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A method has been developed to perform high-frequency deep-water normal-mode calculations in the multikilohertz region in water depths of the order of 5 km. The results from this technique agree with fast field program (FFP) solutions as well as with infinite-frequency solutions in the appropriate limit. The wave-theory calculations presented therefore provide a set of test cases for future numerically more efficient approximations. [ABSTRACT FROM AUTHOR]

An experiment was conducted in a shallow water region of the Mediterranean Sea to study wind-generated noise. In addition to measuring the noise field, propagation-loss data were collected and used in a detailed modeling of the environment. The environmental information was then used as input to a noise model based on wave theory that computes the noise field in the water column for a given (unknown) source strength. By comparing model predictions with data, the influence of the environment on recorded noise levels could be removed and a measure of the noise source spectrum levels obtained as a function of wind speed. It was found that noise levels correlate better with wind speed than with wave height. In addition it was found that the nearfield contribution dominates the noise level, with the result of producing virtually constant noise intensity over depth from moderate to high wind speeds and frequencies above 200 Hz. [ABSTRACT FROM AUTHOR]

The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 47 fragments with diverse scaffolds were identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we performed deep mutational scanning of NS2B-NS3 protease. Merging fragment hits yields an extensive set of 'mergers', defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points that maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development.

The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement., Competing Interests: Declaration of interests A.C. is an employee of GeneDx, LLC. The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing completed at Baylor Genetics Laboratories., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants. Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 ± 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed. MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed. This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Background: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome., Methods: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants., Results: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving., Conclusions: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing., (© 2023. Crown.)

PKDCC encodes a component of Hedgehog signalling required for normal chondrogenesis and skeletal development. Although biallelic PKDCC variants have been implicated in rhizomelic shortening of limbs with variable dysmorphic features, this association was based on just two patients. In this study, data from the 100 000 Genomes Project was used in conjunction with exome sequencing and panel-testing results accessed via international collaboration to assemble a cohort of eight individuals from seven independent families with biallelic PKDCC variants. The allelic series included six frameshifts, a previously described splice-donor site variant and a likely pathogenic missense variant observed in two families that was supported by in silico structural modelling. Database queries suggested that the prevalence of this condition is between 1 of 127 and 1 of 721 in clinical cohorts with skeletal dysplasia of unknown aetiology. Clinical assessments, combined with data from previously published cases, indicate a predominantly upper limb involvement. Micrognathia, hypertelorism and hearing loss appear to be commonly co-occurring features. In conclusion, this study strengthens the link between biallelic inactivation of PKDCC and rhizomelic limb-shortening and will enable clinical testing laboratories to better interpret variants in this gene., (© 2023 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Extreme sea levels (ESLs) affect coastal ecosystems worldwide. Protection and adaptation strategies rely on the characterization of the extreme's occurrence probability in the future. However, knowledge on the occurrence rate and duration of ESLs is also needed to properly characterize the associated future risk. In this paper, we focus on the Venice lagoon, where a system of storm surge barriers can disconnect the lagoon from the sea to mitigate ESLs. Using long-term (96 years) sea level records, we model occurrence rate and duration of ESLs while accounting explicitly for seasonality and mean sea level rise. While historically ESLs occurred in the winter season, we project a significant increase (up to 10-fold with a mean sea level increase of +100 cm) of the occurrence rate also in the summer season, when disconnections from the sea can have profound impacts on the lagoon's ecosystem. We also predict an increase in ESLs durations up to 200 h, leading to longer disconnections of the lagoon from the sea in the future. Therefore, several adaptation strategies will be needed to limit the adverse effects of storm surge barriers on the lagoon ecosystem., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Trichomoniasis, one of the most common non-viral sexually transmitted infections worldwide, is caused by the parasite Trichomonas vaginalis. The pathogen colonizes the human urogenital tract, and the infection is associated with complications such as adverse pregnancy outcomes, cervical cancer, and an increase in HIV transmission. The mechanisms of pathogenicity are multifactorial, and controlling immune responses is essential for infection maintenance. Extracellular purine nucleotides are released by cells in physiological and pathological conditions, and they are hydrolyzed by enzymes called ecto-nucleotidases. The cellular effects of nucleotides and nucleosides occur via binding to purinoceptors, or through the uptake by nucleoside transporters. Altogether, enzymes, receptors and transporters constitute the purinergic signaling, a cellular network that regulates several effects in practically all systems including mammals, helminths, protozoa, bacteria, and fungi. In this context, this review updates the data on purinergic signaling involved in T. vaginalis biology and interaction with host cells, focusing on the characterization of ecto-nucleotidases and on purine salvage pathways. The implications of the final products, the nucleosides adenosine and guanosine, for human neutrophil response and vaginal epithelial cell damage reveal the purinergic signaling as a potential new mechanism for alternative drug targets., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: This prospective cohort study aimed at evaluating patterns of polypharmacy and aggressive and violent behavior during a 1-year follow-up in patients with severe mental disorders., Methods: A total of 340 patients (125 inpatients from residential facilities and 215 outpatients) were evaluated at baseline with the Structured Clinical Interview for DSM-IV Axis I and II, Brief Psychiatric Rating Scale, Specific Levels of Functioning scale, Brown-Goodwin Lifetime History of Aggression, Buss-Durkee Hostility Inventory, Barratt Impulsiveness Scale, and State-Trait Anger Expression Inventory-2. Aggressive behavior was rated every 15 days with the Modified Overt Aggression Scale and treatment compliance with the Medication Adherence Rating Scale., Results: The whole sample was prescribed mainly antipsychotics with high levels of polypharmacy. Clozapine prescription and higher compliance were associated with lower levels of aggressive and violent behavior. Patients with a history of violence who took clozapine were prescribed the highest number of drugs. The patterns of cumulative Modified Overt Aggression Scale mean scores of patients taking clozapine (n = 46), other antipsychotics (n = 257), and no antipsychotics (n = 37) were significantly different (P = .001). Patients taking clozapine showed a time trend at 1-year follow-up (24 evaluations) indicating a significantly lower level of aggressive behavior. Patient higher compliance was also associated with lower Modified Overt Aggression Scale ratings during the 1-year follow-up., Conclusion: Both inpatients and outpatients showed high levels of polypharmacy. Clozapine prescription was associated with lower Modified Overt Aggression Scale ratings compared with any other antipsychotics or other psychotropic drugs. Higher compliance was associated with lower levels of aggressive and violent behavior., (© The Author(s) 2020. Published by Oxford University Press on behalf of CINP.)

