Cl-out is a novel cooperative optogenetic tool for extruding chloride from neurons

Chloride regulation affects brain function in many ways, for instance, by dictating the GABAergic reversal potential, and thereby influencing neuronal excitability and spike timing. Consistent with this, there is increasing evidence implicating chloride in a range of neurological conditions. Investigations about these conditions, though, are made difficult by the limited range of tools available to manipulate chloride levels. In particular, there has been no way to actively remove chloride from neurons; we now describe an optogenetic strategy, \u27Cl- out\u27, to do exactly this. Cl-out achieves its effect by the cooperative action of two different component opsins: the proton pump, Archaerhodopsin and a chloride channel opsin. The removal of chloride happens when both are activated together, using Archaerhodopsin as an optical voltage clamp to provide the driving force for chloride removal through the concurrently opened, chloride channels. We further show that this novel optogenetic strategy can reverse an in vitro epileptogenic phenotype

The encapsulation and controlled release of proteins from “meltable” chemically cross-linked hydrogels

\ua9 2024 The Authors. Journal of Applied Polymer Science published by Wiley Periodicals LLC.Chemically crosslinked hydrogels can be used for the controlled delivery of therapeutic proteins, however, cargo loading can be challenging. A common approach involves performing the hydrogel crosslinking step in the presence of the cargo proteins, however, this can require the utilization of selective chemistries to ensure the cargos do not chemically react with the forming hydrogel network. We demonstrate that chemically crosslinked hydrogels based upon the protein polymer Capsular antigen fragment 1 (Caf1) can be used to encapsulate proteins within a chemically crosslinked hydrogel network. This encapsulation exploits the “meltable” feature of Caf1 hydrogels, where upon heating the Caf1 protein unfolds with concomitant depolymerization of the hydrogel network. Protein cargos can be loaded into the “molten” form of the hydrogel which cools with protein refolding and concomitant reformation of the hydrogel network to entrap the cargos, ensuring a more homogenous dispersion of the cargo. Analysis of release profiles indicated lower burst release in comparison to release from identical hydrogels loaded by an absorption method. This work is significant because it suggest that the “meltable” feature of Caf1 hydrogels may present an improved method of loading protein cargos into crosslinked hydrogel networks whilst avoiding the need for crosslinking chemistries

A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells

Continuing transmissions of highly pathogenic H5N1 viruses in poultry and humans underscores the need for a rapid response to potential pandemic in the form of vaccine. Recombinant technologies for production of immunogenic hemagglutinin (HA) could provide an advantage over the traditional inactivated vaccine manufacturing process. Generation of stably transfected mammalian cells secreting properly folded HA proteins is important for scalable controlled manufacturing.We have developed a Flp-In based 293 stable cell lines through targeted site-specific recombination for expression of secreted hemagglutinin (HA) proteins and evaluated their immunogenicity. H5N1 globular domain HA1(1-330) and HA0(1-500) proteins were purified from the supernatants of 293 Flp-In stable cell lines. Both proteins were properly folded as confirmed by binding to H5N1-neutralizing conformation-dependent human monoclonal antibodies. The HA0 (with unmodified cleavage site) was monomeric, while the HA1 contained oligomeric forms. Upon rabbit immunization, both HA proteins elicited neutralizing antibodies against the homologous virus (A/Vietnam/1203/2004, clade 1) as well as cross-neutralizing antibodies against heterologous H5N1 clade 2 strains, including A/Indonesia/5/2005. These results exceeded the human antibody responses against the inactivated sub-virion H5N1 vaccine.Our data suggest that the 293 Flp-In system could serve as a platform for rapid expression of HA immunogens in mammalian cells from emerging influenza strains

Roles of residues in the interface of transient protein-protein complexes before complexation

Transient protein-protein interactions play crucial roles in all facets of cellular physiology. Here, using an analysis on known 3-D structures of transient protein-protein complexes, their corresponding uncomplexed forms and energy calculations we seek to understand the roles of protein-protein interfacial residues in the unbound forms. We show that there are conformationally near invariant and evolutionarily conserved interfacial residues which are rigid and they account for ∼65% of the core interface. Interestingly, some of these residues contribute significantly to the stabilization of the interface structure in the uncomplexed form. Such residues have strong energetic basis to perform dual roles of stabilizing the structure of the uncomplexed form as well as the complex once formed while they maintain their rigid nature throughout. This feature is evolutionarily well conserved at both the structural and sequence levels. We believe this analysis has general bearing in the prediction of interfaces and understanding molecular recognition

Effects of Once-Weekly Exenatide on Cardiovascular Outcomes in Type 2 Diabetes.

Abstract BACKGROUND: The cardiovascular effects of adding once-weekly treatment with exenatide to usual care in patients with type 2 diabetes are unknown. METHODS: We randomly assigned patients with type 2 diabetes, with or without previous cardiovascular disease, to receive subcutaneous injections of extended-release exenatide at a dose of 2 mg or matching placebo once weekly. The primary composite outcome was the first occurrence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. The coprimary hypotheses were that exenatide, administered once weekly, would be noninferior to placebo with respect to safety and superior to placebo with respect to efficacy. RESULTS: In all, 14,752 patients (of whom 10,782 [73.1%] had previous cardiovascular disease) were followed for a median of 3.2 years (interquartile range, 2.2 to 4.4). A primary composite outcome event occurred in 839 of 7356 patients (11.4%; 3.7 events per 100 person-years) in the exenatide group and in 905 of 7396 patients (12.2%; 4.0 events per 100 person-years) in the placebo group (hazard ratio, 0.91; 95% confidence interval [CI], 0.83 to 1.00), with the intention-to-treat analysis indicating that exenatide, administered once weekly, was noninferior to placebo with respect to safety (P<0.001 for noninferiority) but was not superior to placebo with respect to efficacy (P=0.06 for superiority). The rates of death from cardiovascular causes, fatal or nonfatal myocardial infarction, fatal or nonfatal stroke, hospitalization for heart failure, and hospitalization for acute coronary syndrome, and the incidence of acute pancreatitis, pancreatic cancer, medullary thyroid carcinoma, and serious adverse events did not differ significantly between the two groups. CONCLUSIONS: Among patients with type 2 diabetes with or without previous cardiovascular disease, the incidence of major adverse cardiovascular events did not differ significantly between patients who received exenatide and those who received placebo. (Funded by Amylin Pharmaceuticals; EXSCEL ClinicalTrials.gov number, NCT01144338 .)