Optimization of a Biotechnological Process for Production and Purification of Two Recombinant Proteins: Col G and Col H

Different strategies can be used for increasing production of heterologous recombinant proteins in Escherichia coli. Protein size is often critical for obtaining the best quantity/quality ratio of recombinant protein expression. This study focuses on two recombinant proteins; Class I and class II Collagenases, namely Col G and Col H. Their size is about 150 kDa each. We have developed a method to obtain high levels of cell growth and intracellular expression of each Collagenases in recombinant E. coli BL21(DE3). Batch and Fed-batch fermentation procedures have been performed. Results show that Fed-batch technique was most effective in obtaining the highest cell density for each recombinant bacteria; 28 g/L. We also investigated how to optimize recombinant protein expression; best results were obtained when “multiple shot IPTG induction system” was chosen instead of canonical single shot. By applying a purification protocol based on the use of tangential flow filtration and affinity chromatography we were able to obtain the highest quantity of purified protein: about 13,2 g for Col G and about 12,6 for Col H fermentations. Moreover, by using a stainless steel cooling coil system, we have investigated the effects of low controlled temperature (7°C) during the whole purification process. This system, allowed us to improve the final enzymatic activity of both Collagenases, obtaining 2 fold increase values respect processes performed at room temperature, measured with Pz Grassmann assay. This study shows that, even when the size of a recombinant protein is limiting, is possible to apply a defined Fed-batch protocol to obtain a very high protein production. Moreover these results can be used as a scale up starting step for industrial production and purification of these kind of recombinant enzymes.Different strategies can be used for increasing production of heterologous recombinant proteins in Escherichia coli. Protein size is often critical for obtaining the best quantity/quality ratio of recombinant protein expression. This study focuses on two recombinant proteins; Class I and class II Collagenases, namely Col G and Col H. Their size is about 150 kDa each. We have developed a method to obtain high levels of cell growth and intracellular expression of each Collagenases in recombinant E. coli BL21(DE3). Batch and Fed-batch fermentation procedures have been performed. Results show that Fed-batch technique was most effective in obtaining the highest cell density for each recombinant bacteria; 28 g/L. We also investigated how to optimize recombinant protein expression; best results were obtained when “multiple shot IPTG induction system” was chosen instead of canonical single shot. By applying a purification protocol based on the use of tangential flow filtration and affinity chromatography we were able to obtain the highest quantity of purified protein: about 13,2 g for Col G and about 12,6 for Col H fermentations. Moreover, by using a stainless steel cooling coil system, we have investigated the effects of low controlled temperature (7°C) during the whole purification process. This system, allowed us to improve the final enzymatic activity of both Collagenases, obtaining 2 fold increase values respect processes performed at room temperature, measured with Pz Grassmann assay. This study shows that, even when the size of a recombinant protein is limiting, is possible to apply a defined Fed-batch protocol to obtain a very high protein production. Moreover these results can be used as a scale up starting step for industrial production and purification of these kind of recombinant enzymes

Autonomic cardiovascular control and cardiac arrhythmia in two pregnant women with hypertrophic cardiomyopathy: Insights from ICD monitoring

