Assembly of genetic parts for CRISPR mediated interference against antimicrobial resistance

openThis thesis presents parts of a collaborative project designed by a team of the University of Padua that is going to participate to the iGEM competition on synthetic biology. The project aims to exploit the CRISPR interference system to contrast antimicrobial resistance (AMR). The idea we aim to develop consists in delivering a CRISPR interference system, targeting antibiotic resistance genes, by engineered bacteriophages. These phages will be modified to specifically infect one of the following hosts: Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. For a hypothetical application, the platform would be used in a synergic approach with antibiotic therapy to treat infections caused by antibiotic resistant bacteria. In particular, the thesis deals with the construction of a dCas9 and sgRNAs expression systems able to function in the above mentioned bacteria. Specifically, plasmids described in the scientific literature were engineered, mainly through PCR mutagenesis and the BioBrick RFC [10] standard assembly. The use of this standard required the mutagenesis of the dCas9 used in the project. The creation of these standardized parts, promoted by iGEM itself, aims to simplify updates of target genes and bacterial hosts for future implementations, raising a possible solution to the global issue of AMR.This thesis presents parts of a collaborative project designed by a team of the University of Padua that is going to participate to the iGEM competition on synthetic biology. The project aims to exploit the CRISPR interference system to contrast antimicrobial resistance (AMR). The idea we aim to develop consists in delivering a CRISPR interference system, targeting antibiotic resistance genes, by engineered bacteriophages. These phages will be modified to specifically infect one of the following hosts: Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. For a hypothetical application, the platform would be used in a synergic approach with antibiotic therapy to treat infections caused by antibiotic resistant bacteria. In particular, the thesis deals with the construction of a dCas9 and sgRNAs expression systems able to function in the above mentioned bacteria. Specifically, plasmids described in the scientific literature were engineered, mainly through PCR mutagenesis and the BioBrick RFC [10] standard assembly. The use of this standard required the mutagenesis of the dCas9 used in the project. The creation of these standardized parts, promoted by iGEM itself, aims to simplify updates of target genes and bacterial hosts for future implementations, raising a possible solution to the global issue of AMR

Influence of the Diet on Lipofilling Resorption Rate

Obiettivo dello studio \ue8 verificare l\u2019attecchimento del tessuto adiposo innestato nelle pazienti sottoposte a lipofilling dopo ricostruzione mammaria oncologica. Nel 2015 ci sono state in Italia 692.955 diagnosi di carcinoma mammario. Queste pazienti hanno riferito un forte impatto sulla loro qualit\ue0 di vita (QoL) causato dal cancro al seno e conseguenti trattamenti. Oggi, le pazienti hanno un alto tasso di sopravvivenza e una buona aspettativa di vita. Ai fini di migliorare la QoL sono state significative le possibilit\ue0 offerte dalla ricostruzione mammaria, in particolare dal lipofilling. Il maggiore svantaggio di questa procedura consiste nell\u2019alto tasso di riassorbimento, che rende quindi necessario ripeterla. Infatti, la letteratura descrive la sopravvivenza del tessuto adiposo innestato variabile tra il 55% e l\u201982%. Per questo motivo, diversi autori hanno cercato metodi che aiutassero ad aumentare la sopravvivenza del lipoinfiltrato. Diversi studi hanno ipotizzato una connessione fra il tasso di riassorbimento e la presenza di uno status infiammatorio, in particolare un minor riassorbimento del grasso in presenza di sostanze anti-infiammatorie. Un target stimolante \ue8 riuscire a raggiungere questa condizione tramite la dieta. Esaminando le diverse diete, \ue8 emerso che la dieta mediterranea (MD) \ue8 l\u2019unica che in diversi studi \ue8 stata associata ad un impatto sullo status infiammatorio. L\u2019endpoint primario \ue8 stato individuato nella proporzione di pazienti che ottengono l\u2019attecchimento del tessuto adiposo innestato, definito come l\u2019aumento dello spessore del lembo di mastectomia dopo il lipofilling, valutato per mezzo di un\u2019ecografia pre- e post operatoria. L\u2019endpoint coprimario \ue8 rappresentato dalla verifica dell\u2019influenza dell\u2019aderenza alla MD sul tasso di riassorbimento del tessuto adiposo in termini di minor riassorbimento. Lo studio inoltre valuta l\u2019incidenza delle complicanze. L\u2019aderenza alla MD viene valutata attraverso il questionario dello studio PREDIMED. Purtroppo l\u2019emergenza relata all'infezione COVID-19 e le restrizioni attuate per il suo contrasto hanno comportato una drammatica riduzione delle sedute operatorie e dell'attivit\ue0 ambulatoriale. Sono stati ottenuti dei risultati preliminari con 11 casi completi di controllo pre e post-operatorio. Tre pazienti sono state escluse dallo studio: una ha sviluppato infezione e due hanno rifiutato di recarsi in ospedale. Dai risultati \ue8 emerso che in Friuli Venezia Giulia tutte le pazienti dello studio hanno aderenza buona o ottima alla MD. Non ci sono state pazienti con bassa aderenza. Il 73% delle pazienti ha riferito aderenza media, il 27% delle pazienti ha riferito alta aderenza. Quattro pazienti hanno storia di chemioterapia (CHT) e il 27% ha storia di radioterapia (RT). Tre pazienti sono risultate fumatrici, una paziente ha avuto pregressi lipofilling. La media di tessuto adiposo innestato \ue8 stata di 119 cc, con mediana di 115 cc. Il 64% delle donne \ue8 tuttora in terapia ormonale. Il COVID-19 ha influenzato il tempo del controllo postoperatorio, che ha media e mediana di 5 mesi. Il guadagno di spessore del tessuto adiposo dopo lipofilling \ue8 stato statisticamente significativo, con un valore pari a 0.062 e p= 0.00001. Non si \ue8 dimostrata nessuna associazione fra il guadagno di spessore e la DM, le comorbidit\ue0, CHT, RT, terapia ormonale, fumo, pregressi lipofilling o quantit\ue0 di lipoinfiltrato. A causa della pandemia da COVID-19, lo studio ha arruolato troppe poche pazienti per soddisfare il sample size, influenzando la possibilit\ue0 di trovare associazioni statisticamente significative. Ciononostante, \ue8 stato possibile rilevare una buona aderenza alla DM e molte pazienti hanno riferito modifiche attive del loro stile di vita. Continuare lo studio permetter\ue0 di studiare l\u2019attecchimento del tessuto adiposo e l\u2019associazione con la dieta.The study aims to investigate the fat graft engraftment in patients who undergo breast reconstruction with implant and lipofilling after mastectomy. In 2015 in Italy 692.955 women had a diagnosis of breast cancer. Breast cancer survivals report that cancer has had a great impact on their quality of life (QoL) due to treatments they had undergone. In the last decades, QoL for breast cancer survivors has improved due to the development of different techniques offered for breast reconstruction. Among these, fat grafting (lipofilling) is a widely used technique that has been developed by plastic surgeons. The main disadvantage of lipofilling is the unpredictable resorption rate of the graft, reported as ranging from 55% to 82%. Several studies have observed the connection between fat resorption rate and presence of an inflammatory status. A stimulating target is to be able to reach an anti-inflammatory status by the virtue of the diet. Among the dietary regimens, the Mediterranean Diet (MD) is the one that in different studies has shown promising results in terms of impact on the inflammatory status. The primary endpoint of the study is the rate of patients with successful fat graft engraftment, defined as an increase in mean breast soft tissue thickness after lipofilling. Co-primary endpoint is to evaluate if the adherence to the MD impacts on fat graft resorption rate, in terms of minor fat graft resorption. Patients are investigated in their dietetic habits and their adherence to the MD by means of a questionnaire that examines their usual daily and weekly consumptions, previously employed in the PREDIMED study. Mastectomy skin flap thickness is evaluated by means of an ultrasound pre- and post-operatory. Besides, the study collects the incidence of complications. Due to COVID-19 impact and the consequent reduction in surgeries and postoperatory controls, study is still on course. However, promising results have been given from the first surveys and a total of 11 patients with complete preoperatory and post-operatory controls has been enrolled in the study. Three patients were excluded from the study: one patient developed infection; two patients refused to come to the hospital. In Friuli Venezia Giulia there is a good adherence to the MD: there were no patients with low adherence to the MD. 73% of patients reported an average adherence, 27% of patients described a high adherence. Four patients had preoperatory chemotherapy (CHT), 27% of the patients had preoperatory radiotherapy (RT). Three patients are active smokers and one patient had previous lipofillings. The average amount of fat grafted is 119 cc, median 115 cc. 64% of the patients has currently hormonotherapy. COVID-19 impacted post-operatory control times: the mean time was of 5 months, median 5 months. The gain in breast soft tissue thickness after the lipofilling is statistically significative: 0.062 (p= 0.00001). The study did not find statistically significative association between MD adherence and gain in breast soft tissue thickness. Moreover, no statistically significative association among gain in breast soft tissue thickness and previous lipofillings, comorbidities, previous RT/CHT, smoke habit, hormonotherapy was found. As a result of COVID-19 pandemic, the study has collected really few patients to satisfy the sample size target, and this has impacted the possibility to find statistically significative association. However, the study permits to observe that there is a good adherence to the MD and several patients reported changes in dietary habits after the diagnosis of cancer, with active effort to do their best and modifications of their lifestyle. The study will continue to investigate the association among fat graft survival rate and influence of the diet and identify the characteristics of patients with low lipofilling resorption rate

