomatic (n = 62) individuals associated with nosocomial and sporadic norovirus outbreaks revealed that homozygous nonsense mutation (428G-->A) in FUT2 segregated with complete resistance for the disease. Of all symptomatic individuals, 49% were homozygous (SeSe) and 51% heterozygous (Sese428) secretors, and none were secretor negative (se428se428), in contrast to 20% nonsecretors (se428se428) among Swedish blood donors (n = 104) (P < 0.0002) and 29% for asymptomatic individuals associated with nosocomial outbreaks (P < 0.00001). Furthermore, saliva from secretor-positive and symptomatic patients but not from secretor-negative and asymptomatic individuals bound the norovirus strain responsible for that particular outbreak. This is the first report showing that the FUT2 nonsecretor (se428se428) genotype is associated with resistance to nosocomial and sporadic outbreaks with norovirus.


Unemployment, informal work, precarious employment, child labor, slavery, and health inequalities: pathways and mechanisms.
The study explores the pathways and mechanisms of the relation between employment conditions and health inequalities. A significant amount of published research has proved that workers in several risky types of labor--precarious employment, unemployment, informal labor, child and bonded labor--are exposed to behavioral, psychosocial, and physio-pathological pathways leading to physical and mental health problems. Other pathways, linking employment to health inequalities, are closely connected to hazardous working conditions (material and social deprivation, lack of social protection, and job insecurity), excessive demands, and unattainable work effort, with little power and few rewards (in salaries, fringe benefits, or job stability). Differences across countries in the social contexts and types of jobs result in varying pathways, but the general conceptual model suggests that formal and informal power relations between employees and employers can determine health conditions. In addition, welfare state regimes (unionization and employment protection) can increase or decrease the risk of mortality, morbidity, and occupational injury. In a multilevel context, however, these micro- and macro-level pathways have yet to be fully studied, especially in middle- and low-income countries. The authors recommend some future areas of study on the pathways leading to employment-related health inequalities, using worldwide standard definitions of the different forms of labor, authentic data, and a theoretical framework.


Dietary fish oil is antihypertrophic but does not enhance postischemic myocardial function in female mice.
Clinically and experimentally, a case for omega-3 polyunsaturated fatty acid (PUFA) cardioprotection in females has not been clearly established. The goal of this study was to investigate whether dietary omega-3 PUFA supplementation could provide ischemic protection in female mice with an underlying genetic predisposition to cardiac hypertrophy. Mature female transgenic mice (TG) with cardiac-specific overexpression of angiotensinogen that develop normotensive cardiac hypertrophy and littermate wild-type (WT) mice were fed a fish oil-derived diet (FO) or PUFA-matched control diet (CTR) for 4 wk. Myocardial membrane lipids, ex vivo cardiac performance (intraventricular balloon) after global no-flow ischemia and reperfusion (15/30 min), and reperfusion arrhythmia incidence were assessed. FO diet suppressed cardiac growth by 5% and 10% in WT and TG, respectively (P < 0.001). The extent of mechanical recovery [rate-pressure product (RPP) = beats/min x mmHg] of FO-fed WT and TG hearts was similar (50 +/- 7% vs. 45 +/- 12%, 30 min reperfusion), and this was not significantly different from CTR-fed WT or TG. To evaluate whether systemic estrogen was masking a protective effect of the FO diet, the responses of ovariectomized (OVX) WT and TG mice to FO dietary intervention were assessed. The extent of mechanical recovery of FO-fed OVX WT and TG (RPP, 50 +/- 4% vs. 64 +/- 8%) was not enhanced compared with CTR-fed mice (RPP, 60 +/- 11% vs. 80 +/- 8%, P = 0.335). Dietary FO did not suppress the incidence of reperfusion arrhythmias in WT or TG hearts (ovary-intact mice or OVX). Our findings indicate a lack of cardioprotective effect of dietary FO in females, determined by assessment of mechanical and arrhythmic activity postischemia in a murine ex vivo heart model.


ATP and phosphocreatine utilization in single human muscle fibres during the development of maximal power output at elevated muscle temperatures.
In this study, we examined the effect of muscle temperature (Tm) on adenosine triphosphate (ATP) and phosphocreatine utilization in single muscle fibres during the development of maximal power output in humans. Six male participants performed a 6-s maximal sprint on a friction-braked cycle ergometer under both normal (Tm = 34.3 degrees C, s = 0.6) and elevated (T(m) = 37.3 degrees C, s = 0.2) muscle temperature conditions. During the elevated condition, muscle temperature of the legs was raised, passively, by hot water immersion followed by wrapping in electrically heated blankets. Muscle biopsies were taken from the vastus lateralis before and immediately after exercise. Freeze-dried single fibres were dissected, characterized according to myosin heavy chain composition, and analysed for ATP and phosphocreatine content. Single fibres were classified as: type I, IIA, IIAX25 (1 - 25% IIX isoform), IIAX50 (26 - 50% IIX), IIAX75 (51 - 75% IIX), or IIAX100 (76 - 100% IIX). Maximal power output and pedal rate were both greater (P < 0.05) during the elevated condition by 258 W (s = 110) and 22 rev . min(-1) (s = 6), respectively. In both conditions, phosphocreatine content decreased significantly in all fibre types, with a greater decrease during the elevated condition in type IIA fibres (P < 0.01). Adenosine triphosphate content was also reduced to a greater (P < 0.01) extent in type IIA fibres during the elevated condition. The results of the present study indicate that after passive elevation of muscle temperature, there was a greater decrease in ATP and phosphocreatine content in type IIA fibres than in the normal trial, which contributed to the higher maximal power output.


Divergent unprotected peptide macrocyclisation by palladium-mediated cysteine arylation.
Macrocyclic peptides are important therapeutic candidates due to their improved physicochemical properties in comparison to their linear counterparts. Here we detail a method for a divergent macrocyclisation of unprotected peptides by crosslinking two cysteine residues with bis-palladium organometallic reagents. These synthetic intermediates are prepared in a single step from commercially available aryl bis-halides. Two bioactive linear peptides with cysteine residues at i, i + 4 and i, i + 7 positions, respectively, were cyclised to introduce a diverse array of aryl and bi-aryl linkers. These two series of macrocyclic peptides displayed similar linker-dependent lipophilicity, phospholipid affinity, and unique volume of distributions. Additionally, one of the bioactive peptides showed target binding affinity that was predominantly affected by the length of the linker. Collectively, this divergent strategy allowed rapid and convenient access to various aryl linkers, enabling the systematic evaluation of the effect of appending unit on the medicinal properties of macrocyclic peptides.


