Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

<p>Abstract</p> <p>Background</p> <p>In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in <it>Pseudoalteromonas haloplanktis </it>TAC125. This system makes use of the psychrophilic α-amylase from <it>P. haloplanktis </it>TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, <it>Pseudoalteromonales </it>are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a <it>P. haloplanktis </it>TAC125 mutant strain with reduced extra-cellular proteolytic activity.</p> <p>Results</p> <p><it>P. haloplanktis </it>TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of <it>P. haloplanktis </it>TAC125 in which the <it>gspE </it>gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-β-lactamase chimera.</p> <p>Conclusion</p> <p>Here we report a cell engineering approach to the construction of a <it>P. haloplanktis </it>TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic α-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the <it>gspE </it>mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that <it>P. haloplanktis </it>TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.</p

An Acidic Loop and Cognate Phosphorylation Sites Define a Molecular Switch That Modulates Ubiquitin Charging Activity in Cdc34-Like Enzymes

E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the β4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft

The effect of social media and infodemic on mental health during the COVID-19 pandemic: results from the COMET multicentric trial

On January 30, 2020, the World Health Organization (WHO) declared the status of pandemic due to the COVID-19 infection. The initial phases of the pandemic were characterized by uncertainty and public fears. In order to cope with such unexpected conditions, people adopted different coping strategies, including search for information, accessing Internet, and using social media. The present study based on the COMET collaborative research network aims to: (1) assess use of Internet and of social media among the Italian general population; (2) explore differences in web usage between people with pre-existing mental disorders and the general population; (3) identify changes over time in social media usage along the phase 1 of the pandemic; (4) identify the clinical, socio-demographic and contextual predictors of excessive use of social media. A significant increase in time spent on Internet, with an average time of 4.8 ± 0.02 h per day, was found in the global sample of 20,720 participants. Compared with the general population, Internet use was significantly higher in people with pre-existing mental disorders (5.2 ± 0.1 h vs. 4.9 ± 0.02; p < 0.005). According to the multivariate logistic regression model, the risk of excessive use of social media and Internet was significantly higher in people with moderate levels of depressive symptoms (OR: 1.26, CI 95%: 0.99 to 1.59, p < 0.0.005); while protective factors were being students (OR: 0.72, CI 95%: 0.53 to 0.96, p < 0.0029) and living in central Italy (OR: 0.46, CI 95%: 0.23 to 0.90, p < 0.002). The evaluation of social media and Internet use by the general population represents a first step for developing specific protective and supportive interventions for the general population, including practical suggestions on how to safely use Internet and social media

COVID-19-Related Social Isolation Predispose to Problematic Internet and Online Video Gaming Use in Italy

COVID-19 pandemic and its related containment measures have been associated with increased levels of stress, anxiety and depression in the general population. While the use of digital media has been greatly promoted by national governments and international authorities to maintain social contacts and healthy lifestyle behaviors, its increased access may also bear the risk of inappropriate or excessive use of internet-related resources. The present study, part of the COVID Mental hEalth Trial (COMET) study, aims at investigating the possible relationship between social isolation, the use of digital resources and the development of their problematic use. A cross sectional survey was carried out to explore the prevalence of internet addiction, excessive use of social media, problematic video gaming and binge watching, during Italian phase II (May-June 2020) and III (June-September 2020) of the pandemic in 1385 individuals (62.5% female, mean age 32.5 +/- 12.9) mainly living in Central Italy (52.4%). Data were stratified according to phase II/III and three groups of Italian regions (northern, central and southern). Compared to the larger COMET study, most participants exhibited significant higher levels of severe-to-extremely-severe depressive symptoms (46.3% vs. 12.4%; p &lt; 0.01) and extremely severe anxiety symptoms (77.8% vs. 7.5%; p &lt; 0.01). We also observed a rise in problematic internet use and excessive gaming over time. Mediation analyses revealed that COVID-19-related general psychopathology, stress, anxiety, depression and social isolation play a significant role in the emergence of problematic internet use, social media addiction and problematic video gaming. Professional gamers and younger subjects emerged as sub-populations particularly at risk of developing digital addictions. If confirmed in larger and more homogenous samples, our findings may help in shedding light on possible preventive and treatment strategies for digital addictions

