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

Activation of the AIM2 Receptor in Circulating Cells of Post-COVID-19 Patients With Signs of Lung Fibrosis Is Associated With the Release of IL-1α, IFN-α and TGF-β.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for COVID-19, has caused a global pandemic. Observational studies revealed a condition, herein called as Long-COVID syndrome (PC), that affects both moderately and severely infected patients, reducing quality-of-life. The mechanism/s underlying the onset of fibrotic-like changes in PC are still not well defined. The goal of this study was to understand the involvement of the Absent in melanoma-2 (AIM2) inflammasome in PC-associated lung fibrosis-like changes revealed by chest CT scans. Peripheral blood mononuclear cells (PBMCs) obtained from PC patients who did not develop signs of lung fibrosis were not responsive to AIM2 activation by Poly dA:dT. In sharp contrast, PBMCs from PC patients with signs of lung fibrosis were highly responsive to AIM2 activation, which induced the release of IL-1α, IFN-α and TGF-β. The recognition of Poly dA:dT was not due to the activation of cyclic GMP-AMP (cGAMP) synthase, a stimulator of interferon response (cGAS-STING) pathways, implying a role for AIM2 in PC conditions. The release of IFN-α was caspase-1- and caspase-4-dependent when AIM2 was triggered. Instead, the release of pro-inflammatory IL-1α and pro-fibrogenic TGF-β were inflammasome independent because the inhibition of caspase-1 and caspase-4 did not alter the levels of the two cytokines. Moreover, the responsiveness of AIM2 correlated with higher expression of the receptor in circulating CD14+ cells in PBMCs from patients with signs of lung fibrosis

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

Absent in melanoma 2 (AIM2) positive profile identifies a poor prognosis of lung adenocarcinoma patients.

The absent in melanoma 2 (AIM2) inflammasome has been demonstrated as involved in tumor growth. In this study we used human samples of lung adenocarcinoma (LUAD) patients, taking advantage of a mouse model of smoking cessation. Human samples were stratified according to the smoking status, high-risk factor for this type of tumor. Both public transcriptomic and human samples obtained by a clinical trial proved that AIM2 was upregulated either in terms of mRNA or protein, respectively, in the tumor mass according to the TNM stage, but it did not relate to the smoking status, age and sex. The upregulation of AIM2 was correlated to an immunosuppressive environment according to resting/non-active dendritic cells (DCs) and T regulatory cells, as demonstrated in both human samples and by means of an experimental model of smoking mice. Computational analysis showed that AIM2 upregulation was correlated to both an inflammasome profile, responsible for the poor prognosis of non-smoker and smoker LUAD patients, and to a non-inflammasome profile for former smoker. In conclusion, our study demonstrated that AIM2 is involved in lung carcinogenesis either in a canonical and non-canonical manner due to an immunosuppressive microenvironment associated to a dismal prognosis of LUAD patients

Factors influencing carbon stocks and accumulation rates in eelgrass meadows across New England, USA

Increasing the protection of coastal vegetated ecosystems has been suggested as one strategy to compensate for increasing carbon dioxide (CO2) in the atmosphere as the capacity of these habitats to sequester and store carbon exceeds that of terrestrial habitats. Seagrasses are a group of foundation species that grow in shallow coastal and estuarine systems and have an exceptional ability to sequester and store large quantities of carbon in biomass and, particularly, in sediments. However, carbon stocks (Corg stocks) and carbon accumulation rates (Corg accumulation) in seagrass meadows are highly variable both spatially and temporally, making it difficult to extrapolate this strategy to areas where information is lacking. In this study, Corg stocks and Corg accumulation were determined at 11 eelgrass meadows across New England, representing a range of eutrophication and exposure conditions. In addition, the environmental factors and structural characteristics of meadows related to variation in Corg stocks were identified. The objectives were accomplished by assessing stable isotopes of δ13C and δ15N as well as %C and %N in plant tissues and sediments, measuring grain size and 210Pb of sediment cores, and through assessing site exposure. Variability in Corg stocks in seagrass meadows is well predicted using commonly measured environmental variables such as grain size distribution. This study allows incorporation of data and insights for the northwest Atlantic, where few studies on carbon sequestration by seagrasses have been conducted

Modelling hCDKL5 heterologous expression in bacteria

hCDKL5 refers to the human cyclin-dependent kinase like 5 that is primarily expressed in the brain. Mutations in its coding sequence are often causative of hCDKL5 deficiency disorder, a devastating neurodevelopmental disorder currently lacking a cure. The large-scale recombinant production of hCDKL5 is desirable to boost the translation of preclinical therapeutic approaches into the clinic. However, this is hampered by the intrinsically disordered nature of almost two-thirds of the hCDKL5 sequence, making this region more susceptible to proteolytic attack, and the observed toxicity when the enzyme is accumulated in the cytoplasm of eukaryotic host cells. The bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is the only prokaryotic host in which the full-length production of hCDKL5 has been demonstrated. To date, a system-level understanding of the metabolic burden imposed by hCDKL5 production is missing, although it would be crucial for upscaling of the production process. Here, we combined experimental data on protein production and nutrients assimilation with metabolic modelling to infer the global consequences of hCDKL5 production in PhTAC125 and to identify potential overproduction targets. Our analyses showed a remarkable accuracy of the model in simulating the recombinant strain phenotype and also identified priority targets for optimised protein production