Psychiatric emergencies during, after, and before the COVID-19 lockdown: what happened to our patients? A naturalistic observational study.

Background: Despite concerns on mental health problems related to lockdowns, recent reports revealed a reduction in psychiatric admissions in Emergency Departments (ED) during the lockdown period compared with the previous year in several countries. Most of the existing studies focused on the first lockdown not considering the different phases of the COVID-19 crisis. The present study aimed to analyze differences in ED admission for psychiatric consultation during three different phases of the COVID-19 health crisis in Italy.

Cell-free protein synthesis: advances on production process for biopharmaceuticals and immunobiological products.

Biopharmaceutical products are of great importance in the treatment or prevention of many diseases and represent a growing share of the global pharmaceutical market. The usual technology for protein synthesis (cell-based expression) faces certain obstacles, especially with 'difficult-to-express' proteins. Cell-free protein synthesis (CFPS) can overcome the main bottlenecks of cell-based expression.

On the expression of recombinant Cas9 protein in E. coli BL21(DE3) and BL21(DE3) Rosetta strains.

The CRISPR-Cas9 system is a new tool that has been extensively used for genome editing. The system is composed of a Cas9 endonuclease, which has the function of cleaving DNA at a specific site, and a guide RNA (gRNA), which contains the sequence of the cleavage site that is the target of editing. Despite the great interest that has been generated because of the utility of Cas 9 as a molecular tool and a potential therapeutic protein, the production of the 158 kDa recombinant Cas9 protein derived from Streptococcus pyogenes remains a challenge.

Switching cell penetrating and CXCR4-binding activities of nanoscale-organized arginine-rich peptides.

Arginine-rich protein motifs have been described as potent cell-penetrating peptides (CPPs) but also as rather specific ligands of the cell surface chemokine receptor CXCR4, involved in the infection by the human immunodeficiency virus (HIV). Polyarginines are commonly used to functionalize nanoscale vehicles for gene therapy and drug delivery, aimed to enhance cell penetrability of the therapeutic cargo. However, under which conditions these peptides do act as either unspecific or specific ligands is unknown.

Arginine homopeptides for plasmid DNA purification using monolithic supports.

Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA.

Techno-economic analysis of the industrial production of a low-cost enzyme using : the case of recombinant β-glucosidase.

Background: The enzymatic conversion of lignocellulosic biomass into fermentable sugars is a promising approach for producing renewable fuels and chemicals. However, the cost and efficiency of the fungal enzyme cocktails that are normally employed in these processes remain a significant bottleneck. A potential route to increase hydrolysis yields and thereby reduce the hydrolysis costs would be to supplement the fungal enzymes with their lacking enzymatic activities, such as β-glucosidase.

Protein nanoparticles are nontoxic, tuneable cell stressors.

Aim: Nanoparticle-cell interactions can promote cell toxicity and stimulate particular behavioral patterns, but cell responses to protein nanomaterials have been poorly studied.

Results: By repositioning oligomerization domains in a simple, modular self-assembling protein platform, we have generated closely related but distinguishable homomeric nanoparticles. Composed by building blocks with modular domains arranged in different order, they share amino acid composition.

Intracellular trafficking of a dynein-based nanoparticle designed for gene delivery.

The success of viruses in the delivery of the viral genome to target cells relies on the evolutionary selection of protein-based domains able to hijack the intermolecular interactions through which cells respond to intra- and extracellular stimuli. In an effort to mimic viral infection capabilities during non-viral gene delivery, a modular recombinant protein named T-Rp3 was recently developed, containing a DNA binding domain, a dynein molecular motor interacting domain, and a TAT-derived transduction domain. Here, we analyzed at the microscopic level the mechanisms behind the cell internalization and intracellular trafficking of this highly efficient modular protein vector.

Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X.

Development and characterization of a cationic lipid nanocarrier as non-viral vector for gene therapy.

The aim of the present work was to produce a cationic solid lipid nanoparticle (SLN) as non-viral vector for protein delivery. Cationic SLN were produced by double emulsion method, composed of softisan(®) 100, cetyltrimethylammonium bromide (CTAB), Tween(®) 80, Span(®) 80, glycerol and lipoid(®) S75 loading insulin as model protein. The formulation was characterized in terms of mean hydrodynamic diameter (z-ave), polydispersity index (PI), zeta potential (ZP), stability during storage time, stability after lyophilization, effect of toxicity and transfection ability in HeLa cells, in vitro release profile and morphology.

Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide.

Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT.

Characterization of the human dynein light chain Rp3 and its use as a non-viral gene delivery vector.

Dynein light chains mediate the interaction between the cargo and the dynein motor complex during retrograde microtubule-mediated transport in eukaryotic cells. In this study, we expressed and characterized the recombinant human dynein light chain Rp3 and developed a modified variant harboring an N-terminal DNA-binding domain (Rp3-Db). Our approach aimed to explore the retrograde cell machinery based on dynein to enhance plasmid DNA (pDNA) traffic along the cytosol toward the nucleus.

Impact of plasmid quality on lipoplex-mediated transfection.

This work investigates the impact of quality attributes (impurity content, plasmid charge, and compactness) of plasmid DNA isolated with different purification methodologies on the characteristics of lipoplexes prepared thereof (size, zeta potential, stability) and on their ability to transfect mammalian cells. A 3.7 kb plasmid with a green fluorescence protein (GFP) reporter gene, Lipofectamine®-based liposomes, and Chinese Hamster Ovary (CHO) cells were used as models.

Microfluidic devices for continuous production of pDNA/cationic liposome complexes for gene delivery and vaccine therapy.

To evaluate the process parameters for the production of plasmid DNA/cationic liposome (pDNA/CL) complexes in microfluidic systems, we studied two microfluidic devices: one with simple straight hydrodynamic flow focusing (SMD) and a second one with barriers in the mixing microchannel (patterned walls, PMD). A conventional bulk mixing method was used as a comparison to microfluidic mixing. The CL and the pDNA were combined at a molar positive/negative charge ratio of 6.

Mortality and reintervention following elective abdominal aortic aneurysm repair.

Background: The objective of this study is to provide an up-to-date meta-analysis on the short- and long-term mortality rates of elective repair of abdominal aortic aneurysms (AAAs) via the open and endovascular approaches.

Methods: MEDLINE, EMBASE, and Cochrane Central Register of Controlled trials, conference proceeding from major vascular meetings were searched for randomized trials comparing open vs elective endovascular aneurysm repair (EVAR) of AAAs. A random-effects model was used for analysis.

Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate.

The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography.

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC).

Correlation of the physicochemical and structural properties of pDNA/cationic liposome complexes with their in vitro transfection.

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R(±)) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R(±) = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R(±) is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated.

Structural characterization of the H-NS protein from Xylella fastidiosa and its interaction with DNA.

The nucleoid-associated protein H-NS is a major component of the bacterial nucleoid involved in DNA compaction and transcription regulation. The NMR solution structure of the Xylella fastidiosa H-NS C-terminal domain (residues 56-134) is presented here and consists of two beta-strands and two alpha helices, with one loop connecting the two beta-strands and a second loop connecting the second beta strand and the first helix. The amide (1)H and (15)N chemical shift signals for a sample of XfH-NS(56-134) were monitored in the course of a titration series with a 14-bp DNA duplex.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli.

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal).

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery.

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells.