DNA damage and expression of DNA methylation modulators in urine-derived cells of patients with hypertension and diabetes.
Diabetes and hypertension have become the primary causes of chronic kidney disease worldwide. However, there are no established markers for early diagnosis or predicting renal prognosis. Here, we investigated the expression profiles of DNA repair and DNA methylation factors in human urine-derived cells as a possible diagnostic or renal prognosis-predicting marker. A total of 75 subjects, aged 63.3 ± 1.9 years old, were included in this study. DNA and RNA were extracted from 50 mL of urine samples. We evaluated DNA double-strand breaks (DSBs) by the quantitative long distance-PCR method and performed real-time RT-PCR analysis to analyze the expression of renal cell-specific markers, DNA DSB repair factor KAT5, DNA methyltransferases DNMTs, and demethylation enzymes TETs. In patients with hypertension and diabetes, DNA DSBs of the nephrin gene increased with decreased urine KAT5/nephrin expression, consistent with our previous study (Cell Rep 2019). In patients with hypertension, DNA DSBs of the AQP1 gene were increased with elevated urine DNMTs/AQP1 and TETs/AQP1 expression. Moreover, urine DNMTs/AQP1 expression was significantly correlated with the annual eGFR decline rate after adjustment for age, baseline eGFR, the presence of diabetes and the amount of albuminuria, suggesting a possible role as a renal prognosis predictor.


A waitlist control-group study of cognitive, mood, and quality of life outcome after posteroventral pallidotomy in Parkinson disease.
The aim of this study was to examine neuropsychological outcome from unilateral posteroventral pallidotomy (PVP) in Parkinson disease while controlling for confounding factors such as test practice and disease progression. Participants underwent baseline and 2-month follow-up assessments of cognition, quality of life, mood, and motor functioning. The surgery group (22 patients) underwent PVP (15 left, seven right) after baseline assessment. The waitlist group (14 patients) underwent PVP after follow up. At follow up, the left PVP group exhibited a decline on verbal measures of learning, fluency, working memory, and speeded color naming. The incidence of significant decline on these measures after left PVP ranged from 50 to 86%. The right PVP group did not exhibit a significant cognitive decline, but fluency did decline in 71% of patients who underwent right PVP. Participants who underwent PVP reported better bodily pain and social functioning at follow up than participants in the waitlist group. Improved bodily pain was evident for 62% of the surgery group, and social functioning improved for 19%. Surgery did not alter reported physical functioning or mood. Dyskinesia improved after surgery, but there were no improvements in "on-state" manual dexterity or handwriting. Most patients who underwent left PVP exhibited declines in learning, fluency, working memory, and speeded color naming. Accounting for retesting effects altered the magnitude of these declines by up to one quarter of a standard deviation, but did not increase the breadth of postsurgical neuropsychological decline beyond that typically reported in the literature. It was found that PVP improved dyskinesia, bodily pain, and social functioning, but did not lead to improvement on other objective and self-reported measures of motor functioning.


Detection of COVID-19 by Machine Learning Using Routine Laboratory Tests.
The present study aimed to develop a clinical decision support tool to assist coronavirus disease 2019 (COVID-19) diagnoses with machine learning (ML) models using routine laboratory test results. We developed ML models using laboratory data (n = 1,391) composed of six clinical chemistry (CC) results, 14 CBC parameter results, and results of a severe acute respiratory syndrome coronavirus 2 real-time reverse transcription-polymerase chain reaction as a gold standard method. Four ML algorithms, including random forest (RF), gradient boosting (XGBoost), support vector machine (SVM), and logistic regression, were used to build eight ML models using CBC and a combination of CC and CBC parameters. Performance evaluation was conducted on the test data set and external validation data set from Brazil. The accuracy values of all models ranged from 74% to 91%. The RF model trained from CC and CBC analytes showed the best performance on the present study's data set (accuracy, 85.3%; sensitivity, 79.6%; specificity, 91.2%). The RF model trained from only CBC parameters detected COVID-19 cases with 82.8% accuracy. The best performance on the external validation data set belonged to the SVM model trained from CC and CBC parameters (accuracy, 91.18%; sensitivity, 100%; specificity, 84.21%). ML models presented in this study can be used as clinical decision support tools to contribute to physicians' clinical judgment for COVID-19 diagnoses.


Challenges for resuming normal life after earthquake: a qualitative study on rural areas of iran.
Growing evidence is indicating that some of disaster affected people face challenges to resume normal life several months after an earthquake. However, there is no sufficient in-depth understanding of complex process of resuming normal life after an earthquake in Iran, as one of the most disaster-prone countries in the world, and in rural areas as a particular setting. This study aimed to explore challenges of return to normalcy in rural earthquake-stricken areas of Iran. The study was conducted using qualitative content analysis method (Graneheim approach). Twenty people from the earthquake-stricken areas and seven qualified experts were selected via purposeful sampling .Data was collected through semi-structured interviews, focus group discussions, and field notes from August 2013 to January 2014. Data collection continued to the point of data saturation (no new information was provided by interviewees). Data saturation supported the sample size. Data analysis was based on qualitative content analysis principles. "Social uncertainty and confusion" was the most prominent challenge of return to the normal life after earthquake, which was categorized into six concepts of social vulnerability, lack of comprehensive rehabilitation plan, incomplete reconstruction, ignorance of local social capital, waste of assets, and psychological problems. Findings showed that social uncertainty and confusion occurs as a result of negligence of some important social aspects in process of returning to the normal life. This issue, in turn, can greatly interrupt the normal developmental processes. Understanding the challenges of life recovery after disasters will help policy makers consider social rehabilitation as a key factor in facilitation of return to normal life process after earthquakes. Disaster; earthquake; social rehabilitation; social uncertainty.


Conformal radiotherapy computation by the method of alternating projections onto convex sets.
Synthesis of beam profiles for a given dose prescription is a central problem in radiotherapy. Care must be taken in the beam design to expose the tumour volume at a high level, to avoid significant irradiation of critical organs, and to minimize exposure of all other tissue. Use of the synthesis procedure known as alternating projections onto convex sets (POCS) is shown to be a viable approach to beam design. POCS is a powerful tool for signal and image restoration and synthesis. Convex sets of signals obeying desired constraint sets are first specified. Then, by repeated projections onto these sets, convergence is to a signal obeying all desired constraints if the constraint sets have a finite intersection. In this paper we apply the method of POCS to conformal radiotherapy dose computation. The performance of the method is shown through three representative examples.


Cerebrospinal fluid penetration of targeted therapeutics in pediatric brain tumor patients.
Treatment with small-molecule inhibitors, guided by precision medicine has improved patient outcomes in multiple cancer types. However, these compounds are often not effective against central nervous system (CNS) tumors. The failure of precision medicine approaches for CNS tumors is frequently attributed to the inability of these compounds to cross the blood-brain barrier (BBB), which impedes intratumoral target engagement. This is complicated by the fact that information on CNS penetration in CNS-tumor patients is still very limited. Herein, we evaluated cerebrospinal fluid (CSF) drug penetration, a well-established surrogate for CNS-penetration, in pediatric brain tumor patients. We analyzed 7 different oral anti-cancer drugs and their metabolites by high performance liquid chromatography mass spectrometry (HPLC-MS) in 42 CSF samples obtained via Ommaya reservoirs of 9 different patients. Moreover, we related the resulting data to commonly applied predictors of BBB-penetration including ABCB1 substrate-character, physicochemical properties and in silico algorithms. First, the measured CSF drug concentrations depicted good intra- and interpatient precision. Interestingly, ribociclib, vorinostat and imatinib showed high (> 10 nM), regorafenib and dasatinib moderate (1-10 nM) penetrance. In contrast, panobinostat und nintedanib were not detected. In addition, we identified active metabolites of imatinib and ribociclib. Comparison to well-established BBB-penetrance predictors confirmed low molecular weight, high proportion of free-drug and low ABCB1-mediated efflux as central factors. However, evaluation of diverse in silico algorithms showed poor correlation within our dataset. In summary, our study proves the feasibility of measuring CSF concentration via Ommaya reservoirs thus setting the ground for utilization of this method in future clinical trials. Moreover, we demonstrate CNS presence of certain small-molecule inhibitors and even active metabolites in CSF of CNS-tumor patients and provide a potential guidance for physicochemical and biological factors favoring CNS-penetration.


A Randomized, Open-Label, Controlled Clinical Trial of Azvudine Tablets in the Treatment of Mild and Common COVID-19, a Pilot Study.
Coronavirus disease 2019 (COVID-19) has spread worldwide. To date, no specific drug for COVID-19 has been developed. Thus, this randomized, open-label, controlled clinical trial (ChiCTR2000029853) was performed in China. A total of 20 mild and common COVID-19 patients were enrolled and randomly assigned to receive azvudine and symptomatic treatment (FNC group), or standard antiviral and symptomatic treatment (control group). The mean times of the first nucleic acid negative conversion (NANC) of ten patients in the FNC group and ten patients in the control group are 2.60 (SD 0.97; range 1-4) d and 5.60 (SD 3.06; range 2-13) d, respectively (p = 0.008). The mean times of the first NANC of four newly diagnosed subjects in the FNC group and ten subjects in the control group are 2.50 (SD 1.00; range 2-4) d and 9.80 (SD 4.73; range 3-19) d, respectively (starting from the initial treatment) (p = 0.01). No adverse events occur in the FNC group, while three adverse events occur in the control group (p = 0.06). The preliminary results show that FNC treatment in the mild and common COVID-19 may shorten the NANC time versus standard antiviral treatment. Therefore, clinical trials of FNC treating COVID-19 with larger sample size are warranted.