The Role of Math Anxiety and Disciplinary Self-Efficacy in Math Learning for Children with Special Educational Needs

Students with special educational needs exhibit difficulties in school achievements, especially in mathematics. However, few studies have simultaneously assessed the affective-motivational profiles and mathematical performance of these students. This study aims to examine closely these two aspects. The results show that students with special needs exhibit lower mathematical self-efficacy (both academic and regulatory) compared to typically developing peers, as well as lower mathematical performance. The results underline the need to propose educational interventions aimed at promoting the self-efficacy of special education needs students.In letteratura è ben noto che gli studenti con Bisogni Educativi Speciale (BES) esibiscono difficoltà in ambito educativo, specialmente in matematica. Tuttavia, ad oggi, pochi studi hanno valutato simultaneamente i profili affettivo-motivazionali e la prestazione matematica degli studenti BES rispetto agli studenti a sviluppo tipico. L’obiettivo dello studio era quello di esaminare le differenze nei profili affettivo-motivazionali e di prestazione matematica tra studenti con BES e a sviluppo tipico. I risultati mostrano che gli studenti con BES sembrano presentare minore self-efficacy matematica (sia accademica che regolatoria) rispetto ai pari a sviluppo tipico, nonché prestazioni matematiche inferiori. I risultati sottolineano la necessità di proporre interventi educativi mirati a promuovere la self-efficacy degli studenti BES

EMI and Beyond

This collection presents the state of the art on English-medium instruction (EMI) / Integrating content and language (ICL) in Italian higher education, drawing attention to different critical aspects of the teaching/learning experience and highlighting the perspectives of various educational stakeholders regarding the effectiveness of tertiary study in a foreign language. The chapters draw on a range of methodologies, from multimodal participant observation, to action research, to video-stimulated recall (VSR), to questionnaires and interviews, in examining language policies and practices across various educational settings. Overall, the volume suggests that internationalisation succeeds best when the form of lessons (language) and the content of lessons (disciplinary concepts) are constructively aligned in curriculum planning and delivery. This integration process requires the strategic support of educators to guarantee the quality of learning in multilingual education.

EMI and Beyond

This collection presents the state of the art on English-medium instruction (EMI) / Integrating content and language (ICL) in Italian higher education, drawing attention to different critical aspects of the teaching/learning experience and highlighting the perspectives of various educational stakeholders regarding the effectiveness of tertiary study in a foreign language. The chapters draw on a range of methodologies, from multimodal participant observation, to action research, to video-stimulated recall (VSR), to questionnaires and interviews, in examining language policies and practices across various educational settings. Overall, the volume suggests that internationalisation succeeds best when the form of lessons (language) and the content of lessons (disciplinary concepts) are constructively aligned in curriculum planning and delivery. This integration process requires the strategic support of educators to guarantee the quality of learning in multilingual education.

surgical and oncological outcomes of free dermal fat graft for breast reconstruction after breast conserving surgery

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Convergent Sets of Data from In Vivo and In Vitro Methods Point to an Active Role of Hsp60 in Chronic Obstructive Pulmonary Disease Pathogenesis

BACKGROUND: It is increasingly clear that some heat shock proteins (Hsps) play a role in inflammation. Here, we report results showing participation of Hsp60 in the pathogenesis of chronic obstructive pulmonary diseases (COPD), as indicated by data from both in vivo and in vitro analyses. METHODS AND RESULTS: Bronchial biopsies from patients with stable COPD, smoker controls with normal lung function, and non-smoker controls were studied. We quantified by immunohistochemistry levels of Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and HSF-1, along with levels of inflammatory markers. Hsp10, Hsp40, and Hsp60 were increased during progression of disease. We found also a positive correlation between the number of neutrophils and Hsp60 levels. Double-immunostaining showed that Hsp60-positive neutrophils were significantly increased in COPD patients. We then investigated in vitro the effect on Hsp60 expression in bronchial epithelial cells (16HBE) caused by oxidative stress, a hallmark of COPD mucosa, which we induced with H\u2082O\u2082. This stressor determined increased levels of Hsp60 through a gene up-regulation mechanism involving NFkB-p65. Release of Hsp60 in the extracellular medium by the bronchial epithelial cells was also increased after H\u2082O\u2082 treatment in the absence of cell death. CONCLUSIONS: This is the first report clearly pointing to participation of Hsps, particularly Hsp60, in COPD pathogenesis. Hsp60 induction by NFkB-p65 and its release by epithelial cells after oxidative stress can have a role in maintaining inflammation, e.g., by stimulating neutrophils activity. The data open new scenarios that might help in designing efficacious anti-inflammatory therapies centered on Hsp60 and applicable to COP

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries