Active human full-length CDKL5 produced in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Background: A significant fraction of the human proteome is still inaccessible to in vitro studies since the recombinant production of several proteins failed in conventional cell factories. Eukaryotic protein kinases are difficult-to-express in heterologous hosts due to folding issues both related to their catalytic and regulatory domains. Human CDKL5 belongs to this category. It is a serine/threonine protein kinase whose mutations are involved in CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental pathology still lacking a therapeutic intervention. The lack of successful CDKL5 manufacture hampered the exploitation of the otherwise highly promising enzyme replacement therapy. As almost two-thirds of the enzyme sequence is predicted to be intrinsically disordered, the recombinant product is either subjected to a massive proteolytic attack by host-encoded proteases or tends to form aggregates. Therefore, the use of an unconventional expression system can constitute a valid alternative to solve these issues. Results: Using a multiparametric approach we managed to optimize the transcription of the CDKL5 gene and the synthesis of the recombinant protein in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 applying a bicistronic expression strategy, whose generalization for recombinant expression in the cold has been here confirmed with the use of a fluorescent reporter. The recombinant protein largely accumulated as a full-length product in the soluble cell lysate. We also demonstrated for the first time that full-length CDKL5 produced in Antarctic bacteria is catalytically active by using two independent assays, making feasible its recovery in native conditions from bacterial lysates as an active product, a result unmet in other bacteria so far. Finally, the setup of an in cellulo kinase assay allowed us to measure the impact of several CDD missense mutations on the kinase activity, providing new information towards a better understanding of CDD pathophysiology. Conclusions: Collectively, our data indicate that P. haloplanktis TAC125 can be a valuable platform for both the preparation of soluble active human CDKL5 and the study of structural–functional relationships in wild type and mutant CDKL5 forms. Furthermore, this paper further confirms the more general potentialities of exploitation of Antarctic bacteria to produce “intractable” proteins, especially those containing large intrinsically disordered regions

Monitoring and modeling the invasion of the fast spreading alien Senecio inaequidens DC. in an alpine region

We modeled the distribution of the South African alien Senecio inaequidens DC. in the Aosta Valley, Western Italian Alps, using data extracted from the Regional floristic database and from an intensive field survey carried out in years 2009-2010. The aims of the work were (1) to evaluate whether the species is in the introduction, colonization, or establishment stage of invasion, (2) to detect the environmental factors that drive the invasion process, and (3) to highlight the potential range of distribution of the alien species. The modeling framework was a stepwise generalized linear model (GLM), using gridded presence/absence data and environmental predictors such as topography, climate, land use, and anthropogenic and natural disturbances. GLM were fit both with and without an additional independent variable to take into account current dispersal limitations. S. inaequidens displayed a very fast spread in the Aosta Valley in the years 1990-2010. The species was positively associated with roads and rivers, southern slopes, and negatively with elevation. However, it was found at an elevation of 1600 m, showing the ability to reach higher elevations than those observed for other invasive alien species, and confirming to be pre-adapted to mountain conditions. The difference between the species distribution models, with and without dispersal constraints, suggested that the availability of seed sources still limits the potential distribution of the species, rather than the environmental variables, and that the realized regional niche differs to a great extent from the equilibrium niche. When limitations to the seed source cease (i.e., in the establishment stage), the species will likely invade large areas that are currently characterized by pastures and grasslands with native species of high agricultural importance. The invasion of S. inaequidens should therefore be considered a serious threat, due to its potential to invade mountain regions, and in particular to colonize habitats used for grazing and forage, thus leading to a high risk for cattle and human health. We discuss the relevance of the results both concerning communication with the public and to support local eradication and control activities. The inclusion of S. inaequidens in the "black list" of the regional law for the conservation of alpine flora (L.R. 45/2009) will help to transfer the information and support invasion control, in particular at medium elevations

Indoor/Outdoor Air Quality Assessment at School near the Steel Plant in Taranto (Italy)

This study aims to investigate the air quality in primary school placed in district of Taranto (south of Italy), an area of high environmental risk because of closeness between large industrial complex and urban settlement. The chemical characterization of PM2.5 was performed to identify origin of pollutants detected inside school and the comparison between indoor and outdoor levels of PAHs and metals allowed evaluating intrusion of outdoor pollutants or the existence of specific indoor sources. The results showed that the indoor and outdoor levels of PM2.5, BaP, Cd, Ni, As, and Pb never exceeded the target values issued by World Health Organization (WHO). Nevertheless, high metals and PAHs concentrations were detected especially when school were downwind to the steel plant. TheI/Oratio showed the impact of outdoor pollutants, especially of industrial markers as Fe, Mn, Zn, and Pb, on indoor air quality. This result was confirmed by values of diagnostic ratio as B(a)P/B(g)P, IP/(IP + BgP), BaP/Chry, and BaP/(BaP + Chry), which showed range characteristics of coke and coal combustion. However, Ni and As showedI/Oratio of 2.5 and 1.4, respectively, suggesting the presence of indoor sources

Development of high-copy number plasmids in Pseudoalteromonas haloplanktis TAC125