Naloxone infusion and drainage of cerebrospinal fluid as adjuncts to postoperative care after repair of thoracoabdominal aneurysms.
The mechanisms that produce paraplegia in patients after TAA repair are complex and involve alterations in regional blood flow to the spinal cord, CSF dynamics, and reperfusion. Although neither the minimal level of blood flow nor the maximal spinal cord pressure that can be tolerated by the spinal cord is known, adjuncts such as CSF drainage and naloxone infusions may allow longer durations of aortic cross-clamping before irreversible ischemia occurs. Because paraplegia is multifactorial and none of the recommended adjuncts alone provides complete protection of the spinal cord, a combination of treatments may be necessary to reduce the prevalence of neurological complications after thoracoabdominal aortic reconstruction. Critical care nurses thus must be acquainted with the advanced monitoring techniques and the pathophysiology behind these new treatment modalities. Advanced assessment skills are also essential to recognize the potential neurological complications that may occur in these patients. Care of patients with TAA is a challenge. Critical care nurses must use multidimensional skills in the areas of hemodynamic monitoring, physical assessment, and psychological counseling to effectively manage postoperative care of these patients.


Impaired smooth muscle cell contractility as a novel concept of abdominal aortic aneurysm pathophysiology.
Ruptured abdominal aortic aneurysms (AAA) are associated with overall mortality rates up to 90%. Despite extensive research, mechanisms leading to AAA formation and advancement are still poorly understood. Smooth muscle cells (SMC) are predominant in the aortic medial layer and maintain the wall structure. Apoptosis of SMC is a well-known phenomenon in the pathophysiology of AAA. However, remaining SMC function is less extensively studied. The aim of this study is to assess the in vitro contractility of human AAA and non-pathologic aortic SMC. Biopsies were perioperatively harvested from AAA patients (n = 21) and controls (n = 6) and clinical data were collected. Contractility was measured using Electric Cell-substrate Impedance Sensing (ECIS) upon ionomycin stimulation. Additionally, SMC of 23% (5 out of 21) of AAA patients showed impaired maximum contraction compared to controls. Also, SMC from patients who underwent open repair after earlier endovascular repair and SMC from current smokers showed decreased maximum contraction vs. controls (p = 0.050 and p = 0.030, respectively). Our application of ECIS can be used to study contractility in other vascular diseases. Finally, our study provides with first proof that impaired SMC contractility might play a role in AAA pathophysiology.


Predictors of patterns of pain, fatigue, and insomnia during the first year after a cancer diagnosis in the elderly.
Pain (P), fatigue (F), and insomnia (I) are among the most prevalent, distressing, and undermanaged symptoms experienced by cancer patients. Research has demonstrated that PFI co-occur; what remain unclear are the patterns and stability of PFI and the patient, disease, and treatment characteristics that predict PFI patterns over time. This secondary analysis used a data set composed of 867 elders (46% women) who were newly diagnosed with breast, colorectal, lung, or prostate cancer and followed at 4 points during the year after diagnosis. The university's institutional review board approved this study. Descriptive statistics and multistate transition models using multinomial logistic regression were used. The typical participant was 72.6 years old, who reported 7.9 symptoms and 2.7 comorbidities. Previous PFI pattern was consistently associated with significantly increased risks of subsequent PFI pattern. At observations 1 to 3, lung cancer, treatment, higher comorbidity with breast cancer, and late-stage colorectal cancer were significantly associated with increased risks of PFI patterns. Advancing age was not significantly associated with increased risks of PFI patterns. Pain, fatigue, and insomnia co-occurrence declined over time but was associated with significantly increased risks of death or loss to follow-up and increased reports of other symptoms. Pain, fatigue, and insomnia co-occurrence is associated with adverse outcomes and should be proactively targeted for intervention.


Mechanotransduction current is essential for stability of the transducing stereocilia in mammalian auditory hair cells.
Mechanotransducer channels at the tips of sensory stereocilia of inner ear hair cells are gated by the tension of 'tip links' interconnecting stereocilia. To ensure maximal sensitivity, tip links are tensioned at rest, resulting in a continuous influx of Ca2+ into the cell. Here, we show that this constitutive Ca2+ influx, usually considered as potentially deleterious for hair cells, is in fact essential for stereocilia stability. In the auditory hair cells of young postnatal mice and rats, a reduction in mechanotransducer current, via pharmacological channel blockers or disruption of tip links, leads to stereocilia shape changes and shortening. These effects occur only in stereocilia that harbor mechanotransducer channels, recover upon blocker washout or tip link regeneration and can be replicated by manipulations of extracellular Ca2+ or intracellular Ca2+ buffering. Thus, our data provide the first experimental evidence for the dynamic control of stereocilia morphology by the mechanotransduction current.


Selective Structural Derivatization of Flavonoid Acetamides Significantly Impacts Their Bioavailability and Antioxidant Properties.
Flavonoids show abundant favorable physicochemical and drug related properties, leading to substantial biological applications which are limited by undesirable properties such as poor solubility, high polarity, low bioavailability, and enzymatic degradations. Chemical modification with bioisosteres can be used to address some of these challenges. We report the synthesis and characterization of partial flavonoid acetamide derivatives from quercetin, apigenin and luteolin and the evaluation of their structure-activity relationships based on antioxidant, bioavailability, drug likeness, and toxicity properties. The sequential synthesis was achieved with 76.67-87.23% yield; the structures of the compounds were confirmed using 1H & 13C NMR characterizations. The purity of each compound was determined by HPLC while the molecular weights were determined by mass spectrometry. The % bioavailability was determined using the dialysis tubing procedure and the values were in the range 15.97-38.12%. The antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and expressed as the IC50 values which were in the range 31.52-198.41 µM. The drug likeness and the toxicity properties of compounds 4, 5, 7, 11 and 15 were predicted using computational tools and showed satisfactory results. A structure-activity relationship evaluation reveals that hydroxyl and methylene groups attached on the 2-phenylchromen-4-one structure of the flavonoid play a colossal role in the overall antioxidant and bioavailability properties. The improved bioavailability and excellent drug relevance and toxicity properties present flavonoid acetamide derivatives as prospective drug candidates for further evaluations.


Standardization of diagnostic materials. 2. Problems of standardization in clinical chemistry.
If analytical results in clinical chemistry are to be made meaningful, i.e., accurate, precise, and specific, a systematic approach to their attainment is necessary. Furthermore, because this system is so complex in scope and the need for it is so widespread, it will require international coordination. Agreement on the units of measurement, the production and certification of standard reference materials, and the development of reference methods of demonstrated accuracy will require the support of all segments of clinical chemistry.


Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody.
The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine-scanning mutagenesis overlaps that of b12. These results suggest that m14 is a novel CD4bs cross-reactive HIV-1-neutralizing antibody that exhibits a different inhibitory profile compared to the only known potent broadly neutralizing CD4bs human antibody, b12, and may have implications for our understanding of the mechanisms of immune evasion and for the development of inhibitors and vaccines.


Epigenetics: judge, jury and executioner of stem cell fate.
Emerging evidence is shedding light on a large and complex network of epigenetic modifications at play in human stem cells. This "epigenetic landscape" governs the fine-tuning and precision of gene expression programs that define the molecular basis of stem cell pluripotency, differentiation and reprogramming. This review will focus on recent progress in our understanding of the processes that govern this landscape in stem cells, such as histone modification, DNA methylation, alterations of chromatin structure due to chromatin remodeling and non-coding RNA activity. Further investigation into stem cell epigenetics promises to provide novel advances in the diagnosis and treatment of a wide array of human diseases.


Family history of cancer and the risk of prostate cancer and benign prostatic hyperplasia.
We analysed the relation between family history of cancer in first-degree relatives and risk of prostate cancer (PC) and benign prostatic hyperplasia (BPH) using data from a multicentric case-control study conducted in Italy from 1991 to 2002 on 1,294 cases of incident, histologically confirmed PC, 1,369 cases of BPH and 1,451 men admitted to the same network of hospitals for acute, nonneoplastic conditions. Unconditional logistic regression was used to estimate odds ratios (OR) of PC and BPH, adjusted for age and other confounders. Men with a family history of PC had an OR of PC of 4.0 (95% confidence interval [CI] 2.5-6.5), and the risk was higher when the proband was younger, when 2 or more relatives were affected or when the affected relative was a brother. The risk of PC was also increased in men with a family history of cancer of the ovary (OR = 6.2, 95% CI 1.2-32), bladder (OR = 3.5, 95% CI 1.6-7.4) and kidney (OR = 3.1, 95% CI 1.1-8.5). An involvement of breast/ovarian cancer predisposition genes in a small proportion of PCs was suggested by the cluster of these cancers in female relatives of a few PC cases. The risk of BPH was increased in men with a family history of bladder cancer (OR = 2.2, 95% CI 1.0-5.0) but not PC (OR = 1.2, 95% CI 0.7-2.2). Our study adds further information on the association of family history of cancer and risk of PC and is, to our knowledge, the first comprehensive epidemiologic information on family history of cancer and risk of BPH.


[Effectiveness of parenteral administration of piracetam in acute and chronic consciousness disorders in cerebral arteriosclerosis].
The effectiveness of treatment with piracetam of 32 patients with acute and chronic consciousness disturbances caused by clinical syndromes of cerebrovascular disease (strokes, syndromes of dementia) was studied. The drug was administered in drip infusions in daily doses of 6 g for 10 days. A statistically significant improvement was obtained in the signs belonging to the syndrome of consciousness disturbances. No significant difference was observed in the power of action of the drug in acute and chronic consciousness disturbances.


Involvement of the kynurenine pathway in the pathogenesis of Parkinson's disease.
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by loss of dopaminergic neurons and localized neuroinflammation occurring in the midbrain several years before the actual onset of symptoms. Neuroinflammation leads to microglia activation and release of a large number of proinflammatory mediators. The kynurenine pathway (KP) of tryptophan catabolism is one of the major regulators of the immune response and is also likely to be implicated in the inflammatory and neurotoxic events in Parkinsonism. Several neuroactive compounds are produced through the KP that can be either a neurotoxic, neuroprotective or immunomodulator. Among these metabolites kynurenic acid (KYNA), produced by astrocytes, is considered as neuroprotective whereas quinolinic acid (QUIN), released by activated microglia, can activate the N-methyl-d-aspartate (NMDA) receptor-signalling pathway, leading to excitotoxicity and amplify the inflammatory response. Previous studies have shown that NMDA antagonists can ease symptoms and exert a neuroprotective effect in PD both in vivo and in vitro. There are to date several lines of evidence linking some of the KP intermediates and the neuropathogenesis of PD. Moreover, it is likely that some of the KP metabolites could be used as prognostic biomarkers and that pharmacological modulators of the KP enzymes could represent a new therapeutic strategy for PD.


