Simvastatin protects against multiple low-dose streptozotocin-induced type 1 diabetes in CD-1 mice and recurrence of disease in nonobese diabetic mice.
Statins are drugs well known for their cholesterol-lowering properties. Lately, statins have been shown to possess anti-inflammatory properties that might be attributed to inhibition of leukocyte adhesion and migration to sites of inflammation. Therefore, we have explored the effects of administration of simvastatin (30 mg/kg body weight given i.p. once a day, from days 4-14) on the development of diabetes induced by multiple low-dose streptozotocin (MLDS) in CD-1 mice, a type 1 diabetes model. We found that treatment with simvastatin could delay and in certain mice fully protect against MLDS-induced diabetes. The protective effect could last up to 3 weeks after simvastatin treatment was ended. Morphological examinations of the pancreas suggest that simvastatin might reduce the islet inflammation. Based on experiments in vitro, using isolated pancreatic islets, we conclude that the protective effect of simvastatin is not mediated by a direct effect on streptozotocin action but rather the result of an immunomodulatory effect. This was reinforced by the finding that simvastatin treatment also prolonged islet function in the recurrence of disease model in diabetic nonobese diabetic mice.


Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism.
Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na(+)-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression.


A compendium of inborn errors of metabolism mapped onto the human metabolic network.
Inborn errors of metabolism (IEMs) are hereditary metabolic defects, which are encountered in almost all major metabolic pathways occurring in man. Many IEMs are screened for in neonates through metabolomic analysis of dried blood spot samples. To enable the mapping of these metabolomic data onto the published human metabolic reconstruction, we added missing reactions and pathways involved in acylcarnitine (AC) and fatty acid oxidation (FAO) metabolism. Using literary data, we reconstructed an AC/FAO module consisting of 352 reactions and 139 metabolites. When this module was combined with the human metabolic reconstruction, the synthesis of 39 acylcarnitines and 22 amino acids, which are routinely measured, was captured and 235 distinct IEMs could be mapped. We collected phenotypic and clinical features for each IEM enabling comprehensive classification. We found that carbohydrate, amino acid, and lipid metabolism were most affected by the IEMs, while the brain was the most commonly affected organ. Furthermore, we analyzed the IEMs in the context of metabolic network topology to gain insight into common features between metabolically connected IEMs. While many known examples were identified, we discovered some surprising IEM pairs that shared reactions as well as clinical features but not necessarily causal genes. Moreover, we could also re-confirm that acetyl-CoA acts as a central metabolite. This network based analysis leads to further insight of hot spots in human metabolism with respect to IEMs. The presented comprehensive knowledge base of IEMs will provide a valuable tool in studying metabolic changes involved in inherited metabolic diseases.


[Internal Medicine Residencies in Argentina, 2008].
The acquisition of medical competence in the Internal Medicine Residencies (IMR) is a complex process. In Argentina there are not current data available on the number, organization and the way the residencies work. The objective of this study was to survey the IMR in Argentina and categorize their academic and general structure. From June 2007 to June 2008 we identified all the IMR using a combined searching strategy. We contacted them looking for structural and organizational data. Academic data were collected in a sample of 24 residencies using questionnaires. We also gathered data using qualitative measurements, in a subsample of 15 residencies, by means of direct observation and in-depth interviews. We identified 162 ongoing residencies and a total of 2012 residents. The majority of them (87%) were located in big cities, with a preponderance of public residencies (66%); 13% of these didn't have a residency instructor or coordinator. Most of these didn't have Institutional Internet available. The residents median age was 29 years old (Intercuartil range 2.7), with a 64.5% of women; 24% were under the regimen called "concurrente". From the sample, 230 residents were interviewed; 13% of them (CI95% 9-18.7) did not receive any remuneration. The rest received a monthly payment (in Arg $) ranging from US$ 140 to 552. A 58% (13/22) was involved in Specialist courses. Medians for weekly working hours and for "on duty" were respectively 50 and 8 per month, with no difference between public or private institutions. These findings evidence the need to certify the formative and academic process of IMR.


Analysis of the Micro-RNA-133 and PITX3 genes in Parkinson's disease.
MicroRNAs are small RNA sequences that negatively regulate gene expression by binding to the 3' untranslated regions of mRNAs. MiR-133b has been implicated in Parkinson's disease (PD) by a mechanism that involves the regulation of the transcription factor PITX3. The variation in these genes could contribute to the risk of developing PD. We searched for DNA variants in miR-133 and PITX3 genes in PD patients and healthy controls from Spain. We found common DNA variants in the three miR-133 genes. Genotyping of a first set of patients (n = 777) and controls (n = 650) showed a higher frequency of homozygous for a miR-133b variant (-90 del A) in PD-patients (6/575; 1%) than in healthy controls (0/650) (P = 0.03). However, this association was not confirmed in a second set of patients (1/250; 0.4%) and controls (2/210; 1%). No common PITX3 variants were associated with PD, although a rare missense change (G32S) was found in only one patient and none of the controls. In conclusion, we report the variation in genes of a pathway that has been involved in dopaminergic neuron differentiation and survival. Our work suggests that miR-133 and PITX3 gene variants did not contribute to the risk for PD.


Structural determinants of the ligand-binding site of the human retinoic acid receptor alpha.
The ligand-dependent transactivating properties of retinoic acid receptors are controlled through a complex structure at the C-terminus of these proteins, commonly referred to as the hormone binding domain. This domain is involved not only in ligand recognition but also in protein-protein interactions such as homo- and heterodimerization processes. To identify more precisely regions of the human all-trans-retinoic acid receptor alpha (hRAR alpha) that are involved in ligand binding, we constructed a series of deletion mutants of this molecule and overexpressed them in bacteria. We found that the C-terminal part of the D domain (amino acids 186-198) was necessary for ligand binding. The F domain and the 10 C-terminal amino acids of the E domain were dispensable for high-affinity binding of various natural and synthetic retinoids. A further deletion to position 403 resulted in a moderate decrease in affinity for all-trans-(ATRA) and 9-cis-retinoic acids, whereas the binding of two RAR alpha-specific ligands (Am80 and Am580) was abolished. In addition, hRAR alpha and the minimal hormone binding domain (amino acids 186-410) bound ATRA with a positive, cooperative mechanism. This behavior was not observed with CD367, a conformationally restricted synthetic retinoid. The positive cooperativity could be correlated with stable ATRA binding to RAR homodimers, whose formation was triggered by ligand. In the same conditions, only monomeric CD367-RAR alpha complexes were detected. These data indicate that ligand binding to hRAR alpha requires the presence of part of the D domain, whereas the C-terminal end of the E domain is involved in more subtle ligand recognition processes.(ABSTRACT TRUNCATED AT 250 WORDS)


Circulating tumor cells versus imaging--predicting overall survival in metastatic breast cancer.
The presence of >or=5 circulating tumor cells (CTC) in 7.5 mL blood from patients with measurable metastatic breast cancer before and/or after initiation of therapy is associated with shorter progression-free and overall survival. In this report, we compared the use of CTCs to radiology for prediction of overall survival. One hundred thirty-eight metastatic breast cancer patients had imaging studies done before and a median of 10 weeks after the initiation of therapy. All scans were centrally reviewed by two independent radiologists using WHO criteria to determine radiologic response. CTC counts were determined approximately 4 weeks after initiation of therapy. Specimens were analyzed at one of seven laboratories and reviewed by a central laboratory. Interreader variability for radiologic responses and CTC counts were 15.2% and 0.7%, respectively. The median overall survival of 13 (9%) patients with radiologic nonprogression and >or=5 CTCs was significantly shorter than that of the 83 (60%) patients with radiologic nonprogression and <5 CTCs (15.3 versus 26.9 months; P=0.0389). The median overall survival of the 20 (14%) patients with radiologic progression and <5 CTCs was significantly longer than the 22 (16%) patients with >or=5 CTCs that showed progression by radiology (19.9 versus 6.4 months; P=0.0039). Assessment of CTCs is an earlier, more reproducible indication of disease status than current imaging methods. CTCs may be a superior surrogate end point, as they are highly reproducible and correlate better with overall survival than do changes determined by traditional radiology.


The role of proximity to death in need-based approaches to health care.
This study examines the role of proximity to death (PTD) in need-based approaches to health care by: (1) investigating whether PTD is a statistically significant, independent predictor of health-care use; and (2) estimating PTD's marginal impact on need-based allocation of health-care resources. The primary data source is the Canadian National Population Health Survey (NPHS), a longitudinal survey that uses vital statistics to confirm deaths of the respondents. We use two-part models separately for general practitioner, specialist, and short stay inpatient hospital services. We calculate per-capita allocation, with and without PTD, from the Canadian federal government to its ten provinces and by income groups. PTD is a robust and important predictor of health-care resource use for each service even after adjustment for other need and non-need factors. PTD's marginal impact on allocation is relatively small in the contexts we examined, but failure to include PTD could introduce inequity in allocation by disadvantaging populations with greater need. PTD is an important need indicator when modeling health-care resource requirements. It deserves greater attention in need-based approaches to health-care planning and resource allocation.


