6-Methylquinazolin-4(3H)-one Based Compounds as BRD9 Epigenetic Reader Binders: A Rational Combination of in silico Studies and Chemical Synthesis

6-methylquinazolin-4(3H)-one-based compounds were here identified and synthesized as novel binders of bromodomain-containing protein 9 (BRD9) epigenetic reader. Accounting a fast and efficient synthetic route aimed to easily obtain differently 2- and 8-disubstituted 6-methylquinazolin-4(3H)-one derivatives, a virtual library of synthesizable items was built and submitted to molecular docking experiments. Based on two 3D structure-based pharmacophore models recently developed by us on BRD9, 16 compounds were selected and synthesized, using mild conditions with good yields in relatively short reaction times. Among them, 14, 16, 18, 22, and 26 emerged as the most potent compounds of these series, able to bind BRD9 at the low micromolar range of concentrations. These molecules also showed a promising selective behavior when tested against BRD4 bromodomain. These results highlighted the quinazolin-4(3H)-one chemical core as a valuable scaffold for developing promising BRD9 binders

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

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

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

Stabilities of nanohydrated thymine radical cations: insights from multiphoton ionization experiments and ab initio calculations

Multi-photon ionization experiments have been carried out on thymine-water clusters in the gas phase. Metastable H2O loss from T+(H2O)n was observed at n ≥ 3 only. Ab initio quantum-chemical calculations of a large range of optimized T+(H2O)n conformers have been performed up to n = 4, enabling binding energies of water to be derived. These decrease smoothly with n, consistent with the general trend of increasing metastable H2O loss in the experimental data. The lowest-energy conformers of T+(H2O)3 and T+(H2O)4 feature intermolecular bonding via charge-dipole interactions, in contrast with the purely hydrogen-bonded neutrals. We found no evidence for a closed hydration shell at n = 4, also contrasting with studies of neutral clusters

Databases for Congenital Heart Defect Public Health Studies Across the Lifespan

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139106/1/jah31841_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139106/2/jah31841.pd

Transfusional approach in multi-ethnic Sickle Cell patients: real-world practice data from a Multicenter survey in Italy

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder characterized by recurrent acute vaso-occlusive crises (VOCs and anemia). Gold standard treatments are hydroxycarbamide (HC) and/or different red blood cell (RBC) transfusion regimens to limit disease progression. Here, we report a retrospective study on 1,579 SCD patients (median age 23 years; 802 males/777 females), referring to 34 comprehensive Italian centers for hemoglobinopathies. Although we observed a similar proportion of Caucasian (47.9%) and African (48.7%) patients, Italian SCD patients clustered into two distinct overall groups: children of African descent and adults of Caucasian descent. We found a subset of SCD patients requiring more intensive therapy with a combination of HC plus chronic transfusion regimen, due to partial failure of HC treatment alone in preventing or reducing sickle cell-related acute manifestations. Notably, we observed a higher use of acute transfusion approaches for SCD patients of African descent when compared to Caucasian subjects. This might be related to (i) age of starting HC treatment; (ii) patients' low social status; (iii) patients' limited access to family practitioners; or (iv) discrimination. In our cohort, alloimmunization was documented in 135 patients (8.5%) and was more common in Caucasians (10.3%) than in Africans (6.6%). Alloimmunization was similar in male and female and more frequent in adults than in children. Our study reinforces the importance of donor-recipient exact matching for ABO, Rhesus, and Kell antigen systems for RBC compatibility as a winning strategy to avoid or limit alloimmunization events that negatively impact the clinical management of SCD-related severe complications

Multi-photon and electron impact ionisation studies of reactivity in adenine–water clusters

Multi-photon ionisation (MPI) and electron impact ionisation (EII) mass spectrometry experiments have been carried out to probe unimolecular and intermolecular reactivities in hydrated adenine clusters. The effects of clustering with water on fragment ion production from adenine have been studied for the first time. While the observation of NH4+ fragments indicated the dissociation of protonated adenine, the dominant hydration effects were enhanced C4H4N4+ production and the suppression of dissociative ionisation pathways with high activation energies. These observations can be attributed to energy removal from the excited adenine radical cation via cluster dissociation. Comparisons of MPI and EII measurements provided the first experimental evidence supporting hypoxanthine formation in adenine–water clusters via theoretically predicted barrierless deamination reactions in closed shell complexes

Endothelial Domes Encapsulate Adherent Neutrophils and Minimize Increases in Vascular Permeability in Paracellular and Transcellular Emigration

Local edema, a cardinal sign of inflammation associates closely with neutrophil emigration. Neutrophil emigration has been described to occur primarily through endothelial junctions (paracellular) and more rarely directly through endothelial cells (transcellular). Recently, we reported that unlike in wild-type (wt) mice, Mac-1-/- (CD11b) neutrophils predominantly emigrated transcellularly and was significantly delayed taking 20–30 min longer than the paracellular emigration (wt). In the present study we noted significant anatomical disruption of the endothelium and hypothesized that transcellular emigration would greatly increase vascular permeability. Surprisingly, despite profound disruption of the endothelial barrier as the neutrophils moved through the cells, the changes in vascular permeability during transcellular emigration (Mac-1-/-) were not increased more than in wt mice. Instead increased vascular permeability completely tracked the number of emigrated cells and as such, permeability changes were delayed in Mac-1-/- mice. However, by 60 min neutrophils from both sets of mice were emigrating in large numbers. Electron-microscopy and spinning disk multichannel fluorescence confocal microscopy revealed endothelial docking structures that progressed to dome-like structures completely covering wt and Mac-1-/- neutrophils. These domes completely enveloped the emigrating neutrophils in both wt and Mac-1-/- mice making the mode of emigration underneath these structures extraneous to barrier function. In conclusion, predominantly paracellular versus predominantly transcellular emigration does not affect vascular barrier integrity as endothelial dome-like structures retain barrier function