PARP-1 inhibition influences the oxidative stress response of the human lens.
Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects.


Nuclear inclusions of the androgen receptor protein in spinal and bulbar muscular atrophy.
Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuronopathy caused by the expansion of an unstable CAG repeat in the coding region of the androgen receptor (AR) gene. To study AR protein expression in normal and SBMA individuals, we used several antibodies that recognize AR protein, and analyzed neural and nonneural tissues by immunohistochemistry and western blotting. Both the normal and the mutant AR proteins were widely distributed, predominantly, but not exclusively, in the cytoplasm of neurons regardless of the pathological involvement, and predominantly in the nuclei of the nonneural tissues in both normal and SBMA individuals, with different expression levels of AR protein among different tissues. In the motor neurons of SBMA patients, there were AR-immunoreactive ubiquitinated nuclear inclusions that were detected by antibodies that recognize a small portion of the N terminus of the AR protein. Absence of other immunoreactive AR epitopes within the inclusion may be due to altered AR configuration, or masking of AR epitopes by other proteins, or proteolytic cleavage of the AR. Our data show that, in addition to the normal cellular distribution of the AR protein, mutant AR-bearing nuclear inclusions are present in SBMA.


Continuous DNA replication in the nucleus of the dinoflagellate Prorocentrum micans (Ehrenberg).
The uptake of tritiated thymine into cells of a heterogeneous population of Prorocentrum micans was investigated using light-microscope and electron-microscope autoradiography. Specificity of thymine uptake into DNA was demonstrated by the specific removal of label from wax-embedded material using DNase and by the high degree of localization of nuclear label to chromosomes in the electron-microscope autoradiographs. All nuclei, including both dividing and non-dividing cells, showed a substantial uptake of label, indicating that nuclear DNA synthesis in Prorocentrum micans is a continuous process. The level of DNA synthesis does show considerable variation, however, with very high levels in some interphase nuclei. The continuous replication of nuclear DNA provides further evidence of dinoflagellate affinity to the prokaryotes, and indicates that Prorocentrum micans is a very primitive eukaryote cell.


Tuning the excited state of photoactive building blocks for metal-templated self-assembly.
The reaction of 2,2':4,4'':4',4'''-quaterpyridyl (qtpy), with d(6) ruthenium(II) (Ru(II) ), and rhenium(I) (Re(I) ) metal centers has been investigated. The pendant pyridyl groups on the products have also been methylated to produce a second series of complexes containing coordinated Meqtpy(2+). The absorption spectra of the complexes are dominated by intraligand and charge-transfer bands. The ruthenium(II) complexes display broad unstructured luminescence consistent with emission from a Ru(d)→diimine(π*) manifold in acetonitrile solutions. In aqueous solutions, their emissions are weaker and the lifetimes are shorter. This effect is particularly acute for complexes incorporating coordinated dipyridylpyrazine, dppz, ligands. Although the emission of the ruthenium(II) complexes containing Meqtpy(2+) is generally shorter than their qtpy analogs, it is notable that solvent-dependent effects are much less intense. The rhenium(I) complexes also display broad unstructured luminescence but, compared with the ruthenium(II) systems, they have a relatively short lifetime in acetonitrile. Electrochemical studies reveal that all of the Ru(II) complexes display chemically reversible metal-based oxidations. Re(I) complexes only display irreversible metal-based oxidations. In most cases, the reduction processes were not fully chemically reversible. The electrochemical and optical studies reveal that the nature of the lowest excited state of these complexes--particularly, the systems incorporating dppz--is highly dependent on the nature of the coordinated ligands. Calculations indicate that, although the excited state of most of the complexes is centered on the qtpy or Meqtpy(2+) ligands, the excited state of the complexes containing dppz ligands is switched away from the dppz by qtpy methylation. A crystallographic study on one of the dicationic ruthenium(II) structures reveals that it forms an inclusion complex with benzene.


Alpha 1-and alpha 2-adrenoceptor mediated vasoconstriction in the forearm of normotensive and hypertensive subjects.
alpha 1- And alpha 2-adrenoceptor mediated vasoconstriction were studied in 13 patients with essential hypertension and 13 age-matched normotensive controls. This was done by comparing the changes in forearm blood flow induced by intra-arterial infusion of the selective alpha 1- and alpha 2-adrenoceptor agonists methoxamine and B-HT 933, the catecholamines adrenaline and noradrenaline, and the selective alpha 2-adrenoceptor antagonist yohimbine in both study groups. The catecholamines were infused in the presence of propranolol in order to prevent beta-adrenergic effects. Forearm blood flow was measured by plethysmography. All agonists produced a dose-dependent vasoconstriction which was more pronounced in the hypertensive patients, although the difference was significant only for the infusion of the catecholamines and for the combined effects of methoxamine and B-HT 933. No preference was found for either the alpha 1- or alpha 2-adrenoceptor mediated vasoconstriction in the greater response of the hypertensive patients. These results could well be explained by structural vascular changes, secondary to the elevated blood pressure. No evidence was found for increased alpha 2-adrenoceptor mediated basal vascular tone in patients with essential hypertension, since yohimbine tended to induce a greater vasodilatation in the normotensive subjects.


Renal protective effect of antiplatelet therapy in antiphospholipid antibody-positive lupus nephritis patients without antiphospholipid syndrome.
We sought to evaluate the effect of antiplatelet therapy in addition to conventional immunosuppressive therapy for lupus nephritis (LN) patients positive for antiphospholipid antibodies (aPL) without definite antiphospholipid syndrome (APS). Patients with biopsy-proven LN class III or IV were retrospectively evaluated. We selected patients positive for anticardiolipin antibody (aCL) or lupus anticoagulant (LA) who did not meet the criteria for a diagnosis of APS. The patients were divided into two subgroups according to whether antiplatelet therapy was received. The cumulative complete renal response (CR) rate, relapse-free rate, and change in estimated glomerular filtration rate (eGFR) over 3 years after induction therapy were calculated. We identified 17 patients who received antiplatelet therapy and 21 who did not. Baseline clinicopathological characteristics and immunosuppressive therapy did not show a significant difference between the two groups except for a higher incidence of LN class IV in the treatment group (p = 0.03). There was no difference in cumulative CR rate, relapse-free rate, or eGFR change between these subgroups. However, when data on LA-positive patients were assessed, an improvement in eGFR was found (p = 0.04) in patients receiving antiplatelet treatment. Addition of anti-platelet therapy was associated with an improvement of eGFR in LA-positive patients with LN class III or IV.


Depressive Symptoms among US Adults during the Great Recession and Economic Recovery.
We study the trajectory of depressive symptoms among US adults before, during, and after the 2008/2009 Great Recession. We use repeated cross-sectional surveys of the National Health and Nutrition Examination Survey (NHANES) between 2005 and 2018. Mental health is assessed with the Patient Health Questionnaire-9 (PHQ-9), with the following categorization for depressive symptoms: none or mild (score 0-9), moderate or severe (score 10-27). A parallel time series was calculated from the Behavioral Risk Factor Surveillance System (BRFSS) on self-reported number of days with poor mental health. NHANES data show a statistically significant increase in depressive symptoms from 2005/2006 to 2007/2008 (the beginning of the Great Recession), but there were no significant or consistent changes after 2007/2008. In particular, the deterioration in the adjusted predicted PHQ-9 scores occurred prior to the large increase in unemployment rate (2009/2010). As the macroeconomic situations improved and unemployment rates recovered, mental health did not return to the previous level. In the latest wave of NHANES (2017/2018), unemployment rates were at the lowest level over the analysis period; however, the adjusted predicted PHQ-9 scores were higher than that at the beginning of the Great Recession. Trends of PHQ-9 scores were similar across income groups - all groups had an increase in depressive symptoms after 2005/2006 and PHQ-9 scores were still high in 2017/2018 after controlling for sociodemographic status. Group with the lowest income had higher levels of depressive symptoms at every time point. BRFSS data shows no consistent changes in the number of days with poor mental health that parallel economic conditions. Depressive symptoms at the population level did not match the economic cycle before, during and after the Great Recession. Future research is needed to better understand the lack of correlation between population mental health and macroeconomic conditions.


Effect of enzymatic treatment of citrus by-products on bacterial growth, adhesion and cytokine production by Caco-2 cells.
Citrus by-products are inexpensive sources of polyphenols, important bioactive compounds with wide pharmaceutical and food applications. This study aimed to investigate the effect of enzymatic treatment of citrus by-products on the polyphenolic profile of extracts and assess the influence of extracts on the growth and adhesion of probiotics and foodborne pathogenic bacteria and on the inflammatory response of epithelial cells. Enzyme-assisted extraction altered the polyphenolic profile (as assessed by HPLC-DAD), increasing the content of aglycone flavanones (naringenin and hesperetin). Enzymatic extracts and aglycone flavanones exhibited higher antibacterial and prebiotic activities than non-enzymatic extracts and glycoside flavanones. However, a higher content of aglycones was not associated with higher anti-adhesion activity. Citrus extracts significantly (P ≤ 0.05) decreased the inflammatory response of Caco-2 cells to Salmonella Typhimurium adhesion. These results support the sustainable reuse of citrus agroindustrial wastes and indicate the potential of citrus extracts in preventing infection by foodborne pathogenic bacteria and inducing proliferation of probiotics in foods and the gut environment.