Conceptualizing Health Promotion in Relation to Outpatient Healthcare Building Design: A Scoping Review.
This review explored different conceptualizations of health promotion in the context of, and in relation to, outpatient building design. Today's healthcare organizations are implementing holistic healthcare approaches such as health promotion, while simultaneously increasing their outpatient services. These health promotion approaches, focused on empowering people to take control of their health, are expected to have implications for the outpatient healthcare building design. Yet there is limited knowledge what these may be. A review of the literature on the current state of the art is thus needed to enable and support dialog on future healthcare building design. A scoping review of 4,506 papers, collected from four databases and three scientific journals in 2015, resulted in 14 papers relating health promotion to building design and outpatient healthcare. From the subsequent content analysis, multiple common themes and subthemes emerged. The review reveals diverse range of health promotion interpretations, three health promotion perspectives (health behavior, health equity, and sense of coherence), associated design approaches, design objectives, health-related outcomes, building features, and solutions. While diverse health promotion perspectives might merely represent variations in focus, these differences become problematic when relating to building design. To support further dialogs on development of health promotion in, and in relation to, the build environment, there is a need to strengthen the health promotion vocabulary. Further research is needed to compare different design approaches and how these can be combined to minimize contradicting implications for building design.


Cerebrovascular risk factors do not contribute to genetic variance of cognitive function: the ERF study.
Impaired cognition in later life may result from Alzheimer's disease-related pathology, but also from vascular pathology. We studied to what extent vascular risk explained heritability of cognition in 780 individuals, related in one extended pedigree in a genetically isolated population, in the ERF study. Heritability was estimated using variance components modelling (SOLAR). Univariate analyses included models with and without vascular disease; bivariate analyses included both cognitive and vascular traits, such as blood pressure, serum glucose or lipids. Heritability for immediate and delayed recall, recognition, semantic fluency, Trail making B and Stroop tests was significant, with estimates from 0.16 to 0.36. Vascular factors did not affect cognitive functions, except immediate recall and the Stroop test. Heritability estimates did not change significantly when adjusted for vascular disease. We found no genetic correlation between cognition and vascular traits. Therefore, in this population vascular disease is mildly associated with cognitive dysfunction, and in those with vascular disease, the underlying genetic risk factors are not likely to account for the genetic variation in cognition at adult age.


Changes in sex hormone-binding globulin, insulin, and serum lipids in postmenopausal women on a low-fat, high-fiber diet combined with exercise.
Dietary factors including fat and fiber have been reported to play a role in the development of breast cancer, possibly mediated by changes in estradiol. Diet and exercise have been shown to affect levels of sex hormone-binding globulin (SHBG), which in turn regulate the bioavailability of estradiol. Diet and exercise also affect insulin levels, which play a role in the synthesis of SHBG, and the hormone itself is a potent mitogen for many cancer cell lines. This study was designed to measure the effects of a low-fat, high-fiber diet, combined with regular aerobic exercise, on the levels of SHBG, insulin, and serum lipids in postmenopausal women with or without hormone replacement therapy (HRT). Two groups of postmenopausal women, 11 on HRT and 11 not on HRT, underwent a low-fat (1O% fat calories), high-fiber (65-70 g/day) diet-and-exercise intervention for three weeks. Serum SHBG, insulin, and lipids were measured before and after the regimen. After the intervention, SHBG levels were significantly increased for the women on HRT (44.5 +/- 3.4 vs. 62 +/- 6.4 nmol/l) and the women not on HRT (32.1 +/- 4.6 vs. 45.5 +/- 6.1 nmol/l, both changes p < 0.01). Also after the intervention, insulin levels were significantly reduced for the women on HRT (196 +/- 44.4 vs. 119.8 +/- 28.7 pmol/l) and the women not on HRT (144.2 +/- 17.9 vs. 115.5 +/- 20.8 pmol/l, both changes p < 0.01). Body mass index and total cholesterol were significantly reduced for both groups of women (all changes p < 0.01). Although the exact mechanism for the change in SHBG is not known, the increases in SHBG and reductions in insulin as a result of this lifestyle intervention should reduce the risk for breast cancer in postmenopausal women.


Information following a diagnosis of congenital heart defect: experiences among parents to prenatally diagnosed children.
Prenatal screening of pregnant women in Sweden has improved the detection of major congenital heart defects (CHD). The aim was to explore parental experiences and need for information following a prenatal diagnosis of CHD. Semi-structured interviews conducted with six fathers and five mothers to seven prenatally diagnosed children. Data were analyzed through content analysis. Three themes and 9 categories emerged. Theme 1, Grasping the facts today while reflecting on the future, containing five categories: Difficulties sorting out information when in emotional chaos; Respectful information regarding termination of pregnancy; Early information is crucial; Understanding the facts regarding the anomaly; Preparing for the future. Theme 2, Personal contact with medical specialists who give honest and trustworthy information is valued, containing two categories: Trust in information received from medical specialists and Truth and honesty is valued. Theme 3, An overwhelming amount of information on the Internet, containing two categories: Difficulties in finding relevant information and Easy to focus on cases with a poor outcome when searching the Internet. Early and honest information in line with individual preferences is crucial to support the decisional process regarding whether to continue or terminate the pregnancy. The use of illustrations is recommended, as a complement to oral information, as it increases comprehension and satisfaction with obtained information. Furthermore, the overwhelming amount of information on the Internet calls for compilation of easily accessible and reliable information sources via the Internet.


