MOLECULAR CONTRIBUTION OF THE AURORA-A KINASE AND THE JUNCTIONAL PROTEIN AFADIN TO ORIENTED CELL DIVISIONS

Correct spindle positioning is essential for tissue morphogenesis and homeostasis. The orientation of the mitotic spindle is determined by cortical force generators formed on NuMA:LGN:G\u3b1i complexes, which anchor astral microtubules emanating from the spindle poles at specialized domains of the plasma membrane via direct interaction with the motor proteins Dynein/Dynactin. Cortical polarity cues and actin-associated proteins synergize with extrinsic signals (such as cell-to-cell and cell-to-extracellular matrix contacts) in recruiting NuMA:LGN:G\u3b1i complexes at the cell cortex. In addition, spindle placement is coordinated with mitotic progression by mitotic kinases regulating the timely cortical recruitment of NuMA:LGN:G\u3b1i above the spindle poles. My PhD project focused on the study of the molecular mechanisms accounting for the spindle orientation functions of the Aurora-A kinase, the polarity protein Lgl2, and the junctional protein Afadin. The Aurora-A kinase is known for being implicated in spindle alignment, however the molecular events underlying this function remain to date unclear. To study the spindle orientation functions of Aurora-A, I developed protocols for the partial inhibition of its activity in transformed and non-transformed cells in culture. Under these conditions, in metaphase NuMA and Dynactin accumulate abnormally at the spindle poles without reaching the cortex, while the cortical distribution of LGN remains unperturbed. Fluorescence Recovery After Photobleaching (FRAP) experiments conducted on GFP-NuMA revealed that Aurora-A governs the dynamic exchange between the cytoplasmic and the spindle-pole-localized pools of NuMA. Molecularly, Aurora-A phosphorylates directly the C-terminus of NuMA on three serine residues, among which Ser-1969 is the major determinant for the dynamic behaviour of NuMA at the spindle poles. Most interestingly, we identify a new microtubule-binding domain of NuMA, which does not overlap with the LGN-binding motif, thus suggesting that NuMA can associate concomitantly with LGN and microtubules. This finding indicates that the microtubule-binding activity of NuMA might contribute to anchor microtubule +TIPs at cortical sites with LGN. Collectively, my studies demonstrate that in metaphase the direct phosphorylation of NuMA by Aurora-A controls its cortical enrichment, and that this is the major event underlying the spindle orientation functions of Aurora-A in cultured cells. Phosphorylation of NuMA by Aurora-A does not affect its affinity for microtubules nor for LGN, but rather determines the mobility of the protein at the spindle-poles. Biochemical studies suggested that Lgl2 can associate with LGN, hinting at a possible role of this protein in spindle orientation in mammalian system. On these premises, I found that depletion of Lgl2 misorients the spindle in HeLa cells plated on fibronectin. However, I could not reproduce the Lgl2:LGN interaction in vitro nor ex vivo. Interestingly, by immunoprecipitation experiments I detected an interaction between NuMA and Lgl2, which could explain the phenotype of spindle misorientation resulting from the silencing of Lgl2 in HeLa cells. Further studies will be required to gain a molecular understanding of the relevance Lgl2:NuMA interaction in oriented divisions. Part of my PhD studies addressed the role of Afadin in spindle orientation; I demonstrated that Afadin is required for spindle positioning, and correct epithelial morphogenesis of Caco-2 three-dimensional cysts. At a molecular level, Afadin binds directly and concomitantly to F-actin and to LGN. Indeed, in mitotic HeLa cells, Afadin is required for cortical accumulation of LGN, NuMA and Dynein above the spindle poles, in a F-actin dependent manner. Collectively, these results uncovered a pivotal role of Afadin in governing the enrichment of LGN and NuMA at the lateral cortex of polarized epithelia. They also depict that Afadin as the first mechanical anchor between Dynein and cortical F-actin