Overview of the Baculovirus Expression System.
This unit provides information on the replication cycle of insect baculovirus to provide an understanding of how this virus has been adapted for use as an expression vector for recombinant proteins in insect cells. We provide an overview of the virus structure and its unique bi-phasic replication cycle, which has been exploited in developing the virus as an expression vector. We also review the development of the baculovirus expression vector system (BEVS), from the mid-1980s to the present day in which the BEVS is now an established tool for the production of a range of recombinant proteins and multi-protein complexes including virus-like particles. We describe advances made to the BEVS to allow the rapid and easy production of recombinant viruses and developments to improve protein yield. We finish by describing the application of recombinant BacMam as vectors for the delivery of genes into mammalian and human cells. © 2018 by John Wiley & Sons, Inc.


Improving asthma outcomes in minority children: a randomized, controlled trial of parent mentors.
Because asthma disproportionately affects minorities, we evaluated the effects of parent mentors (PMs) on asthma outcomes in minority children. This randomized, controlled trial allocated minority asthmatic children to the PM intervention or traditional asthma care. Intervention families were assigned PMs (experienced parents of asthmatic children who received specialized training). PMs met monthly with children and families at community sites, phoned parents monthly, and made home visits. Ten asthma outcomes and costs were monitored for 1 year. Outcomes were examined by using both intention-to-treat analyses and stratified analyses for high participants (attending >or=25% of community meetings and completing >or=50% of PM phone interactions). Patients were randomly assigned to PMs (n = 112) or the control group (n = 108). In intention-to-treat analyses, intervention but not control children experienced significantly reduced rapid-breathing episodes, asthma exacerbations, and emergency department (ED) visits. High participants (but not controls or low participants) experienced significantly reduced wheezing, asthma exacerbations, and ED visits and improved parental efficacy in knowing when breathing problems are controllable at home. Mean reductions in missed parental work days were greater for high participants than controls. The average monthly cost per patient for the PM program was $60.42, and net savings of $46.16 for high participants. For asthmatic minority children, PMs can reduce wheezing, asthma exacerbations, ED visits, and missed parental work days while improving parental self-efficacy. These outcomes are achieved at a reasonable cost and with net cost savings for high participants. PMs may be a promising, cost-effective means for reducing childhood asthma disparities.


Purification and characterization of norcoclaurine synthase. The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants.
Norcoclaurine synthase (NCS; EC ) catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in benzylisoquinoline alkaloid biosynthesis in plants. NCS was purified 1590-fold to homogeneity from cell suspension cultures of meadow rue (Thalictrum flavum ssp. glaucum). The purification procedure, which resulted in a 4.2% yield, involved hydrophobic interaction, anion exchange, hydroxyapatite, and gel filtration chromatography. Purified NCS displayed native and denatured molecular masses of approximately 28 and 15 kDa, respectively, suggesting that the enzyme is composed of two subunits. Two-dimensional polyacrylamide gel electrophoresis revealed two major and two minor isoforms with pI values between 5.5 and 6.2. NCS activity was maximal at pH 6.5 to 7.0 and temperatures between 42 and 55 degrees C and was not affected by divalent cations. The enzyme showed hyperbolic saturation kinetics for 4-HPAA (K(m) = 335 microm) but sigmoidal saturation kinetics for dopamine (Hill coefficient = 1.8) suggesting cooperativity between the dopamine binding sites on each subunit; thus, NCS might play a regulatory, or rate-limiting, role in controlling the rate of pathway flux in benzylisoquinoline alkaloid biosynthesis. Product inhibition kinetics performed at saturating levels of one substrate and with norlaudanosoline as the inhibitor showed that NCS follows an iso-ordered bi-uni mechanism with 4-HPAA binding before dopamine. NCS activity was highest in soluble protein extracts from roots followed by stems, leaves, and flower buds.


Coupling during collective cell migration is controlled by a vinculin mechanochemical switch.
The ability of cells to move in a mechanically coupled, coordinated manner, referred to as collective cell migration, is central to many developmental, physiological, and pathophysiological processes. Limited understanding of how mechanical forces and biochemical regulation interact to affect coupling has been a major obstacle to unravelling the underlying mechanisms. Focusing on the linker protein vinculin, we use a suite of Förster resonance energy transfer-based biosensors to probe its mechanical functions and biochemical regulation, revealing a switch that toggles vinculin between loadable and unloadable states. Perturbation of the switch causes covarying changes in cell speed and coordination, suggesting alteration of the friction within the system. Molecular scale modelling reveals that increasing levels of loadable vinculin increases friction, due to engagement of self-stabilizing catch bonds. Together, this work reveals a regulatory switch for controlling cell coupling and describes a paradigm for relating biochemical regulation, altered mechanical properties, and changes in cell behaviors.


Sexual conflict in wing size and shape in Drosophila melanogaster.
Intralocus sexual conflict occurs when opposing selection pressures operate on loci expressed in both sexes, constraining the evolution of sexual dimorphism and displacing one or both sexes from their optimum. We eliminated intralocus conflict in Drosophila melanogaster by limiting transmission of all major chromosomes to males, thereby allowing them to win the intersexual tug-of-war. Here, we show that this male-limited (ML) evolution treatment led to the evolution (in both sexes) of masculinized wing morphology, body size, growth rate, wing loading, and allometry. In addition to more male-like size and shape, ML evolution resulted in an increase in developmental stability for males. However, females expressing ML chromosomes were less developmentally stable, suggesting that being ontogenetically more male-like was disruptive to development. We suggest that sexual selection over size and shape of the imago may therefore explain the persistence of substantial genetic variation in these characters and the ontogenetic processes underlying them.


Bioactive metals: preparation and properties.
Some ceramics, such as Bioglass, sintered hydroxyapatite, and glass-ceramic A-W, spontaneously form a bone-like apatite layer on their surface in the living body, and bond to bone through the apatite layer. These materials are called bioactive ceramics, and are clinically important for use as bone-repairing materials. However, they cannot be used at high-load sites, such as is found in femoral and tibial bones, because their fracture toughness values are not as high as that of human cortical bone. Titanium metal and its alloys have high fracture toughness, and form a sodium titanate layer on its surface when soaked in a 5 M-NaOH solution at 60 degrees C for 24 h, followed by a heat treatment at 600 degrees C for 1 h. On moving toward the metal interior, the sodium titanate layer gradually changes into the pure metal within a distance of 1 microm from the surface. The mechanical strength of the titanium metal or a titanium alloy is not adversely affected by these chemical and thermal treatments. The titanium metal and its alloys resulting from the above treatment can release Na+ ions from its surface into a surrounding body fluid via an ion exchange reaction with H3O+ ions, resulting in many Ti-OH groups forming on its surface. These Ti-OH groups initially combine with Ca2+ ions to form amorphous calcium titanate in the body environment, and later the calcium titanate combines with phosphate ions to form amorphous calcium phosphate. The amorphous calcium phosphate eventually transforms into bone-like apatite, and by this process the titanium metals are soon tightly bonded to the surrounding living bone through the bone-like apatite layer. The treated metals have already been subjected to clinical trials for applications in artificial total hip joints. Metallic tantalum has also been found to bond to living bone after it has been subjected to the NaOH and heat treatment to form a sodium tantalate layer on its surface.


Stress Tolerance of the Endemic Antarctic Brown Alga Desmarestia anceps to UV Radiation and Temperature is Mediated by High Concentrations of Phlorotannins.
The endemic Antarctic brown macroalga Desmarestia anceps is strongly shade-adapted, but shows also a high capacity to cope with different environmental stressors, e.g. UV radiation and temperature. Therefore, this species colonizes wide depth gradients, which are characterized by changing environmental conditions. In this study, we examine whether the different physiological abilities allowing D. anceps to grow across a wide depth range is determined by high levels of phlorotannins. Photosynthesis, measured by PAM-fluorometry, the contents of soluble phlorotannins, antioxidant capacities of field grown were analyzed in response to different conditions of radiation (PAR and PAR + UV) and temperature (2, 7 and 12°C). The results show that maximal quantum of fluorescence (Fv /Fm ) decreased with increasing doses of UV radiation, but remained unaffected by temperature. High levels of soluble phlorotannins were detected and confirmed by microscopic observation revealing the abundance of large physodes. Exposure to UV radiation and elevated temperature showed that phlorotannins were not inducible by UV but increased at 12°C. ROS scavenging capacity was positively correlated with the contents of phlorotannins. In general, highest contents of phlorotannins were correlated with the lowest inhibition of Fv /Fm in all experimental treatments, highlighting the UV-protective role of these compounds in D. anceps.


Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci.
LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants.


The application of molecular biology techniques to haemostasis and thrombosis.
One may no longer consider coagulation research without encompassing the wealth of knowledge available from the application of molecular biology techniques. This innovative form of research has not proceeded to the exclusion of more conventional techniques, and moreover has provided support for basic scientific hypotheses. This paper discusses the molecular biology techniques currently used in the study of haemostatic and thrombotic disorders and gives an outline of the evolution of such techniques which have taken place at a staggering pace. The article includes specific examples of how results from molecular studies are directly applied to hereditary haemostatic and thrombotic diseases to support clinical diagnosis and to increase our understanding of the biochemical processes involved in the disorders. The reader is referred to several reviews for detailed lists of genetic defects revealed by molecular biological techniques.