Physical function of the upper limb after breast cancer surgery. Results from the SOUND (Sentinel node vs. Observation after axillary Ultra-souND) trial.
The SOUND (Sentinel node vs. Observation after axillary Ultra-souND) trial is an ongoing prospective randomized study comparing sentinel node biopsy vs. no axillary surgical staging in patients with small breast cancer and negative pre-operative ultra-sound of the axilla. The first 180 recruited patients were administered the QuickDASH (Disability Arm and Shoulder) questionnaire at different time points (before surgery, 1 week, 6 months and 1 year after surgery) to evaluate the physical function of the ipsilateral upper limb, The QuickDASH score ranges from 0 (no disability) to 100 (complete disability). 176 patients were available for analysis (94 in SNB arm and 82 in observation arm). The two groups were comparable with respect to age, tumor characteristics and treatments. Pre-surgery score values were 3.0% and 2.7% in the SNB arm and observation arm, respectively (P = 0.730). One week after surgery, the score increased to 24.0% in the SNB arm and 10.6% in the observation arm (P < 0.001). After 6 and 12 months, the score decreased in both arms to values similar to baseline values. The overall trend in time of the score was significantly different between the two arms (P < 0.001), even after the exclusion of five patients who received AD in the SNB arm (P < 0.001). Patients who underwent SNB had a significantly higher rate of disability in the early post-operative period compared to patients who did not. The avoidance of SNB might translate into a considerable reduction of physical and emotional distress.


Osteonecrosis of the femoral head associated with pregnancy. A preliminary report.
Osteonecrosis is usually associated with trauma, use of corticosteroids, or alcohol abuse. We investigated the rare association of osteonecrosis of the femoral head and pregnancy, and we defined differences between the disorder in pregnant women and that in women of childbearing age who were not pregnant. The results of treatment with a free vascularized fibular graft were evaluated in terms of relief of pain and improvement of the Harris hip score after a minimum of two years of follow-up. Thirteen women (seventeen hips) had the onset of pain in the hip during pregnancy or within the first four weeks after delivery, and the pain persisted until a diagnosis of osteonecrosis of the femoral head was made on the basis of magnetic resonance imaging. No patient had any other risk factor for this disease. Information was obtained by means of clinical assessment, a review of the records and radiographs, and a telephone survey. Eleven women (fifteen hips) were managed with a free vascularized fibular graft, and nine of them (eleven hips) were evaluated, with regard to relief of pain and the Harris hip score, at a minimum of two years postoperatively. The average age when the pain began was 31.5 years (range, twenty-five to forty-one years). Eleven of the thirteen women were primigravid, and the patients typically first had the pain late in the second trimester or in the third trimester of pregnancy. The women tended to have a small body frame and a relatively large weight gain during the pregnancy. Eight of the thirteen patients had swelling and varicosity of the lower extremities. The diagnosis was delayed an average of 10.3 months, with a range of three to thirty months. A common misdiagnosis was transient osteoporosis of the hip during pregnancy. A correct diagnosis was established for all hips on the basis of the finding of a double-density signal on magnetic resonance imaging or evidence of progression of the disease on plain radiographs. According the system of Marcus et al., the stage at the time of diagnosis ranged from II to V. All women had involvement of the left hip, and four had bilateral involvement. Of the eleven women (fifteen hips) who were managed with a free vascularized fibular graft, nine noted marked or complete relief of the preoperative pain. Two hips in a patient who had progressive pain were treated with a total hip arthroplasty. Two hips (one patient) were lost to follow-up. The nine patients (eleven hips) who were available for follow-up at a minimum of two years had an average improvement in the Harris hip score of 24 points. Occasionally, pain in the hip that begins during pregnancy is caused by osteonecrosis of the femoral head. A high index of suspicion and use of magnetic resonance imaging may lead to an earlier diagnosis and a better prognosis in this population of women. In this study, treatment with a free vascularized fibular graft was a useful option with which to obviate or postpone the need for total hip arthroplasty.


Psychosocial interventions for people with schizophrenia or psychosis on minimal or no antipsychotic medication: A systematic review.
Antipsychotics are the first-line treatment for people with schizophrenia or psychosis. There is evidence that they can reduce the symptoms of psychosis and risk of relapse. However many people do not respond to these drugs, or experience adverse effects and stop taking them. In the UK, clinical guidelines have stressed the need for research into psychosocial interventions without antipsychotics. This systematic review examines the effects of psychosocial interventions for people with schizophrenia or psychosis who are on no/minimal antipsychotics. Databases were searched for empirical studies investigating a psychosocial intervention in people with a schizophrenia spectrum disorder who were not taking antipsychotics or had received an antipsychotic minimisation strategy. We identified nine interventions tested in 17 studies (N = 2250), including eight randomised controlled trials. Outcomes were generally equal to or in a small number of cases better than the control group (antipsychotics/treatment as usual) for Cognitive Behavioural Therapy (CBT), Need Adapted Treatment and Soteria. The remaining interventions provided some encouraging, but overall inconsistent findings and were Psychosocial Outpatient Treatment, Open Dialogue, Psychosocial Inpatient Treatment, Psychoanalysis/Psychodynamic Psychotherapy, Major Role Therapy, and Milieu Treatment. Study quality was generally low with little recent research. In conclusion, nine psychosocial interventions have been studied for patients on no/minimal antipsychotics. The majority of studies reported outcomes for the intervention which were the same as the control group, however, study quality was problematic. Given the adverse effects of antipsychotics and that many people do not want to take them, high quality trials of psychosocial treatments for people on minimal/no antipsychotics are needed.


Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation.
Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV) L1 and L2 DNA vaccination. Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11-88 of 8 different HPV types (11-88×8) or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies. Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2) vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type. Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines.