In women with hypertrophic cardiomyopathy (HCM), pregnancy prompts major changes in hemodynamic and cardiac autonomic function that may precipitate heart failure (HF) or increase the risk of cardiac arrhythmia.We report the clinical follow-up of two patients with non-obstructive HCM implanted with a cardioverter defibrillator (ICD) allowing for continuous analysis of heart rate (HR), heart rate variability (HRV) and cardiac arrhythmia throughout the entire course of pregnancy.Both patients experienced increased HR and decreased HRV from the early stages of pregnancy, which persisted until delivery. Premature ventricular contractions (PVCs) and runs of non-sustained ventricular tachycardia (NSVT) reached a peak in the second and third trimesters, concurrent with sympathetic hyperactivity. In one patient with baseline NYHA class II HF symptoms, increased PVCs and NSVT were consistent with the deterioration of HF, supporting the decision to bring the delivery forward. While both patients experienced a persistent increase in sympathetic tone and ventricular ectopic activity, no life-threatening arrhythmias were documented.During pregnancy, patients with hypertrophic cardiomyopathy develop progressive neuroautonomic imbalance, prompting an increase in non-sustained ventricular arrhythmia. This enhanced arrhythmia burden warrants close follow-up and rhythm assessment during the third trimester, especially in women who have heart failure symptoms before pregnancy. Implantable cardioverter defibrillators provide a continuous analysis of heart rate variability and arrhythmia burden that supports therapeutic decision-making during follow-up. Resumo: Em mulheres com miocardiopatia hipertrófica, a gravidez aumenta as variações hemodinâmicas e as alterações da função autonómica cardíaca que podem provocar insuficiência cardíaca ou aumentar o risco de arritmia. Reportamos o acompanhamento clínico de duas pacientes com miocardiopatia hipertrófica não obstrutiva, ambas implantadas com cardioversor-desfibrilhador (CID). A monitoração com CID permite a análise contínua da frequência cardíaca, da variabilidade da frequência cardíaca (VFC) e da arritmia durante toda a gravidez. As duas pacientes manifestaram aumentos da FC e diminuições da VFC desde o início da gravidez até ao parto. Observou-se um pico de frequência de extrassístoles ventriculares (EV) e de taquicardias ventriculares não sustentadas (TVNS) no segundo e terceiro trimestres da gestação, em correspondência da hiperatividade simpática. Numa das pacientes com classe funcional NYHA II, antes da gravidez, o aumento de EV e de TVNS contemporaneamente ao agravamento da insuficiência cardíaca levou à decisão de antecipar o parto. As duas pacientes demonstraram um aumento persistente da atividade simpática e da atividade ectópica ventricular, não existiram casos de arritmias ventriculares malignas. Durante a gravidez as pacientes com miocardiopatia hipertrófica desenvolvem um progressivo desequilíbrio autonómico que causa um aumento das arritmias ventriculares não sustentadas. O aumento do risco arrítmico necessita de um constante e frequente controle clínico e do ritmo cardíaco durante o terceiro trimestre, especialmente em mulheres com sintomas de insuficiência cardíaca antes da gravidez. O cardioversor-desfibrilhador implantável fornece uma análise continua da variabilidade da frequência cardíaca e das arritmias que podem apoiar as decisões terapêuticas durante a gravidez. Keywords: Hypertrophic cardiomyopathy, Pregnancy, Implantable cardioverter defibrillator, Heart rate variability, Ventricular arrhythmia, Palavras-chave: Miocardiopatia hipertrófica, Gravidez, Cardioversor-desfibrilhador implantável, Variabilidade da frequência cardíaca, Arritmia ventricula

Hydrogen passivation of boron acceptors in as-grown boron-doped CVD diamond epilayers

A homoepitaxial boron-doped diamond single layer is investigated by means of Fourier transformed infrared spectroscopy (FTIR) and cathodoluminescence (CL). Both techniques are shown to be complementary. μ-FTIR mapping allows to determine the location of active boron while CL allows discernability between passivation and compensation. Hydrogen incorporation during chemical vapour deposition (CVD) growth is revealed to passivate boron acceptors. The obtained results highlight that plasma etching can induce a dissociation of B–H centres.4 page

Randomised Italian Sonography for Occiput POSition Trial Ante Vacuum (R.I.S.POS.T.A.)