Cyclin-dependent kinase-like 5 (CDKL5) mutation screening in Rett syndrome and related disorders.
Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting females almost exclusively and is characterized by a wide spectrum of clinical manifestations. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been found in up to 95% of classical RTT cases and a lesser proportion of atypical cases. Recently, mutations in another X-linked gene, CDKL5 (cyclin-dependent kinase-like 5) have been found to cause atypical RTT, in particular the early onset seizure (Hanefeld variant) and one female with autism. In this study we screened several cohorts of children for CDKL5 mutations, totaling 316 patients, including individuals with a clinical diagnosis of RTT but who were negative for MECP2 mutations (n=102), males with X-linked mental retardation (n=9), patients with West syndrome (n=52), patients with autism (n=59), patients with epileptic encephalopathy (n=33), patients with Aicardi syndrome (n=7) and other patients with intellectual disability with or without seizures (n=54). In all, seven polymorphic variations and four de novo mutations (c.586C>T [p.S196L]; c.58G>C [p.G20R]; c.2504delC [p.P835fs]; deletion of exons 1-3) were identified, and in all instances of the latter the clinical phenotype was that of an epileptic encephalopathy. These results suggest that pathogenic CDKL5 mutations are unlikely to be identified in the absence of severe early-onset seizures and highlight the importance of screening for large intragenic and whole gene deletions.


In Silico Studies on Natural Products and Derivatives Against Different Types of Cancer.
According to the World Health Organization (WHO), cancer is the second cause of death worldwide, responsible for almost 10 million deaths and accounting for one in every six deaths. It is a disease that can affect any organ or tissue with rapid progression to the final stage, which is metastasis, in which the disease spreads to different regions of the body. Many studies have been carried out to find a cure for cancer. Early diagnosis contributes to the individual achieving the cure; however, deaths are increasing considerably due to late diagnosis. Thus, this bibliographical review discussed several scientific research works pointing to in silico analyses in the proposition of new antineoplastic agents for glioblastoma, breast, colon, prostate, and lung cancer, as well as some of their respective molecular receptors involved in molecular docking simulations and molecular dynamics. This review involved articles describing the contribution of computational techniques for the development of new drugs or already existing drugs with biological activity; thus, important data were highlighted in each study, such as the techniques used, results obtained in each study, and the conclusion. Furthermore, 3D chemical structures of the molecules with the best computational response and significant interactions between the tested molecules and the PDB receptors were also presented. With this, it is expected to help new research in the fight against cancer, the creation of new antitumor drugs, and the advancement of the pharmaceutical industry and scientific knowledge about studied tumors.


Twin boundary defect engineering in Au cocatalyst to promote alcohol splitting for coproduction of H2 and fine chemicals.
The microstructure of Au metal cocatalyst has been shown to significantly influence its optical and electronic properties. However, the impact of Au defect engineering on photocatalytic activity remains underexplored. In this study, we synthesize different Au-TiO2 composites by in-situ hybridizing face-centered cubic (F-Au) and twin boundary defect Au (T-Au) nanoparticles (NPs) onto the surface of TiO2. We find that T-Au NPs with twin defects serve as highly efficient cocatalysts for converting alcohols into their corresponding aldehydes while also generating H2. The optimized T-Au/TiO2 composite yields an H2 evolution rate of 6850 µmol h-1 g-1 and a BAD formation rate of 6830 µmol h-1 g-1, about 38 times higher than that of blank TiO2. Compared to F-Au/TiO2, the T-Au/TiO2 composite enhances charge separation, extends the lifetime of electrons, and provides more active sites for H2 reduction. The twin defect also improves alcohol reactant adsorption, boosting overall photocatalytic performance. This research paves the way for more studies on defect engineering in metal cocatalysts for enhanced catalytic activities in organic synthesis and H2 evolution.


Association of metallothionein expression and clinical response to cisplatin based chemotherapy in testicular germ cell tumors.
The protective roles of metallothioneins (MT) against metal toxicity suggest that MT may have a functional role in cisplatin resistance. The aim of this study was to investigate the expression of MT in specimens of germ cell tumors and compare it with clinical sensitivity to cisplatin based chemotherapy. Tissue blocks of primary GCT specimens obtained from 39 patients were examined immunohistochemically for MT expression. Staining intensity was evaluated according to the percentage of MT positive cells and graded as [-], [+] and [++]. The staining characteristics were compared with the clinical response to chemotherapy. Of the 39 tumors, 3 evidenced no MT expression while 26 and 10 specimens showed [+] and [++] staining, respectively. Although seminomas tend to stain weaker than non-seminomas, the difference of staining between them was not significant (p = 0.19). Of the 39 patients, 23 underwent cisplatin based chemotherapy. Of those, 6 progressed and 17 achieved complete remission. Of the non-responders, 5 showed [+] and 1 showed [++] staining. Six of the responders showed [+], 10 had [++] and 1 showed no staining. No association was found between MT staining and chemo-sensitivity (p = 0.53). MT expression in primary germ cell tumors did not differ between responding and non-responding patients and therefore may not be useful in predicting response to chemotherapy.


Substrate-Dependent Fermentation of Bamboo in Giant Panda Gut Microbiomes: Leaf Primarily to Ethanol and Pith to Lactate.
The giant panda is known worldwide for having successfully moved to a diet almost exclusively based on bamboo. Provided that no lignocellulose-degrading enzyme was detected in panda's genome, bamboo digestion is believed to depend on its gut microbiome. However, pandas retain the digestive system of a carnivore, with retention times of maximum 12 h. Cultivation of their unique gut microbiome under controlled laboratory conditions may be a valid tool to understand giant pandas' dietary habits, and provide valuable insights about what component of lignocellulose may be metabolized. Here, we collected gut microbiomes from fresh fecal samples of a giant panda (either entirely green or yellow stools) and supplied them with green leaves or yellow pith (i.e., the peeled stem). Microbial community composition was substrate dependent, and resulted in markedly different fermentation profiles, with yellow pith fermented to lactate and green leaves to lactate, acetate and ethanol, the latter to strikingly high concentrations (∼3%, v:v, within 3.5 h). Microbial metaproteins pointed to hemicellulose rather than cellulose degradation. The alpha-amylase from the giant panda (E.C. 220.127.116.11) was the predominant identified metaprotein, particularly in reactors inoculated with pellets derived from fecal samples (up to 60%). Gut microbiomes assemblage was most prominently impacted by the change in substrate (either leaf or pith). Removal of soluble organics from inocula to force lignocellulose degradation significantly enriched Bacteroides (in green leaf) and Escherichia/Shigella (in yellow pith). Overall, different substrates (either leaf or pith) markedly shaped gut microbiome assemblies and fermentation profiles. The biochemical profile of fermentation products may be an underestimated factor contributing to explain the peculiar dietary behavior of giant pandas, and should be implemented in large scale studies together with short-term lab-scale cultivation of gut microbiomes.


[Evaluation of low molecular weight heparin prescription at the Centre Hospitalier Intercommunal de Créteil].
Low-molecular-weight heparin (LMWH) represents a great expense in general hospitals. At the Centre Hospitalier Intercommunal de Créteil (CHIC), the Quality Assessment Council had set up a study on the prescription of LMWH in order to assess concordance between prescriptions and local guidelines on the preventive uses of LMWH. Adherence to the consensus was found in 73 per cent of cases, overprescription in 10 per cent of cases and underprescription in 26 per cent of cases. It is planned to modify the local consensus, taking into account some clinical situations highlighted by this study, and to perform another evaluation in the near future.


Albumin nitrosylated by activated macrophages possesses antiparasitic effects neutralized by anti-NO-acetylated-cysteine antibodies.
Activated macrophages exert an L-arginine-dependent cytostatic effect on the extracellular parasite, Trypanosoma musculi. This effect is not observed in the absence of albumin in the culture medium but is restored by the addition of albumin, indicating the presence of an albumin-nitric oxide (NO) adduct acting as an effector molecule. Since L-cysteine represents a privileged target for NO, an immunochemical approach was performed using an acetylated-cysteine-BSA conjugate. This conjugate was nitrosylated using sodium nitrite as a NO donor. Binding of NO to the conjugated haptens was assayed using spectrophotometry. It was completely abolished by mercuric chloride, confirming the presence of an S-NO bond. Polyclonal Abs were obtained after immunizing rabbits with S-nitroso-acetylated-cysteine (NO-ac-Cys) conjugates. Using the enzyme-linked immunosorbent assay method, Ab avidity and specificity were determined by competition experiments between NO-ac-Cys-conjugated compounds and other nitrosylated or non-nitrosylated compounds. The resulting cross-reactivity ratios showed that conjugated NO-ac-Cys-BSA was the best recognized compound. These Ab were used for an in vitro study of the kinetics of NO-derived compounds from activated murine macrophages. Anti-NO-ac-Cys Ab inhibited the antimicrobial effect of activated macrophages on the extracellular parasite, T. musculi. Moreover, the L-arginine-dependent antiparasitic activity of supernatants from Calmette-Guerin bacillus-activated macrophages required the presence of albumin and was also inhibited by anti-NO-ac-Cys Ab, showing the effector role of S-nitroso-albumin.


Conditional deletion of TrkB but not BDNF prevents epileptogenesis in the kindling model.
Epileptogenesis is the process whereby a normal brain becomes epileptic. We hypothesized that the neurotrophin brain-derived neurotrophic factor (BDNF) activates its receptor, TrkB, in the hippocampus during epileptogenesis and that BDNF-mediated activation of TrkB is required for epileptogenesis. We tested these hypotheses in Synapsin-Cre conditional BDNF(-/-) and TrkB(-/-) mice using the kindling model. Despite marked reductions of BDNF expression, only a modest impairment of epileptogenesis and increased hippocampal TrkB activation were detected in BDNF(-/-) mice. In contrast, reductions of electrophysiological measures and no behavioral evidence of epileptogenesis were detected in TrkB(-/-) mice. Importantly, TrkB(-/-) mice exhibited behavioral endpoints of epileptogenesis, tonic-clonic seizures. Whereas TrkB can be activated, and epileptogenesis develops in BDNF(-/-) mice, the plasticity of epileptogenesis is eliminated in TrkB(-/-) mice. Its requirement for epileptogenesis in kindling implicates TrkB and downstream signaling pathways as attractive molecular targets for drugs for preventing epilepsy.