Development of a loop-mediated isothermal amplification assay for rapid detection of Burkholderia mallei.
The present study was conducted to establish a Loop-mediated isothermal amplification (LAMP) technique for the rapid detection of B. mallei the etiologic agent of glanders, a highly contagious disease of equines. A set of six specific primers targeting integrase gene cluster were designed for the LAMP test. The reaction was optimized using different temperatures and time intervals. The specificity of the assay was evaluated using DNA from B.pseudomallei and Pseudomonas aeruginosa. The LAMP products were analyzed both visually and under UV light after electrophoresis. The optimized conditions were found to be at 63ºC for 60 min. The assay showed high specificity and sensitivity. It was concluded that the established LAMP assay is a rapid, sensitive and practical tool for detection of B. mallei and early diagnosis of glanders.


A cross-sectional analysis of cardiovascular disease in the hemophilia population.
Men with hemophilia were initially thought to be protected from cardiovascular disease (CVD), but it is now clear that atherothrombotic events occur. The primary objective of the CVD in Hemophilia study was to determine the prevalence of CVD and CVD risk factors in US older men with moderate and severe hemophilia and to compare findings with those reported in age-comparable men in the Atherosclerosis Risk in Communities (ARIC) cohort. We hypothesized if lower factor levels are protective from CVD, we would see a difference in CVD rates between more severely affected and unaffected men. Beginning in October 2012, 200 patients with moderate or severe hemophilia A or B (factor VIII or IX level ≤ 5%), aged 54 to 73 years, were enrolled at 19 US hemophilia treatment centers. Data were collected from patient interview and medical records. A fasting blood sample and electrocardiogram (ECG) were obtained and assayed and read centrally. CVD was defined as any angina, any myocardial infarction by ECG or physician diagnosis, any self-reported nonhemorrhagic stroke or transient ischemic attack verified by physicians, or any history of coronary bypass graft surgery or coronary artery angioplasty. CVD risk factors were common in the population. Compared with men of similar age in the ARIC cohort, patients with hemophilia had significantly less CVD (15% vs 25.8%; P < .001). However, on an individual patient level, CVD events occur and efforts to prevent cardiovascular events are warranted. Few men were receiving secondary prophylaxis with low-dose aspirin, despite published opinion that it can be used safely in this patient population.


Death patterns during the 1918 influenza pandemic in Chile.
Scarce information about the epidemiology of historical influenza pandemics in South America prevents complete understanding of pandemic patterns throughout the continent and across different climatic zones. To fill gaps with regard to spatiotemporal patterns of deaths associated with the 1918 influenza pandemic in Chile, we reviewed archival records. We found evidence that multiple pandemic waves at various times of the year and of varying intensities occurred during 1918-1921 and that influenza-related excess deaths peaked during July-August 1919. Pandemic-associated mortality rates were elevated for all age groups, including for adults >50 years of age; elevation from baseline was highest for young adults. Overall, the rate of excess deaths from the pandemic was estimated at 0.94% in Chile, similar to rates reported elsewhere in Latin America, but rates varied ≈10-fold across provinces. Patterns of death during the pandemic were affected by variation in host-specific susceptibility, population density, baseline death rate, and climate.


Exercise reduces circulating biomarkers of cellular senescence in humans.
Cellular senescence has emerged as a significant and potentially tractable mechanism of aging and multiple aging-related conditions. Biomarkers of senescent cell burden, including molecular signals in circulating immune cells and the abundance of circulating senescence-related proteins, have been associated with chronological age and clinical parameters of biological age in humans. The extent to which senescence biomarkers are affected by interventions that enhance health and function has not yet been examined. Here, we report that a 12-week structured exercise program drives significant improvements in several performance-based and self-reported measures of physical function in older adults. Impressively, the expression of key markers of the senescence program, including p16, p21, cGAS, and TNFα, were significantly lowered in CD3+ T cells in response to the intervention, as were the circulating concentrations of multiple senescence-related proteins. Moreover, partial least squares discriminant analysis showed levels of senescence-related proteins at baseline were predictive of changes in physical function in response to the exercise intervention. Our study provides first-in-human evidence that biomarkers of senescent cell burden are significantly lowered by a structured exercise program and predictive of the adaptive response to exercise.


World Health Organization regional assessments of the risks of poliovirus outbreaks.
While global polio eradication requires tremendous efforts in countries where wild polioviruses (WPVs) circulate, numerous outbreaks have occurred following WPV importation into previously polio-free countries. Countries that have interrupted endemic WPV transmission should continue to conduct routine risk assessments and implement mitigation activities to maintain their polio-free status as long as wild poliovirus circulates anywhere in the world. This article reviews the methods used by World Health Organization (WHO) regional offices to qualitatively assess risk of WPV outbreaks following an importation. We describe the strengths and weaknesses of various risk assessment approaches, and opportunities to harmonize approaches. These qualitative assessments broadly categorize risk as high, medium, or low using available national information related to susceptibility, the ability to rapidly detect WPV, and other population or program factors that influence transmission, which the regions characterize using polio vaccination coverage, surveillance data, and other indicators (e.g., sanitation), respectively. Data quality and adequacy represent a challenge in all regions. WHO regions differ with respect to the methods, processes, cut-off values, and weighting used, which limits comparisons of risk assessment results among regions. Ongoing evaluation of indicators within regions and further harmonization of methods between regions are needed to effectively plan risk mitigation activities in a setting of finite resources for funding and continued WPV circulation.


Update on Lorlatinib: Role in Reducing the Risk of Disease Progression in ALK-Positive NSCLC.
Lorlatinib is an oral third-generation inhibitor of anaplastic lymphoma kinase (ALK) with activity in advanced ALK-positive non-small cell lung cancer (NSCLC) in both the first and subsequent line setting. Superior systemic and intracranial efficacy of lorlatinib over crizotinib, a first-generation ALK tyrosine kinase inhibitor (TKI), in treatment-naïve patients with advanced ALK-positive NSCLC was demonstrated by the phase 3 CROWN trial. Lorlatinib retains anti-tumor effect against single and some compound ALK resistance mutations after disease progression on first- and second-generation ALK TKIs. Currently, alectinib, brigatinib, ceritinib, crizotinib and lorlatinib are approved for treatment of advanced ALK-positive NSCLC. However, no head-to-head studies have directly compared lorlatinib to second-generation ALK inhibitors. Herein, we aim to provide an overview of the efficacy and safety of lorlatinib and discuss where lorlatinib stands in the therapeutic approach to advanced ALK-positive NSCLC.


Speech segregation in rooms: effects of reverberation on both target and interferer.
Speech reception thresholds were measured to investigate the influence of a room on speech segregation between a spatially separated target and interferer. The listening tests were realized under headphones. A room simulation allowed selected positioning of the interferer and target, as well as varying the absorption coefficient of the room internal surfaces. The measurements involved target sentences and speech-shaped noise or 2-voice interferers. Four experiments revealed that speech segregation in rooms was not only dependent on the azimuth separation of sound sources, but also on their direct-to-reverberant energy ratio at the listening position. This parameter was varied for interferer and target independently. Speech intelligibility decreased as the direct-to-reverberant ratio of sources was degraded by sound reflections in the room. The influence of the direct-to-reverberant ratio of the interferer was in agreement with binaural unmasking theories, through its effect on interaural coherence. The effect on the target occurred at higher levels of reverberation and was explained by the intrinsic degradation of speech intelligibility in reverberation.


Does trait-based joint species distribution modelling reveal the signature of competition in stream macroinvertebrate communities?
The occupancy and abundance of species are jointly driven by local factors, such as environmental characteristics and biotic interactions, and regional-scale factors, such as dispersal and climate. Recently, it has been shown that biotic interactions shape species occupancies and abundances beyond local extents. However, for small ectothermic animals, particularly for those occurring in freshwater environments, the importance of biotic interactions remains understudied. Species-to-species associations from joint species distribution models (i.e. species associations while controlling for environmental characteristics) are increasingly used to draw hypotheses of which species possibly show biotic interactions. We studied whether species-to-species associations from joint species distribution models show signs of competition using a hypothesis testing framework in stream macroinvertebrate communities at regional extent. We sampled aquatic macroinvertebrates from 105 stream sites in western Finland encompassing a latitudinal gradient of c. 500 kilometres. We hypothesized that if competition drives these associations (H1) functionally, similar species are mostly negatively associated, whereas functionally dissimilar species show random associations. We further hypothesized that the relationship between functional dissimilarity and the strength of association is more pronounced (H2) for abundances rather than occupancies, (H3) at small grain (i.e. stream site) rather than at large grain (i.e. river basin), and (H4) among species having weak dispersal ability than among species with high dispersal ability. Stream macroinvertebrates showed both negative and positive species-to-species associations while controlling for habitat characteristics. However, the negative associations were mostly at large grain (river basin) rather than at small grain (stream site), in occupancy rather than abundance, and not related to species functional dissimilarity or to their dispersal ability. Thus, all our hypotheses considering possible competition (H1-H4) were rejected. Competition does not appear to be a major driving force of stream macroinvertebrate communities at the spatial grain sizes considered. The observed positive associations in occupancy at small grain (stream site) may be attributed to species' similar microhabitat preferences, whereas at large grain (river basin), they may stem from metacommunity dynamics. Our results highlight that species traits were necessary to interpret whether or not species-to-species associations from joint species distribution models resulted from biotic interactions.


