Biochemistry Research Institute of La Plata

Hemocyanins are multifunctional soluble proteins found in most mollusks and some arthropods that may turn into a phenoloxidase-like enzyme (PO) associated with innate immune functions. To expand the structural understanding of gastropod hemocyanins and their physiological implications, here we report the single-particle cryogenic electron microscopy (cryo-EM) structure of Pomacea canaliculata snail hemocyanin (PcH) at 4.4 Å resolution along with its intrinsic and proteolytically induced PO activity. PcH shows a Megathura crenulata (giant keyhole limpet) hemocyanin (KLH)-type structure with a cylindrical shape, comprising 20 protomers assembled as di-pentamers of antiparallel asymmetric dimers, organized in a D5 symmetry. Each protomer comprises eight paralogous functional units (FUs) sharing conserved structural features typical of hemocyanins. The achieved map resolution allowed delineation of interaction networks among adjacent subunits across quaternary structure tiers-dimer, pentamer of dimers (decamer), and di-pentamer of dimers (di-decamer). Additionally, we identified six N-glycosylation sites per protomer, totaling 120 glycan trees in the overall structure. Kinetic analysis of intrinsic PO activity using catechol as substrate revealed a Michaelis constant (KM) of 45.3 mm and a catalytic rate constant (kcat) of 2.87 min-1. This specific activity was enhanced by limited proteolysis using digestive and bacterial proteases. Potential protease cleavage sites were identified in silico, mapped onto the PcH model, and their accessibility assessed. Combined with molecular dynamics simulations, these findings suggest a structural basis for the PO induction mechanism. This study expands our knowledge of KLH-type hemocyanins and provides clues into their PO activation, which is triggered by endogenous and/or pathogen-associated proteases. This further underscores the role of molluscan hemocyanins in the innate immune system.

Empowerment can be thought of as a gain of power and autonomy by an unfavored group, which creates a better situation. Women in Science (WIS) face multiple barriers that ultimately affect their performance. There is an urgent need to reverse the inequalities and disadvantages that WIS face. With this aim, in May 2023, 17 WIS gathered in person for four days in a workshop entitled Empowering Women in Science. As an outcome of the workshop, we cooperatively started to draw up a list of empowerment recommendations. We continued working together for more than a year to improve this list, which we now present as the Good Practice Guide for Empowering Women in Science (GPG). The GPG contains recommendations across the individual, collective and system levels that we believe help address inequalities for WIS. The aim of this debate is twofold: a) to present the GPG and b) to use the GPG to self-evaluate the number of actions we, the authors, undertook before and after participating in the workshop. Results show that one year after ending the workshop, we doubled the number of actions performed across all categories assessed: I) identify, confront and make visible systemic barriers; II) self-promotion; III) promote other women, and IV) foster diversity, equity and inclusion. This suggests that empowerment can be fostered through formal training programs and discussion spaces. We hope that the GPG will inspire WIS across disciplines and backgrounds to: A) act towards empowerment (at the individual, collective and system level); B) embrace the notion that each of us can be part of the change that we want to see happening towards higher equity for WIS, and C) recognize that any action is better than inaction. Finally, the GPG provides a framework that will continue to be improved and expanded with the feedback of readers and users.

Astrocytes are essential for maintaining neuronal health and regulating the brain’s inflammatory environment. In this study, we developed an adeno-associated viral vector (AAV9) designed to selectively overexpress glial cell line-derived neurotrophic factor (GDNF) in astrocytes, using the astrocyte-specific GFAP promoter and TdTomato for transduction tracking. This approach yielded targeted GDNF expression in hippocampal astrocytes. Sholl analysis revealed that GDNF overexpression significantly enhanced astrocytic branching complexity and process length. Using the intracerebroventricular streptozotocin (STZ) model of neurodegeneration, we evaluated the impact of GDNF on astrocytic morphology, neuroinflammation, and hippocampal-dependent memory. Although GDNF prevented astrocytic process length reduction, it did not mitigate neuroinflammation, as evidenced by persistent microglial activation, nor did it improve deficits in the novel object recognition task. However, GDNF + STZ treated animals performed similarly as SHAM controls at exploring the goal sector at the Barnes Maze. These findings demonstrate the capacity of the AAV-GFAP-GDNF-TdTom construct to induce astrocytic branching and partially preserve memory function. They also underscore its partial therapeutic potential in a neuroinflammatory, metabolically compromised and neurodegenerative context.

Insects are the largest animal group in nature, with significant effects on ecology, human health, and indigenous flora and fauna [...].

Cardiovascular (CV) disease is the major cause of mortality. Estrogens (E) exert multiple CV and neuroprotective effects. During menopause, CV and cognitive pathologies increase dramatically. At present, it is known that E exert many of their beneficial effects through the G protein-coupled estrogen receptor (GPER). Exercise reduces the risk of developing CV diseases. Sodium/proton exchanger (NHE-1) is overexpressed in ovariectomized (OVX) rats, probably due to the increase in reactive oxidative species (ROS). Insulin-like growth factor 1 (IGF-1), the main humoral mediator of exercise, inhibits the NHE-1. We aim to explore the subcellular mechanisms involved in the heart and brain impact of physiological exercise in OVX rats. We speculate that physical training, via IGF-1, prevents the increase in ROS, improving heart and brain physiological functions during menopause. Exercise diminished cardiac ROS production and increased catalase (CAT) activity in OVX rats. In concordance, IGF-1 treatment reduces brain ROS, surely contributing to the improvement in brain behavior. Moreover, the aerobic routine was able to prevent, and IGF-1 therapy to revert, NHE-1 hyperactivity in OVX rats. Finally, our results confirm the proposed signaling pathway as IGF-1/PI3K-AKT/NO. Surprisingly, GPER inhibitor (G36) was able to abolish the IGF-1 effect, suggesting that directly or indirectly GPER is part of the IGF-1 pathway. We propose that IGF-1 is the main responsible for the protective effect of aerobic training both in the heart and brain in OVX rats. Moreover, we showed that not only it is possible to prevent but also to revert the menopause-induced NHE-1 hyperactivity by exercise/IGF-1 cascade.

<bold>Introduction:</bold> Patient characteristics affect symptom control, reliever medication (Rmed) use and exacerbation risk in asthma, but the overall impact of these potentially treatable traits is poorly understood. <bold>Aim:</bold> To evaluate how regular treatment (with fluticasone propionate [FP] monotherapy, FP-salmeterol [FP/SAL], or budesonide-formoterol [BUD/FOR]) affects personalised management of moderate-severe asthma. <bold>Methods:</bold> Simulation scenarios used models previously developed from pooled clinical trial data. Individual Rmed use patterns, symptom control levels (ACQ-5) and time to first exacerbation were predicted over 1 year. Switching patients with poor control from FP to FP/SAL or BUD/FOR was assessed, and treatment differences were tested for each endpoint. <bold>Results:</bold> Stepping up FP to FP/SAL or BUD/FOR led to significant reductions in Rmed use. Improvement in symptoms and decrease in Rmed use was less in patients with asthma history >10 years, higher baseline ACQ-5, obesity and in smokers. These patients had greater reductions in Rmed use and lower exacerbation risks with FP/SAL relative to BUD/FOR (p<0.01 for all comparisons; Figure 1). <bold>Conclusions:</bold> Treatment choice and patient baseline characteristics contribute to significant differences in immediate and long-term response. These findings support personalised management. <bold>Funding:</bold> GSK (ID 215310). <fig><object-id>erj;64/suppl_68/PA1181/F1</object-id><object-id>F1</object-id><object-id>F1</object-id><graphic></graphic></fig>