Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly / Novo mapeamento de mutações patogênicas do gene ASPM em famílias consanguíneas com microcefalia primária do Paquistão

Abstract Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing," with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Resumo Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser "causador de doenças", com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly / Novo mapeamento de mutações patogênicas do gene ASPM em famílias consanguíneas com microcefalia primária do Paquistão

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing", with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser "causador de doenças", com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly

Abstract Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be disease causing, with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

Resumo Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser causador de doenças, com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.

Disaster recovery and structural inequalities: A case study of community assertion for justice.

Formal interventions are rationalized to be irreplaceable, especially with marginalized communities that are presumed to lack capacity. It is event centric and differ considerably from the community's experience of disaster risk and recovery within the everyday context. Thus, community engagement with multiple formal institutions that often fail to address recovery needs of the most marginalized, is inevitable. These contradictions lead to varied forms of community assertion towards addressing structural inequalities and injustices. In this paper we explore these contradictions by drawing from the work of scholars who recognize the limits of procedural justice and push for distributive justice, especially by focusing on grassroots processes using the lens of the politics of neo-liberalism and ontology of possibilities. Using a multi-sited instrumental case study approach the paper explores community's lived experiences, factors contributing to the persistence of structural inequality and injustice, and the alternate conception of justice and their assertions, in the disaster recovery context. The two case studies - Vistapit Mukti Vahini and Thayillam, inform an alternate theoretical conception of disaster recovery embedded in structural inequalities and injustices through the following three perspectives: Firstly how disaster risk and recovery emerge from historical and everyday lived reality of marginalized communities, their social relations and resulting material conditions; Secondly how challenging everyday social relations, processes and injustices is central to the community's alternate conception and assertion for disaster recovery; and finally how community assertion and recovery relies on the mobilization of vulnerability, which could mean being exposed and agentic at the same time.

Novel Pathogenic Mutation Mapping of ASPM Gene in Consanguineous Pakistani Families with Primary Microcephaly.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing," with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.

The NeST (Neoadjuvant systemic therapy in breast cancer) study: National Practice Questionnaire of United Kingdom multi-disciplinary decision making.

BACKGROUND: Neoadjuvant systemic therapy (NST) is increasingly used in the treatment of breast cancer, yet it is clear that there is significant geographical variation in its use in the UK. This study aimed to examine stated practice across UK breast units, in terms of indications for use, radiological monitoring, pathological reporting of treatment response, and post-treatment surgical management. METHODS: Multidisciplinary teams (MDTs) from all UK breast units were invited to participate in the NeST study. A detailed questionnaire assessing current stated practice was distributed to all participating units in December 2017 and data collated securely usingREDCap. Descriptive statistics were calculated for each questionnaire item. RESULTS: Thirty-nine MDTs from a diverse range of hospitals responded. All MDTs routinely offered neoadjuvant chemotherapy (NACT) to a median of 10% (range 5-60%) of patients. Neoadjuvant endocrine therapy (NET) was offered to a median of 4% (range 0-25%) of patients by 66% of MDTs. The principal indication given for use of neoadjuvant therapy was for surgical downstaging. There was no consensus on methods of radiological monitoring of response, and a wide variety of pathological reporting systems were used to assess tumour response. Twenty-five percent of centres reported resecting the original tumour footprint, irrespective of clinical/radiological response. Radiologically negative axillae at diagnosis routinely had post-NACT or post-NET sentinel lymph node biopsy (SLNB) in 73.0 and 84% of centres respectively, whereas 16% performed SLNB pre-NACT. Positive axillae at diagnosis would receive axillary node clearance at 60% of centres, regardless of response to NACT. DISCUSSION: There is wide variation in the stated use of neoadjuvant systemic therapy across the UK, with general low usage of NET. Surgical downstaging remains the most common indication of the use of NAC, although not all centres leverage the benefits of NAC for de-escalating surgery to the breast and/or axilla. There is a need for agreed multidisciplinary guidance for optimising selection and management of patients for NST. These findings will be corroborated in phase II of the NeST study which is a national collaborative prospective audit of NST utilisation and clinical outcomes.

Nature of dispensing errors in selected hospitals providing free healthcare: a multi-center study in Sri Lanka.

BACKGROUND: Dispensing errors, known to result in significant patient harm, are preventable if their nature is known and recognized. However, there is a scarcity of such data on dispensing errors particularly in resource poor settings, where healthcare is provided free-of-charge. Therefore, the purpose of this study was to determine the types, and prevalence of dispensing errors in a selected group of hospitals in Sri Lanka. METHODS: A prospective, cross sectional, multi-center study on dispensing errors was conducted, in a single tertiary care, and two secondary care hospitals, in a cohort of 420 patients attending medical, surgical, diabetic and pediatric clinics. The patients were selected according to the population size, through consecutive sampling. The prescription audit was conducted in terms of dispensing errors which were categorized as i) content, ii) labelling, iii) documentation, iv) concomitant, and v) other errors based on in-house developed definitions. RESULTS: A total of 420 prescriptions (1849 medicines) were analyzed (Hospital-I, 248 prescriptions-1010 medicines; Hospital-II, 84 prescriptions-400 medicines; Hospital-III, 88 prescriptions-439 medicines), and a cumulative total of 16,689 dispensing errors (at least one dispensing error in a prescription) were detected. Labelling errors were the most frequent dispensing error (63.1%; N = 10,523; Mostly missing information on the dispensing label), followed by concomitant prescribing and dispensing errors (20.5%; N = 3425; Missing prescribing information overlooked by the pharmacist), documentation errors (10.6%; N = 1772 Missing identification of pharmacist on dispensing label), clinically significant medication interactions overlooked by pharmacists (0.5%; N = 82), content errors (4.9%; N = 812; Discrepancies between medication dispensed and prescription order), medications dispensed in unsuitable packaging (0.4%; N = 74), and lastly medication dispensed to the wrong patient (0.01%; N = 1). CONCLUSIONS: Dispensing errors are frequent in Sri Lankan hospitals which operate with limited resources and provide free healthcare to all citizenry. Over one half of the errors were labeling errors with minimal content errors. Awareness on common types of dispensing errors and emphasis on detecting them could improve medication safety in Sri Lankan hospitals.

Increasing Susceptibility of Drug-Resistant Candida albicans to Fluconazole and Terbinafine by 2(5H)-Furanone Derivative.

The frequency of mycoses caused by drug-resistant fungal pathogen Candida albicans has increased drastically over the last two decades. The spread of drug-resistant strains, along with the limitations of currently available antifungals, complicates the management of fungal infections, thereby representing great challenges for clinical healthcare. Among various antimicrobial pharmacophores, 2(5H)-furanone derivatives have demonstrated antimicrobial, antifungal, and antibiofilm activities. In this study, we report the antifungal activity of the 2(5H)-furanone derivative F105, consisting of three pharmacophores, namely chlorinated 2(5H)-furanone, sulfonyl group, and l-menthol moiety. Although exhibiting moderate antifungal activity alone with the minimum inhibitory concentration (MIC) values of 32-256 µg/mL, F105 potentiates the activity of fluconazole and terbinafine with fractional inhibitory concentration index (FICI) values of 0.27-0.50. Thus, 16 µg/mL of F105 reduced the MICs of these antifungals against fluconazole-resistant C. albicans isolates four-fold, achieving similar values as for the intermediately susceptible phenotype. Confocal laser scanning microscopy revealed that the fluorescent 2(5H)-furanone derivative F145 was also able to penetrate through biofilms formed by C. albicans. Indeed, in the presence of F105, even sub-MIC concentrations of both fluconazole and terbinafine led to significant reduction of C. albicans CFUs in the mature biofilm. Thus, F105 appears to be a promising candidate for the development of novel antifungal agents as well as enhancers of current antifungal agents, particularly for the treatment of drug-resistant C. albicans infections.

Unraveling the Molecular Mechanism of Selective Antimicrobial Activity of 2(5H)-Furanone Derivative against Staphylococcus aureus.