Dynamical density delay maps: simple, new method for visualising the behaviour of complex systems.
Physiologic signals, such as cardiac interbeat intervals, exhibit complex fluctuations. However, capturing important dynamical properties, including nonstationarities may not be feasible from conventional time series graphical representations. We introduce a simple-to-implement visualisation method, termed dynamical density delay mapping ("D3-Map" technique) that provides an animated representation of a system's dynamics. The method is based on a generalization of conventional two-dimensional (2D) Poincaré plots, which are scatter plots where each data point, x(n), in a time series is plotted against the adjacent one, x(n + 1). First, we divide the original time series, x(n) (n = 1,…, N), into a sequence of segments (windows). Next, for each segment, a three-dimensional (3D) Poincaré surface plot of x(n), x(n + 1), h[x(n),x(n + 1)] is generated, in which the third dimension, h, represents the relative frequency of occurrence of each (x(n),x(n + 1)) point. This 3D Poincaré surface is then chromatised by mapping the relative frequency h values onto a colour scheme. We also generate a colourised 2D contour plot from each time series segment using the same colourmap scheme as for the 3D Poincaré surface. Finally, the original time series graph, the colourised 3D Poincaré surface plot, and its projection as a colourised 2D contour map for each segment, are animated to create the full "D3-Map." We first exemplify the D3-Map method using the cardiac interbeat interval time series from a healthy subject during sleeping hours. The animations uncover complex dynamical changes, such as transitions between states, and the relative amount of time the system spends in each state. We also illustrate the utility of the method in detecting hidden temporal patterns in the heart rate dynamics of a patient with atrial fibrillation. The videos, as well as the source code, are made publicly available. Animations based on density delay maps provide a new way of visualising dynamical properties of complex systems not apparent in time series graphs or standard Poincaré plot representations. Trainees in a variety of fields may find the animations useful as illustrations of fundamental but challenging concepts, such as nonstationarity and multistability. For investigators, the method may facilitate data exploration.


Integrative analysis reveals key mRNA and long non-coding RNA interaction in idiopathic pulmonary arterial hypertension.
Idiopathic pulmonary arterial hypertension (IPAH) is a rare and sporadic form of pulmonary arterial hypertension (PAH), characterized by elevated pulmonary arterial resistance leading to right heart failure. However, molecular mechanisms of PAH development are still not completely understood. In this study, we aimed to uncover key mRNAs and long non-coding RNA (lncRNAs), functional modules and pathways. Moreover, to detect the dysregulated pathway or biological function, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. PPI and co-expression networks were constructed to reveal the potential roles of PAH-related mRNAs and lncRNAs. A total of 3,134 genes, including 945 up-regulated and 2,189 down-regulated genes, were identified to be differentially expressed in IPAH by differential expression analysis. We identified T cell differentiation and the T cell receptor signaling pathway as up-regulated in IPAH by using GO and KEGG analysis. Based on the PPI module analysis, we identified that the pro-inflammatory genes, such as OAS1, CXCL10, STAT1 and TLR4, were the hub genes in the PPI modules. To link the lncRNAs to the PPI modules, we calculated the Spearman correlation coefficient for lncRNA-DE-mRNA pairs to identify the modules with high correlation with each lncRNA. Notably, 6 of these lncRNAs were associated with modules characterized by the NOD-like receptor signaling pathway and chemokine signaling pathway, suggesting that these lncRNAs may promote the occurrence of IPAH via participating in the pro-inflammatory pathways. In conclusion, our systematic analysis not only improved our understanding of the molecular mechanism, but also provided potential lncRNA biomarkers for further research.


Penetration of Ciprofloxacin and Amikacin into the Alveolar Epithelial Lining Fluid of Rats with Pulmonary Fibrosis.
We determined the concentration-time profiles of ciprofloxacin and amikacin in serum and alveolar epithelial lining fluid (ELF) of rats with or without pulmonary fibrosis and investigated the effect of pulmonary fibrosis on the capacity for penetration of antimicrobials into the ELF of rats. Pulmonary fibrosis was induced in rats with a single intratracheal instillation of bleomycin. After intravenous injection of ciprofloxacin or amikacin, blood and bronchoalveolar lavage fluid samples were collected. Urea concentrations in serum and lavage fluid were determined using an enzymatic assay. Ciprofloxacin and amikacin concentrations were determined by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry, respectively. The mean ratio of ELF to plasma concentrations of ciprofloxacin at each time point in the normal group did not significantly differ from that in the pulmonary fibrosis group. However, the ratio of the ciprofloxacin area under the concentration-time curve from 0 to 24 h (AUC0-24) in ELF to the AUC0-24 in plasma was 1.02 in the normal group and 0.76 in the pulmonary fibrosis group. The mean ELF-to-plasma concentration ratios of amikacin at each time point in the normal group were higher than those in the pulmonary fibrosis group, reaching a statistically significant difference at 1, 2, and 4 h. The ratio of the AUC0-24 in ELF to the AUC0-24 in plasma was 0.49 in the normal group and 0.27 in the pulmonary fibrosis group. In conclusion, pulmonary fibrosis can influence the penetration of antimicrobials into the ELF of rats and may have a marked effect on the penetration of amikacin than that of ciprofloxacin.


[Diagnostic value of imaging procedures in nephroblastoma before preoperative chemotherapy: initial results].
According to the international SIOP 9 study preoperative chemotherapy for patients between 6 months and 16 years old is a major component of the therapeutic management of Wilms' tumour. Prior to treatment, the diagnosis is established by diagnostic imaging alone, without biopsy. This study was therefore undertaken to verify the accuracy of diagnostic imaging in the German GPO study group. Between July 1988 and November 1990, 156 patients with known histology were registered in the study centre. Of these patients, 67% (105/156) received preoperative chemotherapy. The diagnosis was initially established by US, TVP and CT, and in some cases MRT without prior biopsy. Of these preoperatively treated patients, 92.4% (97/105) had Wilms' tumour or one of its variants. Five patients had a different malignant tumour. Three cases, i.e. 2.8% of the preoperatively treated patients or 1.9% of all registered patients with known histology, had benign tumours of the kidney. Morphologically, the Wilms' tumour was revealed by a characteristic inhomogeneity in sonography and the CT scan. The inhomogeneity in the CT scan increased following injection of contrast medium. Intratumoral bleeding and cystic areas were observed frequently in the native scan. Calcifications were seen in 5% of the cases. The predictive value of pretherapeutic diagnostic imaging was good enough to justify instituting chemotherapy without diagnostic biopsy. It is still difficult in diagnostic imaging to differentiate the very rare benign cystic nephroma from malignant nephroblastoma. Intravenous pyelography proved to be the best method for distinguishing extrarenal from renal tumours.


Presynaptic plasticity at two giant auditory synapses in normal and deaf mice.
Large calyceal synapses are often regarded as simple relay points, built for high-fidelity and high-frequency synaptic transmission and a minimal requirement for synaptic plasticity, but this view is oversimplified. Calyceal synapses can exhibit surprising activity-dependent developmental plasticity. Here we compare basal synaptic transmission and activity-dependent plasticity at two stereotypical calyceal synapses in the auditory pathway, the endbulb and the calyx of Held. Basal synaptic transmission was more powerful at the calyx than the endbulb synapse: the amplitude of evoked AMPA receptor-mediated excitatory postsynaptic currents (eEPSCs) was significantly greater at the calyx, as were the release probability, and the number of release sites. The quantal amplitude was smaller at the calyx, consistent with the smaller amplitude of spontaneous miniature EPSCs at this synapse. High-frequency trains of stimuli revealed that the calyx had a larger readily releasable pool of vesicles (RRP), less tetanic depression and less asynchronous transmitter release. Activity-dependent synaptic plasticity was assessed in congenitally deaf mutant mice (dn/dn). Previously we showed that a lack of synaptic activity in deaf mice increases synaptic strength at the endbulb of Held via presynaptic mechanisms. In contrast, we have now found that deafness does not affect synaptic transmission at the calyx synapse, as eEPSC and mEPSC amplitude, release probability, number of release sites, size of RRP, tetanic depression and asynchronous release were unchanged compared to normal mice. Synaptic transmission at the calyx synapse is more powerful and has less capacity for developmental plasticity compared to the endbulb synapse.


Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis.
Regulation of cellular death is central to nearly all physiological routines and is dysregulated in virtually all diseases. Cell death occurs by two major processes, necrosis which culminates in a pervasive inflammatory response and apoptosis which is largely immunologically inert. As necrosis has long been considered an accidental, unregulated form of cellular death that occurred in response to a harsh environmental stimulus, it was largely ignored as a clinical target. However, recent elegant studies suggest that certain forms of necrosis can be reprogrammed. However, scant little is known about the molecules and pathways that orchestrate calcium-overload-induced necrosis, a main mediator of ischemia/reperfusion (IR)-induced cardiomyocyte cell death. To rectify this critical gap in our knowledge, we performed a novel genome-wide siRNA screen to identify modulators of calcium-induced necrosis in human muscle cells. Our screen identified multiple molecular circuitries that either enhance or inhibit this process, including lysosomal calcium channel TPCN1, mitophagy mediatorTOMM7, Ran-binding protein RanBP9, Histone deacetylase HDAC2, chemokine CCL11, and the Arp2/3 complex regulator glia maturation factor-γ (GMFG). Notably, a number of druggable enzymes were identified, including the proteasome β5 subunit (encoded by PSMB5 gene), which controls the proteasomal chymotrypsin-like peptidase activity. Such findings open up the possibility for the discovery of pharmacological interventions that could provide therapeutic benefits to patients affected by myriad disorders characterized by excessive (or too little) necrotic cell loss, including but not limited to IR injury in the heart and kidney, chronic neurodegenerative disorders, muscular dystrophies, sepsis, and cancers.