Haemodynamic effects of atrial natriuretic peptide in hypoxic chronic obstructive pulmonary disease.
Pulmonary artery pressure is elevated in patients with advanced chronic obstructive pulmonary disease (COPD). Release of atrial natriuretic peptide (ANP) is increased in pulmonary hypertension and this hormone may both selectively vasodilate pulmonary vessels and inhibit pulmonary vascular remodelling. The hypothesis that ANP has a physiological role in protection of the pulmonary circulation from pressure overload, and that it may be beneficial in patients with COPD, has been examined. Ten patients with hypoxic COPD were infused for 30 minute periods with saline followed by ANP at 0.4, 2, and 10 pmol/kg/min respectively via a pulmonary artery catheter whilst monitoring haemodynamics and oxygenation. Levels of immunoreactive ANP (irANP) increased from a mean (SD) of 23 (15) pmol/l to a maximum of 94 (41) pmol/l. Neither systemic blood pressure, cardiac output nor total systemic vascular resistance showed any correlation with irANP levels. There were negative correlations between levels of ANP and mean pulmonary artery pressure which fell from 28.7 to 25.9 mm Hg, pulmonary artery wedge pressure which fell from 6.5 to 4.6 mmHg, and total pulmonary vascular resistance which fell from 489 to 428 dynes s cm-5. There was a small fall in PaCO2 from 6.2 to 5.9 kPa, whilst venous admixture and oxygen delivery both increased non-significantly. At these pathophysiological concentrations there was evidence that ANP selectively reduced right ventricular afterload. These data support the hypotheses that increased plasma levels of ANP may be beneficial in hypoxic COPD, and that endogenous ANP may ameliorate pulmonary hypertension in humans.


Hybrid grammar-based approach to nonlinear dynamical system identification from biological time series.
We introduce a grammar-based hybrid approach to reverse engineering nonlinear ordinary differential equation models from observed time series. This hybrid approach combines a genetic algorithm to search the space of model architectures with a Kalman filter to estimate the model parameters. Domain-specific knowledge is used in a context-free grammar to restrict the search space for the functional form of the target model. We find that the hybrid approach outperforms a pure evolutionary algorithm method, and we observe features in the evolution of the dynamical models that correspond with the emergence of favorable model components. We apply the hybrid method to both artificially generated time series and experimentally observed protein levels from subjects who received the smallpox vaccine. From the observed data, we infer a cytokine protein interaction network for an individual's response to the smallpox vaccine.


Burden of adult-onset Still's disease: A systematic review of health-related quality of life, utilities, costs and resource use.
Adult-onset Still's disease (AOSD) poses a not well estimated burden on patients and healthcare systems. To assess this burden, a systematic review (SR) was undertaken to identify health-related quality of life (HRQoL), utilities, costs and healthcare resource use data. Searches of twelve databases, four conferences, and three key technology assessment and regulatory agency websites were conducted in August 2022. Reference lists of retrieved SRs published since 2017 were also checked. Overall, 16 studies were eligible for inclusion. Eight studies reported HRQoL outcomes, one of which also reported utilities data. Two studies reported direct costs outcomes, and seven reported healthcare resource use data. No indirect costs were identified. A range of outcomes were reported, thus limiting the comparability of results across studies. SF-36 data were impaired in AOSD on most scales, especially those concerning physical activity. Mean SF-36 data were lower across all subscales in patients with active AOSD compared with inactive AOSD. Biologic therapy showed improvements in the SF-36 physical health summary. Utility scores (one study) were significantly lower for AOSD than for healthy controls. Limited direct economic costs data were identified but were substantial where reported. Hospital length of stay ranged from 6.1 to 23.5 days. The SR showed there is a paucity of research reporting the HRQoL and cost burden of AOSD.


DEEP MR IMAGE SUPER-RESOLUTION USING STRUCTURAL PRIORS.
High resolution magnetic resonance (MR) images are desired for accurate diagnostics. In practice, image resolution is restricted by factors like hardware, cost and processing constraints. Recently, deep learning methods have been shown to produce compelling state of the art results for image superresolution. Paying particular attention to desired hi-resolution MR image structure, we propose a new regularized network that exploits image priors, namely a low-rank structure and a sharpness prior to enhance deep MR image superresolution. Our contributions are then incorporating these priors in an analytically tractable fashion in the learning of a convolutional neural network (CNN) that accomplishes the super-resolution task. This is particularly challenging for the low rank prior, since the rank is not a differentiable function of the image matrix (and hence the network parameters), an issue we address by pursuing differentiable approximations of the rank. Sharpness is emphasized by the variance of the Laplacian which we show can be implemented by a fixed feedback layer at the output of the network. Experiments performed on two publicly available MR brain image databases exhibit promising results particularly when training imagery is limited.


Adverse Radiation Therapy Effects in the Treatment of Head and Neck Tumors.
Whether used as a single modality or as part of a combined approach, radiation therapy (RT) plays an essential role in the treatment of several head and neck malignancies. Despite the improvement in radiation delivery techniques, normal structures in the vicinity of the target area remain susceptible to a wide range of adverse effects. Given their high incidence, some of these effects are referred to as expected postradiation changes (eg, mucositis, sialadenitis, and edema), while others are considered true complications, meaning they should not be expected and can even represent life-threatening conditions (eg, radionecrosis, fistulas, and radiation-induced neoplasms). Also, according to their timing of onset, these deleterious effects can be divided into four groups: acute (during RT), subacute (within weeks to months), delayed onset (within months to years), and very delayed onset (after several years).The authors provide a comprehensive review of the most important radiation-induced changes related to distinct head and neck sites, focusing on their typical cross-sectional imaging features and correlating them with the time elapsed after treatment. Radiologists should not only be familiar with these imaging findings but also actively seek essential clinical data at the time of interpretation (including knowledge of the RT dose and time, target site, and manifesting symptoms) to better recognize imaging findings, avoid pitfalls and help guide appropriate management. © RSNA, 2022.


[Latex sensitization in 28 patients].
We present a review of 28 cases of sensitization to latex. Skin prick test for latex was performed in subjects suspected of having allergy to latex and many fruits. Recently, many publications have evidenced the importance of sensitization to latex. There are well documented cases of intraoperatory anaphylaxis. Frequently, this happens to children with congenital urologic malformations, spina bifida or myelomeningocele, who undergo numerous surgical operations and vesical catheterizations. Latex-induced allergy also affects health professionals, since they are continuously in contact with latex-derived materials. There are other studies that relate latex-induced allergy to several fruit allergies (mainly chestnuts, bananas and avocados). In recent literature some discrepancy exists about the reliability of the prick test and the sensibility of in vitro diagnostic methods. We analyze 28 cases of latex sensitization from our clinic, comparing the most severe cases with the remaining subjects. Atopia antecedents were present in 82%, sub-clinical sensitization in 10 cases, urticaria/angioderma in 5, anaphylaxis in 9, and contact dermatitis in 4. Women were majority, with 22 cases versus 6 males. In patients with latex-induced anaphylaxis, the significantly most frequent feature was previous professional exposure to latex (p = 0.004). Specific IgE was measured by radioallergosorbent test (RAST), finding the highest values in the most severe clinical cases (p=0.0016), comparable to the results of the prick test, which still is the best diagnostic method. In our experience, banana and chestnut-induced allergies were the allergies most frequently associated to latex-induced allergy. There were no adverse reactions in any patient after the prick test.


Factorial estimating assembly base errors using k-mer abundance difference (KAD) between short reads and genome assembled sequences.
Genome sequences provide genomic maps with a single-base resolution for exploring genetic contents. Sequencing technologies, particularly long reads, have revolutionized genome assemblies for producing highly continuous genome sequences. However, current long-read sequencing technologies generate inaccurate reads that contain many errors. Some errors are retained in assembled sequences, which are typically not completely corrected by using either long reads or more accurate short reads. The issue commonly exists, but few tools are dedicated for computing error rates or determining error locations. In this study, we developed a novel approach, referred to as k-mer abundance difference (KAD), to compare the inferred copy number of each k-mer indicated by short reads and the observed copy number in the assembly. Simple KAD metrics enable to classify k-mers into categories that reflect the quality of the assembly. Specifically, the KAD method can be used to identify base errors and estimate the overall error rate. In addition, sequence insertion and deletion as well as sequence redundancy can also be detected. Collectively, KAD is valuable for quality evaluation of genome assemblies and, potentially, provides a diagnostic tool to aid in precise error correction. KAD software has been developed to facilitate public uses.


The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids.
Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.


UMSBP2 is chromatin remodeler that functions in regulation of gene expression and suppression of antigenic variation in trypanosomes.
Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of kinetoplastids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and to play an essential role in chromosome end protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1. DNA decondensation is mediated via protein-protein interactions between TbUMSBP2 and these histones, independently of its previously described DNA binding activity. Silencing of the TbUMSBP2 gene resulted in a significant decrease in the disassembly of nucleosomes in T. brucei chromatin, a phenotype that could be reverted, by supplementing the knockdown cells with TbUMSBP2. Transcriptome analysis revealed that silencing of TbUMSBP2 affects the expression of multiple genes in T. brucei, with a most significant effect on the upregulation of the subtelomeric variant surface glycoproteins (VSG) genes, which mediate the antigenic variation in African trypanosomes. These observations suggest that UMSBP2 is a chromatin remodeling protein that functions in the regulation of gene expression and plays a role in the control of antigenic variation in T. brucei.


