Pseudophosphorylation of single residues of the J-domain of DNAJA2 regulates the holding/folding balance of the Hsc70 system.

The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position.

Comparison of Adding Sildenafil Versus Estradiol to Clomiphene Citrate on the Applebaum Score and Pregnancy Rate in Patients With Unexplained Infertility: A Double-Blind Randomized Controlled Trial.

BACKGROUND: Both sildenafil and estradiol are seen to improve endometrial thickness in patients with infertility who are undergoing clomiphene induction cycles. However, the correlation between endometrial thickness and pregnancy rate is debatable. This study investigated the effect of adding oral sildenafil to clomiphene citrate (CC), compared to adding estradiol valerate, on the uterine biophysical profile (Applebaum score) and pregnancy rate. METHODS: This was a double-blinded, randomized controlled trial conducted in Kisangani in the Democratic Republic of the Congo from October 1, 2021, to October 31, 2023. Patients with unexplained infertility were randomly assigned to one of two groups: the interventional, which was given CC (2 x 50 mg/day from day 3 to day 7 of the menstrual cycle) + sildenafil (2 x 25 mg/day orally from day 8 to day 12) or (ii) the control group, which was given CC (similar dosage as the intervention group) + EV (2 x 2 mg/day orally from day 8 to day 12), for a maximum of three cycles. Applebaum scores and clinical pregnancy rates were measured. RESULTS: Patients in the sildenafil and EV groups were similar in mean age (29.04 versus 28.89 years). Of the 74 patients enrolled in each group, 71 in the sildenafil group and 72 in the EV group received treatment and were followed to completion. The Applebaum scores were significantly higher in the sildenafil group than in the EV group (17.05 versus 15.14, respectively, P=0.000). In the sildenafil group, the clinical pregnancy rate was also significantly higher, at 28.92% versus 20.83% in the EV group (P = 0.04). CONCLUSION: As compared to EV, the oral addition of sildenafil to CC is associated with a good Applebaum score and a high rate of clinical pregnancy in patients with unexplained infertility.

De Novo Engineering of Pd-Metalloproteins and Their Use as Intracellular Catalysts.

The development of transition metal-based catalytic platforms that promote bioorthogonal reactions inside living cells remains a major challenge in chemical biology. This is particularly true for palladium-based catalysts, which are very powerful in organic synthesis but perform poorly in the cellular environment, mainly due to their rapid deactivation. We now demonstrate that grafting Pd(II) complexes into engineered ß-sheets of a model WW domain results in cell-compatible palladominiproteins that effectively catalyze depropargylation reactions inside HeLa cells. The concave shape of the WW domain ß-sheet proved particularly suitable for accommodating the metal center and protecting it from rapid deactivation in the cellular environment. A thorough NMR and computational study confirmed the formation of the metal-stapled peptides and allowed us to propose a three-dimensional structure for this novel metalloprotein motif.

Protein-Protein Stabilization in VIVO/8-Hydroxyquinoline-Lysozyme adduct: A Spectroscopic, Crystallographic, and Computational Study.

The binding of the potential drug [VIVO(8-HQ)2], where 8-HQ is 8-hydroxyquinolinato, with hen egg white lysozyme (HEWL) was evaluated through spectroscopic (electron paramagnetic resonance, EPR, and UV-visible), spectrometric (electrospray ionization-mass spectrometry, ESI-MS), crystallographic (X-ray diffraction, XRD), and computational (DFT and docking) studies. ESI-MS indicates the interaction of [VIVO(8-HQ)(H2O)]+ and [VIVO(8-HQ)2(H2O)] species with HEWL. Room temperature EPR spectra suggest both covalent and non-covalent binding of the two different V-containing fragments. XRD analyses confirm these findings, showing that [VIVO(8-HQ)(H2O)]+ interacts covalently with the solvent exposed Asp119, while cis-[VIVO(8-HQ)2(H2O)] non-covalently with Arg128 and Lys96 from a symmetry mate. The covalent binding of [VIVO(8-HQ)(H2O)]+ to Asp119 is favored by a π-π contact with Trp62 and a H-bond with Asn103 of a symmetry-related molecule. Additionally, the covalent binding of VVO2+ to Asp48 and non-covalent binding of other V-containing fragments to Arg5, Cys6, and Glu7 is revealed. Molecular docking indicates that, in the absence of the interactions occurring at the protein-protein interface close to Asp119, the binding to Glu35 or Asp52 should be preferred. Such a protein-protein stabilization could be more common than what believed up today, at least in the solid state, and should be considered in the characterization of metal-protein adducts.

