In-vitro characterization of promiscuous biotinylation by biotin ligase

Protein-protein interaction networks represent useful system to better understand the organisation of the proteome and, moreover, provide a support for identifying protein functions. Thus, identification of these interactions would help to reach an improved comprehension of the entire cell. Most popular methods for detecting protein interactions are either in vitro assays or are based on heterologous systems, both of which do not represent true cellular environment. Thus, a lot of research is directed towards development of new methods that detect interactions under in vivo conditions, so as to preserve the spatio-temporal integrity of these interactions. One of such methods involve the biotin protein ligase (BirA) from Escherichia coli, which is responsible for catalysing transfer of a small molecule, biotin, to a specific lysine residue (within a specific conserved sequence) of acceptor proteins. This reaction is highly specific, with only one protein in E.coli capable of being biotinylated in vivo. A mutation in BirA (R118G; BirA*), however, reduces this specificity such that the intermediate of reaction (biotinoyl-AMP) is released from the active site of the protein and can biotinylate any protein that it encounters. Based on this principle, if we fuse a protein with BirA*, the proteins that come in proximity of the fusion construct should be biotinylated and could further be identified by a streptavidin pull-down. In order to develop this technique in plants, the enzymatic properties of BirA* need to be characterized. The current project was focused on purifying BirA* (codon optimized for expression in Arabidopsis thaliana) from bacteria and assessing its enzymatic properties under different physiological conditions. To this end, GST-6X Histidine fused BirA* was purified using inducible bacterial overexpression system and checked for promiscuous biotinylation of non-native target, i,e., free GST protein. Further, the substrate concentration, temperature, reaction time and pH conditions for the progress of reaction were optimised. The results generated by the current study verify that BirA* can catalyse promiscuous biotinylation, therefore it would be worthwhile to extend this system in plants for detection of protein-protein interactions. Moreover, if BirA* is selectively overexpressed in a specific cellular compartment, it has potential to biotinylate most of the proteins within this compartment. As different cellular compartments differ in pH, the present study also helped to short-list the compartments whose proteome could be deciphered using BirA*. To summarize, the present study verified that BirA* based proximity-biotinylation technique could be used to develop an in vivo assay for detecting protein interactions and to decipher organellar proteome. Also, we were able to optimize the reaction conditions at which the enzyme is most active

La Rete Lucca BioDinamica: analisi dei vincoli e opportunità di sviluppo secondo l'approccio di network

Sul territorio dei Comuni di Lucca e Capannori si sono avviate interessanti e importanti iniziative per favorire, nell’ambito di una strategia di sviluppo rurale integrato, il rafforzamento di metodi di produzione sostenibili e delle filiera corta. In particolare, con i due progetti “Nutrire la Lucchesìa” e “Orti di Demetra” sono state evidenziate le potenzialità (e, al tempo stesso, i fabbisogni) delle imprese agricolo locale. Questo lavoro si è concentrato sull’analisi e lo sviluppo del progetto “Orti di Demetra”, che si è posto l’obiettivo di sviluppare e diffondere l'orticoltura biologica e biodinamica nella Piana di Lucca, attraverso lo svolgimento di interviste in profondità ad alcuni degli attori coinvolti in questa iniziativa. In particolare, in una prima fase, sono state intervistate sei aziende che praticano agricoltura biologica e biodinamica per la produzione di ortaggi. Le informazioni raccolte dalle interviste e durante situazioni di osservazione partecipata sono state vagliate con analisi SWOT e analisi dei fabbisogni al fine di evidenziare le necessità concrete del settore agricolo della Piana di Lucca. Inoltre, cogliendo l'opportunità di poter osservare da vicino gli incontri per la costituzione della Rete Lucca BioDinamica (LBD), si è potuto indagare l'adeguatezza del nuovo strumento per favorire l’integrazione delle aziende agricole per rispondere alle specifiche esigenze degli agricoltori e del territorio: il contratto di rete. Anche la Rete LBD è stata sottoposta ad analisi SWOT ed, inoltre, tramite l’uso del software NodeXL, si sono valutate le specifiche caratteristiche del network. Infine, con il metodo di analisi qualitativa proposto da Van der Ploeg (Van der Ploeg, 2008) per le reti rurali, si è cercato di capire il ruolo potenziale della Rete LBD per lo sviluppo rurale nella Piana di Lucca. In conclusione si è appurato che il contratto di rete è uno strumento giuridico interessante e innovativo che può risolvere molti dei problemi che le piccole aziende agricole devono affrontare (commercializzazione, impiego di manodopera, ecc.) e, in tal senso, la Rete LBD oltre a confermarne la validità, può rappresentare un valido esempio per altre iniziative simili già presenti sul territorio a livello di network informali

Systemic Lupus Erythematosus and Thyroid Autoimmunity

Most of the studies present in the literature show a high prevalence, and incidence, of new cases of hypothyroidism and autoimmune thyroiditis (AT) in systemic lupus erythematosus (SLE) patients, overall in female gender. A limited number of cases of Graves' disease have been also reported in SLE patients, in agreement with the higher prevalence of thyroid autoimmunity. It has been also demonstrated that a Th1 predominance is associated with AT in SLE patients. Furthermore, a higher prevalence of papillary thyroid cancer has been recently reported in SLE, in particular in the presence of thyroid autoimmunity. However, studies in larger number of SLE patients are needed to confirm findings about thyroid cancer. On the whole, data from literature strongly suggest that female SLE patients, with a high risk (a normal but at the higher limit thyroid-stimulating hormone value, positive antithyroid peroxidase antibodies, a hypoechoic pattern, and small thyroid), should undergo periodic thyroid function follow-up, and appropriate treatments when needed. A careful thyroid monitoring would be opportune during the follow-up of these patients

Surgical management and oncological follow-up of cutaneous squamous cell carcinomas arising in epidermolysis bullosa patients

Background Hereditary epidermolysis bullosa (EB) is a rare genodermatosis characterized by skin fragility and blistering of the skin and mucous membranes in reaction to minimal traumas. The development of cutaneous squamous cell carcinomas (cSCCs) is one of the most common medical complications in junctional and dystrophic forms of the disease. Complete surgical excision of cutaneous tumors represents the gold standard of treatment. However, not only recognition of cSCCs can be challenging in the affected skin but also wound closure after surgical excision poses a great therapeutic challenge in EB patients. The aim of our study was to analyze the postoperative outcomes of such patients in order to have a better knowledge of the main critical issues in their surgical management and oncological follow-up. Methods We retrospectively identified a cohort of five EB patients treated at Modena University Hospital. Collected data included patient age and sex, date of cSCC diagnosis, relapses/recurrences, site of the neoplasm, number of surgical interventions, use of dermal substitutes, and postoperative infections. Results A total of 26 cSCCs were detected in our cohort. Forty-one surgical interventions were necessary to achieve excision of cSCCs with clear margins, varying from 1 to 4 surgical sessions per cSCC. Dermal substitutes were used in most cases but carried a higher infectious risk. Conclusions EB patients tend to develop numerous cSCCs that often relapse even after complete excision with clear margins. These results stress the importance of early cSCC diagnosis and strict postsurgical follow-up

Una procedura sistematica per osservare la variabilit\ue0 nell'interlingua

In questo lavoro viene presentato un protocollo di raccolta dati messo a punto per ottenere campioni significativi di parlato utili a indagare fenomeni di variazione in italiano L2

Evaluation of the potential of Near Infrared Hyperspectral Imaging for monitoring the invasive brown marmorated stink bug