Antimicrobial stewardship programs; a two-part narrative review of step-wise design and issues of controversy Part I: step-wise design of an antimicrobial stewardship program.
Regardless of one's opinion of antimicrobial stewardship programs (ASPs), it is hardly possible to work in hospital care and not be exposed to the term or its practical effects. Despite the term being relatively new, the number of publications in the field is vast, including several excellent reviews of general and specific aspects. Work in antimicrobial stewardship is complex, and includes not only aspects of infectious disease and microbiology, but also of epidemiology, genetics, behavioural psychology, systems science, economics and ethics, to name a few. This review aims to take several of these aspects and the scientific evidence of antimicrobial stewardship studies and merge them into two questions: How should we design ASPs based on what we know today? And which are the most essential unanswered questions regarding antimicrobial stewardship on a broader scale? This narrative review is written in two separate parts aiming to provide answers to the two questions. This first part is written as a step-wise approach to designing a stewardship intervention based on the pillars of unmet need, feasibility, scientific evidence and necessary core elements. It is written mainly as a guide to someone new to the field. It is sorted into five distinct steps: (a) focusing on designing aims; (b) assessing performance and local barriers to rational antimicrobial use; (c) deciding on intervention technique; (d) practical, tailored design including core element inclusion; and (e) evaluation and sustainability. The second part, published separately, formulates ten critical questions on controversies in the field of antimicrobial stewardship. It is aimed at clinicians and researchers with stewardship experience and strives to promote discussion, not to provide answers.


Hereditary Gastrointestinal Cancer Syndromes: Role of Imaging in Screening, Diagnosis, and Management.
Hereditary gastrointestinal (GI) cancer syndromes due to specific germline mutations are characterized by an increased risk of GI tract malignancies, extra-GI tract cancers, and benign abnormalities. These syndromes include Lynch syndrome, familial adenomatous polyposis, juvenile polyposis syndrome, Peutz-Jeghers syndrome, Cowden syndrome, hereditary diffuse gastric cancer, and hereditary pancreatic cancer. Timely identification of the responsible genes will help predict future cancer risks in these patients and their family members. Early detection of cancers is possible with appropriate screening methods; risk-reducing measures will help in cancer prevention. Select malignancies and benign conditions associated with these syndromes have distinctive imaging features that can aid in classifying the syndromes. Imaging also plays a pivotal role in screening and surveillance of patients as well as their at-risk relatives and is invaluable for follow-up of treated malignancies. The American College of Gastroenterology has established specific guidelines for diagnosis and management of hereditary GI cancer syndromes. Knowledge of the imaging features of various pathologic conditions and screening strategies will guide appropriate management of patients and at-risk family members. ©RSNA, 2019.


The Escherichia coli MG1655 in silico metabolic genotype: its definition, characteristics, and capabilities.
The Escherichia coli MG1655 genome has been completely sequenced. The annotated sequence, biochemical information, and other information were used to reconstruct the E. coli metabolic map. The stoichiometric coefficients for each metabolic enzyme in the E. coli metabolic map were assembled to construct a genome-specific stoichiometric matrix. The E. coli stoichiometric matrix was used to define the system's characteristics and the capabilities of E. coli metabolism. The effects of gene deletions in the central metabolic pathways on the ability of the in silico metabolic network to support growth were assessed, and the in silico predictions were compared with experimental observations. It was shown that based on stoichiometric and capacity constraints the in silico analysis was able to qualitatively predict the growth potential of mutant strains in 86% of the cases examined. Herein, it is demonstrated that the synthesis of in silico metabolic genotypes based on genomic, biochemical, and strain-specific information is possible, and that systems analysis methods are available to analyze and interpret the metabolic phenotype.


A Scoping Review of the Quality and the Design of Evaluations of Mobile Health, Telehealth, Smart Pump and Monitoring Technologies Performed in a Pharmacy-Related Setting.
Background: There is currently a need for high quality evaluations of new mobile health, telehealth, smart pump and monitoring technologies undertaken in a pharmacy-related setting. We aim to evaluate the use of these monitoring technologies performed in this setting. Methods: A systematic searching of English articles that examined the quality and the design of technologies conducted in pharmacy-related facilities was performed using the following databases: MEDLINE and Cumulative index to Nursing and Allied Health Literature (CINAHL) to identify original studies examining the quality and the design of technologies and published in peer-reviewed journals. Extraction of articles and quality assessment of included articles were performed independently by two authors. Quality scores over 75% are classed as being acceptable using a "relatively conservative" quality benchmark. Scores over 55% are included using a "relatively liberal" cut-off point. Results: Screening resulted in the selection of 40 formal evaluations. A substantial number of studies (32, 80.00%) were performed in the United States, quantitative in approach (33, 82.50%) and retrospective cohort (24, 60.00%) in study design. The most common pharmacy-related settings were: 22 primary care (55.00%); 10 hospital pharmacy (25.00%); 7 community pharmacy (17.50%); one primary care and hospital pharmacy (2.50%). The majority of the evaluations (33, 82.50%) reported clinical outcomes, six (15.00%) measured clinical and economic outcomes, and one (2.50%) economic only. Twelve (30.00%) quantitative studies and no qualitative study met objective criteria for "relatively conservative" quality. Using a lower "relatively liberal" benchmark, 27 quantitative (81.82%) and four qualitative (57.41%) studies met the lower quality criterion. Conclusion: Worldwide, few evaluations of mobile health, telehealth, smart pump and monitoring technologies in pharmacy-related setting have been published.Their quality is often below the standard necessary for inclusion in a systematic review mainly due to inadequate study design.


National incidence and prevalence of TSH-secreting pituitary adenomas in Sweden.
TSH-secreting pituitary adenomas (TSHomas) are rare. Epidemiological data are scant and there are no reports on national incidence. The objective of the study was to estimate the national Swedish incidence and prevalence of TSHomas. This was an observational study. The study was conducted at tertiary referral centers. The Swedish Pituitary Registry and World Health Organization International Statistical Classification of Diseases and Related Health Problems coding at all university hospitals were used to identify patients diagnosed with TSHomas 1990-2010. The identified patients' medical records were studied until the latest follow-up [median 5.0 years (range < 1-20 years)]. Incidence, prevalence, demographics, tumor characteristics, treatment outcome, and thyroid hormone level at diagnosis were measured. The age-standardized national incidence of 28 TSHoma patients was 0.15 per 1 million inhabitants per year, with an increasing incidence over time (0.05 per 1 million per year in 1990-1994 to 0.26 per 1 million per year in 2005-2009). The national prevalence in 2010 was 2.8 per 1 million inhabitants, in which 0.85 per 1 million had active disease. Most patients (n = 22) underwent pituitary surgery, 5 had radiotherapy, and 6 had somatostatin analogues. Eighteen patients were considered cured at the latest follow-up; 25% remained uncontrolled. Subjects treated for putative primary hyperthyroidism prior to diagnosis had TSH levels more than double those with intact thyroid at diagnosis (P = .013). The median time to diagnosis was longer for women than men (4 vs < 1 year, P = .026). More women than men were treated surgically (94.1% vs 54.5%, P = .022). This is the first estimate of a national incidence of TSHoma. Additional epidemiological studies are needed to compare these results with other geographical areas. This study suggests an increased incidence of TSHomas, in agreement with reports on other pituitary adenomas.


Melanocytes and iris color. Electron microscopic findings.
To quantitatively associate iris color with melanocyte pigment content. Autopsy eyes were classified as uniform-blue, uniform-hazel, or uniform-brown or showing a darker peripupillary ring. Using electron microscopic images and computerized image analysis, area, number, and size of mature melanosomes within the perinuclear cytoplasmic area only or within perinuclear and peripheral cytoplasmic areas of the superficial stromal melanocytes combined were measured. Average melanosomal area per perinuclear cytoplasmic area (AMAC) and average number of melanosomes per perinuclear area (AMNC) significantly differed across iris color groups (overall P<.001). This result reflects the large difference between blue-uniform and all other color groups. A marginally significant (nominal) trend from blue-ring through brown-ring was also detected (P=.06 for AMAC and P=.07 for AMNC). The average perinuclear cytoplasmic area was larger in the central iris zone (within 1 mm around the pupillary margin) than in the intermediate iris zone (between 1 and 2 mm around the pupillary margin) (P=.002), but AMAC and AMNC did not significantly differ between zones. The average melanosome size did not differ significantly across color groups (P=.11). Differences in iris colors are at least partially attributed to variable AMNC and AMAC within superficial melanocytes.


Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation.
Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying.


Traditional and Complementary Medicine in Pediatric Oncology and Low-Middle Income Countries: Recommendations from the International Society of Pediatric Oncology (SIOP), T&CM Collaborative.
Significant strides have been made in the treatment of childhood cancer. Improvements in survival have led to increased attention toward supportive care indications; including the use of traditional and complementary medicine (T&CM). The use of T&CM among children and adolescents with cancer is well documented in both high-income countries (HICs) and low-middle income countries (LMICs). A higher incidence of the use of T&CM has been reported among children undergoing treatment in LMICs, which has elevated concerns related to drug interactions, adherence to therapy, and treatment-related toxicities. These observations have underscored the need for effective models of integrative care that are culturally sensitive yet sustainable in an LMIC setting. We present considerations inclusive of the clinical care, educational opportunities, governmental policy, and research priorities necessary for the development of models of integrative care for pediatric cancer units in an LMIC setting.