[Diabetic nephropathy and cardiac disease].
Patients in different stages of diabetic nephropathy (DN) frequently present cardiac disease expressed by myocardial ischemia and/or diabetic cardiomyopathy. These changes are already present at early stages of DN, probably even before urinary albumin excretion (UAE) reaches the traditionally diagnostic levels of microalbuminuria. The cardiac changes are responsible for a significant proportion of the increased death rates in patients with DN and can be reduced through multiple intervention on the several risk factors present in these patients. Cardiac disease assessment should ideally be performed in every patient, irrespective of renal status, through specific methods to detect ischemia and myocardial dysfunction, besides routinely performing 24-h ambulatory blood pressure monitoring. In patients with advanced atherosclerosis, other arteries (aorta, carotid, renal) should be evaluated as well. Intensive treatment of arterial hypertension, and use of cardioprotective drugs, correction of the associated dyslipidemia and anemia, and use of antiplatelet agents can reduce the elevated cardiovascular mortality in patients with DN.


Cx40 polymorphism in human atrial fibrillation.
Previous studies have shown that two linked polymorphisms within regulatory regions of the gene for connexin40 (Cx40), at nucleotides -44 (G --> A) and +71 (A --> G) occur in about 7% of the general population. Cx40 is abundant in the atrium, and homozygosity for the linked polymorphisms combined with an SCN5A mutation appeared to be responsible for familial atrial standstill. We hypothesized that these polymorphisms are associated with the atrial electrophysiologic substrate favoring reentrant mechanisms for initiation of atrial fibrillation (AF). Reentry is promoted by spatial dispersion of refractoriness that can be expressed as a coefficient of dispersion (CD). CD was calculated from the standard deviation of 12 local mean fibrillatory intervals recorded at right atrial sites during induced AF in 30 patients without structural heart disease (14 sporadic AF episodes, 16 no AF history). CD <or= 3.0 was considered normal. Cx40 genotypes were determined by DNA sequencing. Mean CD in AF patients was 5.96 +/- 0.70 and without AF 1.59 +/- 0.18 (p < 0.001). Thirteen of fourteen patients with AF had enhanced CD. Carriers of -44 AA genotype had higher CD compared with those with -44 GG genotype (6.37 +/- 1.21 vs. 2.38 +/- 0.39, p = 0.018), whereas heterozygotes showed intermediate values (3.95 +/- 1.38, NS). The rare linked Cx40 polymorphisms are associated with enhanced CD and thus with the substrate for reentry in AF.


Mutations in SLC35A3 cause autism spectrum disorder, epilepsy and arthrogryposis.
The heritability of autism spectrum disorder is currently estimated at 55%. Identification of the molecular basis of patients with syndromic autism extends our understanding of the pathogenesis of autism in general. The objective of this study was to find the gene mutated in eight patients from a large kindred, who suffered from autism spectrum disorder, arthrogryposis and epilepsy. By linkage analysis and exome sequencing, we identified deleterious mutations in SLC35A3 in these patients. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter. In Golgi vesicles isolated from patient fibroblasts the transport of the respective nucleotide sugar was significantly reduced causing a massive decrease in the content of cell surface expressed highly branched N-glycans and a concomitant sharp increase of lower branched glycoforms. Spontaneous mutation in SLC35A3 has been discovered in cattle worldwide, recapitulating the human phenotype with arthrogryposis and additional skeletal defects known as Complex Vertebral Malformation syndrome. The skeletal anomalies in the mutant cattle and in our patients, and perhaps even the neurological symptoms are likely the consequence of the lack of high-branched N-glycans and the concomitant abundance of lower-branched glycoforms at the cell surface. This pattern has previously been associated with growth arrest and induction of differentiation. With this study, we add SLC35A3 to the gene list of autism spectrum disorders, and underscore the crucial importance of UDP-GlcNAc in the regulation of the N-glycan branching pathway in the Golgi apparatus.


Interactions of signal peptides with signal-recognition particle.
The mechanisms whereby isolated or synthetic signal peptides inhibit processing of newly synthesized prolactin in microsome-supplemented lysates from reticulocytes and wheat-germ were investigated. At a concentration of 5 microM, a consensus signal peptide reverses the elongation arrest imposed by the signal-recognition particle (SRP), and at higher concentrations in addition inhibits elongation of both secretory and non-secretory proteins. A photoreactive form of a synthetic signal peptide cross-links under u.v. illumination to the 54 kDa and 68 kDa subunits of SRP, whereas the major cross-linked protein produced after photoreaction of rough microsomes is of 45 kDa. As SRP-mediated elongation arrest is unlikely to be essential for translocation, it is suggested that signal peptides may interact with components other than SRP in the translation system in vitro.


A semiautomated image analysis procedure for the quantification of dental microwear II.
Paleontologists often examine microscopic scratches and pits that form on teeth for indications of the diets of past animals. Most researchers count and measure scratches and pits from photomicrographs to identify microwear patterns for comparison among samples. This paper describes an affordable, available semiautomated image analysis procedure for microwear quantification. An image is downloaded from a scanning electron microscope frame buffer to a microcomputer, and the user identifies microwear features with a mouse-driven pointer. Microwear feature density, dimensions, and orientations are then computed and stored as ASCII files for subsequent data analysis.


3D spatially encoded and accelerated TE-averaged echo planar spectroscopic imaging in healthy human brain.
Several different pathologies, including many neurodegenerative disorders, affect the energy metabolism of the brain. Glutamate, a neurotransmitter in the brain, can be used as a biomarker to monitor these metabolic processes. One method that is capable of quantifying glutamate concentration reliably in several regions of the brain is TE-averaged (1) H spectroscopic imaging. However, this type of method requires the acquisition of multiple TE lines, resulting in long scan durations. The goal of this experiment was to use non-uniform sampling, compressed sensing reconstruction and an echo planar readout gradient to reduce the scan time by a factor of eight to acquire TE-averaged spectra in three spatial dimensions. Simulation of glutamate and glutamine showed that the 2.2-2.4 ppm spectral region contained 95% glutamate signal using the TE-averaged method. Peak integration of this spectral range and home-developed, prior-knowledge-based fitting were used for quantitation. Gray matter brain phantom measurements were acquired on a Siemens 3 T Trio scanner. Non-uniform sampling was applied retrospectively to these phantom measurements and quantitative results of glutamate with respect to creatine 3.0 (Glu/Cr) ratios showed a coefficient of variance of 16% for peak integration and 9% for peak fitting using eight-fold acceleration. In vivo scans of the human brain were acquired as well and five different brain regions were quantified using the prior-knowledge-based algorithm. Glu/Cr ratios from these regions agreed with previously reported results in the literature. The method described here, called accelerated TE-averaged echo planar spectroscopic imaging (TEA-EPSI), is a significant methodological advancement and may be a useful tool for categorizing glutamate changes in pathologies where affected brain regions are not known a priori. Copyright © 2016 John Wiley & Sons, Ltd.


Opposite environmental and genetic influences on body size in North American Drosophila pseudoobscura.
Populations of a species often differ in key traits. However, it is rarely known whether these differences are associated with genetic variation and evolved differences between populations, or are instead simply a plastic response to environmental differences experienced by the populations. Here we examine the interplay of plasticity and direct genetic control by investigating temperature-size relationships in populations of Drosophila pseudoobscura from North America. We used 27 isolines from three populations and exposed them to four temperature regimes (16°C, 20°C, 23°C, 26°C) to examine environmental, genetic and genotype-by-environment sources of variance in wing size. By far the largest contribution to variation in wing size came from rearing temperature, with the largest flies emerging from the coolest temperatures. However, we also found a genetic signature that was counter to this pattern as flies originating from the northern, cooler population were consistently smaller than conspecifics from more southern, warmer populations when reared under the same laboratory conditions. We conclude that local selection on body size appears to be acting counter to the environmental effect of temperature. We find no evidence that local adaptation in phenotypic plasticity can explain this result, and suggest indirect selection on traits closely linked with body size, or patterns of chromosome inversion may instead be driving this relationship.


Complement and Coagulation Cascades are Potentially Involved in Dopaminergic Neurodegeneration in α-Synuclein-Based Mouse Models of Parkinson's Disease.
Parkinson's disease (PD) is the second most common neurodegenerative disorder that results in motor dysfunction and, eventually, cognitive impairment. α-Synuclein protein is known as a central protein to the pathophysiology of PD, but the underlying pathological mechanism still remains to be elucidated. In an effort to understand how α-synuclein underlies the pathology of PD, various PD mouse models with α-synuclein overexpression have been developed. However, systemic analysis of the brain proteome of those mouse models is lacking. In this study, we established two mouse models of PD by injecting α-synuclein preformed fibrils (PFF) or by inducing overexpression of human A53T α-synuclein to investigate common pathways in the two different types of the PD mouse models. For more accurate quantification of mouse brain proteome, the proteins were quantified using the method of stable isotope labeling with amino acids in mammals . We identified a total of 8355 proteins from the two mouse models; ∼6800 and ∼7200 proteins from α-synuclein PFF-injected mice and human A53T α-synuclein transgenic mice, respectively. Through pathway analysis of the differentially expressed proteins common to both PD mouse models, it was discovered that the complement and coagulation cascade pathways were enriched in the PD mice compared to control animals. Notably, a validation study demonstrated that complement component 3 (C3)-positive astrocytes were increased in the ventral midbrain of the intrastriatal α-synuclein PFF-injected mice and C3 secreted from astrocytes could induce the degeneration of dopaminergic neurons. This is the first study that highlights the significance of the complement and coagulation pathways in the pathogenesis of PD through proteome analyses with two sophisticated mouse models of PD.