Trastuzumab is an effective therapeutic treatment for Her2-like breast cancer; despite this most of these tumors develop resistance to therapy due to specific gene mutations or alterations in gene expression. Understanding the mechanisms of resistance to Trastuzumab could be a useful tool in order to identify combinations of drugs that elude resistance and allow a better response for the treated patients. Twelve primary biopsies of Her2+/hormone receptor negative (ER-/PgR-) breast cancer patients were selected based on the specific response to neoadjuvant therapy with Trastuzumab and their whole exome was sequenced leading to the identification of 18 informative gene mutations that discriminate patients selectively based on response to treatment. Among these genes, we focused on the study of the ANKRD44 gene to understand its role in the mechanism of resistance to Trastuzumab. The ANKRD44 gene was silenced in Her2-like breast cancer cell line (BT474), obtaining a partially Trastuzumab-resistant breast cancer cell line that constitutively activates the NF-kb protein via the TAK1/AKT pathway. Following this activation an increase in the level of glycolysis in resistant cells is promoted, also confirmed by the up-regulation of the LDHB protein and by an increased TROP2 protein expression, found generally associated with aggressive tumors. These results allow us to consider the ANKRD44 gene as a potential gene involved in Trastuzumab resistance.

Background: Leigh Syndrome (LS, OMIM 256000) is an early-onset, progressive neurodegenerative disorder characterized by broad clinical and genetic heterogeneity; it is the most frequent disorder of mitochondrial energy production in children. LS inheritance is complex because patients may present mutations in mitochondrial DNA (mtDNA) or in nuclear genes, which predominantly encode proteins involved in respiratory chain structure and assembly or in coenzyme Q10 biogenesis. However, during the last 15 years, the discovery of several genetic mutations and improved knowledge of the natural history of LS has significantly increased our understanding of this mitochondrial disorder., Case Presentation: Here we describe a 19-year-old male with clinical and neuroimaging LS diagnosed at 3 years of age. Genetic analyses of the whole mtDNA for maternally inherited LS (MILS) and neuropathy ataxia retinitis pigmentosa (NARP) syndrome failed to reveal any pathogenic mutations., Conclusions: Recently, a missense mutation in ECHS1 and a ~ 35 kb deletion in 10q26.3 involving the region including the gene were identified by WES (whole exome sequencing), uncovering the genetic diagnosis clinically hypothesized for 15 years. We also report the long-term follow-up of this patient, showing a comparison with classical LS or other Leigh-like pictures.

Glioblastoma is a devastating disease that despite all the information gathered so far, its optimal management remains elusive due to the absence of validated targets from clinical studies. A better clarification of the molecular mechanisms is needed. In this study, having access to IDH1 wild-type glioblastoma of patients with exceptionally long recurrence free survival (RFS), we decided to compare their mutational and gene expression profile to groups of IDH1 wild-type glioblastoma of patients with shorter RFS, by using NGS technology. The exome analysis revealed that Long-RFS tumors have a lower mutational rate compared to the other groups. A total of 158 genes were found differentially expressed among the groups, 112 of which distinguished the two RFS extreme groups. Overall, the exome data suggests that shorter RFS tumors could be, chronologically, in a more advanced state in the muli-step tumor process of sequential accumulation of mutations. New players in this kind of cancer emerge from the analysis, confirmed at the RNA/DNA level, identifying, therefore, possible oncodrivers or tumor suppressor genes., Competing Interests: CONFLICTS OF INTEREST There is no conflicts of interest in this study.

Directed evolution has emerged as one of the most effective protein engineering methods in basic research as well as in applications in synthetic organic chemistry and biotechnology. The successful engineering of protein activity, allostery, binding affinity, expression, folding, fluorescence, solubility, substrate scope, selectivity (enantio-, stereo-, and regioselectivity), and/or stability (temperature, organic solvents, pH) is just limited by the throughput of the genetic selection, display, or screening system that is available for a given protein. Sometimes it is possible to analyze millions of protein variants from combinatorial libraries per day. In other cases, however, only a few hundred variants can be screened in a single day, and thus the creation of smaller yet smarter libraries is needed. Different strategies have been developed to create these libraries. One approach is to perform mutational scanning or to construct "mutability landscapes" in order to understand sequence-function relationships that can guide the actual directed evolution process. Herein we provide a protocol for economically constructing scanning mutagenesis libraries using a cytochrome P450 enzyme in a high-throughput manner. The goal is to engineer activity, regioselectivity, and stereoselectivity in the oxidative hydroxylation of a steroid, a challenging reaction in synthetic organic chemistry. Libraries based on mutability landscapes can be used to engineer any fitness trait of interest. The protocol is also useful for constructing gene libraries for deep mutational scanning experiments.

Acid ceramidase (AC) is a lysosomal cysteine hydrolase that catalyzes the conversion of ceramide into fatty acid and sphingosine. This reaction lowers intracellular ceramide levels and concomitantly generates sphingosine used for sphingosine-1-phosphate (S1P) production. Since increases in ceramide and consequent decreases of S1P reduce proliferation of various cancers, AC might offer a new target for anti-tumor therapy. Here we used CrispR-Cas9-mediated gene editing to delete the gene encoding for AC, ASAH1, in human A375 melanoma cells. ASAH1-null clones show significantly greater accumulation of long-chain saturated ceramides that are substrate for AC. As seen with administration of exogenous ceramide, AC ablation blocks cell cycle progression and accelerates senescence. Importantly, ASAH1-null cells also lose the ability to form cancer-initiating cells and to undergo self-renewal, which is suggestive of a key role for AC in maintaining malignancy and self-renewal of invasive melanoma cells. The results suggest that AC inhibitors might find therapeutic use as adjuvant therapy for advanced melanoma.

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α-latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro These findings indicate that the CXCL12α-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers., Competing Interests: There is no conflict of interest.