Snapshot PCR surveillance for SARS-CoV-2 in hospital staff in England

Background: Significant nosocomial transmission of SARS-CoV-2 has been demonstrated. Understanding the prevalence of SARS-CoV-2 carriage amongst HCWs at work is necessary to inform the development of HCW screening programmes to control nosocomial spread. / Methods: Cross-sectional ‘snapshot’ survey from April-May 2020; HCWs recruited from six UK hospitals. Participants self-completed a health questionnaire and underwent a combined viral nose and throat swab, tested by Polymerase Chain Reaction (PCR) for SARS-CoV-2 with viral culture on majority of positive samples. / Findings: Point prevalence of SARS-CoV-2 carriage across the sites was 2.0% (23/1152 participants), median cycle threshold value 35.70 (IQR:32.42–37.57). 17 were previously symptomatic, two currently symptomatic (isolated anosmia and sore throat); the remainder declared no prior or current symptoms. Symptoms in the past month were associated with threefold increased odds of testing positive (aOR 3.46, 95%CI 1.38–8.67; p = 0.008). SARS-CoV-2 virus was isolated from only one (5%) of nineteen cultured samples. A large proportion (39%) of participants reported symptoms in the past month. / Interpretation: The point-prevalence is similar to previous estimates for HCWs in April 2020, though a magnitude higher than in the general population. Based upon interpretation of symptom history and testing results including viral culture, the majority of those testing positive were unlikely to be infectious at time of sampling. Development of screening programmes must balance the potential to identify additional cases based upon likely prevalence, expanding the symptoms list to encourage HCW testing, with resource implications and risks of excluding those unlikely to be infectious with positive tests. / Funding: Public Health England

Combined effects of franchise management strategies and employee service performance on customer loyalty: a multilevel perspective

Although franchisee performance is likely to be influenced by franchisors’ management strategies, little is known about whether and how franchisors’ strategies affect franchisee employees’ performance. This study examines the combined effects of three franchisor management strategies, namely innovative culture, support services and autonomy on service performance of the franchisee store employees and the loyalty of their customers. Data were collected from a total of 38 employees and 679 customers of 25 franchisee stores. The study employs multilevel analysis on a nested data-set created by matching customer data with employee data for each store. The results reveal that customer loyalty of a franchisee store is positively influenced by the service performance of its employees and the support services received by the employees of the store from its franchisor. On the other hand, it has been found that franchisor management strategy such as innovative culture and autonomy negatively influence customer loyalty of the franchisee store. The paper discusses relevant theoretical and managerial implications of the findings

Lucro líquido do franqueado: um sinal para a escolha de franquia em época de crise

Este artículo aborda desde la perspectiva de la Teoría de Señales la elección de una franquicia por un potencial franquiciado que elige este canal de distribución por primera vez. El objetivo es analizar la relación entre algunas señales enviadas por el franquiciador y la elección de una franquicia por el potencial franquiciado. Concluimos que los efectos de las variables macroeconómicas de España en el periodo 2006-2013 influyeron en los ingresos netos de los franquiciados para que se convirtiera en una señal empleada por éstos a la hora de elegir la franquicia donde abrir un establecimiento.This paper adopts the perspective of Theory of Signals to discuss how someone starting a business as a franchisee for the first time can choose a suitable franchise brand. The aim was to analyze the relationship between certain signals sent by the franchisor and the choice of a franchise brand by the prospective franchisee. Using panel data, we found that the effects of macroeconomic variables in Spain for the 2006-2013 crisis period influenced franchisees' net income, which thus became a relevant signal in their process of choosing a franchise brand to start a business.Sob a perspectiva da Teoria de Sinais, este artigo aborda a seleção de uma franquia por um franqueado que elege esse potencial canal de distribuição pela primeira vez. O objetivo é analisar a relação entre alguns sinais enviados pelo franqueador e a escolha de uma franquia pelo potencial franqueado. Usando a metodologia de dados em painel, os resultados obtidos nos permitiram concluir que os efeitos das variáveis macroeconômicas da Espanha no período de crise econômica (2006-2013) influenciaram as receitas líquidas dos franqueados, tornando-se um sinal para eles elegerem onde abrir um estabelecimento franqueado

The plants, rituals and spells that 'cured' helminthiasis in Sicily

<p>Abstract</p> <p>Background</p> <p>The author reports on the plants, rituals and spells used against worms and the so-called <it>scantu </it>(fright) in some areas of Sicily. The work is based on ethnobotanical research carried out, prevalently, between 2002-2006, in some areas of Eastern, South-Eastern, North-Central and South-Central Sicily.</p> <p>Methods</p> <p>This research is based on dialogue. Senior 'healers' were contacted; furthermore, doctors, teachers, farmers and in general 'experts' with herbs and 'magic' rituals. Information was collected about the way the plants of folk medicine are prepared. The interviewees were also invited to recite prayers and spells against helminthiasis.</p> <p>Results</p> <p>The author has highlighted the importance of how, in some parts of Sicily, some ailments like helminthiasis and other correlated pathologies like <it>scantu </it>are 'treated' and, especially within the rural social classes, by folk medicine remedies, herbal practises, particular prayers, rituals and spells.</p> <p>Conclusion</p> <p>As regards health/illness, it should be noted that in the last ten years conventional medicine has provided very satisfactory results even resolving potentially mortal pathologies. However, in certain social classes, there is no real collaboration between conventional and folk medicine; so for some senior citizens, the 'healer' with his rituals and empirical and magical herbs is still the person to turn to for the 'cure' of particular ailments. Interest in these practises from ancestral heritage in an advanced country like Italy, is only relevant if the aim is to recoup a cultural identity which is already in decline.</p> <p>It is significant to report a piece: on 14 October 2007 the news on a well-known national Italian TV channel reported an interview with a 94 year-old man from Arbatax (Sardinia) referred to as a 'healer' because both his townspeople and others from all over the world go to him for his cures. He is not paid except in kind and has been known to cure St. Anthony's fire, burns, scalding and marine fungal infections, by smearing his saliva over the infected part and reciting 'special words'.</p