Phylogenetic signal, evolutionary process, and rate.
A recent advance in the phylogenetic comparative analysis of continuous traits has been explicit, model-based measurement of "phylogenetic signal" in data sets composed of observations collected from species related by a phylogenetic tree. Phylogenetic signal is a measure of the statistical dependence among species' trait values due to their phylogenetic relationships. Although phylogenetic signal is a measure of pattern (statistical dependence), there has nonetheless been a widespread propensity in the literature to attribute this pattern to aspects of the evolutionary process or rate. This may be due, in part, to the perception that high evolutionary rate necessarily results in low phylogenetic signal; and, conversely, that low evolutionary rate or stabilizing selection results in high phylogenetic signal (due to the resulting high resemblance between related species). In this study, we use individual-based numerical simulations on stochastic phylogenetic trees to clarify the relationship between phylogenetic signal, rate, and evolutionary process. Under the simplest model for quantitative trait evolution, homogeneous rate genetic drift, there is no relation between evolutionary rate and phylogenetic signal. For other circumstances, such as functional constraint, fluctuating selection, niche conservatism, and evolutionary heterogeneity, the relationship between process, rate, and phylogenetic signal is complex. For these reasons, we recommend against interpretations of evolutionary process or rate based on estimates of phylogenetic signal.


Termination of intractable hiccups with digital rectal massage.
A 60-year-old man with acute pancreatitis developed persistent hiccups after insertion of a nasogastric tube. Removal of the latter did not terminate the hiccups which had also been treated with different drugs, and several manoeuvres were attempted, but with no success. Digital rectal massage was then performed resulting in abrupt cessation of the hiccups. Recurrence of the hiccups occurred several hours later, and again, they were terminated immediately with digital rectal massage. No other recurrences were observed. This is the second reported case associating cessation of intractable hiccups with digital rectal massage. We suggest that this manoeuvre should be considered in cases of intractable hiccups before proceeding with pharmacological agents.


Ischemic stroke in the elderly: an overview of evidence.
Stroke mostly occurs in elderly people and patient outcomes after stroke are highly influenced by age. A better understanding of the causes of stroke in the elderly might have important practical implications not only for clinical management, but also for preventive strategies and future health-care policies. In this Review, we explore the evidence from both human and animal studies relating to the effect of old age-in terms of susceptibility, patient outcomes and response to treatment-on ischemic stroke. Several aging-related changes in the brain have been identified that are associated with an increase in vulnerability to ischemic stroke in the elderly. Furthermore, risk factor profiles for stroke and mechanisms of ischemic injury differ between young and elderly patients. Elderly patients with ischemic stroke often receive less-effective treatment and have poorer outcomes than younger individuals who develop this condition. Neuroprotective agents for ischemic stroke have been sought for decades but none has proved effective in humans. One contributing factor for this translational failure is that most preclinical studies have used young animals. Future research on ischemic stroke should consider age as a factor that influences stroke prevention and treatment, and should focus on the management of acute stroke in the elderly to reduce the incidence and improve outcomes in this vulnerable group.


Toxicity associated to uptake and depuration of carbamazepine in the clam Scrobicularia plana under a chronic exposure.
Carbamazepine (CBZ) is an antiepileptic drug commonly detected in aquatic systems, with toxic effects to inhabiting organisms. Limited information is known on stress response biomarkers associated to bioconcentration and depuration of CBZ in aquatic organisms. Moreover, few studies addressed if the response and recovery of organisms to a contaminant can change when they are collected in a contaminated site. This study intended to understand the bioconcentration and depuration of CBZ combined with its toxicological impact in Scrobicularia plana clams collected from two contrasting areas (MIRA, Mira channel, non-contaminated and LAR, Laranjo bay, anthropogenically impacted) from the Ria de Aveiro (Portugal). The clams were exposed for 14days to environmentally relevant CBZ concentrations (0.0, 4.0 and 8.0μg/L), followed by a 14day depuration period. CBZ concentrations in S. plana tissues were rapidly bioconcentrated during the exposure period. In the depuration period CBZ was eliminated, in some extent. The main toxic effects occurred at the highest concentration (8.0μg/L) after 14days of exposure in which the clams from LAR accumulated a higher CBZ concentration (LAR: ~10ng/g FW) than clams from MIRA (MIRA: ~7ng/g FW). LAR clams exhibited higher oxidative damage at this concentration, demonstrated by higher LPO levels over time (increase of ~1.4% relative to control) and, in comparison with MIRA clams (LAR: 17.7nmol/g FW; MIRA: 11.4nmol/g FW). After the depuration period, LAR clams recovered from the stress induced by CBZ. A decrease in LPO for LAR (decrease of ~40% in relation to the end of the exposure period) was accompanied by a decrease in CBZ tissue concentrations (decrease of ~61% relative to the end of the exposure period). MIRA clams were not oxidatively injured (low LPO levels remained unchanged after the depuration and CBZ decreased ~80% relative to the end of the exposure period).


[Evaluation of antibiotic resistance of Escherichia coli in urinary tract infections in Primary Care Barbastro Sector (Huesca)].
Evaluate the resistance of community-uropathogen, Escherichia coli to several antibiotics in our health sector and deduce empirical treatment options. E. coli strains isolated from urine cultures of patients from Primary Care Barbastro Sector, between January 2011 and December 2013, were studied. The resistances rates for nine common antibiotics were determined, and differences in sensitivity were analyzed, comparing confidence intervals for proportions by the method of Wilson. E. coli was the most frequently isolated bacteria (61.08% of positive urine cultures sent from Primary Care). Overall, there has been an increase in resistance of E. coli isolates in all antimicrobials studied. Still, resistance has remained below 4% compared to fosfomycin and nitrofurantoin and below 10% in cephalosporins second and third generation. Resistance to amoxicillin-clavulanate has increased progressively reaching 21.5% in 2013; only this antibiotic has presented a statistically significant increase. The maximum levels of resistance (over 30%) were found in the antibiotics administered orally and often indicated in uncomplicated urinary tract infections: trimethoprim-sulfamethoxazole, ciprofloxacin and ampicillin. Update knowledge susceptibility patterns of microorganisms most commonly isolated in urine samples in each health area allows to choose the most suitable and effective treatments trough empirical knowledge.