A realist evaluation of loneliness interventions for older people.
the aim of this study was to develop a programme theory to inform the design of loneliness interventions and guide any future evaluations. we undertook a mixed-method evaluation, informed by the realist approach, of different approaches to loneliness in one health and social care system in the East Midlands, UK. We used a combination of documentary analysis, interviews/focus groups with service providers and users and quantitative analysis to develop an initial programme theory. common aims of local interventions included enhancing social connectivity, providing emotional support and advice/information; recurring interventions included social activities, emotional support, advice and information, lunch clubs, learning new skills and practical support. None were robustly evaluated.Fifty-six service user or providers were involved in interviews or focus groups, which highlighted the causes of loneliness, preferred services, access to services, thoughts about intervention configuration and desired outcomes from services.The themes emerging from the interviews/focus groups from both service provider and service user perspectives were combined with all of the previous emerging data to create an overarching programme theory. Statements were constructed to allow service providers to think about which interventions might be useful to achieve specific outcomes in different contexts. the causes and consequences of loneliness vary widely between individuals, so a personalised approach is required to identify the causes and potential solutions. This study provides some high-level principles that can help commissioners and providers to tailor interventions to the individual needs of service users.


Mechanochromic Wide-Spectrum Luminescence Based on a Monoboron Complex.
A reversible mechanochromic luminescent material based on a simple tetrahedral monoboron complex (B-1) is described. Interestingly, in addition to amorphous powders (P), the compound could exist in three unique crystal states (A, B, and C), showing efficient green-to-red luminescent colors, which is a result of wane and wax of dual emissions of the compound. Surprisingly, one of the emissions increases significantly with increasing temperature, fully offsetting the quenching effect of temperature-assisted internal conversion process. The four states are fully interconvertible through grinding and heating, allowing color writing/painting with a single ink.


Effects of dietary supplementation of fulvic acid on lipid metabolism of finishing pigs.
The experiment was conducted to investigate the effects of dietary supplementation of fulvic acid on lipid metabolism of finishing pigs. One hundred eighty crossbred barrows (Landrace × Yorkshire, 60 ± 2.5 kg) were randomly allotted to 5 dietary treatments (36 pigs/treatment) and fed a basal diet supplemented with 0, 0.2%, 0.4%, 0.6%, and 0.8% fulvic acid for 42 d. Thirty pigs (6 pigs/treatment) were slaughtered at the end of the experiment. Blood samples and adipose tissue were collected for determination of blood parameters and lipid metabolic enzymes. The results showed that compared with the control group, dietary supplementation of 0.2%, 0.4%, and 0.6% fulvic acid significantly reduced mean backfat thickness of pigs (P < 0.05). The serum concentrations of low-density lipoprotein, leptin, growth hormone, insulin, and triiodothyronine were significantly increased by adding fulvic acid in diets (P < 0.05). With the raised concentration of dietary fulvic acid, hormone sensitive lipase (HSL) activity was significantly increased (P < 0.05), and lipoprotein lipase (LPL) activity was significantly decreased (P < 0.05) in adipose tissue. In conclusion, dietary supplementation of fulvic acid reduced the mean backfat thickness of pigs. This change related to the increased activity of HSL and the decreased activity of LPL in adipose tissue.


Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase.
In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.


The elementome of calcium-based urinary stones and its role in urolithiasis.
Urolithiasis affects around 10% of the US population with an increasing rate of prevalence, recurrence and penetrance. The causes for the formation of most urinary calculi remain poorly understood, but obtaining the chemical composition of these stones might help identify key aspects of this process and new targets for treatment. The majority of urinary stones are composed of calcium that is complexed in a crystalline matrix with organic and inorganic components. Surprisingly, mitigation of urolithiasis risk by altering calcium homeostasis has not been very effective. Thus, studies to identify other therapeutic stone-specific targets, using proteomics, metabolomics and microscopy techniques, have been conducted, revealing a high level of complexity. The data suggest that numerous metals other than calcium and many nonmetals are present within calculi at measurable levels and several have distinct distribution patterns. Manipulation of the levels of some of these elemental components of calcium-based stones has resulted in clinically beneficial changes in stone chemistry and rate of stone formation. The elementome--the full spectrum of elemental content--of calcium-based urinary calculi is emerging as a new concept in stone research that continues to provide important insights for improved understanding and prevention of urinary stone disease.


The Novel Landscape of Long Non-Coding RNAs in Response to Human Foamy Virus Infection Characterized by RNA-Seq.
Human foamy virus (HFV) is a complex and unique retrovirus with the longest genomes among retroviruses that are used as vectors for gene therapy. Long non-coding RNAs (lncRNAs) are regarded as key regulators that are involved in diverse biological processes during viral infection. However, the role of lncRNAs in HFV infection remains unknown. In this study, we utilized next-generation sequencing to first characterize lncRNAs in 293T cells after HFV infection, evaluating length distribution, exon number distribution, volcano picture, and lncRNA class distribution. We identified 11,336 lncRNAs (4,729 upregulated lncRNAs and 6,588 downregulated lncRNAs) and 61,367 mRNAs (30,133 upregulated mRNAs and 31,220 downregulated mRNAs), which were differentially expressed in the HFV-infected 293T cells. Subsequently, six differentially expressed lncRNAs characterized using RNA-seq were confirmed by quantitative real-time polymerase chain reaction assays. Interestingly, Gene Ontology (GO)/Gene Ontology Tree Machine (GOTM) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analyses indicated that positive regulation of interleukin 8 (IL8) production and cytokine-cytokine receptor interaction might be involved in the functional enrichment of lncRNAs. Moreover, cis-acting and trans-acting regulatory networks show that NR_028036 may target the fas gene in a cis-acting manner and that ENST00000354838 may target the IL18 gene in a trans-acting manner. Overall, these results not only provide novel insights into the relationship between HFV and lncRNAs in the host response to infection but also have implications for the future wider application of HFV as a vector.


A nationwide validation of the prognostic impact of pathological response and the distribution of recurrence patterns in responders after neoadjuvant chemotherapy for esophageal squamous cell carcinoma.
We previously reported that postoperative recurrence in responders occurred in the regional field mostly as a solitary lesion without distant failure. However, further validation is necessary due to the low percentage of pathological responders, especially those with pCR. This study aimed to validate the prognostic impact of pathological response and the distribution of residual tumors in pathological responders using a nationwide database from 85 Japanese esophageal centers. We retrospectively reviewed patients with esophageal squamous cell carcinoma (ESCC) who underwent subtotal esophagectomy at 85 authorized institutes for esophageal cancer between 2010 and 2015. The recurrence free survival (RFS), overall survival (OS), and recurrent tumor patterns were compared among the pathological responses. Of 4781 patients initially enrolled, 3840 were selected for subsequent analysis, including 237 patients with pathological complete response (pCR, 6%). The RFS and OS were significantly correlated with pathological response. When the recurrence pattern was classified into regional or distant recurrence, the incidence of distant failure was significantly lower in patients with pCR in cT1/2. Three percent of all patients with pCR in cT1/2 encountered postoperative recurrence in distant organs. The prognostic impact of pathological response was reproduced in the nationwide data. pCR in ESCC patients with cT1/2 provides a favorable prognosis with less incidence of distant failure. This finding may contribute to selecting appropriate candidates for an organ preservation approach based on the response to induction therapy.


Anatomically informed basis functions in multisubject studies.
We describe the use of anatomically informed basis functions (AIBF) in the analysis of multisubject functional imaging studies. AIBF are used to specify an anatomically informed spatial model that embodies anatomical knowledge for the statistical analysis of neuroimaging data. In a previous communication, we showed how AIBF can be used to incorporate prior anatomical constraints in single subject functional magnetic resonance image (fMRI) analyses to augment their anatomical precision. In this paper, we extend AIBF such that it can be applied to multisubject studies using fMRI or PET. The key concept is that, after spatial normalization, a canonical cortical surface can be used to generate a forward model of signal sources for all subjects. By estimating the hemodynamic signal in this canonical AIBF-space and then projecting it back into the voxel-space, one effectively extracts functional activity that is smooth, within and only within, the cortical sheet while attenuating other components unrelated to the physiological process of interest. The ensuing procedure can be considered as a highly non-stationary, anisotropic anatomically informed [de]convolution or smoothing. It is shown that this procedure offers various advantages compared to existing conventional methods for the analysis of multisubject studies, in particular it is more sensitive to underlying activations.


Thyroid transcription factor 1 and p63 expression is associated with survival outcome in patients with non-small cell lung cancer treated with erlotinib.
While erlotinib is primarily administered to patients with non-small cell lung cancer with sensitizing epidermal growth factor receptor (EGFR) mutations, it is also prescribed to patients with wild type (wt) EGFR in higher lines of treatment. However, there is no predictive marker for erlotinib efficacy in patients with EGFR wt. Certain immunohistochemical (IHC) parameters, including thyroid transcription factor 1 (TTF1) and p63, have been reported to indicate predictive power in patients with EGFR wt. The present study focused on retrospective data from the University Hospital in Pilsen using the TULUNG register. TTF1 and p63 expression data were extracted from the hospital information system and merged with registry data to calculate progression-free survival (PFS) and overall survival (OS) rates. A cohort of 345 patients with adenocarcinoma (ADC) or squamous cell carcinoma (SCC) exhibited similar erlotinib efficacies when TTF1 and p63 were ignored. However, significant differences were reported in PFS and OS rates of a subgroup of 126 patients where TTF1 and p63 parameters were known. In a univariate analysis, group A (ADC TTF1+/p63-) achieved PFS of 2.6 months, group B (SSC TTF1-/p63+) 1.9 months and group C (did not fit into groups A or B, i.e., ADC TTF1-/p63+ or SCC TTF1+/p63-) 1.4 months (P=0.006). Median OS was 14.2, 19.1 and 5.3 months for A, B and C, respectively (P=0.002). Furthermore, a multivariate analysis demonstrated IHC markers to be the only significant parameters for PFS and OS. Group C had a negative prognostic factor for PFS [hazard ratio (HR), 1.812; P=0.02] and OS (HR=2.367; P=0.01). In conclusion, patients with EGFR wt and lung carcinomas without TTF1 and p63 expression typical for ADC (TTF1+/p633-) or SCC (TTF1-/p63+) do not appear to be suitable candidates for erlotinib treatment.