Virtual brain twins: from basic neuroscience to clinical use.

Virtual brain twins are personalized, generative and adaptive brain models based on data from an individual's brain for scientific and clinical use. After a description of the key elements of virtual brain twins, we present the standard model for personalized whole-brain network models. The personalization is accomplished using a subject's brain imaging data by three means: (1) assemble cortical and subcortical areas in the subject-specific brain space; (2) directly map connectivity into the brain models, which can be generalized to other parameters; and (3) estimate relevant parameters through model inversion, typically using probabilistic machine learning. We present the use of personalized whole-brain network models in healthy ageing and five clinical diseases: epilepsy, Alzheimer's disease, multiple sclerosis, Parkinson's disease and psychiatric disorders. Specifically, we introduce spatial masks for relevant parameters and demonstrate their use based on the physiological and pathophysiological hypotheses. Finally, we pinpoint the key challenges and future directions.

Computational modelling of supramolecular metallopeptide assemblies.

Among the important questions in supramolecular peptide self-assemblies are their interactions with metallic compounds and ions. In the last decade, intensive efforts have been devoted to understanding the structural properties of these interactions including their dynamical and catalytic impact in natural and de novo systems. Since structural insights from experimental approaches could be particularly challenging, computational chemistry methods are interesting complementary tools. Here, we present the general multiscale strategies we developed and applied for the study of metallopeptide assemblies. These strategies include prediction of metal binding site, docking of metallic moieties, classical and accelerated molecular dynamics and finally QM/MM calculations. The systems of choice for this chapter are, on one side, peptides involved in neurodegenerative diseases and, on the other, de novo fibrillar systems with catalytic properties. Both successes and remaining challenges are highlighted so that the protocol could be apply to other system of this kind.

Rationalization of a Streptavidin Based Enantioselective Artificial Suzukiase: An Integrative Computational Approach.

An Artificial Metalloenzyme (ArM) built employing the streptavidin-biotin technology has been used for the enantioselective synthesis of binaphthyls by means of asymmetric Suzuki-Miyaura cross-coupling reactions. Despite its success, it remains a challenge to understand how the length of the biotin cofactors or the introduction of mutations to streptavidin leads the preferential synthesis of one atropisomer over the other. In this study, we apply an integrated computational modeling approach, including DFT calculations, protein-ligand dockings and molecular dynamics to rationalize the impact of mutations and length of the biotion cofactor on the enantioselectivities of the biaryl product. The results unravel that the enantiomeric differences found experimentally can be rationalized by the disposition of the first intermediate, coming from the oxidative addition step, and the entrance of the second substrate. The work also showcases the difficulties facing to control the enantioselection when engineering ArM to catalyze enantioselective Suzuki-Miyaura couplings and how the combination of DFT calculations, molecular dockings and MD simulations can be used to rationalize artificial metalloenzymes.

Stringent criteria needed for germline genome editing of human IVF embryos.

Germline genome editing of IVF embryos is controversial because it is not directly health or lifesaving but is intended to prevent genetic diseases in yet-unborn future offspring. The following criteria are thus proposed for future clinical trials: (i) Due to medical risks, there should be cautious and judicious application while avoiding any non-essential usage, with rigorous patient counseling. (ii) Genome editing should only be performed on the entire batch of IVF embryos without initial PGT screening if all of them are expected to be affected by genetic disease. (iii) When there is a fair chance that some IVF embryos will not be affected by genetic diseases, initial PGT screening must be performed to identify unaffected embryos for transfer. (iv) IVF embryos with carrier status should not undergo germline genome editing. (v) If patients fail to conceive after the transfer of unaffected embryos, they should undergo another fresh IVF cycle rather than opt for genome editing of their remaining affected embryos. (vi) Only if the patient is unable to produce any more unaffected embryos in a fresh IVF cycle due to advanced maternal age or diminished ovarian reserves, can the genome editing of remaining affected embryos be permitted as a last resort.

Counseling Elective Egg Freezing Patients considering Donation of Unused Surplus Frozen Eggs for Fertility Treatment.