Role of accelerated aging in limb muscle wasting of patients with COPD.
Skeletal muscle wasting is an independent predictor of health-related quality of life and survival in patients with COPD, but the complexity of molecular mechanisms associated with this process has not been fully elucidated. We aimed to determine whether an impaired ability to repair DNA damage contributes to muscle wasting and the accelerated aging phenotype in patients with COPD. The levels of phosphorylated H2AX (γH2AX), a molecule that promotes DNA repair, were assessed in vastus lateralis biopsies from 10 COPD patients with low fat-free mass index (FFMI; COPDL), 10 with preserved FFMI and 10 age- and gender-matched healthy controls. A panel of selected markers for cellular aging processes (CDKN2A/p16ink4a, SIRT1, SIRT6, and telomere length) were also assessed. Markers of oxidative stress and cell damage and a panel of pro-inflammatory and anti-inflammatory cytokines were evaluated. Markers of muscle regeneration and apoptosis were also measured. We observed a decrease in γH2AX expression in COPDL, which occurred in association with a tendency to increase in CDKN2A/p16ink4a, and a significant decrease in SIRT1 and SIRT6 protein levels. Cellular damage and muscle inflammatory markers were also increased in COPDL. These data are in keeping with an accelerated aging phenotype as a result of impaired DNA repair and dysregulation of cellular homeostasis in the muscle of COPDL. These data indicate cellular degeneration via stress-induced premature senescence and associated inflammatory responses abetted by the senescence-associated secretory phenotype and reflect an increased expression of markers of oxidative stress and inflammation.


Foreign matter identification from solid dosage forms.
Despite the increased request for robust quality systems, the end product may contain unidentified defects or discoloured regions. The foreign matter has to be monitored, identified and its source defined in order to prevent further contamination. However, the identification task can be complicated, since the origin and nature of foreign matter are various. The aim of this study is to provide an efficient foreign matter identification procedure for various substances possibly originating from pharmaceutical manufacturing environment. The surface or cross-section of the uncoated and coated tablets was analysed by utilization of different analytical techniques, such as light microscopy (LM), scanning electron microscopy in combination with energy dispersive X-ray microanalysis (SEM/EDX), Fourier transform infrared spectroscopy (FT-IR) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The results indicate that the combination of different analytical techniques proved to be a powerful approach in foreign matter identification. Light microscopy and SEM generate information on the morphology of foreign matter particles. EDX provides elemental analysis, which most often serves as final confirmation of the identification. However, FT-IR can be used to obtain information on the compounds chemical structure and conformation, and ToF-SIMS provides sensitivity in cases, where the entire solid dosage form is contaminated with foreign matter.


miR-16 and miR-26a target checkpoint kinases Wee1 and Chk1 in response to p53 activation by genotoxic stress.
The tumour suppressor p53 is a crucial regulator of cell cycle arrest and apoptosis by acting as a transcription factor to regulate a variety of genes. At least in part, this control is exerted by p53 via regulating expression of numerous microRNAs. We identified two abundantly expressed microRNAs, miR-16 and miR-26a, whose expression is regulated by p53 during the checkpoint arrest induced by the genotoxic drug, doxorubicin. Importantly, among the targets of these miRs are two critical checkpoint kinases, Chk1 and Wee1. The p53-dependent augmentation of miR-16 and miR-26a expression levels led to the cell cycle arrest of tumour cells in G1/S and increased apoptosis. Strikingly, the bioinformatics analysis of survival times for patients with breast and prostate cancers has revealed that co-expression of mir-16 and miR-26a correlated with a better survival outcome. Collectively, our data provide a novel mechanism whereby p53 represses Chk1 and Wee1 expression, at least partially, via upregulation of miR-16 and miR-26a and thus sensitizes tumour cells to genotoxic therapies.


Spatiotemporal variation of chromatic and achromatic contrast thresholds.
Moving the retinal image of a sinusoidal grating at a constant velocity (compensated for eye movements) provides controlled spatial and temporal frequencies at every point in the stimulus field. Using this controlled-velocity technique, we have measured the detection threshold for isoluminance, red/green gratings as a function of their spatial and temporal frequencies. The chromatic contrast-threshold surface obtained in this way is analogous to the achromatic contrast-threshold surface measured previously, but the results are quite different. For very low temporal frequencies (below 0.2 Hz), the chromatic sensitivity decreases steadily with decreasing temporal frequency. Below 0.01 Hz, chromatic patterns disappear completely even at maximum contrast (although achromatic or homochromatic patterns do not). In the region above 0.2 Hz, both achromatic and chromatic thresholds can be explained by the same receptive-field-like model. When the center and the surround components of this model are additively combined, they form the chromatic threshold surface; when the sign of either component is reversed, they form the achromatic one.


Molecular dynamics characterization of the structures and induction mechanisms of a reverse phenotype of the tetracycline receptor.
Molecular-dynamics simulations have been used to investigate the mechanism of induction of a mutant (revTetR) of the tetracycline repressor protein (TetR) that shows the reverse phenotype (i.e., it is induced in the absence of tetracyclines and not in their presence). Low-frequency, normal-mode analyses demonstrate that the reverse phenotype is reproduced by the simulations on the basis of criteria established for wild-type TetR. The reverse phenotype is caused by the fact that the DNA-binding heads in revTetR are closer than the ideal distance needed for DNA-binding when no inducer is present. This distance increases on binding an inducer. Whereas this distance increase makes the interhead distance too large in wild-type TetR, it increases to the ideal value in revTetR. Thus, the mechanism of induction is the same for the two proteins, but the consequences are reversed because of the smaller interhead distance in revTetR when no inducer is present.


Effectiveness of sequential v. standard triple therapy for treatment of Helicobacter pylori infection in children in Nairobi, Kenya.
Once the diagnosis of Helicobacter pylori is confirmed, treatment requires at least two antibiotics and an acid inhibitor for a minimum of seven days. Unfortunately, treatment failures are being frequently reported. Treatment regimens that include sequential administration of antibiotics with acid inhibitors have been developed to try and increase the rate of eradication. To determine the effectiveness of a novel 10-day sequential therapy compared with the standard 10-day triple therapy for treatment of H. pylori infection in children. A double-blinded, randomised, controlled trial was conducted. Children under the age of 16 years with recurrent abdominal pain associated with dyspepsia and diagnosed with H. pylori by histology were randomly allocated either to a 10-day sequential treatment regimen or to a 10-day conventional triple therapy. Analysis of the outcome of this study was based on clinical improvement and confirmed H. pylori eradication based on stool H. pylori antigen detection and/or repeat endoscopy. Of the 71 patients included in the analysis, 45 (63.4%) were given the 10-day conventional treatment while 26 (36.6%) received the 10-day sequential treatment. There was no difference in clinical improvement after treatment in the two therapies. However, there was a significant difference in the eradication of H. pylori between the conventional v. sequential regimens (48.8% v. 84.6%, respectively; p=0.02, odds ratio 0.19). The sequential treatment had a significantly higher H. pylori eradication rate than the conventional treatment.


Removal of self-expandable metallic stents: is it possible?
Self-expandable metallic stents (SEMS) have become an important component of the management of central airway disease. These stents are not, however, without problems and stent-related complications (SRCs) can be more formidable to treat than the original indication for SEMS placement. As use of SEMS has increased, the number of patients returning to hospital for management of SRCs has similarly risen. A small group of these patients will have, as their only therapeutic option, stent removal Thus far limited experience exists with late stent removal. Stent extraction is difficult and requires expertise in a variety of techniques. A multidisciplinary approach to both SEMS placement and extraction is strongly advocated.


Comparison of primary care doctors and dentists in the referral of oral cancer: a systematic review.
Oral cancer is referred to specialists by both general practitioners (GPs) and dentists, with varying proportions reported in different studies. However, some have noted that dentists more commonly refer oral cancer in the absence of patient-perceived symptoms and may refer at an earlier stage. Unfortunately, approximately half the UK adult population do not receive regular dental care. We have conducted a systematic review of studies that compare GPs and dentists in the referral of oral cancer and have focused on three aspects: the proportion of diagnosed oral cancers, stage on presentation, and delay. Searches of the databases Medline, Embase, Scopus, Google Scholar, Web of Science, and CINAHL, together with additional searches of reference lists, authors, and conference proceedings, found 22 studies from 10 countries, which included a total of 4953 oral cancers. The percentage of medical referrals ranged from 13% to 86%; dental referrals ranged from 15% to 80%. Random-effects meta-analysis indicated a combined relative risk of medical referral to dental referral of 1.36 (95% CI: 0.99 to 1.86). For UK-based studies, the relative risk was also 1.36 (95% CI: 1.05 to 1.76). There was considerable heterogeneity for all studies and for a subgroup of UK studies: I296.4% (95% CI 95.4 to 97.1) and 81.0% (95% CI 63.3 to 90.1), respectively. Several studies showed a lower stage for dentally-referred cancers; the combined risk for dentists and GPs referring early (stages 1 and 2) disease was 1.37 (95% CI: 1.17 to 1.60), and one cause may be the much higher number of cases referred by dentists in the absence of symptoms. No studies showed a significant difference in delay. Oral cancer is referred by both GPs and dentists, typically about 50% and 40%, respectively, although there is a wide range, probably depending on local circumstances. Both groups require skills in oral examination, recognition of lesions, and knowledge of the risk factors. Effectively, regular dental attenders are a select group that is regularly screened for oral cancer, and it is likely that screening is not delivered to those with the highest risk. We suggest that further work is required on how to access high-risk individuals both for possible screening and preventive interventions.


High metal reactivity and environmental risks at a site contaminated by glass waste.
This study addresses the reactivity and risks of metals (Ba, Cd, Co, Cr, Cu, Ni, Pb, Zn, As and Sb) at a Swedish site with large glass waste deposits. Old glassworks sites typically have high total metal concentrations, but as the metals are mainly bound within the glass waste and considered relatively inert, environmental investigations at these kinds of sites are limited. In this study, soil and landfill samples were subjected to a sequential chemical extraction procedure. Data from batch leaching tests and groundwater upstream and downstream of the waste deposits were also interpreted. The sequential extraction revealed that metals in <2 mm soil/waste samples were largely associated with geochemically active fractions, indicating that metals are released from pristine glass and subsequently largely retained in the surrounding soil and/or on secondary mineral coatings on fine glass particles. From the approximately 12,000 m(3) of coarse glass waste at the site, almost 4000 kg of Pb is estimated to have been lost through corrosion, which, however, corresponds to only a small portion of the total amount of Pb in the waste. Metal sorption within the waste deposits or in underlying soil layers is supported by fairly low metal concentrations in groundwater. However, elevated concentrations in downstream groundwater and in leachates of batch leaching tests were observed for several metals, indicating on-going leaching. Taken together, the high metal concentrations in geochemically active forms and the high amounts of as yet uncorroded metal-rich glass, indicate considerable risks to human health and the environment.


DNA interaction with DAPI fluorescent dye: Force spectroscopy decouples two different binding modes.
In this work, we use force spectroscopy to investigate the interaction between the DAPI fluorescent dye and the λ-DNA molecule under high (174 mM) and low (34 mM) ionic strengths. Firstly, we have measured the changes on the mechanical properties (persistence and contour lengths) of the DNA-DAPI complexes as a function of the dye concentration in the sample. Then, we use recently developed models in order to connect the behavior of both mechanical properties to the physical chemistry of the interaction. Such analysis has allowed us to identify and to decouple two main binding modes, determining the relevant physicochemical (binding) parameters for each of these modes: minor groove binding, which saturates at very low DAPI concentrations ( CT ∼ 0.50 μM) and presents equilibrium binding constants of the order of ∼107 M-1 for the two ionic strengths studied; and intercalation, which starts to play a significant role only after the saturation of the first mode, presenting much smaller equilibrium binding constants (∼105 M-1 ).