Pseudoinvasion of benign squamous epithelium following cervical biopsy: a pseudoneoplastic phenomenon mimicking invasive squamous carcinoma.
To describe an unusual and hitherto unreported pseudoneoplastic phenomenon that is characterised by the entrapment of benign squamous epithelium following cervical loop excision or punch biopsy and that may mimic invasive squamous carcinoma. The authors report six cases in patients aged 25-45 years in whom benign squamous epithelium is incorporated within the cervical stroma as a result of the healing process following prior loop excision (four cases) or punch biopsy (two cases). The entrapped nests of squamous epithelium typically have a hypereosinophilic appearance and are associated with a granulation tissue-like and inflammatory stromal response and sometimes with retraction artefact mimicking lymphovascular permeation. The overall morphological appearances, especially in the context of a prior or current diagnosis of cervical intraepithelial neoplasia, may result in misdiagnosis as invasive squamous carcinoma. Pathologists should be aware of this pseudoneoplastic artefact so that an erroneous diagnosis of malignancy is not made.


Definition of 'close contacts' in leprosy studies: protocol for a scoping review.
Despite difficulties to document transmission pathways (assumed to be airborne), increased risk of leprosy infection has been shown for individuals living in close contact with patients. However, variations in the concept of 'close contacts' are used in different settings and studies. We conduct this review to identify criteria of space (location, geographical variables, distance, indoor vs outdoor), time (including frequency and duration), physical exposure (skin to skin, sexual), and relationship (familial, occupational, social) involved in the definition of 'close contacts' in leprosy studies. We expect this review to provide an overview of the (lack of) conceptualization of this term and its variations across settings. Primary studies and reviews are eligible for inclusion in this review. The main source of records will be the PubMed interface. Secondary searches will be conducted in Google Scholar, as well as through the reference lists of selected publications. The search strategy is based on the combination of the condition of interest (leprosy) and the concept under study ('contact'). The findings of this review will be presented using thematic narrative synthesis, tables, and figures. The protocol is written in line with the Prisma Extension for Scoping reviews (PRISMA-ScR).


[Lithiasis of the transplanted kidney: therapeutical potential].
The formation of calculus in the transplanted kidney is an uncommon complication. Metabolic derangements, infectious or obstructive processes, factors related with the surgical technique and the presence of ureteral catheters have been implicated in its etiopathogenesis. The therapeutic possibilities have changed in the last decade. The different factors related with stone formation in the transplanted kidney, the indications and treatment utilized in each case are analyzed. We analyzed the cases of lithiasis following renal transplantation in our series of 800 renal transplants. The metabolic anomalies and other associated lithogenic factors in 5 cases that required treatment are described. All 5 patients were treated by ESWL. Complete resolution of the lithiasis was achieved in 4 cases whose kidney graft is currently stone free with preserved renal function, except one patient with hyperuricemia and hyperuricosuria who is again on hemodialysis for chronic rejection. In the fifth case fragmentation of the caliceal stone was not achieved after 4 sessions of ESWL. Subsequem ultrasound control evaluations have disclosed no changes in stone size or location. In our view, the approach to renal lithiasis in the transplanted kidney is similar to that of patients with solitary kidney, although stone size for treatment by ESWL should be limited to 2 cms. If stone size is between 1 and 2 cms, placement of a double-J catheter prior to ESWL is recommended, whenever possible. For stones larger than 2 cms, percutaneous nephrolithotomy is more effective and has less complications. Surgery is reserved for those patients in whom these techniques are unamenable or have failed.


Effect of Avocado/Soybean Unsaponifiables (ASU) on Osseointegration in Rats with Experimental Arthritis.
To evaluate the influence of the use of avocado/soybean unsaponifiables (ASU) on osseointegration of implants in animals with experimental arthritis. One hundred twenty rats were randomly divided into four groups: CTR, healthy animals and saline solution administration; ASU, healthy animals and ASU administration; ART, arthritic animals and saline solution administration; and ART/ASU, arthritic animals and ASU administration. The solutions were administered daily by gavage, beginning 7 days before the surgical procedures until the completion of the experimental period (15, 30, and 60 days after the placement of the implants in the tibia). The osseointegration of the implants was evaluated by histometric analysis (bone-to-implant contact [% BIC], bone area between the threads [% BBT]) and biomechanical analysis. Microcomputed tomography (micro-CT) analysis was used to assess bone volume in the vicinity of the implant. Immunohistochemistry analysis was performed to assess the expression of osteocalcin and transforming growth factor beta 1 (TGF-β1). The ART/ASU group showed a decreased percentage of bone in the area around the implant compared with the ASU and ART groups (15 and 30 days). The ART/ASU group showed increased removal torque values (30 days) and % BIC and % BBT (30 to 60 days) compared with the ART group. The ASU group had increased % BIC values compared with the ART and CTR groups (60 days). The CTR group had the highest expression of osteocalcin, while the ASU group presented the highest expression of TGF-β1 at 60 days. The ASU administration improved the osseointegration, particularly in animals with induced arthritis.


From the Soft to the Hard: Changes in Microchemistry During Cell Wall Maturation of Walnut Shells.
The walnut shell is a hard and protective layer that provides an essential barrier between the seed and its environment. The shell is based on only one unit cell type: the polylobate sclerenchyma cell. For a better understanding of the interlocked walnut shell tissue, we investigate the structural and compositional changes during the development of the shell from the soft to the hard state. Structural changes at the macro level are explored by X-ray tomography and on the cell and cell wall level various microscopic techniques are applied. Walnut shell development takes place beneath the outer green husk, which protects and delivers components during the development of the walnut. The cells toward this outer green husk have the thickest and most lignified cell walls. With maturation secondary cell wall thickening takes place and the amount of all cell wall components (cellulose, hemicelluloses and especially lignin) is increased as revealed by FTIR microscopy. Focusing on the cell wall level, Raman imaging showed that lignin is deposited first into the pectin network between the cells and cell corners, at the very beginning of secondary cell wall formation. Furthermore, Raman imaging of fluorescence visualized numerous pits as a network of channels, connecting all the interlocked polylobate walnut shells. In the final mature stage, fluorescence increased throughout the cell wall and a fluorescent layer was detected toward the lumen in the inner part. This accumulation of aromatic components is reminiscent of heartwood formation of trees and is suggested to improve protection properties of the mature walnut shell. Understanding the walnut shell and its development will inspire biomimetic material design and packaging concepts, but is also important for waste valorization, considering that walnuts are the most widespread tree nuts in the world.


[Clinical and epidemiological study of multiple sclerosis in Volgograd city].
Descriptive and analytical study of multiple sclerosis (MS) has been first-ever carried out in population of Volgograd city, South region of Russian Federation. At the period of the study (1996-2000) the population was estimated as 814 100 adult persons. An average age-adjusted level of MS prevalence was 31.9 per 100,000, MS incidence--9.8, mortality 1,8. Thus, the city is at a moderate risk for MS, however the incidence of the disease appeared to be rather high that indicates a further increase of MS patients number in this population. The highest level of MS prevalence was registered in two districts with poor ecological characteristics and numerous industries: Krasnoarmeysky (52.4 per 100,000 persons) and Krasnooktiabrsky (46.4). An analytical case-control study included data collected from 178 pairs (73.6% female) of MS patients and controls matched for age, sex and ethnic origin. Significant differences between patients and controls were detected as follows: patients more often had blue eyes as compared to dark ones; in the patient group mother's age at birth was above 30 years; patients more often lived near chemical and/or oil factories at age before 15 years; they more often reported a presence of stress factors in the family and chronic tonsillitis. Association with other factors, including infections, nutrition, acute and chronic diseases, poisoning, traumas, family history of different diseases, contact with animals etc, has not been found. A possible association between environmental factors and features of MS course were analyzed. Living near chemical factories at age before 15 was associated with more active MS course, i.e. high frequency of progressive course, short period from MS onset to confirmed EDSS=3, short duration of first remission. The same, though less significant, influence may exert the presence of herpes infection and chronic tonsillitis at age under 15.