The majority of women who freeze their eggs for non-medical or social reasons, commonly referred to as elective egg freezing (EEF), do not eventually utilize their frozen eggs. This would result in an accumulated surplus of unused frozen eggs in fertility clinics worldwide, which represents a promising source of donation to infertile women undergoing IVF treatment. Rigorous and comprehensive counseling is needed, because the process of donating one's unused surplus frozen eggs involves complex decision-making. Prospective EEF donors can be broadly categorized into those who have achieved motherhood and those who remained childless and have given up on motherhood aspirations. A two-step systematic counseling protocol is proposed. Firstly, it is imperative to verify and ensure that these women do not want to conceive any children with their surplus frozen eggs before proceeding with further counseling and signing of consent forms. Secondly, various motivating and dissuading factors in the donation of unused surplus frozen eggs should then be comprehensively discussed with egg freezers to facilitate informed decision-making. Key motivating factors for donation include reciprocity in wanting to share the joys of motherhood among egg freezers who already have children, goodwill to help others in need, and avoiding the wastage of surplus frozen eggs after expending so much money, time, and effort. Key dissuading factors include fear of accidental incest between natural and unknown donor-conceived offspring, as well as apprehension of unexpected future contact with unknown donor-conceived offspring due to either donor anonymity being abolished in their jurisdiction or widespread consumer DNA testing.

Computational Study of Amyloidß42 Familial Mutations and Metal Interaction: Impact on Monomers and Aggregates Dynamical Behaviors.

One of the main hallmarks of Alzheimer's Disease is the formation of ß-amyloid plaques, whose formation may be enhanced by metal binding or the appearance of familial mutations. In the present study, the simultaneous effect of familial mutations (E22Q, E22G, E22K, and D23N) and binding to metal ions (Cu(II) or Al(III)) is studied at the Aß42 monomeric and fibrillar levels. With the application of GaMD and MD simulations, it is observed that the effects of metal binding and mutations differ in the monomeric and fibrillar forms. In the monomeric structures, without metal binding, all mutations reduce the amount of α-helix and increase, in some cases, the ß-sheet content. In the presence of Cu(II) and Al(III) metal ions, the peptide becomes less flexible, and the ß-sheet content decreases in favor of forming α-helix motifs that stabilize the system through interhelical contacts. Regarding the fibrillar structures, mutations decrease the opening of the fiber in the vertical axis, thereby stabilizing the S-shaped structure of the fiber. This effect is, in general, enhanced upon metal binding. These results may explain the different Aß42 aggregation patterns observed in familial mutations.

[Overview of the implementation of pharmaceutical interviews in pharmacies in France: Analysis of responses to a questionnaire]. / État des lieux de la mise en place en France des entretiens pharmaceutiques en officine : analyse des réponses issues d'un questionnaire.

OBJECTIVE: To evaluate the development of pharmaceutical interviews in pharmacies in France, in order to understand the organization implemented, any limitations and the expansion of eligible pathologies. METHOD: A dematerialized questionnaire was designed and distributed between November 2022 and February 2023 to pharmacists and pharmacy students in France (mainland and overseas) via a link to a Google Form. RESULTS: Ninety-four pharmacists from 8 different regions of France responded to the survey. The 94 responses showed that 56% of pharmacists practiced pharmaceutical interviews. Among pharmacists who practiced interviews, pharmacy owners practiced significantly more interviews than other statuses within the pharmacy (67% vs. 38% P=0.014). No other factor, such as dispensary size or geographical area of practice, had a significant impact on whether or not pharmaceutical interviews were carried out. These talks are often carried out at the patient's request, and 89% of them are accompanied by documents for the patient's attention. For pharmacists who do not carry out interviews, time, staffing and remuneration are the 3 main blocking factors found in both quantitative and verbatim variables. Whether or not pharmacists carry out pharmaceutical interviews, this activity received 87% approval from the 94 respondents, and 84% of them would like to include more chronic disease themes. CONCLUSION: The survey shows that pharmacists approve of the pharmaceutical interviewing activity, but it also highlights obvious logistical obstacles linked to a lack of resources. Thus, even among pharmacists who carry out pharmaceutical interviews, this activity is still carried out relatively infrequently on a routine basis, and often by the incumbent pharmacist, who takes on the responsibility of carrying out this activity.