Does the Jarman-Bell principle at intra-specific level explain sexual segregation in polygynous ungulates? Sex differences in forage digestibility in Soay sheep.
The Jarman-Bell principle states that large-bodied mammalian herbivores can subsist on lower quality diets because of their lower metabolism requirement/gut capacity ratio. Two major hypotheses for sexual segregation (the behaviour in which animals of the same species aggregate by sex) base their foundations on extending this principle to the intraspecific level, despite the lack of experimental evidence to support this. The first proposes that the larger males can process fibre (low-quality diet) more efficiently than the smaller females, leading to sexual segregation by habitat partitioning due to selection of different food quality and/or quantity (sexual dimorphism-body size hypothesis). The second suggests that the longer time and extra rumination required to digest low-quality food will cause asynchrony of behaviour between males and females, which then leads to sexual segregation (activity budget hypothesis). To provide experimental evidence for the Jarman-Bell principle at the intraspecific level we carried out a set of digestibility trials in Soay sheep (Ovis aries) using grass hay as the diet to test whether sexual dimorphism in body mass can produce significant sexual differences in the efficiency of food digestion. Males were slightly more efficient in digesting forage than females that were at least 30% smaller than the males. Overall, there was a decrease in faecal output of 1 g/kg body mass in favour of males. These differences were not due to differences in food selection, passage rates or faecal particle size and it was not clear why males were more efficient in digesting forage. Although these results do not directly support arguments for either the sexual dimorphism-body size or activity budget hypotheses, they do indicate that the physiological argument upon which the Jarman-Bell principle is founded also operates at the intraspecific level and may be an important factor influencing sexual segregation.


Fam83h null mice support a neomorphic mechanism for human ADHCAI.
Truncation mutations in FAM83H (family with sequence similarity 83, member H) cause autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI), but little is known about FAM83H function and the pathogenesis of ADHCAI. We recruited three ADHCAI families and identified two novel (p.Gln457*; p.Lys639*) and one previously documented (p.Q452*) disease-causing FAM83H mutations. We generated and characterized Fam83h-knockout/lacZ-knockin mice. Surprisingly, enamel thickness, density, Knoop hardness, morphology, and prism patterns were similar in Fam83h (+/+), Fam83h (+/-), and Fam83h (-/-) mice. The histology of ameloblasts in all stages of development, in both molars and incisors, was virtually identical in all three genotypes and showed no signs of pathology, although the Fam83h (-/-) mice usually died after 2 weeks and rarely survived to 7 weeks. LacZ expression in the knockin mice was used to report Fam83h expression in the epithelial tissues of many organs, notably in skin and hair follicles, which manifested a disease phenotype. Pull-down studies determined that FAM83H dimerizes through its N-terminal phospholipase D-like (PLD-like) domain and identified potential FAM83H interacting proteins. Casein kinase 1 (CK1) interacts with the FAM83H PLD-like domain via an F(270)-X-X-X-F(274)-X-X-X-F(278) motif. CK1 can phosphorylate FAM83H in vitro, and many phosphorylation sites were identified in the FAM83H C-terminus. Truncation of FAM83H alters its subcellular localization and that of CK1. Our results support the conclusion that FAM83H is not necessary for proper dental enamel formation in mice, but may act as a scaffold protein that localizes CK1. ADHCAI is likely caused by gain-of-function effects mediated by truncated FAM83H, which potentially mislocalizes CK1 as part of its pathological mechanism.


Role of mechanical-stress inducible protein Hcs24/CTGF/CCN2 in cartilage growth and regeneration: mechanical stress induces expression of Hcs24/CTGF/CCN2 in a human chondrocytic cell line HCS-2/8, rabbit costal chondrocytes and meniscus tissue cells.
Mechanical stress plays an important role in the cartilage metabolism. The aim of this study is to determine the influence of mechanical load magnitude and frequency on cartilage metabolism in terms of the expression of hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), as an essential mediator of extracellular matrix (ECM) production. When a human chondrocytic cell line, HCS-2/8 was exposed to uni-axial cyclic mechanical force (6% elongation, 10 times/min) only for 30 min, the expression level of Hcs24/CTGF/CCN2 (CCN2) increased, and c-Jun N-terminal protein kinase (JNK) was activated. These findings suggest that stretch-induced CCN2 may be mediated by the JNK pathway. When HCS-2/8 cells were subjected to cyclic tension force at 15 kPa, 30 cycles/min, which has been reported to be a degradation force for HCS-2/8 cells, the expressions of CCN2 and aggrecan were inhibited, and such expressions remained unchanged in rabbit hyaline costal cartilage cells. However, these expressions increased in rabbit meniscus tissue cells. These findings suggest that the sensitivity of mechanical stretch may be different depending on the type of cells. Furthermore, CCN2 was co-localized with aggrecan in this meniscus tissue region exposed to mechanical stress in vivo. These findings suggest that CCN2 induced by mechanical stress may therefore play some role in meniscus growth and regeneration.


A comprehensive review with potential significance during skull base and neck operations, Part II: glossopharyngeal, vagus, accessory, and hypoglossal nerves and cervical spinal nerves 1-4.
Knowledge of the possible neural interconnections found between the lower cranial and upper cervical nerves may prove useful to surgeons who operate on the skull base and upper neck regions in order to avoid inadvertent traction or transection. We review the literature regarding the anatomy, function, and clinical implications of the complex neural networks formed by interconnections between the lower cranial and upper cervical nerves. A review of germane anatomic and clinical literature was performed. The review is organized into two parts. Part I discusses the anastomoses between the trigeminal, facial, and vestibulocochlear nerves or their branches and other nerve trunks or branches in the vicinity. Part II deals with the anastomoses between the glossopharyngeal, vagus, accessory and hypoglossal nerves and their branches or between these nerves and the first four cervical spinal nerves; the contribution of the autonomic nervous system to these neural plexuses is also briefly reviewed. Part II is presented in this article. Extensive and variable neural anastomoses exist between the lower cranial nerves and between the upper cervical nerves in such a way that these nerves with their extra-axial communications can be collectively considered a plexus.