Glioblastoma (GB) is the most aggressive type of primary brain tumor. Despite the progress in recent years regarding the diagnosis and treatment of GB, the recurrence rate remains high, due to the infiltrative and dispersive nature of the tumor, which typically results in poor patient prognosis. In the present study, 19 formalin-fixed, paraffin-embedded GB samples were selected from patients with GB tumors. The samples were classified into a short or long recurrence-free survival (RFS) group, based on the time of first recurrence of the disease in the patients. The 19 samples were molecularly characterized for mutations in the isocitrate dehydrogenase 1 (IDH1) gene, amplification of the epidermal growth factor receptor (EGFR) gene, presence of the EGFR variant III, and methylation of the promoter region of the O 6 -methylguanine-DNA methyltransferase (MGMT) gene. Then, the expression of 84 genes involved in cell-cell and cell-matrix interactions, and that of 84 microRNAs (miRNAs) associated with brain cancer, was profiled. In addition, a copy number variation analysis of 23 genes reported to undergo frequent genomic alterations in human glioma was also performed. Differences in the expression levels of a number of genes were detected across the short and long RFS groups. Among these genes, 5 in particular were selected, and a 5-genes combination approach was developed, which was able to differentiate between patients with short and long RFS outcome. The high levels of sensitivity and precision displayed by this 5-genes combination approach, which were confirmed with a cross-validation method, provide a strong foundation for further validation of the involvement of the aforementioned genes in GB in a larger patient population. In conclusion, the present study has demonstrated how the expression pattern of miRNAs and mRNAs in patients with GB defines a particular molecular hallmark that may increase or reduce the aggressive behavior of GB tumors, thus influencing the survival rates of patients with GB, their response to therapy and their tendency to suffer a relapse.

The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the deubiquitination enzyme gene OTU1, the nuclear pore protein POM152 and the SNT1 that encodes for the Set3C subunit of the histone deacetylase complex. In these strains the PARP-1 level was roughly the same as in the wild type. PARP-1 localized in the nucleus more in the snt1Δ than in the wild type strain; after UV radiation, PARP-1 localized in the nucleus more in hho1 and pom152 deletion strains than in the wild type indicating that these functions may have a role on regulating PARP-1 level and activity in the nucleus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: Desire thinking is a voluntary cognitive process involving verbal and imaginary elaboration of a desired target. Recent research has highlighted the role of desire thinking in predicting addictive behaviours independent of other psychological constructs including negative affect and craving. The goal of this research project was to explore the role of desire thinking across the continuum of drinking behaviour., Methods: A sample of alcohol-dependent drinkers (n = 43), problem drinkers (n = 59), and social drinkers (n = 68) completed self-report instruments of desire thinking, negative affect, craving and drinking behaviour., Results: Analyses revealed that alcohol-dependent drinkers and problem drinkers scored higher than social drinkers on imaginal prefiguration, and that alcohol-dependent drinkers scored higher than problem drinkers who in turn scored higher than social drinkers on verbal perseveration. A multi-group discriminant analysis showed that craving, imaginal prefiguration and verbal perseveration loaded on a first function whilst age loaded on a second function. The variables correctly classified 75.9% of cases., Conclusions: The findings suggest that desire thinking may be a risk factor across the continuum of drinking behaviour and that treatment may benefit from specifically targeting this cognitive process., (Copyright © 2011 S. Karger AG, Basel.)

Alternative, non-electrochemistry-based technologies for continuous glucose monitoring are needed for eventual use in diabetes mellitus. As part of a programme investigating fluorescent glucose sensors, we have developed fibre-optic biosensors using glucose/galactose binding protein (GBP) labelled with the environmentally sensitive fluorophore, Badan. GBP-Badan was attached via an oligohistidine-tag to the surface of Ni-nitrilotriacetic acid (NTA)-functionalized agarose or polystyrene beads. Fluorescence lifetime increased in response to glucose, observed by fluorescence lifetime imaging microscopy of the GBP-Badan-beads. Either GBP-Badan agarose or polystyrene beads were loaded into a porous chamber at the end of a multimode optical fibre. Fluorescence lifetime responses were recorded using pulsed laser excitation, high speed photodiode detection and time-correlated single-photon counting. The maximal response was at 100 mM glucose with an apparent K(d) of 13 mM (agarose) and 20 mM (polystyrene), and good working-day stability was demonstrated. We conclude that fluorescence lifetime fibre-optic glucose sensors based on GBP-Badan are suitable for development as clinical glucose monitors.

Objectives: Health Information systems training is one of the bottlenecks in clinical systems implementation. In this article, a strategy to massively create and train interdisciplinary coordinating teams is described for a project in Uruguay at FEMI, a non-academic setting which includes 23 health care institutions across the country and a tertiary referral center in Montevideo., Methods: A series of educational activities were designed for the local coordinating teams. They included both onsite and online formats, site visits, integrated with some of the project tasks., Results: In total, 128 professionals from all the Institutions participated in one or more of the training sessions (onsite and online) and 87 of them accomplished one of the forms of training., Conclusions: Massive basic health informatics training was possible in Uruguay through collaboration with academic institutions at the country and regional level. Next steps include an active involvement of nurses in the educational events and planning of massive training of end users.