Mitochondrial Function in Enamel Development.
Enamel is the most calcified tissue in vertebrates. Enamel formation and mineralization is a two-step process that is mediated by ameloblast cells during their secretory and maturation stages. In these two stages, ameloblasts are characterized by different morphology and function, which is fundamental for proper mineral growth in the extracellular space. Ultrastructural studies have shown that the mitochondria in these cells localize to different subcellular regions in both stages. However, limited knowledge is available on the role/s of mitochondria in enamel formation. To address this issue, we analyzed mitochondrial biogenesis and respiration, as well as the redox status of rat primary enamel cells isolated from the secretory and maturation stages. We show that maturation stage cells have an increased expression of PGC1α, a marker of mitochondrial biogenesis, and of components of the electron transport chain. Oxygen consumption rate (OCR), a proxy for mitochondrial function, showed a significant increase in oxidative phosphorylation during the maturation stage, promoting ATP production. The GSH/GSSG ratio was lower in the maturation stage, indicative of increased oxidation. Because higher oxidative phosphorylation can lead to higher ROS production, we tested if ROS affected the expression of AmelX and Enam genes that are essential for enamel formation. The ameloblast cell line LS8 treated with H2O2 to promote ROS elicited significant expression changes in AmelX and Enam. Our data highlight important metabolic and physiological differences across the two enamel stages, with higher ATP levels in the maturation stage indicative of a higher energy demand. Besides these metabolic shifts, it is likely that the enhanced ETC function results in ROS-mediated transcriptional changes.


Effect of amine content and chemistry on long-term, three-dimensional hepatocyte spheroid culture atop aminated elastin-like polypeptide coatings.
Culture conditions that induce hepatic spheroidal aggregates sustain liver cells with metabolism that mimics in vivo hepatocytes. Here we present an array of elastin-like polypeptide conjugate coating materials (Aminated-ELPs) that are biocompatible, have spheroid-forming capacity, can be coated atop traditional culture surfaces, and maintain structural integrity while ensuring adherence of spheroids over long culture period. The Aminated-ELPs were synthesized either by direct conjugation of ELP and various polyelectrolytes or by conjugating both ELP and various small electrolytes to the reactive polymer poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA). Spheroid morphology, cellular metabolic function, and liver-specific gene expression over the long-term, 20-day culture period were assessed through optical microscopy, measurement of total protein content and albumin and urea production, and quantitative real-time (qRT) PCR. We found that the amine content of the Aminated-ELP coatings dictated the initial hepatocyte attachment, but not the subsequent hepatocyte spheroid formation and their continued attachment. A lower amine content was generally found to sustain higher albumin production by the spheroids. Out of the 19 Aminated-ELP coatings tested, we found that the lysine-containing substrates comprising ELP-polylysine or ELP-PVDMA-butanediamine proved to consistently culture productive spheroidal hepatocytes. We suggest that the incorporation of lysine functional groups in Aminated-ELP rendered more biocompatible surfaces, increasing spheroid attachment and leading to increased liver-specific function. Taken together, the Aminated-ELP array presented here has the potential to create in vitro hepatocyte culture models that mimic in vivo liver functionality and thus, lead to better understanding of liver pathophysiology and superior screening methods for drug efficacy and toxicity. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 377-388, 2017.


Dynamic alignment models for neural coding.
Recently, there have been remarkable advances in modeling the relationships between the sensory environment, neuronal responses, and behavior. However, most models cannot encompass variable stimulus-response relationships such as varying response latencies and state or context dependence of the neural code. Here, we consider response modeling as a dynamic alignment problem and model stimulus and response jointly by a mixed pair hidden Markov model (MPH). In MPHs, multiple stimulus-response relationships (e.g., receptive fields) are represented by different states or groups of states in a Markov chain. Each stimulus-response relationship features temporal flexibility, allowing modeling of variable response latencies, including noisy ones. We derive algorithms for learning of MPH parameters and for inference of spike response probabilities. We show that some linear-nonlinear Poisson cascade (LNP) models are a special case of MPHs. We demonstrate the efficiency and usefulness of MPHs in simulations of both jittered and switching spike responses to white noise and natural stimuli. Furthermore, we apply MPHs to extracellular single and multi-unit data recorded in cortical brain areas of singing birds to showcase a novel method for estimating response lag distributions. MPHs allow simultaneous estimation of receptive fields, latency statistics, and hidden state dynamics and so can help to uncover complex stimulus response relationships that are subject to variable timing and involve diverse neural codes.


Visual adaptation of the perception of causality.
We easily recover the causal properties of visual events, enabling us to understand and predict changes in the physical world. We see a tennis racket hitting a ball and sense that it caused the ball to fly over the net; we may also have an eerie but equally compelling experience of causality if the streetlights turn on just as we slam our car's door. Both perceptual and cognitive processes have been proposed to explain these spontaneous inferences, but without decisive evidence one way or the other, the question remains wide open. Here, we address this long-standing debate using visual adaptation-a powerful tool to uncover neural populations that specialize in the analysis of specific visual features. After prolonged viewing of causal collision events called "launches", subsequently viewed events were judged more often as noncausal. These negative aftereffects of exposure to collisions are spatially localized in retinotopic coordinates, the reference frame shared by the retina and visual cortex. They are not explained by adaptation to other stimulus features and reveal visual routines in retinotopic cortex that detect and adapt to cause and effect in simple collision stimuli.


Hemoglobin and Erythropoietin After Commercial Saturation Diving.
Saturation divers are exposed to elevated partial pressure of oxygen (ppO2) in their hyperbaric work environment. Experimental studies indicate that oxygen transport is altered, and we have previously reported a drop in hematocrit and extensive downregulation of genes involved in blood oxygen transport capacity after decompression from professional saturation diving. Here we investigate the initial period of hematological adjustment back to normobaric air after professional saturation diving. Erythropoietin (EPO) and hemoglobin (Hb) were measured in blood from 13 divers at two time-points after saturation assignments lasting up to 4 weeks; first immediately after decompression and again 24 h later. Pre-dive levels defined baselines. The ppO2 varied from 40 kPa in the saturation chambers during storage, 50 to 80 kPa during bell excursions, and gradually reduced to 21 kPa during decompression to surface pressure. EPO was similar to baseline immediately after saturation diving (P = 0.4), and markedly increased within the next 24 h (99%, P < 0.0002). Hb levels remained slightly reduced at both time-points (4% immediately after; P = 0.02, 8% 24 h after; P < 0.001). The results imply that the hematological acclimatization back to normobaric air was ongoing, but not completed, during the first 24 h after professional saturation diving.


Parental Alienation Syndrome in Italian legal judgments: an exploratory study.
The present study highlights the characteristics of separated families in Italy for whom Parental Alienation Syndrome (PAS) has been diagnosed during court custody evaluations. The study analyzed the psychological reports of 12 court-appointed expert evaluations of families for whom PAS had been diagnosed. Twelve evaluations that did not receive the PAS diagnosis served as a control group. A specific coding system was used for data analysis. The results indicated that the alienating parents were always the parents who had custody of the children. Children who were diagnosed with PAS were predominantly the only child in the family, had identity problems and manifested manipulative behavior. The consultant in these cases suggested individual psychotherapy for the children and recommended foster care to the Social Services agency.


Primary osteoarthrosis of the knee in men and women as a result of lifelong physical load from work.
This study investigated the effect of lifelong physical load from work on the development of knee osteoarthrosis (OA) leading to prosthetic surgery among men and women. In a population-based case-referent study, men and women (N=625) who had had prosthetic surgery due to primary tibiofemoral OA were compared with referents (N=548) as to job titles and exposure to physical load in occupational work, housework, and leisure-time activities from 15 to 50 years of age. Male forestry and construction workers, and both male and female farmers ran the highest risk of knee OA. The men had considerably higher exposure to lifting at work, and also to jumps and vibration, than the women. Among the men there was an association between lifting at work [odds ratio (OR) 3.0, 95% confidence interval (95% CI) 1.6-5.5], squatting or knee bending (OR 2.9, 95% CI 1.7-4.9), kneeling (OR 2.1, 95% CI 1.4-3.3), and jumping (OR 2.7, 95% CI 1.7-4.1) with knee OA. Exposure to physically demanding tasks at home, such as taking care of an elderly or handicapped person, was associated with knee OA among the women (OR 2.2, 95% CI 1.3-3.6). Working as a farmer or as a construction worker could be associated with the development of knee OA and lead to prosthetic surgery. Men and women differ in the quality and quantity of reported physical load and also in the strength of the risk estimates. A reduction of high physical load at work and at home could probably lower the risk of knee OA later in life.


Covenantal relationships: grounding for the nursing ethic.
This article is in reaction to recent articles in Advances In Nursing Science by Yarling and McElmurry and Bishop and Scudder, which interpret the nursing ethic as grounded in the social reform of the institution or in the role of the nurse on the health care team. It is argued here that covenantal relationships between the nurse and the patient provide a more substantial foundation for the nursing ethic. Arising from the moral principle of fidelity, this model furnishes guidance for the nurse, a link for the nursing ethic with traditional moral theory, and, most important, a grounding in the unique experience of the nurse-patient relationship.


Urbanization, regime type and durability, and environmental degradation in Ghana.
This study examines the effect of urbanization, income, trade openness, and institutional quality (i.e., regime type and durability) on environmental degradation in Ghana over the period 1965-2011. Using the bounds test approach to cointegration and the Fully Modified Phillip-Hansen (FMPH) technique, the findings show that urbanization, income, trade openness, and institutional quality have long-run cointegration with environmental degradation. Further, the results show that income, trade openness, and institutional quality are negatively associated with environmental degradation. This suggests that income, trade openness, and institutional quality enhance environmental performance. Urbanization, however, is positively related to environmental degradation. Additionally, long-run estimates conditioned on institutional quality reveal that the extent to which trade openness and urbanization enhance environmental performance is largely due to the presence of quality institutions (or democratic institutions). Finally, controlling for structural breaks, we find that trade openness, urbanization, and regime type (i.e., democracy) improve environmental performance significantly after the 1970s except for income.