Acute stress among adolescents and female rape victims measured by ASC-Kids: a pilot study.
Rape is considered a stressful trauma and often with durable consequences. How the aftermath of rape is for young adolescents' girls considering acute stress is an overlooked field and remains to be studied. In this study, we wanted to investigate acute stress among adolescent victims of rape and the psychometric properties of the Acute Stress Checklist for Children (ASC-Kids). A clinical sample (n = 79) of raped girls, 13-17 years old who had turned to a special rape victim unit for treatment, answered the ASC-Kids. ASC-Kids was also given to a group of minor stressed, non-raped adolescents in the same age range (n = 154) together with the University of California at Los Angeles Post-traumatic Stress Disorder Reaction Index (UCLA PTSD RI), and the Sense of Coherence Scale 13 (SOC-13). The scores from the groups were compared and showed significant differences in mean values on all the diagnostic criteria of acute stress disorder. In the clinical group, 36.7% obtained full ASD criteria. ASC-Kids could discriminate well between groups. Cronbach's alpha was found to be excellent, and the correlation between the UCLA PTSD RI and ASC-Kids found to be good; both ASC-Kids and UCLA PTSD RI had a good and moderate negative correlation with SOC-13. Adolescent female rape victims were shown to have a very high level of acute stress, and the ASC-Kids was found to have sound psychometrics and can be a valuable screening instrument to support clinicians in their assessments of an indication of adolescents after potentially stressful events such as rape.


Using a quartz paraboloid for versatile wide-field TIR microscopy with sub-nanometer localization accuracy.
Illumination based on objective-type total internal reflection (TIR) is nowadays widely used in high-performance fluorescence microscopy. However, the desirable application of such setups for dark-field imaging of scattering entities is cumbersome due to the spatial overlap of illumination and detection light, which cannot be separated spectrally. Here, we report a novel TIR approach based on a parabolically shaped quartz prism that allows for the detection of single-molecule fluorescence as well as single-particle scattering with high signal-to-noise ratios. We demonstrate homogeneous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, we quantitatively compare the fluorescence performance of our setup to objective-type TIR and demonstrate sub-nanometer localization accuracies for the scattering of 40 nm gold nanoparticles (AuNPs). When bound to individual kinesin-1 motors, the AuNPs reliably report on the characteristic 8 nm stepping along microtubules.


Strikingly diverse reactivity of structurally identical silylene and stannylene.
The reactivity of structurally identical silylene and stannylene [PhC(NtBu)2EN(SiMe3)2] (E = Si (1) and Sn (2)) towards coinage metals has been explored. While 1 has the propensity to form an adduct with coinage metals (4 and 5), 2 undergoes a ligand exchange reaction with copper halides and silver triflate leading to PhC(NtBu)2SnX (X = Br (6), Cl (7), and OSO2CF3 (8)) with concomitant formation of [M{N(SiMe3)2}] (M = Cu, Ag). However, with AgSbF6 both 1 and 2 led to ion pairs, 9+·SbF6-and 10+·SbF6- displaying weaker Ag·F interactions in the latter.


Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia.
Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate (PP(i)), and regulated generation and homeostasis of extracellular PP(i) is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular PP(i) generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular PP(i) mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate (P(i)) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular PP(i). Attenuated PP(i) accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades PP(i). Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular P(i) alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated P(i) induced decreases in the ATP release pathway(s) that supports ENPP1 activity; this resulted in markedly reduced rates of PP(i) accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) alpha-actin, SM22alpha, and calponin. Notably, addition of exogenous ATP (or PP(i) per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular PP(i) generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification.


New DArT markers for oat provide enhanced map coverage and global germplasm characterization.
Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT). Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' x 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure. These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity.


On resolving simultaneous congruences using belief propagation.
Graphical models and related algorithmic tools such as belief propagation have proven to be useful tools in (approximately) solving combinatorial optimization problems across many application domains. A particularly combinatorially challenging problem is that of determining solutions to a set of simultaneous congruences. Specifically, a continuous source is encoded into multiple residues with respect to distinct moduli, and the goal is to recover the source efficiently from noisy measurements of these residues. This problem is of interest in multiple disciplines, including neural codes, decentralized compression in sensor networks, and distributed consensus in information and social networks. This letter reformulates the recovery problem as an optimization over binary latent variables. Then we present a belief propagation algorithm, a layered variant of affinity propagation, to solve the problem. The underlying encoding structure of multiple congruences naturally results in a layered graphical model for the problem, over which the algorithms are deployed, resulting in a layered affinity propagation (LAP) solution. First, the convergence of LAP to an approximation of the maximum likelihood (ML) estimate is shown. Second, numerical simulations show that LAP converges within a few iterations and that the mean square error of LAP approaches that of the ML estimation at high signal-to-noise ratios.


CAB39L elicited an anti-Warburg effect via a LKB1-AMPK-PGC1α axis to inhibit gastric tumorigenesis.
Metabolic dysfunction is a hallmark of gastric cancer (GC). In this study, we reported the identification of Calcium Binding Protein 39-Like (CAB39L) as a novel regulator of tumor metabolism in GC. CAB39L mRNA was frequently silenced by promoter methylation in GC cell lines and tissues. Functional studies suggested that CAB39L functions as a tumor suppressor, as overexpression of CAB39L elicited suppression of multiple cancer phenotypes both in GC cells and an orthotopic mouse model; whilst its knockdown promoted tumorigenesis. Mechanistically, CAB39L interacted with LKB1-STRAD complex and induced LKB1, leading to the phosphorylation and activation of AMPKα/β. LKB1-AMPK activation in GC cell lines was tumor suppressive, as metformin (an AMPK activator) inhibited GC cell growth in the CAB39L-silenced cells. Moreover, knockdown of LKB1 reversed growth inhibitory effect of CAB39L, indicating that tumor suppression by CAB39L depended on LKB1-AMPK. RNAseq and gene set enrichment analysis revealed that CAB39L was closely correlated with oxidative phosphorylation and mitochondrial biogenesis. Consistently, CAB39L-induced p-AMPK elicited PGC1α phosphorylation and increased the expression of genes involved in mitochondrial respiration complexes. Accordingly, CAB39L reversed the Warburg effect in GC, as evidenced by enhanced oxygen consumption rate and reduced extracellular acidification rate; inversely, CAB39L knockdown promoted a metabolic shift towards the Warburg phenotype. In GC patients, CAB39L promoter hypermethylation was correlated with poor prognosis. Our data demonstrate that CAB39L is a novel tumor suppressor which suppresses tumorigenesis by promoting LKB1-AMPK-PGC1α axis, thereby preventing a metabolic shift that drives carcinogenesis. CAB39L methylation is a potential prognostic biomarker for GC patients.