Morphologic analysis of gastroesophageal reflux diseases in patients after distal gastrectomy.
The precise mechanisms that cause gastroesophageal reflux after distal gastrectomy remain unclear. We analyzed the endoscopic findings of the cardia and the position of the remnant stomach, which are related to gastroesophageal reflux. We retrospectively examined the records of 45 patients with Billroth I (B-I) and 39 patients with Roux-en-Y (R-Y) procedure for gastric cancer. Esophagitis was evaluated by the Los Angeles (LA) classification. The endoscopic findings of hiatus hernia were classified according to the criteria of the Keio Cancer Detection Center form (K-form). The valvular appearance of the cardia was classified according to V-grades. The height of the remnant stomach was measured on computed tomography scans. The postoperative findings of esophagitis in the B-I group were significantly worse than the preoperative findings, but no significant change was observed in the R-Y group. The postoperative V-grades and K-forrn findings in the B-I group were worse than their preoperative findings. In the R-Y group, however, there was no significant change in the V-grades or K-form findings. In addition, the height of the remnant stomach was significantly higher in the B-I group than in the R-Y group. This study suggested that an aggravated cardia is associated with the B-I procedure and that the position of the remnant stomach may therefore play an important role in the occurrence of postoperative reflux esophagitis. In contrast, the R-Y operation was shown to preserve the cardia and the position of the remnant stomach better. As a result, R-Y might help prevent not only duodenogastric reflux but also gastroesophageal reflux.


Channelling of dipolar molecules through carbon nanotubes.
We study the dynamic polarization of carbon nanotubes caused by the propagation of fast electric dipoles under channelling conditions. We specifically analyse the position and orientation dependences of the dipole self-energy, stopping force, and the torque about the dipole centre. It is found that a dipole is strongly attracted to the nanotube wall and shows a tendency to orient itself perpendicular to the direction of motion. The stopping force shows more complex behaviour, but is generally found to be larger close to the nanotube wall and when oriented in the perpendicular direction at higher speeds.


Transcriptomic changes and signalling pathways induced by arsenic stress in rice roots.
Arsenic (As) is considered the most common toxic metalloid, but its molecular mode of action is not well understood. We investigated whether arsenate [As(V)] can induce intracellular reactive oxygen species production and calcium oscillation in rice roots. To better understand the molecular basis of plant cell responses to As, we performed a large-scale analysis of the rice transcriptome during As(V) stress. As(V) induced genes involved in abiotic stress, detoxification pathways and secondary metabolic process. Genes involved in secondary cell wall biogenesis, cell cycle and oligopeptide transport were mainly downregulated. Genes encoding signalling components such as receptor-like cytoplasmic kinases protein kinase, APETALA2/ethylene response factor, heat shock factor, MYB and zinc-finger protein expressed in inflorescence meristem transcription factors were increased in expression. The expression of GARP-G2-like and C3H transcription factors was specifically modulated by As(V) stress. The predominant families of As(V)-regulated transporters belonged to the ATP-binding cassette superfamily and telurite-resistance/dicarboxylate transporters. Several factors involved in signaling, such as mitogen-activated protein kinase (MAPK), MAPK kinase kinase and calcium-dependent protein kinase (CDPK), were also upregulated. Moreover, As(V) markedly increased the activity of MAPKs and CDPK-like kinases, and CDPK and NADPH oxidases were involved in As-induced MAPK activation. Further characterization of these As(V)-responsive genes and signalling pathways may help better understand the mechanisms of metalloid uptake, tolerance and detoxification in plants.


Lysophosphatidylcholine-induced lysis of erythrocytes in Duchenne and myotonic dystrophies and in Huntington's disease.
Erythrocytes from Duchenne dystrophy patients lysed more readily than red cells from age-matched normal boys when lysophosphatidylcholine (LPC) concentrations that caused 50% lysis were compared. Erythrocytes from myotonic dystrophy patients appeared to be more resistant than cells from age-matched normal adults at certain medium LPC concentrations. Erythrocytes from patients with Huntington's disease showed no significant differences from erythrocytes of normal adults. Thus, the manner in which erythrocytes respond to LPC may reflect the putative membrane alterations in these diseases. Inhibition of LPC-induced lysis by 0.1 mM dipyridamole was observed in all groups. Since this agent did not inhibit LPC lysis at 0 degrees C, its action at 37 degrees C could be related to activation of a membrane enzyme. On the other hand, dipyridamole decreased osmotic fragility at 0 degrees C and 37 degrees C indicating that a physical change in membrane structure may be the primary alteration produced by this agent.


Bacteriome and mycobiome associations in oral tongue cancer.
Squamous cell carcinoma of the oral (mobile) tongue (OMTC), a non-human papilloma virus-associated oral cancer, is rapidly increasing without clear etiology. Poor oral hygiene has been associated with oral cancers, suggesting that oral bacteriome (bacterial community) and mycobiome (fungal community) could play a role. While the bacteriome is increasingly recognized as an active participant in health, the role of the mycobiome has not been studied in OMTC. Tissue DNA was extracted from 39 paired tumor and adjacent normal tissues from patients with OMTC. Microbiome profiling, principal coordinate, and dissimilarity index analyses showed bacterial diversity and richness, and fungal richness, were significantly reduced in tumor tissue (TT) compared to their matched non-tumor tissues (NTT, P<0.006). Firmicutes was the most abundant bacterial phylum, which was significantly increased in TT compared to NTT (48% vs. 40%, respectively; P=0.004). Abundance of Bacteroidetes and Fusobacteria were significantly decreased in TT compared to matched NTT (P≤0.003 for both). Abundance of 22 bacterial and 7 fungal genera was significantly different between the TT and NTT, including Streptococcus, which was the most abundant and significantly increased in the tumor group (34% vs. 22%, P<0.001). Abundance of fungal genus Aspergillus in TT correlated negatively with bacteria (Actinomyces, Prevotella, Streptococcus), but positively with Aggregatibacter. Patients with high T-stage disease had lower mean differences between TT and NTT compared with patients with low T-stage disease (0.07 vs. 0.21, P=0.04). Our results demonstrate differences in bacteriome and mycobiome between OMTC and their matched normal oral epithelium, and their association with T-stage.


A time for change: an exploration of attitudes toward part-time work in academia among women internists and their division chiefs.
Increasing numbers of clinicians desire part-time work, and many will opt out of academic medicine if the barriers to part-time work are too great. Purposeful sampling was used to investigate the experiences of part-time academic physicians and their division leaders to understand (1) how each identified the negative and positive consequences of part-time work, and (2) how each conceptualize part-time work. In 2004, the authors interviewed the Society of General Medicine Horn Scholars Program applicants and their division chiefs from the 2001 and 2004 award cycles and performed a qualitative analysis of the one- to two-hour audiotaped interviews. Seven out of nine eligible applicants and six of seven division chiefs participated. All applicants were female junior faculty clinician educators in academic internal medicine from seven institutions. All division chiefs were male full-time faculty members. Both applicants and division chiefs identified multiple specific negative and positive consequences of part-time work. Analysis of interviews suggested that both groups tended to frame part-time work as a decision to "work less" or to "work differently." Self-reflection and articulation of values helped some faculty determine where they derive the greatest happiness and fulfillment personally and professionally. As more academics seek work-life balance and consider part-time work as a tool to achieve that balance, academic medicine will be challenged to develop creative models for integrating successful part-time physicians, or it will lose that segment of the workforce. This study's findings suggest that one such model may require that physicians and their leaders reconceptualize work altogether.


Marginal Time-Dependent Causal Effects in Mediation Analysis With Survival Data.
The main aim of mediation analysis is to study the direct and indirect effects of an exposure on an outcome. To date, the literature on mediation analysis with multiple mediators has mainly focused on continuous and dichotomous outcomes. However, the development of methods for multiple mediation analysis of survival outcomes is still limited. Here we extend to survival outcomes a method for multiple mediation analysis based on the computation of appropriate weights. The approach considered has the advantages of not requiring specific models for mediators, allowing nonindependent mediators of any nature, and not relying on the assumption of rare outcomes. Simulation studies show good performance of the proposed estimator in terms of bias and coverage probability. The method is further applied to an example from a published study on prostate cancer mortality aimed at understanding the extent to which the effect of DNA methyltransferase 3b (DNMT3b) genotype on mortality was explained by DNA methylation and tumor aggressiveness. This approach can be used to quantify the marginal time-dependent direct and indirect effects carried by multiple indirect pathways, and software code is provided to facilitate its application.


Understanding patient-reported outcome measures in Huntington disease: at what point is cognitive impairment related to poor measurement reliability?
Symptom progression in Huntington disease (HD) is associated with cognitive decline which may interfere with the self-report of symptoms. Unfortunately, data to support or refute the psychometric reliability of patient-reported outcomes (PROs) as HD progresses are limited. This is problematic given that PROs are increasingly recognized as important measures of efficacy for new treatments. We examined PRO data from the HDQLIFE Measurement System (Speech Difficulties; Swallowing Difficulties; Chorea) in 509 individuals with premanifest, early-stage, or late-stage HD. Clinician-administered assessments of motor functioning (items from the UHDRS) and standardized objective assessments of cognition (Stroop, Symbol Digit Modalities) were also collected. We examined item bias using differential item functioning (DIF) across HD stage (premanifest, early-, late-) and relative to cognitive performance. We also examined the correlations between self-report and clinician ratings. Regression models that considered total cognitive ability were utilized to determine psychometric reliability of the PROs. Most PRO items were free from DIF for both staging and cognition. There were modest correlations between PROs and clinician report (ranged from - 0.40 to - 0.60). Modeling analyses indicated that psychometric reliability breaks down with poorer cognition and more progressed disease stage; split-half reliability was compromised (i.e., split-half reliability < 0.80) when scores were < 136 for Chorea, < 109 for Speech Difficulties, and < 179 for Swallowing Difficulties. Results indicate that the psychometric reliability of PROs can be compromised as HD symptoms progress and cognition declines. Clinicians should consider PROs in conjunction with other types of assessments when total cognition scores exceed critical thresholds.