The effect of social media and infodemic on mental health during the COVID-19 pandemic: results from the COMET multicentric trial

On January 30, 2020, the World Health Organization (WHO) declared the status of pandemic due to the COVID-19 infection. The initial phases of the pandemic were characterized by uncertainty and public fears. In order to cope with such unexpected conditions, people adopted different coping strategies, including search for information, accessing Internet, and using social media. The present study based on the COMET collaborative research network aims to: (1) assess use of Internet and of social media among the Italian general population; (2) explore differences in web usage between people with pre-existing mental disorders and the general population; (3) identify changes over time in social media usage along the phase 1 of the pandemic; (4) identify the clinical, socio-demographic and contextual predictors of excessive use of social media. A significant increase in time spent on Internet, with an average time of 4.8  ±  0.02 h per day, was found in the global sample of 20,720 participants. Compared with the general population, Internet use was significantly higher in people with pre-existing mental disorders (5.2  ± 0.1 h vs. 4.9  ±  0.02; p &lt; 0.005). According to the multivariate logistic regression model, the risk of excessive use of social media and Internet was significantly higher in people with moderate levels of depressive symptoms (OR: 1.26, CI 95%: 0.99 to 1.59, p &lt; 0.0.005); while protective factors were being students (OR: 0.72, CI 95%: 0.53 to 0.96, p &lt; 0.0029) and living in central Italy (OR: 0.46, CI 95%: 0.23 to 0.90, p &lt; 0.002). The evaluation of social media and Internet use by the general population represents a first step for developing specific protective and supportive interventions for the general population, including practical suggestions on how to safely use Internet and social media

Common genetic variation and susceptibility to partial epilepsies: a genome-wide association study

Partial epilepsies have a substantial heritability. However, the actual genetic causes are largely unknown. In contrast to many other common diseases for which genetic association-studies have successfully revealed common variants associated with disease risk, the role of common variation in partial epilepsies has not yet been explored in a well-powered study. We undertook a genome-wide association-study to identify common variants which influence risk for epilepsy shared amongst partial epilepsy syndromes, in 3445 patients and 6935 controls of European ancestry. We did not identify any genome-wide significant association. A few single nucleotide polymorphisms may warrant further investigation. We exclude common genetic variants with effect sizes above a modest 1.3 odds ratio for a single variant as contributors to genetic susceptibility shared across the partial epilepsies. We show that, at best, common genetic variation can only have a modest role in predisposition to the partial epilepsies when considered across syndromes in Europeans. The genetic architecture of the partial epilepsies is likely to be very complex, reflecting genotypic and phenotypic heterogeneity. Larger meta-analyses are required to identify variants of smaller effect sizes (odds ratio <1.3) or syndrome-specific variants. Further, our results suggest research efforts should also be directed towards identifying the multiple rare variants likely to account for at least part of the heritability of the partial epilepsies. Data emerging from genome-wide association-studies will be valuable during the next serious challenge of interpreting all the genetic variation emerging from whole-genome sequencing studies

NEK1 variants confer susceptibility to amyotrophic lateral sclerosis

To identify genetic factors contributing to amyotrophic lateral sclerosis (ALS), we conducted whole-exome analyses of 1,022 index familial ALS (FALS) cases and 7,315 controls. In a new screening strategy, we performed gene-burden analyses trained with established ALS genes and identified a significant association between loss-of-function (LOF) NEK1 variants and FALS risk. Independently, autozygosity mapping for an isolated community in the Netherlands identified a NEK1 p.Arg261His variant as a candidate risk factor. Replication analyses of sporadic ALS (SALS) cases and independent control cohorts confirmed significant disease association for both p.Arg261His (10,589 samples analyzed) and NEK1 LOF variants (3,362 samples analyzed). In total, we observed NEK1 risk variants in nearly 3% of ALS cases. NEK1 has been linked to several cellular functions, including cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity. Our results provide new and important insights into ALS etiopathogenesis and genetic etiology

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

Author Correction:Study of 300,486 individuals identifies 148 independent genetic loci influencing general cognitive function

Christina M. Lill, who contributed to analysis of data, was inadvertently omitted from the author list in the originally published version of this article. This has now been corrected in both the PDF and HTML versions of the article