Postnatal elongation of eye size in DBA/2J mice compared with C57BL/6J mice: in vivo analysis with whole-eye OCT.
To characterize postnatal changes in eye size in glaucomatous DBA/2J (D2) mice and in nonglaucomatous C57BL/6J mice (B6) in vivo by means of whole-eye optical coherence tomography (OCT). D2 (n = 32) and B6 (n = 36) mice were tested between 2 and 20 months of age in eight age bins. A custom time-domain OCT system with a center wavelength of 825 nm and an axial scan length of 7.1 mm produced axial A-scan interferograms at a rate of 20 A-lines/s with a resolution of 8 μm. Axial length (AL), corneal thickness (CT), anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and retinal thickness (RT) were measured in the optical axis and adjusted with corresponding refractive indices. Corneal curvature (CC) and IOP were also measured. AL increased (P < 0.001) more in the D2 (21%) than in the B6 (9%) mice. There was an interaction effect (two-way ANOVA, P < 0.001) between age and strain for AL, CT, ACD, and VCD. In the D2 mice, the lens became dislocated posteriorly. Multiple regression analysis in the D2 mice revealed an independent effect of age and IOP (P ≤ 0.01) on axial length. CC steepened in the older D2 mice, whereas it flattened in the B6 mice. In D2 mice, postnatal elongation of AL is larger than that in B6 mice and is associated with a greater increase in ACD and IOP, which seems to be a causal factor. The ease of use, short acquisition time, and noninvasiveness of whole-eye OCT make it suitable for routine use in longitudinal studies of mouse models.


Association between molecular subtypes of colorectal cancer and patient survival.
Colorectal cancer (CRC) is a heterogeneous disease that can develop via several pathways. Different CRC subtypes, identified based on tumor markers, have been proposed to reflect these pathways. We evaluated the significance of these previously proposed classifications to survival. Participants in the population-based Seattle Colon Cancer Family Registry were diagnosed with invasive CRC from 1998 through 2007 in western Washington State (N = 2706), and followed for survival through 2012. Tumor samples were collected from 2050 participants and classified into 5 subtypes based on combinations of tumor markers: type 1 (microsatellite instability [MSI]-high, CpG island methylator phenotype [CIMP] -positive, positive for BRAF mutation, negative for KRAS mutation); type 2 (microsatellite stable [MSS] or MSI-low, CIMP-positive, positive for BRAF mutation, negative for KRAS mutation); type 3 (MSS or MSI low, non-CIMP, negative for BRAF mutation, positive for KRAS mutation); type 4 (MSS or MSI-low, non-CIMP, negative for mutations in BRAF and KRAS); and type 5 (MSI-high, non-CIMP, negative for mutations in BRAF and KRAS). Multiple imputation was used to impute tumor markers for those missing data on 1-3 markers. We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations of subtypes with disease-specific and overall mortality, adjusting for age, sex, body mass, diagnosis year, and smoking history. Compared with participants with type 4 tumors (the most predominant), participants with type 2 tumors had the highest disease-specific mortality (HR = 2.20, 95% CI: 1.47-3.31); subjects with type 3 tumors also had higher disease-specific mortality (HR = 1.32, 95% CI: 1.07-1.63). Subjects with type 5 tumors had the lowest disease-specific mortality (HR = 0.30, 95% CI: 0.14-0.66). Associations with overall mortality were similar to those with disease-specific mortality. Based on a large, population-based study, CRC subtypes, defined by proposed etiologic pathways, are associated with marked differences in survival. These findings indicate the clinical importance of studies into the molecular heterogeneity of CRC.


Azithromycin in DuraSite for the treatment of blepharitis.
Blepharitis is a common inflammatory disease of the eyelid. Posterior blepharitis affects the posterior lamella of the eyelid and involves inflammation of the meibomian glands, whereas anterior blepharitis affects the anterior lamella of the eyelid and the eyelashes; either version can be inflammatory or infectious in nature. Each of these conditions can incite or propagate the other; anterior blepharitis, if not treated, can lead to meibomian gland disease, and vice versa. Blepharitis is typically chronic, and can be associated with a variety of systemic diseases such as dermatitis, as well as ocular diseases such as dry eye, conjunctivitis, or keratitis. The standard treatment regimen historically consists of lid hygiene with warm compresses and eyelid scrubs, although these treatment modalities may have limited efficacy for many patients, especially those with more severe disease. Adjunctive treatment includes systemic and topical antibiotics, topical corticosteroids, and tear replacement therapy. Topical antibiotics are recommended to decrease the bacterial load, and topical corticosteroids may help in cases of severe inflammation. Azithromycin ophthalmic solution 1% in DuraSite((R)) (AzaSite((R)); Inspire Pharmaceuticals, Durham, North Carolina, USA) has been proposed as a novel treatment for posterior blepharitis, based on its well-known anti-infective profile, its anti-inflammatory properties, its excellent tissue penetration, and its regulatory approval for the treatment of bacterial conjunctivitis. This review focuses on an off-label indication for topical azithromycin 1% in DuraSite for the treatment of blepharitis.


Synthesis, structural studies, and oxidation catalysis of the late-first-row-transition-metal complexes of a 2-pyridylmethyl pendant-armed ethylene cross-bridged cyclam.
The first 2-pyridylmethyl pendant-armed ethylene cross-bridged cyclam ligand has been synthesized and successfully complexed to Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+) cations. X-ray crystal structures were obtained for all six complexes and demonstrate pentadentate binding of the ligand with the requisite cis-V configuration of the cross-bridged cyclam ring in all cases, leaving a potential labile binding site cis to the pyridine donor for interaction of the complex with oxidants and/or substrates. The electronic properties of the complexes were evaluated using solid-state magnetic moment determination and acetonitrile solution electronic spectroscopy, which both agree with the crystal structure determination of high-spin divalent metal complexes in all cases. Cyclic voltammetry in acetonitrile revealed reversible redox processes in all but the Ni(2+) complex, suggesting that catalytic reactivity involving electron-transfer processes is possible for complexes of this ligand. Kinetic studies of the dissociation of the ligand from the copper(II) complex under strongly acidic conditions and elevated temperatures revealed that the pyridine pendant arm actually destabilizes the complex compared to the parent cross-bridged cyclam complex. Screening for oxidation catalysis using hydrogen peroxide as the terminal oxidant for the most biologically relevant Mn(2+), Fe(2+), and Cu(2+) complexes identified the Mn(2+) complex as a potential mild oxidation catalyst worthy of continued development.


Feeding ecology and trophic comparisons of six shark species in a coastal ecosystem off southern Brazil.
The diets of six shark species, Sphyrna lewini, Sphyrna zygaena, Carcharhinus obscurus, Carcharhinus limbatus, Rhizoprionodon lalandii and Galeocerdo cuvier, were investigated in a subtropical coastal ecosystem of southern Brazil. Stomach content data were obtained to assess foraging niche segregation and ontogenetic shifts in the diets of these sharks. Five of the shark species off the Paraná coast were ichthyophagous, with the exception of S. zygaena, which was teutophagous. With the exception of G. cuvier, which had a generalist diet, the other five species displayed specialization in their feeding. Ontogenetic shifts were observed in C. obscurus and S. lewini with large individuals consuming elasmobranchs. Owing to the diet overlap between C. obscurus and S. lewini, C. obscurus and C. limbatus and R. lalandii and C. limbatus, future studies on the spatial and temporal distributions of these species are needed to understand the extent of competitive interactions.


Visual signs and symptoms of progressive supranuclear palsy.
Progressive supranuclear palsy is a rare, degenerative brain disorder and the second most common syndrome in which the patient exhibits 'parkinsonism', that is, a variety of symptoms involving problems with movement. General symptoms include difficulties with gait and balance; the patient walking clumsily and often falling backwards. The syndrome can be difficult to diagnose and visual signs and symptoms can help to separate it from closely related movement disorders such as Parkinson's disease, multiple system atrophy, dementia with Lewy bodies and corticobasal degeneration. A combination of the presence of vertical supranuclear gaze palsy, fixation instability, lid retraction, blepharospasm and apraxia of eyelid opening and closing may be useful visual signs in the identification of progressive supranuclear palsy. As primary eye-care practitioners, optometrists should be able to identify the visual problems of patients with this disorder and be expected to work with patients and their carers to manage their visual welfare.


Subclinical thyroid dysfunction and risk of carotid atherosclerosis.
The effect of subclinical thyroid dysfunction on vascular atherosclerosis remains uncertain. The objective of this study was to elucidate the association between sustained subclinical thyroid dysfunction and carotid plaques, which are an early surrogate marker of systemic atherosclerosis. The study included 21,342 adults with consistent thyroid hormonal status on serial thyroid function tests (TFTs) and carotid artery duplex ultrasonography at a health screening center between 2007 and 2014. The effect of subclinical thyroid dysfunction on baseline carotid plaques and newly developed carotid plaques during 5-year follow-up was determined by logistic regression analyses and GEE (Generalized Estimating Equations), respectively. Carotid plaques were more common in the subclinical hypothyroidism (55.6%) than the euthyroidism (47.8%) at baseline. However, in multivariable analysis, thyroid status was not a significant risk for the carotid plaques at baseline. Instead, traditional cardiovascular risk factors, such as age (P <0.001), systolic blood pressure (P = 0.023), fasting blood glucose (P = 0.030), and creatinine (P = 0.012) were associated with baseline carotid plaques in subclinical hypothyroidism. In longitudinal analyses of subjects who were followed up for more than 5 years, there was no significant difference in the cumulative incidence of new carotid plaques according to time between subjects with subclinical hypothyroidism and those with euthyroidism (P = 0.392). Sustained subclinical thyroid dysfunction did not affect the baseline or development of carotid plaques in healthy individuals.


The effect of finasteride on spermatogenesis of Mesocricetus auratus.
To study the effect of finasteride on the spermatogenesis of adult Mesocricetus auratus. Twenty adult hamsters were evaluated. The animals were one year-older, and were randomly divided in 2 different groups: control group with ten animals (n=10) and experimental group also with ten animals (n=10). The animals in the experimental group were shot 7.14 ng/mL (0.5mL) of finasteride by 100mg/Kg, subcutaneously in the dorsal region three times per week during 90 days. This dose corresponds to 5mg of the drug used in adult men for the treatment of benign prostatic hyperplasia (BPH). After three months, the animals were anesthetized through association of 200mg/kg ketamine chloridrate and 2.5 mg/kg of diazepan and were dead through hypovolemia.. The testis removed along with the whole genitourinary apparel were fixed with 10% formalin and submitted to histological analysis by optical microscopy. The hematoxilin-eosin (HE) method was used to stain the slides. The mean weight of animals in the control group before death was 129.0+/-18.8gr. The mean weight of animals in experimental group was 145.0+/-15.25gr. The mean age of animals in control group before death was 15.2+/-1.13 months. The mean age of animals in experimental group before death was 17.16+/-0.82 months. The mean difference in weight between both groups was not statistical significant (p=0.0514). The totality of animals in control group (100%) presented no tubular alterations and showed no disturbancy in the spermatogenesis stages. Four animals (40%) in the experimental group showed hypotrophy of the seminiferous tubules and six (60%) showed normal spermatogenesis, however reduced compared to control group. There was statistically significant difference (p=0.043) between the control and experimental group related to testicular alterations. The animals that were administered finasteride showed significant tubules atrophy and spermatogenesis reduction compared to control group.