The brown marmorated stink bug (BMSB), Halyomorpha halys, is an invasive insect pest of global importance that damages several crops, compromising agri-food production. Field monitoring procedures are fundamental to perform risk assessment operations, in order to promptly face crop infestations and avoid economical losses. To improve pest management, spectral cameras mounted on Unmanned Aerial Vehicles (UAVs) and other Internet of Things (IoT) devices, such as smart traps or unmanned ground vehicles, could be used as an innovative technology allowing fast, efficient and real-time monitoring of insect infestations. The present study consists in a preliminary evaluation at the laboratory level of Near Infrared Hyperspectral Imaging (NIR-HSI) as a possible technology to detect BMSB specimens on different vegetal backgrounds, overcoming the problem of BMSB mimicry. Hyperspectral images of BMSB were acquired in the 980-1660 nm range, considering different vegetal backgrounds selected to mimic a real field application scene. Classification models were obtained following two different chemometric approaches. The first approach was focused on modelling spectral information and selecting relevant spectral regions for discrimination by means of sparse-based variable selection coupled with Soft Partial Least Squares Discriminant Analysis (s-Soft PLS-DA) classification algorithm. The second approach was based on modelling spatial and spectral features contained in the hyperspectral images using Convolutional Neural Networks (CNN). Finally, to further improve BMSB detection ability, the two strategies were merged, considering only the spectral regions selected by s-Soft PLS-DA for CNN modelling.Comment: Accepted manuscrip

Circulating CXCL10 is increased in non-segmental vitiligo, in presence or absence of autoimmune thyroiditis

Recently the importance of CXCL10 in the pathogenesis of non-segmental vitiligo (NSV) and autoimmune thyroid disorders (AITD) has been shown. No data are present about chemokines CXCL10 (Th1 prototype) and CCL2 (Th2 prototype) circulating levels in NSV patients with/without thyroiditis (AT). Serum CXCL10 and CCL2 have been measured in 50 consecutive NSV patients, in 40 consecutive patients with NSV and AT (NSV+AT), in 50 sex- and age-matched controls without AT (control 1) and in 40 sex- and age-matched patients with AT without NSV (control 2). Serum CXCL10 levels were significantly higher in control 2, than in control 1 (P=0.001; ANOVA). NSV patients have serum CXCL10 levels significantly higher than control 1, or control 2 (P=0.001). NSV+AT patients have serum CXCL10 levels higher than control 1, or 2 (P<0.001), and than NSV (P=0.01). In conclusion, we first demonstrate high serum CXCL10 in NSV patients, overall in presence of AT and hypothyroidism, suggesting the importance of a common Th1 immune response in their immune-pathogenesis. To evaluate if serum CXCL10 might be used as a clinical marker of NSV and/or AT further studies are needed

Surgical complications following sentinel lymph node biopsy guided by γ‐probe and methylene blue in 113 tumour‐bearing dogs

Sentinel lymph node biopsy (SLNB) is an accepted veterinary surgical procedure given the impact of early detection of nodal metastases on staging of several canine malignancies. This study aims at reporting the incidence and risk factors for surgical complications of SLNB in tumour-bearing dogs. A total of 113 client-owned dogs that underwent tumour excision and SLNB guided by γ-probing and blue dye were retrospectively enrolled. Recorded variables included: signalment, location and number of extirpated lymphocenters and nodes, time for SLNB, histopathological status of excised nodes. Incidence of SLNB complications was calculated. They were classified as minor and major based on severity and required treatment, and as short-term (0–30 days) and long-term (31–90 days). Univariate analysis with generalized linear model with binomial error estimated the association between variables and incidence of SLNB complications. Significance was set at 5%. Median overall time for SLNB was 25 min. Surgeons excised one node in 38% of dogs and multiple nodes in 62% of cases, belonging to one (62%) or multiple (38%) lymphocenters. Metastases were detected in 45% of nodes. No intraoperative complications occurred. The overall incidence of postoperative complications of SLNB was 21,24%, the majority of which (91.67%) were minor. Only increasing dogs' weight was associated with an increased incidence of SLNB complications (p = .00976). Sentinel lymphadenectomy was associated with a relatively low incidence of complications, most of which were self-limiting. The low morbidity and previously reported impact on staging of SLNB justify its implementation to collect data for prognostic studies