Staphylococcus aureus causes various infectious diseases, from skin impetigo to life-threatening bacteremia and sepsis, thus appearing an important target for antimicrobial therapeutics. In turn, the rapid development of antibiotic resistance and biofilm formation makes it extremely robust against treatment. Here, we unravel the molecular mechanism of the antimicrobial activity of the recently unveiled F105 consisting of three pharmacophores: chlorinated 2(5H)-furanone, sulfone, and l-menthol moieties. F105 demonstrates highly selective activity against Gram-positive bacteria and biofilm-embedded S. aureus and exhibits low risk of resistance development. We show explicitly that the fluorescent analogue of F105 rapidly penetrates into Gram-positive bacteria independently of their cell integrity and viability and accumulates there. By contrast, Gram-negative bacteria remain impermeable and, therefore, insusceptible to F105. Apparently, in bacterial cells, F105 induces reactive oxygen species (ROS) formation and nonspecifically interacts with a number of proteins, including ROS-utilizing ones. Using native and 2D PAGE, we confirm that F105 changes the charge of some proteins by either oxidation or direct interaction with them. Therefore, it seems justified to conclude that being simultaneously a ROS inducer and damaging proteins responsible for ROS utilization, F105 impairs the cellular anti-ROS defense representing a prospective ROS-inducing antibacterial agent.

Targeting Bacillus cereus cells: increasing efficiency of antimicrobials by the bornylpossessing 2(5Н)-furanone derivative.

Among a variety of antimicrobial compounds, the derivatives of 2(5H)-furanone exhibit different effects on Firmicutes and Proteobacteria. While inhibiting quorum-dependent biofilm formation and virulence factor expression by Gram-negative bacteria through specific interference with the AI-2 signaling pathways, these compounds demonstrate bactericidal effects against Gram-positive bacteria. Here we report that 3,4-dichloro-5(S)-[(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yloxy]-2(5H)-furanone designed as F123 inhibits growth and biofilm formation by the food-poisoning bacterium Bacillus cereus at 8 µg/ ml and kills bacteria at 16 µg/ml. While the growth of Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis were also inhibited at 8-16 µg/ml of F123, no bactericidal effect on these strains was observed at concentrations up to 128 µg/ml, suggesting pronounced specificity of F123 for B. cereus. In a checker-board assay F123 increased the efficacy of amikacin, gentamicin and benzalkonium chloride against B. cereus with medians of fractional inhibitory concentration index of 0.38, 0.56 and 0.56, respectively. Moreover, the number of viable B. cereus cells in biofilm was reduced by more than 3 orders of magnitude at 64 µg/ml of F123, suggesting its chemotype as a promising enhancer for specific treatment of B. cereus-associated topical infections, including biofilm-embedded bacteria.

The NeST (neoadjuvant systemic therapy in breast cancer) study - Protocol for a prospective multi-centre cohort study to assess the current utilization and short-term outcomes of neoadjuvant systemic therapies in breast cancer.

INTRODUCTION: Neoadjuvant systemic therapy (NST) has several potential advantages in the treatment of breast cancer. However, there is currently considerable variation in NST use across the UK. The NeST study is a national, prospective, multicentre cohort study that will investigate current patterns of care with respect to NST in the UK. METHODS AND ANALYSIS: Phase 1 - a national practice questionnaire (NPQ) to survey current practice.Phase 2 - a multi-centre prospective cohort study of breast cancer patients, undergoing NST.Women undergoing NST as their MDT recommended primary breast cancer treatment between December 2017 and May 2018 will be included. The breast surgery and oncological professional associations and the trainee research collaborative networks will encourage participation by all breast cancer centres.Patient demographics, radiological, oncological, surgical and pathological data will be collected, including complications and the need for further intervention/treatment. Data will be collated to establish current practice in the UK, regarding NST usage and variability of access and provision of these therapies. Prospective data on 600 patients from ~50 centres are anticipated.Trial registration: ISRCTN11160072. ETHICS AND DISSEMINATION: Research ethics approval is not required for this study, as per the online Health Research Authority decision tool. The information obtained will provide valuable insights to help patients make informed decisions about their treatment. These data should establish current practice in the UK concerning NST, inform future service delivery as well as identifying further research questions.This protocol will be disseminated through the Mammary Fold Academic Research Collaborative (MFAC), the Reconstructive Surgery Trials Network and the Association of Breast Surgery. Participating units will have access to their own data and collective results will be presented at relevant conferences and published in appropriate peer-reviewed journals, as well as being made accessible to relevant patient groups.

Fast and simple tool for the quantification of biofilm-embedded cells sub-populations from fluorescent microscopic images.

Fluorescent staining is a common tool for both quantitative and qualitative assessment of pro- and eukaryotic cells sub-population fractions by using microscopy and flow cytometry. However, direct cell counting by flow cytometry is often limited, for example when working with cells rigidly adhered either to each other or to external surfaces like bacterial biofilms or adherent cell lines and tissue samples. An alternative approach is provided by using fluorescent microscopy and confocal laser scanning microscopy (CLSM), which enables the evaluation of fractions of cells subpopulations in a given sample. For the quantitative assessment of cell fractions in microphotographs, we suggest a simple two-step algorithm that combines single cells selection and the statistical analysis. To facilitate the first step, we suggest a simple procedure that supports finding the balance between the detection threshold and the typical size of single cells based on objective cell size distribution analysis. Based on a series of experimental measurements performed on bacterial and eukaryotic cells under various conditions, we show explicitly that the suggested approach effectively accounts for the fractions of different cell sub-populations (like the live/dead staining in our samples) in all studied cases that are in good agreement with manual cell counting on microphotographs and flow cytometry data. This algorithm is implemented as a simple software tool that includes an intuitive and user-friendly graphical interface for the initial adjustment of algorithm parameters to the microphotographs analysis as well as for the sequential analysis of homogeneous series of similar microscopic images without further user intervention. The software tool entitled BioFilmAnalyzer is freely available online at https://bitbucket.org/rogex/biofilmanalyzer/downloads/.

AllergoOncology: Opposite outcomes of immune tolerance in allergy and cancer.

While desired for the cure of allergy, regulatory immune cell subsets and nonclassical Th2-biased inflammatory mediators in the tumour microenvironment can contribute to immune suppression and escape of tumours from immunological detection and clearance. A key aim in the cancer field is therefore to design interventions that can break immunological tolerance and halt cancer progression, whereas on the contrary allergen immunotherapy exactly aims to induce tolerance. In this position paper, we review insights on immune tolerance derived from allergy and from cancer inflammation, focusing on what is known about the roles of key immune cells and mediators. We propose that research in the field of AllergoOncology that aims to delineate these immunological mechanisms with juxtaposed clinical consequences in allergy and cancer may point to novel avenues for therapeutic interventions that stand to benefit both disciplines.

Antimicrobial Effects of Sulfonyl Derivative of 2(5H)-Furanone against Planktonic and Biofilm Associated Methicillin-Resistant and -Susceptible Staphylococcus aureus.

The gram-positive opportunistic bacterium Staphylococcus aureus is one of the most common causatives of a variety of diseases including skin and skin structure infection or nosocomial catheter-associated infections. The biofilm formation that is an important virulence factor of this microorganism renders the antibiotic therapy ineffective, because biofilm-embedded bacteria exhibit strongly increased tolerance to antimicrobials. Here, we describe a novel 3-chloro-5(S)-[(1R,2S,5R)-2-isopropyl-5-methylcyclohexyloxy]-4-[4-methylphenylsulfonyl]-2(5H)-furanone (F105), possessing a sulfonyl group and l-menthol moiety. Minimal inhibitory and bactericidal concentration values (MIC and MBC) of F105 were 10 and 40 mg/L, respectively, suggesting F105 biocidal properties. F105 exhibits pronounced activity against biofilm-embedded S. aureus and increases the efficacy of aminoglycosides (amikacin, gentamicin, and kanamycin) and benzalkonium chloride with fractional inhibitory concentration index values of 0.33-0.44 and 0.29, respectively, suggesting an alternative external treatment option, e.g., for wound infections. Moreover, low concentrations (0.5-1.3 mg/L) of F105 reduced the MICs of these antimicrobials twofold. By using confocal laser scanning microscopy and CFU counting, we show explicitly that F105 also restores the antimicrobial activity of gentamicin and ampicillin against S. aureus biofilms by several orders of magnitude. Biofilm structures were not destroyed but sterilized, with embedded cells being almost completely killed at twofold MBC. While F105 is quite toxic (CC50/MBC ratio 0.2), our data suggest that the F105 chemotype might be a promising starting point for the development of complex topical agents for combined anti-staphylococcal biofilm-therapies restoring the efficacy of some antibiotics against difficult to treat S. aureus biofilm.

Inhibition of biofilm formation in Bacillus subtilis by new halogenated furanones.

Gram-positive bacteria can cause various infections including hospital-acquired infections. While in the biofilm, the resistance of bacteria to both antibiotics and the human immune system is increased causing difficulties in the treatment. Bacillus subtilis, a non-pathogenic Gram-positive bacterium, is widely used as a model organism for studying biofilm formation. Here we investigated the effect of novel synthesized chloro- and bromo-containing 2(5H)-furanones on biofilm formation by B. subtilis. Mucobromic acid (3,4-dibromo-5-hydroxy-2(5H)-furanone) and the two derivatives of mucochloric acid (3,4-dichloro-5-hydroxy-2(5H)-furanone)-F8 and F12-were found to inhibit the growth and to efficiently prevent biofilm formation by B. subtilis. Along with the low production of polysaccharide matrix and repression of the eps operon, strong repression of biofilm-related yqxM also occurred in the presence of furanones. Therefore, our data confirm that furanones affect significantly the regulatory pathway(s) leading to biofilm formation. We propose that the global regulator, Spo0A, is one of the potential putative cellular targets for these compounds.

The use of molecular imaging combined with genomic techniques to understand the heterogeneity in cancer metastasis.

Tumour heterogeneity has, in recent times, come to play a vital role in how we understand and treat cancers; however, the clinical translation of this has lagged behind advances in research. Although significant advancements in oncological management have been made, personalized care remains an elusive goal. Inter- and intratumour heterogeneity, particularly in the clinical setting, has been difficult to quantify and therefore to treat. The histological quantification of heterogeneity of tumours can be a logistical and clinical challenge. The ability to examine not just the whole tumour but also all the molecular variations of metastatic disease in a patient is obviously difficult with current histological techniques. Advances in imaging techniques and novel applications, alongside our understanding of tumour heterogeneity, have opened up a plethora of non-invasive biomarker potential to examine tumours, their heterogeneity and the clinical translation. This review will focus on how various imaging methods that allow for quantification of metastatic tumour heterogeneity, along with the potential of developing imaging, integrated with other in vitro diagnostic approaches such as genomics and exosome analyses, have the potential role as a non-invasive biomarker for guiding the treatment algorithm.

Assessment of microtubule-associated protein (MAP)-Tau expression as a predictive and prognostic marker in TACT; a trial assessing substitution of sequential docetaxel for FEC as adjuvant chemotherapy for early breast cancer.

The TACT trial is the largest study assessing the benefit of taxanes as part of adjuvant therapy for early breast cancer. The goal of this translational study was to clarify the predictive and prognostic value of Tau within the TACT trial. Tissue microarrays (TMA) were available from 3,610 patients. ER, PR, HER2 from the TACT trial and Tau protein expression was determined by immunohistochemistry on duplicate TMAs. Two parallel scoring systems were generated for Tau expression ('dichotomised' vs. 'combined' score). The positivity rate of Tau expression was 50 % in the trial population (n = 2,483). Tau expression correlated positively with ER (p < 0.001) and PR status (p < 0.001); but negatively with histological grade (p < 0.001) and HER2 status (p < 0.001). Analyses with either scoring systems for Tau expression demonstrated no significant interaction between Tau expression and efficacy of docetaxel. Contrary to the hypothesis that taxane benefit would be enriched in Tau negative/low patients, the only groups with a suggestion of a reduced event rate in the taxane group were the HER2-positive, Tau positive subgroups. Tau expression was seen to be a prognostic factor on univariate analysis associated with an improved DFS, independent of the treatment group (p < 0.001). It had no prognostic value in ER-negative tumours and the weak prognostic effect of Tau in ER-positive tumours (p = 0.02) diminished, when considering ER as an ordinal variable. On multivariable analyses, Tau had no prognostic value in either group. In addition, no significant interaction between Tau expression and benefit from docetaxel in patients within the PR-positive and negative subsets was seen. This is now the second large adjuvant study, and the first with quantitative analysis of ER and Tau expression, failing to show an association between Tau and taxane benefit with limited utility as a prognostic marker for Tau in ER-positive early breast cancer patients.