[The practice guideline 'Depressive disorder' (first revision) from the Dutch College of General Practitioners; a response from the perspective of psychiatry].
The first revision of the Dutch guidelines for the treatment of depressive disorders by general practitioners adapted the international psychiatric nomenclature for the diagnosis of depressive disorders. This enhances collaboration between general practitioners and psychiatrists. The use of selective serotonin reuptake inhibitors in this guideline is an advantage compared to the earlier version. The distinction between the indication for cognitive therapy and for antidepressant treatment is not very clear in this guideline. Overall the guideline represents a scientific and conservative stance in the treatment of depressed patients, which is commendable. The use of target plasma level concentrations with tricyclic antidepressants, better guidelines for the use of antidepressants or cognitive therapy, and the use of severity rating scales during treatment could further improve the next version of this guideline.


Food availability and predation risk, rather than intrinsic attributes, are the main factors shaping the reproductive decisions of a long-lived predator.
Deciphering the causes of variation in reproductive success is a fundamental issue in ecology, as the number of offspring produced is an important driver of individual fitness and population dynamics. Little is known, however, about how different factors interact to drive variation in reproduction, such as whether an individual's response to extrinsic conditions (e.g. food availability or predation) varies according to its intrinsic attributes (e.g. age, previous allocation of resources towards reproduction). We used 29 years of reproductive data from marked female tawny owls and natural variation in food availability (field vole) and predator abundance (northern goshawk) to quantify the extent to which extrinsic and intrinsic factors interact to influence owl reproductive traits (breeding propensity, clutch size and nest abandonment). Extrinsic and intrinsic factors appeared to interact to affect breeding propensity (which accounted for 83% of the variation in owl reproductive success). Breeding propensity increased with vole density, although increasing goshawk abundance reduced the strength of this relationship. Owls became slightly more likely to breed as they aged, although this was only apparent for individuals who had fledged chicks the year before. Owls laid larger clutches when food was more abundant. When owls were breeding in territories less exposed to goshawk predation, 99·5% of all breeding attempts reached the fledging stage. In contrast, the probability of breeding attempts reaching the fledging stage in territories more exposed to goshawk predation depended on the amount of resources an owl had already allocated towards reproduction (averaging 87·7% for owls with clutches of 1-2 eggs compared to 97·5% for owls with clutches of 4-6 eggs). Overall, our results suggested that changes in extrinsic conditions (predominantly food availability, but also predator abundance) had the greatest influence on owl reproduction. In response to deteriorating extrinsic conditions (fewer voles and more goshawks), owls appeared to breed more frequently, but allocated fewer resources per breeding attempt. However, intrinsic attributes also appeared to have a relatively small influence on how an individual responded to variation in extrinsic conditions, which indicates that owl reproductive decisions were shaped by a complex series of extrinsic and intrinsic trade-offs.


Application of ionic liquids based microwave-assisted simultaneous extraction of carnosic acid, rosmarinic acid and essential oil from Rosmarinus officinalis.
An ionic liquid based microwave-assisted simultaneous extraction and distillation (ILMSED) method has been developed for the effective extraction of carnosic acid (CA), rosmarinic acid (RA) and essential oil (EO) from Rosmarinus officinalis. A series of 1-alkyl-3-methylimidazolium ionic liquids differing in composition of anion and cation were evaluated for extraction yield in this work. The results obtained indicated that the anions and cations of ionic liquids had influences on the extraction of CA and RA, 1.0M 1-octyl-3-methylimidazolium bromide ([C8mim]Br) solution was selected as solvent. In addition, the ILMSED procedures for the three target ingredients were optimized and compared with other conventional extraction techniques. ILMSED gave the best result due to the highest extraction yield within the shortest extraction time for CA and RA. The novel process developed offered advantages in term of yield and selectivity of EO and shorter isolation time (20 min in comparison of 4h of hydrodistillation), and provides a more valuable EO (with high amount of oxygenated compounds). The microstructures and chemical structures of rosemary samples before and after extraction were also investigated. Moreover, the proposed method was validated by the stability, repeatability and recovery experiments. The results indicated that the developed ILMSED method provided a good alternative for the both extraction of non-volatile compounds (CA and RA) and EO from rosemary as well as other herbs.


Ablation of Dnmt3b in chondrocytes suppresses cell maturation during embryonic development.
DNA methylation is a major mode of epigenetic regulation in the mammalian genome and is essential for embryonic development. The three catalytic DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, catalyze the methylation of cytosine. Dnmt3b is highly expressed in chondrocytes and global knockout of Dnmt3b led to skeletal deformations and embryonic lethality, suggesting an essential role of Dnmt3b in endochondral bone formation. To further define the role of Dnmt3b in skeletal development, Dnmt3b was deleted in Col2 positive chondrocyte lineage cells. Both axial and appendicular skeletal size were reduced and bone mineralization was delayed in Col2Cre+ ;Dnmt3bf/f (Dnmt3bCol2 ) mice at E14.5 and E18.5. While Alcian Blue Hematoxylin/Orange G (ABH/OG) staining showed normal chondrocyte columns in control growth plates, the length of hypertrophic chondrocyte zone and type X collagen expression were decreased in E18.5 growth plates from Dnmt3bCol2 mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3bCol2 growth plates was not secondary to altered chondrocyte apoptosis or proliferation. Complementary in vitro experiments were performed on primary sternal chondrocytes isolated from control and Dnmt3bCol2 mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3bCol2 chondrocytes. In addition, alkaline phosphatase (ALP) and Alizarin Red staining confirmed that Dnmt3b deletion in chondrocytes delays in vitro chondrocyte hypertrophic differentiation and matrix mineralization. Mechanistically, Dnmt3b gene deletion resulted in decreased BMP signaling through reduction of Smad1 phosphorylation. These findings show that epigenetic factor, Dnmt3b is necessary for normal chondrocyte hypertrophic maturation and limb development.


[The tyrosine kinase activity of the internalized epidermal growth factor receptor in A-431 cells].
The interaction between epidermal growth factor (EGF) and specific membrane receptors in A-431 cells results in stimulation of its tyrosine kinase (TK) activity. The intrinsic tyrosine kinase phosphorylates both the receptor itself and different intracellular substrates. Upon EGF binding, clustering and internalization of EGF-receptor (EGF-R) complexes are observed. In the present study by incubating A-431 cells with EGF at 4 or at 37 degrees C, eluting surface-bound EGF we were able to compare the TK-activity of internalized EGF receptors to those localized on the plasma membrane. By the method of the phosphorylation of exogenic substrate poly (Glu/Tyr) 4:1 in vitro it was shown that the total TK-activity was equal to 3 microM PO4- incorporated/mg protein/min. Under conditions, when 40% EGF-R complexes were internalized their activity consisted of 46% of the total after inactivation 60% surface-bound receptors. We draw a conclusion that the TK-activity of EGF-receptors is remained during their internalization.


Localization of nicotine-14C, cotinine-14C, and nicotine-1'-N-oxide-14C in tissues of the mouse.
The distrubutions of nicotine-14C (2'- or methyl-labeled), cotinine-14C, and nicotine-1'-N-oxide-14C were studied by whole-body autoradiography in mice and in perfused rabbit lung. The compounds were administered either iv, sc, ip, or by inhalation. Blank sections were also incubated with nicotine-14C and cotinine-14C in vitro. The distributions were compared in a black (C57BL/6J), brown (C57L/J), and albino (A/HeJ) strain and in CD-1 germ-free mice. After administration of nicotine-14C in vivo, radioactivity was localized in all strains in bronchi, nasal mucosa, salivary gland, Harder's gland, liver, kidney, stomach, spleen, pancreas, intestine, bone, gallbladder, and adrenal medulla. In the pigmented strains, it was also localized in melanin in the eye, brain, and hair. Radioactivity did not accumulate in the bronchi of late-term fetuses or the 1-day-old newborn but was present in the 2-day-old and older. Cotinine-14C and nicotine-1'-N-oxide-14C, after iv administration, did not localize in bronchi or nasal mucosa at short time intervals after injection. Frozen sections incubated in vitro did not accumulate either nicotine-14C or cotinine-14C in the bronchi; whereas the affinity for melanin was unchanged. Incubation of fresh, nonfrozen mouse lung and perfusion of rabbit lung in vitro with nicotine-14C produced the same localization of radioactivity in bronchi as that seen after in vivo administration. Preheating the frozen sections or lungs in a microwave oven before incubation did not change the localization of radioactivity in the bronchi or other tissues. The localization of radioactivity in bronchi may be due to metabolism, active transport, or binding of nicotine; this mechanism is destroyed by freezing the tissue.


Deregulation of the c-myc oncogene in virus-induced thymic lymphomas of AKR/J mice.
A high frequency (greater than or equal to 65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, S1 nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.


Repair of phage lambda DNA damaged by near ultraviolet light plus 8-methoxypsoralen.
Treatment of phage lambda with 8-methoxypsoralen plus near ultraviolet light (PUVA) and its subsequent infection and growth on various mutant and non-mutant hosts were investigated. A number of Escherichia coli DNA repair-deficient mutants, particularly those deficient in genes producing proteins known to participate in interstrand crosslink repair, were used as hosts to assess the roles of these gene products in the activation of phage affected by PUVA. Results show that puvA, uvrA, uvrD, recA, recO, sulA and recN of E. coli are involved in the repair process. Based on the data presented it is proposed that phage lambda DNA is repaired, following PUVA damage, using the recombinational repair process. This may be in agreement with the recombinational model of the repair of E. coli DNA.