Treatment of vaginal discharge with yoghurt.
A preliminary trial was conducted, using yoghurt to assess its potential for the management of vaginal discharge. The results were encouraging in non-specific vaginal discharge and warrant further investigation. The treatment was ineffective in reducing Trichomonas vaginalis infestation, and in this condition no further investigation is warranted.


Association of the Sweet-Liking Phenotype and Craving for Alcohol With the Response to Naltrexone Treatment in Alcohol Dependence: A Randomized Clinical Trial.
Identification of moderators of the response to naltrexone hydrochloride treatment for alcohol dependence could improve clinical care for patients with alcohol use disorders. To investigate the preliminary finding that the sweet-liking (SL) phenotype interacts with a high level of craving for alcohol and is associated with an improved response to naltrexone in alcohol dependence. This 12-week double-blind, randomized, placebo-controlled clinical trial was conducted from February 1, 2010, to April 30, 2012, in an academic outpatient medical center. Eighty actively drinking patients were randomized by the SL (n = 22) or the sweet-disliking (SDL) (n = 58) phenotype and by pretreatment high (n = 40) or low (n = 40) craving for alcohol, with high craving defined as greater than the median. Patients and staff were blinded to categorization. Patients were excluded for unstable medical or psychiatric illness, including dependence on drugs other than nicotine. Four patients (2 in the placebo arm and 2 in the naltrexone arm) stopped medication therapy because of adverse effects. Data were analyzed from January 15, 2013, to May 15, 2016, based on intention to treat. Oral naltrexone hydrochloride, 50 mg/d, or daily placebo with weekly to biweekly brief counseling. The a priori hypothesis tested SL/SDL phenotype, pretreatment craving, and their interaction as moderators of frequency of abstinent and heavy drinking days during treatment, assessed with the timeline follow-back method. Eighty patients were randomized (57 men [71%]; 23 women [29%]; mean [SD] age, 47.0 [8.6] years). A nonsignificant effect of naltrexone on heavy drinking was noted (4.8 fewer heavy drinking days; Cohen d = 0.45; 95% CI, -0.01 to 0.90; F1,67 = 3.52; P = .07). The SL phenotype moderated the effect of naltrexone on heavy drinking (6.1 fewer heavy drinking days; Cohen d = 0.58; 95% CI, 0.12-1.03; F1,67 = 5.65; P = .02) and abstinence (10.0 more abstinent days; Cohen d = 0.57; 95% CI, 0.11-1.02; F1,67 = 5.36; P = .02), and high craving moderated heavy drinking (7.1 fewer heavy drinking days; Cohen d = 0.66; 95% CI, 0.20-1.11; F1,67 = 7.37; P = .008). The combination of the SL phenotype and high craving was associated with a strong response to naltrexone, with 17.1 fewer heavy drinking days (Cohen d = 1.07; 95% CI, 0.58-1.54; F1,67 = 19.33; P < .001) and 28.8 more abstinent days (Cohen d = 0.72; 95% CI, 0.25-1.17; F1,67 = 8.73; P = .004) compared with placebo. The SL phenotype and a high craving for alcohol independently and particularly in combination are associated with a positive response to naltrexone. The SL/SDL phenotype and a high craving for alcohol merit further investigation as factors to identify patients with alcohol dependence who are responsive to naltrexone. clinicaltrials.gov Identifier: NCT01296646.


Calcium-sensing receptor and CPAP-induced neonatal airway hyperreactivity in mice.
Continuous positive airway pressure (CPAP) in preterm infants is initially beneficial, but animal models suggest longer term detrimental airway effects towards asthma. We used a neonatal CPAP mouse model and human fetal airway smooth muscle (ASM) to investigate the role of extracellular calcium-sensing receptor (CaSR) in these effects. Newborn wild type and smooth muscle-specific CaSR-/- mice were given CPAP for 7 days via a custom device (mimicking CPAP in premature infants), and recovered in normoxia for another 14 days (representing infants at 3-4 years). Airway reactivity was tested using lung slices, and airway CaSR quantified. Role of CaSR was tested using NPS2143 (inhibitor) or siRNA in WT mice. Fetal ASM cells stretched cyclically with/without static stretch mimicking breathing and CPAP were analyzed for intracellular Ca2+ ([Ca2+]i) responses, role of CaSR, and signaling cascades. CPAP increased airway reactivity in WT but not CaSR-/- mice, increasing ASM CaSR. NPS2143 or CaSR siRNA reversed CPAP effects in WT mice. CPAP increased fetal ASM [Ca2+]I, blocked by NPS2143, and increased ERK1/2 and RhoA suggesting two mechanisms by which stretch increases CaSR. These data implicate CaSR in CPAP effects on airway function with implications for wheezing in former preterm infants. Neonatal CPAP increases airway reactivity to bronchoconstrictor agonist. CPAP increases smooth muscle expression of the extracellular calcium-sensing receptor (CaSR). Inhibition or absence of CaSR blunts CPAP effects on contractility. These data suggest a causal/contributory role for CaSR in stretch effects on the developing airway. These data may impact clinical recognition of the ways that CPAP may contribute to wheezing disorders of former preterm infants.


Alpha 2-adrenergic model of gastroparesis: validation with renzapride, a stimulator of motility.
Motility-stimulating drugs can increase gastric antral and intestinal contractions but do not usually enhance emptying unless gastroparesis is present. An alpha 2-adrenergic agonist (SC-39585A) was used to inhibit antroduodenal motility and simulate gastroparesis in dogs. SC-39585A caused dose-related inhibition of emptying of solid and liquid meals as well as the antral and duodenal motility responses to the solid meal. The motility-enhancing agent renzapride (100 micrograms/kg iv) did not enhance emptying of the solid meal under nondelayed conditions. However, at the same dose it partially reversed the delay in solid emptying but only when antroduodenal motility was incompletely (30 micrograms/kg sc) and not totally (100 micrograms/kg sc) inhibited by SC-39585A. This was done in part by antagonizing antral but not duodenal inhibition of motility. Renzapride was also effective orally in reversing the delay in solid emptying. Similarly, renzapride reversed the delay in liquid emptying caused by SC-39585A (30 micrograms/kg sc). An alpha 2-adrenergic agonist can be used to model gastroparesis in dogs by inhibiting antroduodenal motility and can also be used to examine the actions of motor stimulants, such as renzapride, which promote gastric emptying.


Changing agglutination activities of salivary immunoglobulin A preparations against oral streptococci.
Immunoglobulin A (IgA) was prepared from samples of parotid saliva collected from two individuals over a 150-day period. Standardized solutions of the IgA were tested for agglutinating antibody activity against strains of streptococcus salivarius, S. sanguis, S. mitis, and S. mutans that had been isolated from a homologous individual on day 30 and preserved in the lyophilized state. The agglutinating activity of the standardized IgA preparations against these strains was found to change over time, whereas the agglutination patterns of IgA prepared from saliva samples collected 3 days apart were generally similar. Differences in agglutination titers were also observed between IgA samples prepared from saliva collected on day 0 and day 150. The data indicate that the oral cavity may harbor streptococci of different antigenic composition representing the same species at a given time and that parotid IgA agglutinating antibody against these serotypes changes over time.


[The role of peripheral cholinergic and adrenergic receptor systems in the action of histamine on the isolated coronary arteries].
The action of peripheral cholinergic and adrenergic receptor systems on the histamine coronary vasospastic effects are investigated in the experiments on the isolated ring-shaped strips of coronary arteries of pigs with the aid of specific agonists and antagonists. It has been demonstrated that neuromediator systems play a significant role in the regulation of contractile activity of the coronary spasm pathogenesis. When medicines are prescribed to patients with ischemic heart disease it is necessary to take into account the character of the preparations interaction which affect the peripheral neuromediator processes such as histaminergic, cholinergic, adrenergic and others.


Factors affecting complement activation by Staphylococcus aureus cell walls, their components, and mutants altered in teichoic acid.
In a previous study, Staphylococcus aureus purified cell walls (PCW), consisting of peptidoglycan (PG) plus covalently linked teichoic acid (TA), were found to be more active in complement consumption than isolated PG. Isolated TA has now been shown to be capable of activating complement. Mild sonication markedly increased the ability of PG to activate complement but had essentially no effect on the activities of PCW and TA. Optimal sonication of PG did not yield activities equal to those of PCW in dose-response and kinetic studies, which may imply that TA plays some role in complement consumption. Sonication did not lead to solubilization of PCW or PG but may have enhanced the activity of PG in complement consumption by better dispersing PG particles, thereby exposing more surface area. Lysostaphin solubilization of PCW and PG markedly decreased their activities in complement consumption. The PCW of an S. aureus TA-deficient mutant, which were mostly PG, caused similar amounts of complement consumption as the parent strain PCW. Of the treatments of PCW commonly used to isolate PG, formamide and periodate extractions in particular led to PG preparations with lower activities in complement consumption than the PCW from which they were prepared, although these activities were stimulated by sonication. When whole organisms were studied by using a TA-deficient mutant, a mutant with an additional cell surface polymer, and the TA-containing parent strains and complement consumption by these strains was compared, no difference was found in either the rate or the degree of complement activation. This led to experiments demonstrating that both material released extracellularly from staphylococci and the cytoplasmic fraction of S. aureus were active in complement consumption. The results of these experiments indicate that both physical and chemical factors must be considered in studies of complement activation by isolated bacterial cell wall components. Under certain conditions, staphylococcal TA may enhance complement activation, but studies with whole organisms clearly show that this cell wall constituent does not play an essential role in this process. In addition, studies of complement consumption with intact organisms have demonstrated that there may be contributions both from cell surface components and from material released by the cells.