A Prescription for Improving Drug Formulary Decision Making

Gordon Schiff and colleagues present a new tool and checklist to help formularies make decisions about drug inclusion and to guide rational drug use

Fluoride Depletes Acidogenic Taxa in Oral but Not Gut Microbial Communities in Mice

ABSTRACT Fluoridation of drinking water and dental products prevents dental caries primarily by inhibiting energy harvest in oral cariogenic bacteria (such as Streptococcus mutans and Streptococcus sanguinis), thus leading to their depletion. However, the extent to which oral and gut microbial communities are affected by host fluoride exposure has been underexplored. In this study, we modeled human fluoride exposures to municipal water and dental products by treating mice with low or high levels of fluoride over a 12-week period. We then used 16S rRNA gene amplicon and shotgun metagenomic sequencing to assess fluoride’s effects on oral and gut microbiome composition and function. In both the low- and high-fluoride groups, several operational taxonomic units (OTUs) belonging to acidogenic bacterial genera (such as Parabacteroides, Bacteroides, and Bilophila) were depleted in the oral community. In addition, fluoride-associated changes in oral community composition resulted in depletion of gene families involved in central carbon metabolism and energy harvest (2-oxoglutarate ferredoxin oxidoreductase, succinate dehydrogenase, and the glyoxylate cycle). In contrast, fluoride treatment did not induce a significant shift in gut microbial community composition or function in our mouse model, possibly due to absorption in the upper gastrointestinal tract. Fluoride-associated perturbations thus appeared to have a selective effect on the composition of the oral but not gut microbial community in mice. Future studies will be necessary to understand possible implications of fluoride exposure for the human microbiome. IMPORTANCE: Fluoride has been added to drinking water and dental products since the 1950s. The beneficial effects of fluoride on oral health are due to its ability to inhibit the growth of bacteria that cause dental caries. Despite widespread human consumption of fluoride, there have been only two studies of humans that considered the effect of fluoride on human-associated microbial communities, which are increasingly understood to play important roles in health and disease. Notably, neither of these studies included a true cross-sectional control lacking fluoride exposure, as study subjects continued baseline fluoride treatment in their daily dental hygiene routines. To our knowledge, this work (in mice) is the first controlled study to assess the independent effects of fluoride exposure on the oral and gut microbial communities. Investigating how fluoride interacts with host-associated microbial communities in this controlled setting represents an effort toward understanding how common environmental exposures may potentially influence health

Pediatric Intensive Care Unit admission criteria for haemato-oncological patients: a basis for clinical guidelines implementation

Recent advances in supportive care and progress in the development and use of chemotherapy have considerably improved the prognosis of many children with malignancy, thus the need for intensive care admission and management is increasing, reaching about 40% of patients throughout the disease course. Cancer remains a major death cause in children, though outcomes have considerably improved over the past decades. Prediction of outcome for children with cancer in Pediatric Intensive Care Unit (PICU) obviously requires clinical guidelines, and these are not well defined, as well as admission criteria. Major determinants of negative outcomes remain severe sepsis/septic shock association and respiratory failure, deserving specific approach in children with cancer, particularly those receiving a bone marrow transplantation. A nationwide consensus should be achieved among pediatric intensivists and oncologists regarding the threshold clinical conditions requiring Intensive Care Unit (ICU) admission as well as specific critical care protocols. As demonstrated for the critically ill non-oncologic child, it appears unreasonable that pediatric patients with malignancy can be admitted to an adult Intensive Care Unit ICU. On a national basis a pool of refecence institutions should be identified and early referral to an oncologic PICU is warranted

Sustainable Financing of Innovative Therapies: A Review of Approaches

The process of innovation is inherently complex, and it occurs within an even more complex institutional environment characterized by incomplete information, market power, and externalities. There are therefore different competing approaches to supporting and financing innovation in medical technologies, which bring their own advantages and disadvantages. This article reviews value- and cost-based pricing, as well direct government funding, and cross-cutting institutional structures. It argues that performance-based risk-sharing agreements are likely to have little effect on the sustainability of financing; that there is a role for cost-based pricing models in some situations; and that the push towards longer exclusivity periods is likely contrary to the interests of industry