Abstract: The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is considered an interesting alternative host for the recombinant protein production, that can be explored when the conventional bacterial expression systems fail. Indeed, the manufacture of all the difficult-to-express proteins produced so far in this bacterial platform gave back soluble and active products. Despite these promising results, the low yield of recombinant protein production achieved is hampering the wider and industrial exploitation of this psychrophilic cell factory. All the expression plasmids developed so far in PhTAC125 are based on the origin of replication of the endogenous pMtBL plasmid and are maintained at a very low copy number. In this work, we set up an experimental strategy to select mutated OriR sequences endowed with the ability to establish recombinant plasmids at higher multiplicity per cell. The solution to this major production bottleneck was achieved by the construction of a library of psychrophilic vectors, each containing a randomly mutated version of pMtBL OriR, and its screening by fluorescence-activated cell sorting (FACS). The selected clones allowed the identification of mutated OriR sequences effective in enhancing the plasmid copy number of approximately two orders of magnitude, and the production of the recombinant green fluorescent protein was increased up to twenty times approximately. Moreover, the molecular characterization of the different mutant OriR sequences allowed us to suggest some preliminary clues on the pMtBL replication mechanism that deserve to be further investigated in the future. Key points: • Setup of an electroporation procedure for Pseudoalteromonas haloplanktis TAC125. • Two order of magnitude improvement of OriR-derived psychrophilic expression systems. • Almost twenty times enhancement in Green fluorescent protein production

An investigation of bedside laparoscopy in the ICU for cases of non-occlusive mesenteric ischemia

Background: Acute mesenteric ischemia is a rare affection with high related mortality. NOMI presents the most important diagnostic problems and is related with the higher risk of white laparotomy. This study wants to give a contribution for the validation of laparoscopic approach in case of NOMI. Methods: Thirty-two consecutive patients were admitted in last 10 years in ICU of Paolo Giaccone University Hospital of Palermo for AMI. Diagnosis was obtained by multislice CT and selective angiography was done if clinical conditions were permissive. If necrosis was already present or suspected, surgical approach was done. Endovascular or surgical embolectomy was performed when necessary. Twenty NOMI patients underwent medical treatment performing laparoscopy 24 h later to verify the evolution of AMI. A three-port technique was used. In all patients we performed a bed side procedure 48-72 h later in both non-resected and resected group. Results: In 14 up 20 case of NOMI the disease was extended throughout the splanchnic district, in 6 patients it involved the ileum and the colon; after a first look, only 6 patients underwent resection. One patient died 35 h after diagnosis of NOMI. The second look, 48 h later, demonstrated 4 infarction recurrences in the group of resected patients and onset signs of necrosis in 5 patients of non-resected group. A total of 15 resections were performed on 11 patients. Mortality rate was 6/20-30% but it was much higher in resected group (5/11-45,5%). Non-therapeutic laparotomy was avoided in 9/20 patients and in this group mortality rate was 1/9-11%. No morbidity was recorded related to laparoscopic procedure. Conclusions: Laparoscopy could be a feasible and safety surgical approach for management of patient with NOMI. Our retrospective study demonstrates that laparoscopy don't increase morbidity, reduce mortality avoiding non-therapeutic laparotomy

Is it really useful the harmonic scalpel in axillary dissection for locally advanced breast cancer? A case series

Background. The seroma is one of the most common complications in the axillary lymph nodal dissection (different surgical approaches have been tried to reduce the seroma incidence). In our study we evaluate the outcome of patients using or not the ultrasonic scalpel (Harmonic scalpel) according to a standardized surgical technique. Patients and Methods. From January 2011 to December 2015 120 patients underwent axillary dissection for breast cancer. Patients were divided in two groups: patients belonging to the first group underwent Harmonic scalpel dissection and patients belonging to the second group underwent classical dissection. Each group consisted of 60 patients. Quadrantectomy (QUAD) was performed in 54 patients, 66 women underwent mastectomy. In all patients axillary dissection included the I, II and III level. We compared two groups in terms of: time of surgery, hematoma, drainage volume, days of sealing drainage, seroma formation, number of post-seroma aspirations, upper limb lymphedema, wound infections, post-operative pain. Results. Statistically significant results were obtained in terms of the total volume of the breast and axillary drainage in the two techniques. There were no significant differences in the two samples in terms of operative time incidence of seroma, post-operative hematoma, wound infection, and lymphedema of the upper limb. Conclusion. The small number of cases did not allow us to reach definitive conclusions. The use of Harmonic scalpel seems to show smaller incidence of seroma and reduction of the amount of both breast and axillary drainages. Further studies are needed to define the real advantage in terms of cost benefit of using these devices in the axillary surgery

Minimal invasive surgery in craniostenosis

En el presente trabajo se describe la experiencia en craneoestenosis con cirugía mínimamente invasiva, evaluando el diseño y eficacia de un nuevo craneotomo en cadáveres así como su aplicación clínica en un caso de sinostósis sagital con instrumentación endoscópica. Este procedimiento es sin duda un gran recurso en el tratamiento de las craneoestenosis brindando los beneficios de la cirugía mínimamente invasiva, eliminando la necesidad de grandes incisiones, disminuyendo el sangrado quirúrgico, reduciendo estancia hospitalaria y disminuyendo la morbilidad operatoria In this paper, we describe the experience with the use of endoscopic craniofacial procedures, evaluating the design and the efficacy of a new craniotome in cadavers and his clinical application in a case of sagittal synostosis for an endoscopic assisted cranioplasty. This procedure is a great option in the treatment of craniosynostosis, giving the benefits of minimal invasive surgery and eliminating the needing of big incisions, long hospital stay and reducing the postoperative morbidit