Health effects associated with exposure to intimate partner violence against women and childhood sexual abuse: a burden of proof study.
The health impacts of intimate partner violence against women and childhood sexual abuse are not fully understood. Here we conducted a systematic review by comprehensively searching seven electronic databases for literature on intimate partner violence-associated and childhood sexual abuse-associated health effects. Following the burden of proof methodology, we evaluated the evidence strength linking intimate partner violence and/or childhood sexual abuse to health outcomes supported by at least three studies. Results indicated a moderate association of intimate partner violence with major depressive disorder and with maternal abortion and miscarriage (63% and 35% increased risk, respectively). HIV/AIDS, anxiety disorders and self-harm exhibited weak associations with intimate partner violence. Fifteen outcomes were evaluated for their relationship to childhood sexual abuse, which was shown to be moderately associated with alcohol use disorders and with self-harm (45% and 35% increased risk, respectively). Associations between childhood sexual abuse and 11 additional health outcomes, such as asthma and type 2 diabetes mellitus, were found to be weak. Although our understanding remains limited by data scarcity, these health impacts are larger in magnitude and more extensive than previously reported. Renewed efforts on violence prevention and evidence-based approaches that promote healing and ensure access to care are necessary.


Outpatient Intravenous Diuretic Clinic: An Effective Strategy for Management of Volume Overload and Reducing Immediate Hospital Admissions.
Heart failure (HF) readmissions pose a major burden to patients and the healthcare system. We evaluated whether outpatient intravenous (IV) diuretic clinic is a safe and effective strategy to reduce HF hospitalizations. We reviewed 34 clinic encounters with 27 unique patients (median age 72) who had volume overload refractory to oral diuretics that were treated with IV furosemide in the outpatient clinic. One patient (2.9%) was admitted to the hospital directly, and the rest were discharged home. More than 80% of the patients had continued weight loss for 7 days (median weight loss 5.4 lb). During the median follow-up period of 15 months, 15 patients (56%) had subsequent HF hospitalizations. HF admission was delayed by a median of 22 days from the clinic visit and 138 days from the previous HF admission prior to clinic visit. Estimated cost saving per admission avoided was $10,395. One patient developed severe hypokalemia (< 3.0 mmol/L), and the remaining had no adverse events. Outpatient IV diuresis is effective and well tolerated. It leads to significant weight loss, persisting in the majority of patients for 7 days. In select patients, it should be considered as a strategy to rapidly improve symptoms, reduce hospitalizations and decrease costs.


Assessment of Orofacial Strength in Patients with Dysarthria.
Assessment of orofacial weakness is common during the evaluation of patients with suspected dysarthria. This study addressed the validity of clinical assessments of orofacial weakness by comparing clinical (subjective) ratings to instrumental (objective) measures. Forty-four adults referred to a speech pathology clinic for dysarthria evaluation were tested for strength of the tongue during elevation, lateralization, and protrusion, and for the strength of the muscles of the lower face during buccodental and interlabial compression. Subjective assessment of weakness involved rating maximum resistance against a firmly held tongue depressor, using a 5-point scale. Objective assessment involved the Iowa Oral Performance Instrument (IOPI), measured as the maximal pressure generated against an air-filled bulb. A recent adaptation to the IOPI permitted testing of tongue and cheek strength using tasks that are comparable to the subjective tasks. Moderate correlations were found between the objective and subjective evaluations, with the strongest correlations for tongue lateralization. Lower pressure values were associated with higher subjective ratings of weakness for each task, although there was substantial overlap in the data. These results, combined with the notion that examiner bias is inherent to clinical assessment, support the use of instrumentation to improve objectivity and precision of measurement in the clinic.


Nitric oxide suppresses cerebral vasomotion by sGC-independent effects on ryanodine receptors and voltage-gated calcium channels.
In cerebral arteries, nitric oxide (NO) release plays a key role in suppressing vasomotion. Our aim was to establish the pathways affected by NO in rat middle cerebral arteries. In isolated segments of artery, isometric tension and simultaneous measurements of either smooth muscle membrane potential or intracellular [Ca(2+)] ([Ca(2+)](SMC)) changes were recorded. In the absence of L-NAME, asynchronous propagating Ca(2+) waves were recorded that were sensitive to block with ryanodine, but not nifedipine. L-NAME stimulated pronounced vasomotion and synchronous Ca(2+) oscillations with close temporal coupling between membrane potential, tone and [Ca(2+)](SMC). If nifedipine was applied together with L-NAME, [Ca(2+)](SMC) decreased and synchronous Ca(2+) oscillations were lost, but asynchronous propagating Ca(2+) waves persisted. Vasomotion was similarly evoked by either iberiotoxin, or by ryanodine, and to a lesser extent by ODQ. Exogenous application of NONOate stimulated endothelium-independent hyperpolarization and relaxation of either L-NAME-induced or spontaneous arterial tone. NO-evoked hyperpolarization involved activation of BK(Ca) channels via ryanodine receptors (RYRs), with little involvement of sGC. Further, in whole cell mode, NO inhibited current through L-type voltage-gated Ca(2+) channels (VGCC), which was independent of both voltage and sGC. NO exerts sGC-independent actions at RYRs and at VGCC, both of which normally suppress cerebral artery myogenic tone.


Biological therapy in refractory cases of uveitis and scleritis: An analysis of 18 cases from a tertiary eye care center from South India.
To evaluate the effectiveness of biologic therapy in a cohort of patients with various types of refractory non-infectious uveitis and scleritis. A retrospective observational study on patients with non-infectious uveitis and scleritis who were not responding or had a high recurrence rate with the conventional treatment and had received biologic therapy. We studied 18 patients (33 eyes) who received biological therapy between January 2017 and November 2019. The mean age was 30 ± 17 years and mean duration of uveitis was 36.8 months (range 1-120 months). Anterior uveitis (27.7%) was most commonly observed followed by scleritis, panuveitis, posterior, and intermediate uveitis. The most common etiology was Behçet's disease (4 patients, 22.2%) followed by juvenile idiopathic arthritis (3 patients, 16.6%), granulamotosis polyangitis, and idiopathic (2 patients each, 11.1%). Majority had trialled one or more immunosuppressive and were refractory in nature. Maximum patients had received adalimumab (61%) followed by infliximab (22%), rituximab (12%), and golimumab (6%). The median prednisolone dose was reduced from 30 mg (range 7.5-60 mg) to 5 mg (range 0-10 mg) after biological therapy (P = 0.002). Significant visual improvement was observed post biologic therapy (mean log mar VA 0.41 ± 0.62 improved to 0.23 ± 0.48 at the final visit, P = 0.008). Maximum number of patients (16 patients, 89%) responded well with biological therapy. Three patients developed recurrence and systemic complications were observed in two patients. Biologic therapy is effective in non-infectious refractory uveitis who were resistant to conventional therapy and may prolong disease recurrence.


In vivo evaluation of highly macroporous ceramic scaffolds for bone tissue engineering.
During the last decades, different materials of both natural and synthetic origin have been developed with the aim of inducing and controlling osteogenic differentiation of mesenchymal stem cells (MSCs). In order for that to happen, it is necessary that the material to be implanted obey a series of requirements, namely: osteoconduction, biocompatibility, and biodegradability. Additionally, they must be low-priced, easy to produce, shape, and store. Hydroxyapatite (HA) is a well known ceramic with a composition similar to the mineral component of bone and is highly biocompatible and easy to obtain and/or process. On the other hand, collagen is the main structural protein present in the human body and bone. In this study, a polymer replication method was applied and a highly porous HA scaffold was produced. Collagen was later incorporated to improve the biological properties of the scaffold while resembling the bone composition. The scaffolds were characterized by means of scanning electron microscopy, Fourier transform infrared spectroscopy and energy dispersive spectroscopy. In vitro and in vivo testing was performed in all scaffolds produced. The goal of this study was to evaluate the in vivo osteogenic potential of MSCs from two different species seeded on the different HA basedporous scaffolds with collagen type I. The resultsindicate that all scaffolds exhibit relevant bone formation, being more prominent in the case of the HA scaffolds.


Markers of successful recruitment of students to family practice residency programs.
With limited numbers of medical students choosing a primary care specialty, family practice residency programs have had to complete to fill available residency positions. The purpose of this study was to determine the characteristics of the recruiting process used by US family practice residencies and to compare recruiting strategies used by successful and unsuccessful programs. We surveyed program directors of all 361 nonmilitary, accredited, US family practice residency programs in existence for more than 2 years. The questionnaire elicited information on program descriptors, recruitment process, and fill rate in the National Resident Matching Program. Seventy-eight percent (282) of the program directors returned usable questionnaires. Success in filling available positions through the Match was significantly associated with high quality of current housestaff and faculty and current residents' positive feelings about the program (P < .01). Program directors perceived successful programs as having an outstanding reputation and being regarded positively by other departments within the institution (P < .01). Programs in the Pacific and Mountain regions of the United States are more successful in recruitment. Programs not filling available positions through the Match spent more effort in recruiting strategies such as mailing and marketing materials (P < .01). Having high-quality faculty, housestaff, and residents with good attitudes are markers of success in the Match. Less successful programs appear to try harder to attract residents.


LED-Induced fluorescence and image analysis to detect stink bug damage in cotton bolls.
Stink bugs represent a major agricultural pest complex attacking more than 200 wild and cultivated plants, including cotton in the southeastern US. Stink bug feeding on developing cotton bolls will cause boll abortion or lint staining and thus reduced yield and lint value. Current methods for stink bug detection involve manual harvesting and cracking open of a sizable number of immature cotton bolls for visual inspection. This process is cumbersome, time consuming, and requires a moderate level of experience to obtain accurate estimates. To improve detection of stink bug feeding, we present here a method based on fluorescent imaging and subsequent image analyses to determine the likelihood of stink bug damage in cotton bolls. Damage to different structures of cotton bolls including lint and carpal wall can be observed under blue LED-induced fluorescence. Generally speaking, damaged regions fluoresce green, whereas non-damaged regions with chlorophyll fluoresce red. However, similar fluorescence emission is also observable on cotton bolls that have not been fed upon by stink bugs. Criteria based on fluorescent intensity and the size of the fluorescent spot allow to differentiate between true positives (fluorescent regions associated with stink bug feeding) and false positives (fluorescent regions due to other causes). We found a detection rates with two combined criteria of 87% for true-positive marks and of 8% for false-positive marks. The imaging technique presented herein gives rise to a possible detection apparatus where a cotton boll is imaged in the field and images processed by software. The unique fluorescent signature left by stink bugs can be used to determine with high probability if a cotton boll has been punctured by a stink bug. We believe this technique, when integrated in a suitable device, could be used for more accurate detection in the field and allow for more optimized application of pest control.


Probiotic characteristics of bacteriocin-producing Enterococcus faecium strains isolated from human milk and colostrum.
As potential probiotic traits of human milk-isolated bacteria have increasingly been recognized, this study aimed to evaluate the probiotic properties of bacteriocin-producing Enterococcus faecium strains isolated from human milk and colostrum. Among 118 human milk- and colostrum-isolated lactic cocci, only 29 were identified as Enterococcus. Of these, only four Enterococcus faecium isolates exhibited bacteriocigenic activity against several pathogenic Gram-positive bacteria, including Listeria monocytogenes. These isolates exhibited high acid (up to pH 3.0) and bile tolerance (0.5% oxgall) in simulated gastrointestinal conditions, demonstrating their ability to survive through the upper gastrointestinal tract. All of the E. faecium strains were shown to be sensitive to most of the antibiotics including vancomycin, tetracycline, rifampicin, and erythromycin, while they were resistant to kanamycin and chloramphenicol. None of the strains showed any virulence (gelE, agg2, clyA, clyB, clyM) and antibiotic resistance genes (vanA, vanB, ermB, tetM, and aac(6')-le-aph(2″)-la). In addition, all the strains were able to assimilate cholesterol, ranging between 25.2-64.1% and they exhibited variable adherence (19-36%) to Caco-2 cells. Based on the overall results of this in vitro study, four of the E. faecium strains isolated from human milk and colostrum can be considered as promising probiotic candidates; however, further in vivo evaluations are required.


The CCC system in two teaching hospitals: a progress report.
Computing systems developed by the Center for Clinical Computing (CCC) have been in operation in Beth Israel and Brigham and Women's hospitals for over 10 years. Designed to be of direct benefit to doctors, nurses, and other clinicians in the care of their patients, the CCC systems give the results of diagnostic studies immediately upon request; offer access to the medical literature: give advice, consultation, alerts, and reminders; assist in the day-to-day practice to medicine, and participate directly in the education of medical students and house officers. The CCC systems are extensively used, even by physicians who are under no obligation to use them. Studies have shown that the systems are well received and that they help clinicians improve the quality of patient care. In addition, the CCC systems have had a beneficial impact on the finances of the two hospitals, and they have cost less than what many hospitals spend for financial computing alone.


Pathogens and insect herbivores drive rainforest plant diversity and composition.
Tropical forests are important reservoirs of biodiversity, but the processes that maintain this diversity remain poorly understood. The Janzen-Connell hypothesis suggests that specialized natural enemies such as insect herbivores and fungal pathogens maintain high diversity by elevating mortality when plant species occur at high density (negative density dependence; NDD). NDD has been detected widely in tropical forests, but the prediction that NDD caused by insects and pathogens has a community-wide role in maintaining tropical plant diversity remains untested. We show experimentally that changes in plant diversity and species composition are caused by fungal pathogens and insect herbivores. Effective plant species richness increased across the seed-to-seedling transition, corresponding to large changes in species composition. Treating seeds and young seedlings with fungicides significantly reduced the diversity of the seedling assemblage, consistent with the Janzen-Connell hypothesis. Although suppressing insect herbivores using insecticides did not alter species diversity, it greatly increased seedling recruitment and caused a marked shift in seedling species composition. Overall, seedling recruitment was significantly reduced at high conspecific seed densities and this NDD was greatest for the species that were most abundant as seeds. Suppressing fungi reduced the negative effects of density on recruitment, confirming that the diversity-enhancing effect of fungi is mediated by NDD. Our study provides an overall test of the Janzen-Connell hypothesis and demonstrates the crucial role that insects and pathogens have both in structuring tropical plant communities and in maintaining their remarkable diversity.


Dangerous jellyfish blooms are predictable.
The potentially fatal Irukandji syndrome is relatively common in tropical waters throughout the world. It is caused by the sting of the Irukandji jellyfish, a family of box jellyfish that are almost impossible to detect in the water owing to their small size and transparency. Using collated medical records of stings and local weather conditions, we show that the presence of Irukandji blooms in coastal waters can be forecast on the basis of wind conditions. On the Great Barrier Reef, blooms largely coincide with relaxation of the prevailing southeasterly trade winds, with average conditions corresponding to near zero alongshore wind on the day prior to the sting. These conditions are consistent with hypotheses long held by local communities and provide a basis for designing management interventions that have the potential to eliminate the majority of stings.


A critical role for the g protein-coupled receptor mFPR2 in airway inflammation and immune responses.
The formylpeptide receptor-like 1, now officially termed FPR2, in human and its mouse homolog mFPR2 mediate leukocyte migration in response to agonists associated with inflammation and immune responses. To clarify the in vivo role of the receptor, we generated mice deficient in mFPR2. mFPR2(-/-) mice showed markedly reduced severity in OVA/alum-induced allergic airway inflammation. This was associated with diminished recruitment of CD11c(+) dendritic cells into the airway mucosa and secondary lymphoid organs, as well as reduced production of Type 2 cytokines and Igs. We also found that the bronchoalveolar lavage fluid from wild type mice with airway inflammation contained mFPR2 agonist activity. This study reveals a critical role for mFPR2 in the progression of allergic airway inflammation and immune responses.


Familial occurrence of mitral regurgitation in patients with mitral valve prolapse undergoing mitral valve surgery.
Initial studies have suggested the familial clustering of mitral valve prolapse, but most of them were either community based among unselected individuals or applied non-specific diagnostic criteria. Therefore little is known about the familial distribution of mitral regurgitation in a referral-type population with a more severe mitral valve prolapse phenotype. The objective of this study was to evaluate the presence of familial mitral regurgitation in patients undergoing surgery for mitral valve prolapse, differentiating patients with Barlow's disease, Barlow forme fruste and fibro-elastic deficiency. A total of 385 patients (62 ± 12 years, 63% men) who underwent surgery for mitral valve prolapse were contacted to assess cardiac family history systematically. Only the documented presence of mitral regurgitation was considered to define 'familial mitral regurgitation'. In the probands, the aetiology of mitral valve prolapse was defined by surgical observations. A total of 107 (28%) probands were classified as having Barlow's disease, 85 (22%) as Barlow forme fruste and 193 (50%) patients as fibro-elastic deficiency. In total, 51 patients (13%) reported a clear family history for mitral regurgitation; these patients were significantly younger, more often diagnosed with Barlow's disease and also reported more sudden death in their family as compared with 'sporadic mitral regurgitation'. In particular, 'familial mitral regurgitation' was reported in 28 patients with Barlow's disease (26%), 15 patients (8%) with fibro-elastic deficiency and eight (9%) with Barlow forme fruste (P < 0.001). In a large cohort of patients operated for mitral valve prolapse, the self-reported prevalence of familial mitral regurgitation was 26% in patients with Barlow's disease and still 8% in patients with fibro-elastic deficiency, highlighting the importance of familial anamnesis and echocardiographic screening in all mitral valve prolapse patients.


Inhibitory Control, but Not Prolonged Object-Related Experience Appears to Affect Physical Problem-Solving Performance of Pet Dogs.
Human infants develop an understanding of their physical environment through playful interactions with objects. Similar processes may influence also the performance of non-human animals in physical problem-solving tasks, but to date there is little empirical data to evaluate this hypothesis. In addition or alternatively to prior experiences, inhibitory control has been suggested as a factor underlying the considerable individual differences in performance reported for many species. Here we report a study in which we manipulated the extent of object-related experience for a cohort of dogs (Canis familiaris) of the breed Border Collie over a period of 18 months, and assessed their level of inhibitory control, prior to testing them in a series of four physical problem-solving tasks. We found no evidence that differences in object-related experience explain variability in performance in these tasks. It thus appears that dogs do not transfer knowledge about physical rules from one physical problem-solving task to another, but rather approach each task as a novel problem. Our results, however, suggest that individual performance in these tasks is influenced in a complex way by the subject's level of inhibitory control. Depending on the task, inhibitory control had a positive or a negative effect on performance and different aspects of inhibitory control turned out to be the best predictors of individual performance in the different tasks. Therefore, studying the interplay between inhibitory control and problem-solving performance will make an important contribution to our understanding of individual and species differences in physical problem-solving performance.


Alteration of sequence specificity of the type II restriction endonuclease HincII through an indirect readout mechanism.
The functional and structural consequences of a mutation of the DNA intercalating residue of HincII, Q138F, are presented. Modeling has suggested that the DNA intercalation by Gln-138 results in DNA distortions potentially used by HincII in indirect readout of its cognate DNA, GTYRAC (Y = C or T, R = A or G) (Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47). Kinetic data presented here indicate that the mutation of glutamine 138 to phenylalanine (Q138F) results in a change in sequence specificity at the center two base pairs of the cognate recognition site. We show that the preference of HincII for cutting, but not binding, the three cognate sites differing in the center two base pairs has been altered by the mutation Q138F. Five new crystal structures are presented including Q138F HincII bound to GTTAAC and GTCGAC both with and without Ca2+ as well as the structure of wild type HincII bound to GTTAAC. The Q138F HincII/DNA structures show conformational changes in the protein, bound DNA, and at the protein-DNA interface, consistent with the formation of adaptive complexes. Analysis of these structures and the effect of Ca2+ binding on the protein-DNA interface illuminates the origin of the altered specificity by the mutation Q138F in the HincII enzyme.


Mobile Medical Education (MoMEd) - how mobile information resources contribute to learning for undergraduate clinical students - a mixed methods study.
Mobile technology is increasingly being used by clinicians to access up-to-date information for patient care. These offer learning opportunities in the clinical setting for medical students but the underlying pedagogic theories are not clear. A conceptual framework is needed to understand these further. Our initial questions were how the medical students used the technology, how it enabled them to learn and what theoretical underpinning supported the learning. 387 medical students were provided with a personal digital assistant (PDA) loaded with medical resources for the duration of their clinical studies. Outcomes were assessed by a mixed-methods triangulation approach using qualitative and quantitative analysis of surveys, focus groups and usage tracking data. Learning occurred in context with timely access to key facts and through consolidation of knowledge via repetition. The PDA was an important addition to the learning ecology rather than a replacement. Contextual factors impacted on use both positively and negatively. Barriers included concerns of interrupting the clinical interaction and of negative responses from teachers and patients. Students preferred a future involving smartphone platforms. This is the first study to describe the learning ecology and pedagogic basis behind the use of mobile learning technologies in a large cohort of undergraduate medical students in the clinical environment. We have developed a model for mobile learning in the clinical setting that shows how different theories contribute to its use taking into account positive and negative contextual factors.The lessons from this study are transferable internationally, to other health care professions and to the development of similar initiatives with newer technology such as smartphones or tablet computers.


Molecularly imprinted polymers for bisphenol A for HPLC and SPE from water and milk.
Molecularly imprinted polymers (MIPs) for bisphenol A (BPA) were prepared by two synthetic routes: semi-covalent and noncovalent methodology. The molecular imprinting effect was evaluated using the polymers in HPLC and SPE. Polymers prepared with noncovalent mode were proven more effective. These polymers were applied in SPE facilitating selective retention of BPA from bottled water and milk. The developed sample preparation was simple and efficient comprising only dilution of milk and MISPE prior to LC-MS analysis. Overall MISPE enhanced sample clean-up. Compared with control nonimprinted polymers and conventional C18 SPE cartridges, the MIPs exhibited selective analyte recognition. The method provided quantitative BPA recoveries, very good reproducibility (% RSDs lower than 7%), and low LOD (0.2 ng/g). MIP interacts similarly with deuterated BPA allowing its use as internal standard in LC-MS. The most critical parameters of MISPE were the organic content in loading-washing medium and the washing volume. Low flow rates in the elution step enhanced extraction recovery. Important advantages of the MIP were: the high breakthrough volumes (> 500 mL of water), high mass capacity (> 10 ng/mg of MIP sorbent), good linearity, and good stability in performance for over 35 cycles of use.


Individualized chemotherapy for patients with non-small cell lung cancer determined by prospective identification of neuroendocrine markers and in vitro drug sensitivity testing.
We attempted to prospectively select individualized chemotherapy for 165 non-small cell lung cancer patients based on in vitro analysis of neuroendocrine (NE) markers and drug sensitivity testing (DST) using fresh tumor. The chemotherapy used for small cell lung cancer (SCLC) was selected when NE marker expression determined by L-dopa decarboxylase assay was documented. Selection of chemotherapy for other patients was guided by DST results using a modified dye exclusion assay when available; otherwise etoposide and cisplatin was administered. A total of 112 of 165 (68%) specimens were assayed for L-dopa decarboxylase and 36 patients (22%) had DST. In vitro data directed management for 27 of 96 (28%) patients given chemotherapy: 6 with NE markers were treated with the SCLC regimen; and 21 (58% of those with DST) received their DST-selected chemotherapy regimen. There were no significant differences in response rate among all 3 treatment arms (P = 0.076). However, response to chemotherapy for the patients treated prospectively with a SCLC regimen was 3 of 6 (50%), marginally better than patients given their DST-selected chemotherapy regimen (2 of 21; 9%; P = 0.056) or those treated with etoposide and cisplatin (10 of 69; 14%; P = 0.061). When patients whose NE markers were identified retrospectively are included, 4 of 9 (44%) responded to administered chemotherapy, compared to 7 of 55 (13%) with no NE markers present (P = 0.04). There were no differences in survival among the three treatment groups. Cisplatin and etoposide comprised the most active regimen in vitro for tumors from 16 of 36 (44%) patients, potentially limiting the benefit of DST since this is often the empiric therapy for non-SCLC. Furthermore, the correlation between in vitro and clinical response is nonsignificant for all drugs tested, highlighting the overall relative resistance of non-SCLC tumors to currently available chemotherapy.


Risk Factors for Forced, Incapacitated, and Coercive Sexual Victimization Among Sexual Minority and Heterosexual Male and Female College Students.
Although college students are at high risk for sexual victimization, the majority of research has focused on heterosexual students and often does not differentiate by victimization type. Thus, little is known about prevalence rates and risk factors for sexual victimization among sexual minority college students and whether the interaction between gender and sexual orientation differs by victimization type. To address these gaps, we examine whether risk factors for three types of sexual victimization (i.e., forced, incapacitated, and coerced) differ by gender (n = 681 males; n = 732 females) and sexual orientation (n = 1,294 heterosexual; n = 119 sexual minority) and whether the intersection of gender and sexual orientation is correlated with these three types of sexual victimization among 1,413 college students. Prevalence rate results revealed significant differences between gender and sexual orientation: Sexual minority females had the highest rates of coerced sexual victimization (58%), and their mean was significantly different from the other three groups (i.e., heterosexual females, heterosexual males, and sexual minority males). For both forced and incapacitated sexual victimization, heterosexual males had significantly lower means than the other three groups. Logistic regression results revealed that child sexual abuse increased the odds of experiencing both forced and coerced sexual victimization for both heterosexual and sexual minority students, whereas increased rates of risky sexual behavior were associated with forced and incapacitated sexual victimization but only for heterosexuals. Finally, heavy drinking increased the odds of experiencing incapacitated sexual victimization for both heterosexuals and sexual minorities.


Symmetry breakage in the vertebrate embryo: when does it happen and how does it work?
Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.


Cognitive, adaptive, and behavioral features in Joubert syndrome.
Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by a distinctive cerebellar and brainstem malformation recognizable on brain imaging, the so-called molar tooth sign. The full spectrum of cognitive and behavioral phenotypes typical of JS is still far from being elucidated. The aim of this multicentric study was to define the clinical phenotype and neurobehavioral features of a large cohort of subjects with a neuroradiologically confirmed diagnosis of JS. Fifty-four patients aged 10 months to 29 years were enrolled. Each patient underwent a neurological evaluation as well as psychiatric and neuropsychological assessments. Global cognitive functioning was remarkably variable with Full IQ/General Quotient ranging from 32 to 129. Communication skills appeared relatively preserved with respect to both Daily Living and Socialization abilities. The motor domain was the area of greatest vulnerability, with a negative impact on personal care, social, and academic skills. Most children did not show maladaptive behaviors consistent with a psychiatric diagnosis but approximately 40% of them presented emotional and behavioral problems. We conclude that intellectual disability remains a hallmark but cannot be considered a mandatory diagnostic criterion of JS. Despite the high variability in the phenotypic spectrum and the extent of multiorgan involvement, nearly one quarter of JS patients had a favorable long-term outcome with borderline cognitive deficit or even normal cognition. Most of JS population also showed relatively preserved communication skills and overall discrete behavioral functioning in everyday life, independently from the presence and/or level of intellectual disability. © 2016 Wiley Periodicals, Inc.


Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris.
Secretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation. Analysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa). Rapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.