Objective: To assess whether sonographic diagnosis of fetal head position before instrumental vaginal delivery can reduce the risk of failed vacuum extraction and improve delivery outcome. Methods: Randomised Italian Sonography for occiput POSition Trial Ante vacuum (R.I.S.POS.T.A.) is a randomized controlled trial of term (37 + 0 to 41 + 6 weeks' gestation) singleton pregnancies with cephalic presentation requiring instrumental delivery by vacuum extraction, which was conducted between April 2014 and June 2017 and involved 13 Italian maternity hospitals. Patients were randomized to assessment of fetal head position before attempted instrumental delivery by either vaginal examination (VE) alone or VE plus transabdominal sonography (TAS). Primary outcome was incidence of emergency Cesarean section due to failed vacuum extraction. A sample size of 653 women per group was planned to compare the primary outcome between the two groups. The sample size estimation was based on the hypothesis that the risk of failed vacuum delivery in the VE group would be 5% and that ultrasound assessment of fetal position prior to vacuum extraction would decrease this risk to 2%. Results: On interim analysis, the trial was stopped for futility. During this period, 222 women were randomized and 221 were included in the final data analysis, of whom 132 (59.7%) were randomized to evaluation of fetal head position by VE only and 89 (40.3%) to assessment by VE plus TAS prior to vacuum extraction. No significant differences were observed between the two groups with respect to incidence of emergency Cesarean section due to failed instrumental delivery and other maternal and fetal outcomes. Women randomized to assessment by VE plus TAS showed higher incidence of non-occiput anterior position of the fetal head at randomization and lower incidence of incorrect diagnosis of occiput position compared with women undergoing assessment by VE alone. A higher rate of episiotomy was noted in the women undergoing both VE and TAS compared with those in the VE-only group. Conclusions: Our prematurely discontinued randomized controlled trial did not demonstrate any benefit in terms of reduced risk of failed instrumental delivery or maternal and fetal morbidity in women undergoing sonographic assessment of fetal head position prior to vacuum extraction. Copyright \ua9 2018 ISUOG. Published by John Wiley & Sons Ltd

Human exploration of cis-lunar space via assets tele-operated from EML2 (HECATE)

This paper presents the preliminary design of the international space mission HECATE (Human Exploration of Cis-lunar space via Assets Tele-operated from EML-2), aimed at exploring the far side of the Moon via tele-robotic activities during the 2020s. The exploration is realized by astronauts from HOPE (Human Orbiting Protected Environment), a space habitat in a halo orbit around the Earth-Moon Lagrange Point 2, a critical staging location for future robotic and human deep space missions. Inside the habitat, astronauts have access to tele-robotic hardware and instruments, used to tele-operate rovers and scientific equipment on the surface of the Moon. Plans to resupply and maintain HOPE for future missions, using a solar electric tug, are given. Ultimately, HOPE represents an energetically favorable intermediate locations for missions to Mars, Near-Earth Asteroids, and beyond

Non-neural phenotype of spinal and bulbar muscular atrophy: Results from a large cohort of Italian patients

Objective: To carry out a deep characterisation of the main androgen-responsive tissues involved in spinal and bulbar muscular atrophy (SBMA). Methods: 73 consecutive Italian patients underwent a full clinical protocol including biochemical and hormonal analyses, genitourinary examination, bone metabolism and densitometry, cardiological evaluation and muscle pathology. Results: Creatine kinase levels were slightly to markedly elevated in almost all cases (68 of the 73; 94%). 30 (41%) patients had fasting glucose above the reference limit, and many patients had total cholesterol (40; 54.7%), low-density lipoproteins cholesterol (29; 39.7%) and triglyceride (35; 48%) levels above the recommended values. Although testosterone, luteinising hormone and follicle-stimulating hormone values were generally normal, in one-third of cases we calculated an increased Androgen Sensitivity Index reflecting the presence of androgen resistance in these patients. According to the International Prostate Symptom Score (IPSS), 7/70 (10%) patients reported severe lower urinal tract symptoms (IPSS score >19), and 21/73 (30%) patients were moderately symptomatic (IPSS score from 8 to 19). In addition, 3 patients were carriers of an indwelling bladder catheter. Videourodynamic evaluation indicated that 4 of the 7 patients reporting severe urinary symptoms had an overt prostate-unrelated bladder outlet obstruction. Dual-energy X-ray absorptiometry scan data were consistent with low bone mass in 25/61 (41%) patients. Low bone mass was more frequent at the femoral than at the lumbar level. Skeletal muscle biopsy was carried out in 20 patients and myogenic changes in addition to the neurogenic atrophy were mostly observed. Conclusions: Our study provides evidence of a wide non-neural clinical phenotype in SBMA, suggesting the need for comprehensive multidisciplinary protocols for these patients. \ua9 2016 Published by the BMJ Publishing Group Limited

Targeting prolyl-isomerase Pin1 prevents mitochondrial oxidative stress and vascular dysfunction: insights in patients with diabetes

The present study demonstrates that Pin1 is a common activator of key pathways involved in diabetic vascular disease in different experimental settings including primary human endothelial cells, knockout mice, and diabetic patients. Gene silencing and genetic disruption of Pin1 prevent hyperglycaemia-induced mitochondrial oxidative stress, endothelial dysfunction, and vascular inflammation. Moreover, we have translated our findings to diabetic patients. In line with our experimental observations, Pin1 up-regulation is associated with impaired flow-mediated dilation, increased oxidative stress, and plasma levels of adhesion molecules. In perspective, these findings may provide the rationale for mechanism-based therapeutic strategies in patients with diabete

Impact of image filtering and assessment of volume-confounding effects on CT radiomic features and derived survival models in non-small cell lung cancer

BACKGROUND No evidence supports the choice of specific imaging filtering methodologies in radiomics. As the volume of the primary tumor is a well-recognized prognosticator, our purpose is to assess how filtering may impact the feature/volume dependency in computed tomography (CT) images of non-small cell lung cancer (NSCLC), and if such impact translates into differences in the performance of survival modeling. The role of lesion volume in model performances was also considered and discussed. METHODS Four-hundred seventeen CT images NSCLC patients were retrieved from the NSCLC-Radiomics public repository. Pre-processing and features extraction were implemented using Pyradiomics v3.0.1. Features showing high correlation with volume across original and filtered images were excluded. Cox proportional hazards (PH) with least absolute shrinkage and selection operator (LASSO) regularization and CatBoost models were built with and without volume, and their concordance (C-) indices were compared using Wilcoxon signed-ranked test. The Mann Whitney U test was used to assess model performances after stratification into two groups based on low- and high-volume lesions. RESULTS Radiomic models significantly outperformed models built on only clinical variables and volume. However, the exclusion/inclusion of volume did not generally alter the performances of radiomic models. Overall, performances were not substantially affected by the choice of either imaging filter (overall C-index 0.539-0.590 for Cox PH and 0.589-0.612 for CatBoost). The separation of patients with high-volume lesions resulted in significantly better performances in 2/10 and 7/10 cases for Cox PH and CatBoost models, respectively. Both low- and high-volume models performed significantly better with the inclusion of radiomic features (P<0.0001), but the improvement was largest in the high-volume group (+10.2% against +8.7% improvement for CatBoost models and +10.0% against +5.4% in Cox PH models). CONCLUSIONS Radiomic features complement well-known prognostic factors such as volume, but their volume-dependency is high and should be managed with vigilance. The informative content of radiomic features may be diminished in small lesion volumes, which could limit the applicability of radiomics in early-stage NSCLC, where tumors tend to be small. Our results also suggest an advantage of CatBoost models over the Cox PH models

A novel enzyme blend for efficient tissue dissociation and primary cells isolation

Tissue dissociation/primary cell isolation and cell harvesting are principal appli- cations for enzymes in tissue culture research and cell biology studies. The goal of a cell isolation procedure is to maximize the yield of functionally viable dissoci- ated cells. Among the parameters which affect the outcome of any particular dissociating procedure there are enzyme(s) used and related impurities presents in crude enzyme preparation. ABIEL srl recently produced the recombinant collagenase class I (Col G) and II (Col H) from Clostridium histolyticum (PCT WO 2011/073925 A9). The enzymes were produced in Escherichia coli and purified by affinity chromatography. The method of production adopted allows absolute control of the final composition of these enzymes, as well as their stability, purity, activity, absence of toxicity and higher reproducibility of batches. The two collagenases produced separately have been used in conjunction according to precise proportions to dissociate calvaria, liver, pancreas, retina of the BALB/c mouse; and bovine hoof. The analyses carried out on all isolated cell populations suggest that the cells maintain the structural and functional integrity of specific tissues/organs originating. Recombinant Col G and Col H enzymes produced by ABIEL are promising in the context of the tissue/cells dissociation, with the aim to make innovation in the fields of tissue engineering and transplantation medicine