Lipoprotein(a) and the risk of cardiovascular disease in the European population: results from the BiomarCaRE consortium.
As promising compounds to lower Lipoprotein(a) (Lp(a)) are emerging, the need for a precise characterization and comparability of the Lp(a)-associated cardiovascular risk is increasing. Therefore, we aimed to evaluate the distribution of Lp(a) concentrations across the European population, to characterize the association with cardiovascular outcomes and to provide high comparability of the Lp(a)-associated cardiovascular risk by use of centrally determined Lp(a) concentrations. Based on the Biomarkers for Cardiovascular Risk Assessment in Europe (BiomarCaRE)-project, we analysed data of 56 804 participants from 7 prospective population-based cohorts across Europe with a maximum follow-up of 24 years. All Lp(a) measurements were performed in the central BiomarCaRE laboratory (Biokit Quantia Lp(a)-Test; Abbott Diagnostics). The three endpoints considered were incident major coronary events (MCE), incident cardiovascular disease (CVD) events, and total mortality. We found lower Lp(a) levels in Northern European cohorts (median 4.9 mg/dL) compared to central (median 7.9 mg/dL) and Southern European cohorts (10.9 mg/dL) (Jonckheere-Terpstra test P < 0.001). Kaplan-Meier curves showed the highest event rate of MCE and CVD events for Lp(a) levels ≥90th percentile (log-rank test: P < 0.001 for MCE and CVD). Cox regression models adjusted for age, sex, and cardiovascular risk factors revealed a significant association of Lp(a) levels with MCE and CVD with a hazard ratio (HR) of 1.30 for MCE [95% confidence interval (CI) 1.15‒1.46] and of 1.25 for CVD (95% CI 1.12‒1.39) for Lp(a) levels in the 67‒89th percentile and a HR of 1.49 for MCE (95% CI 1.29‒1.73) and of 1.44 for CVD (95% CI 1.25‒1.65) for Lp(a) levels ≥ 90th percentile vs. Lp(a) levels in the lowest third (P < 0.001 for all). There was no significant association between Lp(a) levels and total mortality. Subgroup analysis for a continuous version of cube root transformed Lp(a) identified the highest Lp(a)-associated risk in individuals with diabetes [HR for MCE 1.31 (95% CI 1.15‒1.50)] and for CVD 1.22 (95% CI 1.08‒1.38) compared to those without diabetes [HR for MCE 1.15 (95% CI 1.08‒1.21; HR for CVD 1.13 (1.07-1.19)] while no difference of the Lp(a)- associated risk were seen for other cardiovascular high risk states. The addition of Lp(a) levels to a prognostic model for MCE and CVD revealed only a marginal but significant C-index discrimination measure increase (0.001 for MCE and CVD; P < 0.05) and net reclassification improvement (0.010 for MCE and 0.011 for CVD). In this large dataset on harmonized Lp(a) determination, we observed regional differences within the European population. Elevated Lp(a) was robustly associated with an increased risk for MCE and CVD in particular among individuals with diabetes. These results may lead to better identification of target populations who might benefit from future Lp(a)-lowering therapies.


The biochemical investigation of cases of hypoglycaemia: an assessment of the clinical effectiveness of analytical services.
To assess the extent to which biochemical analytical services contribute to the diagnosis and management of clinical cases of hypoglycaemia. All cases of confirmed hypoglycaemia, referred during a six month period, were included in the survey. Questionnaires were sent to each referring laboratory requesting information on the clinical progress and current status of the patient. The level of influence exerted by analytical data was assigned in each case and those with similar outcomes combined. Identifiable case groups were: (1) Results not recorded in the patients' notes (15.7%). (2) Inappropriate requesting of insulin and C peptide measurements in cases of diabetes (11.4%). (3) Patient died soon after investigation (20.0%). (4) Patient recovered spontaneously (17.1%). (5) Patient received effective medical or surgical treatment (12.9%). (6) Patient awaiting or not requiring pathology based treatment (31.4%). (7) Inconclusive outcome prompting further investigation (5.7%). Within the timescale of the survey (approximately 12 months), positive progress had been made towards diagnosis and subsequent treatment in only 10% of cases. Another 30% were either awaiting some form of treatment or further diagnostic tests. The remaining 60% did not appear to benefit in any way from the biochemical investigations.


In vitro assessment of the influence of intravenous extension set materials on insulin aspart drug delivery.
Insulin is a frequently prescribed drug in hospitals and is usually administered by syringe pumps with an extension line which can be made of various materials. Two insulin solutions were studied: an insulin analogue, Novorapid® which contains insulin aspart and two phenolic preservatives (e.g. phenol and metacresol) and Umuline rapide® with human insulin and metacresol as preservative. Some studies have indicated interactions between insulin, polyvinyl chloride (PVC) and polyethylene (PE). The aim of this work was to study such interactions between Novorapid® or Umuline rapide® and infusion extension line materials (PVC, PE and coextruded (PE/PVC)). Insulin solution at 1 IU/mL was infused at 2 mL/h over 24 hours with 16 different extension lines (8 in PVC, 3 in PE and 5 in PE/PVC). Ultra-Fast Liquid Chromatography with diode array detection (UFLC-DAD) was performed to quantify insulin (human and aspart) and preservatives (metacresol and phenol). Limited human insulin sorption was observed thirty minutes after the onset of infusion: 24.3 ± 12.9%, 3.1 ± 1.6% and 18.6 ± 10.0% for PVC, PE and PE/PVC respectively. With insulin aspart, sorption of about 5% was observed at the onset of infusion for all materials. However, there were interactions between phenol and especially metacresol with PVC, but no interactions with PE and PE/PVC. This study shows that insulin interacts with PVC, PE and PE/PVC at the onset of infusion. It also demonstrates that insulin preservatives interact with PVC, which may result in problems of insulin conservation and conformation. Some more studies are required to understand the clinical impact of the latter during infusion.


Surveillance for catheter-associated bloodstream infection in hematology units: quantifying the characteristics of a practical case definition.
We evaluated 66 patients in a hematology unit, who used a total of 106 central venous catheters (CVCs), to identify CVC-associated bloodstream infections using standard and modified surveillance case definitions. Compared with the National Nosocomial Infection Surveillance system criteria, a modified case definition used by treating physicians demonstrated 100.0% sensitivity and 94.3% specificity. This case definition provides a practical method for effectively excluding CVC-associated bloodstream infection.


Characterization of caveolins from human knee joint catilage: expression of caveolin-1, -2, and -3 in chondrocytes and association with integrin beta1.
Interactions between the extracellular matrix (ECM) and chondrocytes are of great importance for structure and function of cartilage. The present study was undertaken to answer the question whether caveolins take part in integrin-mediated cell-ECM interactions in the human cartilage. In samples of human knee joint cartilage, we detected the caveolin subtypes -1, -2, and -3 by immunohistochemical methods. Double-label experiments revealed a colocalization of caveolin with beta1-integrin. Results of immunoprecipitation and immunoblotting assays show that beta1-integrins associate with all three caveolin subtypes in human chondrocytes and indicate that they are part of the same complexes. Furthermore, immunoelectron microscopy shows the localization of beta1-integrin in caveolae-like structures of the cell membrane. The data stimulate further investigations on the role of the caveolin-integrin complex for integrin-mediated signaling pathways in chondrocytes.


5-Bromodeoxyuridine-induced formation of virus-like particles in Naegleria gruberi EGs.
Exposure of axenic cultures of the amoeboflagellate Naegleria gruberi EGs to the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) resulted in the induction of virus-like particles (VLP) and various structures associated with their development and presumed transmission. Previously, VLP induction could be accomplished only by growing amoebae in the presence of living bacteria as a food source. Addition of excess thymidine along with BrdU did not block induction of particles. This account demonstrates that the EGs-VLP system responded to BrdU as do a number of mammalian cell lines harbouring latent viruses, and provides the basis for future work on the infectivity of the VLPs for other amoebae as well as tissue culture cells.


Structure of the retinoblastoma protein bound to adenovirus E1A reveals the molecular basis for viral oncoprotein inactivation of a tumor suppressor.
The adenovirus (Ad) E1A (Ad-E1A) oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the retinoblastoma protein (pRb) tumor suppressor. In this study we determined the crystal structure of the pRb pocket domain in complex with conserved region 1 (CR1) of Ad5-E1A. The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transactivation domain use similar conserved nonpolar residues to engage overlapping sites on pRb, implicating a novel molecular mechanism for pRb inactivation by a viral oncoprotein.


Avian eggshell thinning caused by transovarian exposure to o,p'-DDT: changes in histology and calcium-binding protein production in the oviduct uterus.
Reproductive disorders in birds are the most characteristic effects of DDT contamination of wildlife. Experimental exposure of avian eggs to the estrogenic substance o,p'-DDT causes abnormal development of the reproductive tract (shortening of the left oviduct and aberrant development of the right oviduct) and eggshell thinning in mature birds, but it is still not known how eggshell thinning occurs in the abnormal oviduct. To fill this information gap, we examined the histology of the uterine part of the oviduct in Japanese quail treated in ovo with o,p'-DDT or a synthetic estrogen, diethylstilbestrol (DES), and we performed immunohistochemical staining for the calcium-binding proteins CALB1, SPP1, and TRPV6. Both o,p'-DDT-treated and DES-treated quail had few, and scattered, gland cells in the left uterus, unlike vehicle controls, in which gland cells tightly occupied the lamina propria. The aberrantly developed right uterus retained all the components of the normal left uterus, but in immature form. Immunostaining for CALB1, SPP1, and TRPV6 was greatly reduced by both o,p'-DDT and DES; SPP1 and TRPV6 immunostaining patterns, in particular, differed distinctly from those in the controls. These findings suggest that CALB1, SPP1, and TRPV6 are molecular factors, decreased production of which is responsible for eggshell thinning. Our findings also could contribute to understanding of the eggshell formation mechanism in birds.