Nuclear localization of factor inhibitor hypoxia-inducible factor in prostate cancer is associated with poor prognosis.
We determined the role of factor inhibiting hypoxia-inducible factor-1 in prostate cancer specimens. A tissue microarray of 152 prostate cancers was constructed and stained for factor inhibiting hypoxia-inducible factor-1, hypoxia-inducible factor-1α and 2α, and glucose transporter 1 as a prototypical downstream target of hypoxia-inducible factor-1α. Correlation analysis was done between these variables, and between factor inhibiting hypoxia-inducible factor-1, and clinical and pathological variables, including prostate specific antigen as a surrogate of recurrence. Factor inhibiting hypoxia-inducible factor-1 was expressed in the cytoplasm and/or the nucleus in 86.5% of tumors, including exclusive cytoplasmic expression in 51.3% and exclusive nuclear expression in 5.3%. Any nuclear and exclusive expression of factor inhibiting hypoxia-inducible factor was associated with poor prognosis on univariate analysis (p = 0.007 and 0.042, respectively). On multivariate analysis men with nuclear expression in tumors were twice as likely to experience recurrence (p = 0.034). Factor inhibiting hypoxia-inducible factor-1 is widely expressed in prostate tumors. Its differential subcellular expression suggests that regulation of its expression is an important factor in the activity of the hypoxia-inducible factor pathway. Its modulation may help treat hypoxia-inducible factor driven aggressive prostate cancer.


Motility and ramification of human fetal microglia in culture: an investigation using time-lapse video microscopy and image analysis.
Microglia are mononuclear phagocytes of the central nervous system and are considered to derive from circulating bone marrow progenitors that colonize the developing human nervous system in the second trimester. They first appear as ameboid forms and progressively differentiate to process-bearing "ramified" forms with maturation. Signals driving this transformation are known to be partly derived from astrocytes. In this investigation we have used cocultures of astrocytes and microglia to demonstrate the relationship between motility and morphology of microglia associated with signals derived from astrocytes. Analysis of progressive cultures using time-lapse video microscopy clearly demonstrates the dynamic nature of microglia. We observe that ameboid microglial cells progressively ramify when cocultured with astrocytes, mirroring the "differentiation" of microglia in situ during development. We further demonstrate that individual cells undergo morphological transformations from "ramified" to "bipolar" to "tripolar" and "ameboid" states in accordance with local environmental cues associated with astrocytes in subconfluent cultures. Remarkably, cells are still capable of migration at velocities of 20-35 microm/h in a fully ramified state overlying confluent astrocytes, as determined by image analysis of motility. This is in keeping with the capacity of microglia for a rapid response to inflammatory cues in the CNS. We also demonstrate selective expression of the chemokines MIP-1alpha and MCP-1 by confluent human fetal astrocytes in cocultures and propose a role for these chemotactic cytokines as regulators of microglial motility and differentiation. The interchangeable morphological continuum of microglia supports the view that these cells represent a single heterogeneous population of resident mononuclear phagocytes capable of marked plasticity.


Hypomethylation promotes autonomous endosperm development and rescues postfertilization lethality in fie mutants.
In most flowering plants, fertilization is necessary for development of the central cell into endosperm, but in the fie-1 mutant of Arabidopsis, the central cell can proliferate autonomously. However, autonomous fie-1 endosperms do not develop completely: They have fewer nuclei than sexually produced endosperms, cellularization does not take place, and no clear distinction is seen between the different endosperm compartments. Here, we show that autonomous endosperm develop much further in hypomethylated than normally methylated fie-1 mutants, undergoing cellularization and regional specification to resemble endosperm in sexually produced wild-type seeds. Therefore, the combination of maternal hypomethylation and loss of FIE function enables formation of differentiated endosperm without fertilization. A maternal fie-1 mutation is also lethal to sexual seeds, even if the pollen donor is wild type. We report that sexual mutant fie-1 endosperms fail to cellularize and overproliferate, consistent with the hypothesis that embryo abortion may be due, at least in part, to a defect in endosperm development. Finally, we show that pollen from hypomethylated plants rescues fie-1 mutant seeds provided that it also donates a wild-type paternal FIE allele. These results are discussed in light of models for parent-of-origin effects on seed development.


Unilateral hippocampal CA3-predominant damage and short latency epileptogenesis after intra-amygdala microinjection of kainic acid in mice.
Mesial temporal lobe epilepsy is the most common, intractable seizure disorder in adults. It is associated with an asymmetric pattern of hippocampal neuron loss within the endfolium (hilus and CA3) and CA1, with limited pathology in extra-hippocampal regions. We previously developed a model of focally-evoked seizure-induced neuronal death using intra-amygdala kainic acid (KA) microinjection and characterized the acute hippocampal pathology. Here, we sought to characterize the full extent of hippocampal and potential extra-hippocampal damage in this model, and the temporal onset of epileptic seizures. Seizure damage assessed at four stereotaxic levels by FluoroJade B staining was most prominent in ipsilateral hippocampal CA3 where it extended from septal to temporal pole. Minor but significant neuronal injury was present in ipsilateral CA1. Extra-hippocampal neuronal damage was generally limited in extent and restricted to the lateral septal nucleus, injected amygdala and select regions of neocortex ipsilateral to the seizure elicitation side. Continuous surface EEG recorded with implanted telemetry units in freely-moving mice detected spontaneous, epileptic seizures by five days post-KA in all mice. Epileptic seizure number averaged 1-4 per day. Hippocampi from epileptic mice 15 days post-KA displayed unilateral CA3 lesions, astrogliosis and increased neuropeptide Y immunoreactivity suggestive of mossy fiber rearrangement. These studies characterize a mouse model of unilateral hippocampal-dominant neuronal damage and short latency epileptogenesis that may be suitable for studying the cell and molecular pathogenesis of human mesial temporal lobe epilepsy.


Etiology, pathogenesis and prevention of neural tube defects.
Spina bifida, anencephaly, and encephalocele are commonly grouped together and termed neural tube defects (NTD). Failure of closure of the neural tube during development results in anencephaly or spina bifida aperta but encephaloceles are possibly post-closure defects. NTD are associated with a number of other central nervous system (CNS) and non-neural malformations. Racial, geographic and seasonal variations seem to affect their incidence. Etiology of NTD is unknown. Most of the non-syndromic NTD are of multifactorial origin. Recent in vitro and in vivo studies have highlighted the molecular mechanisms of neurulation in vertebrates but the morphologic development of human neural tube is poorly understood. A multisite closure theory, extrapolated directly from mouse experiments highlighted the clinical relevance of closure mechanisms to human NTD. Animal models, such as circle tail, curly tail, loop tail, shrm and numerous knockouts provide some insight into the mechanisms of NTD. Also available in the literature are a plethora of chemically induced preclosure and a few post-closure models of NTD, which highlight the fact that CNS malformations are of hetergeneitic nature. No Mendelian pattern of inheritance has been reported. Association with single gene defects, enhanced recurrence risk among siblings, and a higher frequency in twins than in singletons indicate the presence of a strong genetic contribution to the etiology of NTD. Non-availability of families with a significant number of NTD cases makes research into genetic causation of NTD difficult. Case reports and epidemiologic studies have implicated a number of chemicals, widely differing therapeutic drugs, environmental contaminants, pollutants, infectious agents, and solvents. Maternal hyperthermia, use of valproate by epileptic women during pregnancy, deficiency and excess of certain nutrients and chronic maternal diseases (e.g. diabetes mellitus) are reported to cause a manifold increase in the incidence of NTD. A host of suspected teratogens are also available in the literature. The UK and Hungarian studies showed that periconceptional supplementation of women with folate (FA) reduces significantly both the first occurrence and recurrence of NTD in the offspring. This led to mandatory periconceptional FA supplementation in a number of countries. Encouraged by the results of clinical studies, numerous laboratory investigations focused on the genes involved in the FA, vitamin B12 and homocysteine metabolism during neural tube development. As of today no clinical or experimental study has provided unequivocal evidence for a definitive role for any of these genes in the causation of NTD suggesting that a multitude of genes, growth factors and receptors interact in controlling neural tube development by yet unknown mechanisms. Future studies must address issues of gene-gene, gene-nutrient and gene-environment interactions in the pathogenesis of NTD.


Risk-adapted donor lymphocyte infusion based on chimerism and donor source in pediatric leukemia.
Donor lymphocyte infusion (DLI) is commonly used to treat leukemia relapse following stem cell transplantation. In florid relapse, however, the efficacy of DLI is limited with substantial risk of severe graft-versus-host disease (GvHD). Here, we develop a novel risk-adapted strategy characterized by pre-emptive DLI initiated at the time of mixed chimerism, a small starting dose based on donor source, dose-escalation guided by real-time chimerism monitoring and withholding of DLI immediately in patients achieving full donor chimerism. A total of 178 DLIs were given to 38 patients with mixed chimerism; thereafter, 33 patients (86.8%) had donor chimerism successfully increased, including 30 (78.9%) who had chimerism fully converted back to 100% donor. Cumulative incidence of relapse was significantly lower (P=0.00004) and overall survival higher (P=0.0003) in patients with chimerism fully corrected as compared with those of patients whose chimerism remained mixed. Only 13.2% of the patients developed acute grade III-IV GvHD with no associated mortality. In conclusion, the risk-adapted DLI strategy is useful in minimizing the risk of childhood leukemia relapse, GvHD and death.


[Megakaryopoietic cytokine levels in patients with essential thrombocythemia and their relationship with clinical and biochemical features].
Megakaryopoiesis and platelet production are driven by transcription factors and cytokines present in bone marrow environment. Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by high platelet count and megakaryocytic hyperplasia. In the present work we evaluated plasmatic levels of cytokines involved in megakaryocytic development in a group of patients with ET that were not on treatment, as well as thrombopoietin (TPO) levels before and during anagrelide treatment. The assays were carried out using ELISA techniques. Among the cytokines mainly involved in proliferation of megakaryocytic progenitors, interleukin 3 (IL-3) levels were found increased in patients compared to normal controls (p = 0.0383). Granulocyte-macrophage colony stimulating factor and stem cell factor levels were normal. Interleukin 6, as well as interleukin 11 and erythropoietin (EPO), cytokines mainly related to megakaryocytic maturation, were normal. Plasma TPO levels before treatment were within the normal range and increased during treatment but the difference was not statistically significant. Patients who displayed spontaneous platelet aggregation had higher plasma TPO levels compared to those who did not (p = 0.049). We did not find any relationship between cytokine levels and clinical or laboratory parameters. The high IL-3 levels seen in some patients with ET could contribute to megakaryocytic proliferation. The simultaneous occurrence of higher TPO levels and elevated platelet count could be a predisposing factor for the development of spontaneous platelet aggregation in ET patients.


NCIPLOT: a program for plotting non-covalent interaction regions.
Non-covalent interactions hold the key to understanding many chemical, biological, and technological problems. Describing these non-covalent interactions accurately, including their positions in real space, constitutes a first step in the process of decoupling the complex balance of forces that define non-covalent interactions. Because of the size of macromolecules, the most common approach has been to assign van der Waals interactions (vdW), steric clashes (SC), and hydrogen bonds (HBs) based on pairwise distances between atoms according to their van der Waals radii. We recently developed an alternative perspective, derived from the electronic density: the Non-Covalent Interactions (NCI) index [J. Am. Chem. Soc. 2010, 132, 6498]. This index has the dual advantages of being generally transferable to diverse chemical applications and being very fast to compute, since it can be calculated from promolecular densities. Thus, NCI analysis is applicable to large systems, including proteins and DNA, where analysis of non-covalent interactions is of great potential value. Here, we describe the NCI computational algorithms and their implementation for the analysis and visualization of weak interactions, using both self-consistent fully quantum-mechanical, as well as promolecular, densities. A wide range of options for tuning the range of interactions to be plotted is also presented. To demonstrate the capabilities of our approach, several examples are given from organic, inorganic, solid state, and macromolecular chemistry, including cases where NCI analysis gives insight into unconventional chemical bonding. The NCI code and its manual are available for download at http://www.chem.duke.edu/~yang/software.htm.


Dimensions of functional social support and psychological symptoms.
In the summer following graduation a sample of 125 female college graduates (mean age = 28) completed Cohen & Wills' ISEL (1985) which includes scales measuring four social support functions: belonging (social companionship), appraisal (availability of confidants), tangible (instrumental), and self-esteem support. In the summer and fall subject status on two outcome scales was ascertained: the Psychophysiologic Symptom Scale and the Center for Epidemiologic Studies Depression Scale (CES-D). Reliability of the difference scores suggested that the ISEL scales do not measure entirely different constructs and the ISEL Self-esteem Scale is operationally redundant with the Rosenberg Self-esteem scale and the CES-D. Cross-sectional analyses indicated that the ISEL scales were related to symptoms. By contrast, standard longitudinal and prospective MLR analyses indicated that only the Belonging Scale was significantly related to future symptoms. The issues of confounding support with symptoms and the dimensionality of the subscales were discussed. The study suggests that specific functions of support take on greater importance during major life transitions and that any one supportive behaviour often serves multiple functions.


Homeless near a thousand homes: outcomes of homeless youth in a crisis shelter.
Clients who received crisis services at a homeless shelter for transition-aged youth were recruited for a study to describe the youth served, to track outcomes of care, and to examine factors associated with differing outcomes. Participants were 202 men and women who completed a battery of interviews and self-report measures at intake and at 3 follow-up points. Youth served had experienced high levels of adversity and trauma and typically had poor educational and vocational preparation. A multidisciplinary array of services was provided, and overall, participants showed significant improvement from intake to discharge and in the 6 months after discharge. Background, service, and psychological factors did not predict housing outcomes. Better vocational outcome was associated with more recent work experience. Results point to the need for providers of services to the homeless to be aware of the distinct needs and characteristics of transition-aged youth.


Peach Fruit Development: A Comparative Proteomic Study Between Endocarp and Mesocarp at Very Early Stages Underpins the Main Differential Biochemical Processes Between These Tissues.
Peach (Prunus persica) is an important economically temperate fruit. The development follows double sigmoid curve with four phases (S1-S4). We centered our work in the early development. In addition to S1, we studied the very early stage (E) characterized by the lag zone of the exponential growing phase S1, and the second stage (S2) when the pit starts hardening. "Dixiland" peach fruit were collected at 9 (E), 29 (S1), and 53 (S2) days after flowering (DAF) and endocarp and mesocarp were separated. There was a pronounced decrease in total protein content along development in both tissues. Quantitative proteomic allowed the identification of changes in protein profiles across development and revealed the main biochemical pathways sustaining tissue differentiation. Protein metabolism was the category most represented among differentially proteins in all tissues and stages. The decrease in protein synthesis machinery observed during development would be responsible of the protein fall, rather than a proteolytic process; and reduced protein synthesis during early development would reroute cell resources to lignin biosynthesis. These changes were accompanied by net decrease in total amino acids in E1-S1 and increase in S1-S2 transitions. Amino acid profiling, showed Asn parallels this trend. Concerted changes in Asn and in enzymes involved in its metabolism reveal that increased synthesis and decreased catabolism of Asn may conduct to an Asn increase during very early development and that the β-Cyano-Alanine synthase/β-Cyano-Alanine hydratase could be the pathway for Asn synthesis in "Dixiland" peach fruit. Additionally, photosynthetic machinery decays during early development in mesocarp and endocarp. Proteins related to photosynthesis are found to a higher extent in mesocarp than in endocarp. We conclude mesocarpic photosynthesis is possible to occur early on the development, first providing both carbon and reductive power and latter only reductive power. Together with proteomic, histological tests and anatomical analysis help to provide information about changes and differences in cells and cell-walls in both tissues. Collectively, this work represents the first approach in building protein databases during peach fruit development focusing on endocarp and mesocarp tissues and provides novel insights into the biology of peach fruit development preceding pit hardening.


A rapid, PCR based test for differential molecular diagnosis of Prader-Willi and Angelman syndromes.
Approximately 98% of Prader-Willi syndrome (PWS) and 80% of Angelman syndrome (AS) cases have deletions at a common region in chromosome 15q11-13, uniparental disomy for chromosomes 15 (UPD15), or mutations affecting gene expression in this region. The resulting clinical phenotype (PWS or AS) in each class of mutation depends upon the parent of origin. Both disorders are characterised at the molecular level by abnormal methylation of imprinted genes at 15q11-q13 including the small nuclear ribonucleoprotein N gene (SNRPN). Current diagnostic strategies include high resolution cytogenetics, fluorescence in situ hybridisation (FISH), Southern blot hybridisation, or microsatellite typing. We have developed a novel and rapid diagnostic test for PWS and AS based on differential digestion of expressed (paternally imprinted) SNRPN sequences by the methylation sensitive endonuclease NotI or repressed (maternally imprinted) SNRPN sequences by the methylation requiring nuclease McrBC, followed by PCR amplification of the SNRPN promoter. We have evaluated this test by blinded analysis of 60 characterised DNA samples (20 PWS, 20 AS, and 20 unaffected controls). SNRPN sequences could not be amplified from PWS patient DNA which had been digested with McrBC, nor from AS patient DNA which had been digested with NotI. We were able to make a correct diagnosis of PWS, AS, or unaffected in all 60 samples tested. This novel test is rapid and has a high specificity and sensitivity for deletion and UPD15 cases. These features make this new test suitable as the initial step in a molecular diagnostic strategy for PWS/AS.


The psychosocial impact of human papillomavirus testing in primary cervical screening-a study within a randomized trial.
The purpose of the study was to assess the psychosocial impact of human papillomavirus (HPV) testing as an adjunct to cytology in routine primary cervical screening. A controlled study of the psychosocial impact of HPV testing within a randomized trial of HPV testing to assess its efficacy in cervical screening was carried out. The trial provides a randomized setting of revealed HPV results versus concealed results permitting valid comparisons for assessing true psychosocial impact. The setting comprised a large number of general practices in primary care. Women aged 20-64 years who underwent routine cervical screening participated in the study. The intervention was a revealed high-risk HPV test result in addition to cervical cytology. The main outcome was measured using General Health Questionnaire (GHQ-28), Spielberger State-Trait Anxiety Inventory, and Sexual Rating Scale (SRS). Among women with either mildly abnormal or normal cytology, receiving an HPV(+ve) result did not impact significantly on GHQ caseness and mean scores or on Spielberger State and Trait scores when compared with women in whom the HPV(+ve) test result was concealed. Among women with normal cytology, receiving an HPV(+ve) result was associated with a reduction in the Sexual Rating Scale compared with similar women whose HPV(+ve) result was concealed. HPV testing does not add significant psychologic distress when combined with cytology in routine primary cervical screening.