Correction: Whose shoulders is health research standing on? Determining the key actors and contents of the prevailing biomedical research agenda.

[This corrects the article DOI: 10.1371/journal.pone.0249661.].

Academic dependency: the influence of the prevailing international biomedical research agenda on Argentina's CONICET.

Background: The prevailing health and biomedical sciences (HBMS) research agenda, not only determined by leading academic institutions but also by large pharmaceutical companies, has been shown to prioritize the exploration of novel pharmacological interventions over the study of the socio-environmental factors influencing illness onset and progression. The aim of this investigation is to quantitatively explore whether and to what extent the prevailing international HBMS research agenda and the key actors setting this agenda influence research in non-core countries. Methods: We used the Web of Science database and the CorText platform to proxy the HBMS research agenda of a prestigious research institution from Latin America: Argentina's National Research Council (CONICET). We conducted a bibliometric and lexical analysis of 16,309 HBMS academic articles whereby CONICET was among the authors' affiliations. The content of CONICET's agenda was represented through co-occurrence network maps of the most frequent concatenation of terms found in titles, keywords, and abstracts. We compared our findings with previous reports on the international HBMS research agenda. Results: In line with the results previously reported for the prevailing international agenda, we found that terms linked to molecular biology and cancer research hegemonize CONICET's HBMS research agenda, whereas terms connecting HBMS research with socio-environmental cues are marginal. However, we also found differences with the international agenda: CONICET's HBMS agenda shows a marginal presence of terms linked to translational medicine, while terms associated with categories such as pathogens, plant research, agrobiotechnology, and food industry are more represented than in the prevailing agenda. Conclusions: CONICET's HBMS research agenda shares topics, priorities, and methodologies with the prevailing HBMS international research agenda. However, CONICET's HBMS research agenda is internally heterogeneous, appearing to be mostly driven by a combination of elements that not only reflect academic dependency (the adoption of the prevailing research agenda by non-core research institutions) but also local economic determinants associated with Argentina's place in the international division of labor as an exporter of primary goods.

Live and let die: signaling AKTivation and UPRegulation dynamics in SARS-CoVs infection and cancer.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the coronavirus disease 2019 (COVID-19) pandemic. Of particular interest for this topic are the signaling cascades that regulate cell survival and death, two opposite cell programs whose control is hijacked by viral infections. The AKT and the Unfolded Protein Response (UPR) pathways, which maintain cell homeostasis by regulating these two programs, have been shown to be deregulated during SARS-CoVs infection as well as in the development of cancer, one of the most important comorbidities in relation to COVID-19. Recent evidence revealed two way crosstalk mechanisms between the AKT and the UPR pathways, suggesting that they might constitute a unified homeostatic control system. Here, we review the role of the AKT and UPR pathways and their interaction in relation to SARS-CoV-2 infection as well as in tumor onset and progression. Feedback regulation between AKT and UPR pathways emerges as a master control mechanism of cell decision making in terms of survival or death and therefore represents a key potential target for developing treatments for both viral infection and cancer. In particular, drug repositioning, the investigation of existing drugs for new therapeutic purposes, could significantly reduce time and costs compared to de novo drug discovery.

Dynamic Distribution of ASIC1a Channels and Other Proteins within Cells Detected through Fractionation.

Proteins in eukaryotic cells reside in different cell compartments. Many studies require the specific localization of proteins and the detection of any dynamic changes in intracellular protein distribution. There are several methods available for this purpose that rely on the fractionation of the different cell compartments. Fractionation protocols have evolved since the first use of a centrifuge to isolate organelles. In this study, we described a simple method that involves the use of a tabletop centrifuge and different detergents to obtain cell fractions enriched in cytosolic (Cyt), plasma membrane (PM), membranous organelle (MO), and nuclear (Nu) proteins and identify the proteins in each fraction. This method serves to identify transmembrane proteins such as channel subunits as well as PM-embedded or weakly associated proteins. This protocol uses a minute amount of cell material and typical equipment present in laboratories, and it takes approximately 3 h. The process was validated using endogenous and exogenous proteins expressed in the HEK293T cell line that were targeted to each compartment. Using a specific stimulus as a trigger, we showed and quantified the shuttling of a protein channel (ASIC1a, acid sensing ion channel) from the MO fraction to the PM fraction and the shuttling of a kinase from a cytosolic location to a nuclear location.

Correction: Whose shoulders is health research standing on? Determining the key actors and contents of the prevailing biomedical research agenda.

[This corrects the article DOI: 10.1371/journal.pone.0249661.].

Whose shoulders is health research standing on? Determining the key actors and contents of the prevailing biomedical research agenda.

BACKGROUND: Conflicts of interest in biomedical research can influence research results and drive research agendas away from public health priorities. Previous agenda-setting studies share two shortfalls: they only account for direct connections between academic institutions and firms, as well as potential bias based on researchers' personal beliefs. This paper's goal is to determine the key actors and contents of the prevailing health and biomedical sciences (HBMS) research agenda, overcoming these shortfalls. METHODS: We performed a bibliometric and lexical analysis of 95,415 scientific articles published between 1999 and 2018 in the highest impact factor journals within HBMS, using the Web of Science database and the CorText platform. HBMS's prevailing knowledge network of institutions was proxied with network maps where nodes represent affiliations and edges the most frequent co-authorships. The content of the prevailing HBMS research agenda was depicted through network maps of prevalent multi-terms found in titles, keywords, and abstracts. RESULTS: The HBMS research agendas of large private firms and leading academic institutions are intertwined. The prevailing HBMS agenda is mostly based on molecular biology (40% of the most frequent multi-terms), with an inclination towards cancer and cardiovascular research (15 and 8% of the most frequent multi-terms, respectively). Studies on pathogens and biological vectors related to recent epidemics are marginal (1% of the most frequent multi-terms). Content of the prevailing HBMS research agenda prioritizes research on pharmacological intervention over research on socio-environmental factors influencing disease onset or progression and overlooks, among others, the study of infectious diseases. CONCLUSIONS: Pharmaceutical corporations contribute to set HBMS's prevailing research agenda, which is mainly focused on a few diseases and research topics. A more balanced research agenda, together with epistemological approaches that consider socio-environmental factors associated with disease spreading, could contribute to being better prepared to prevent and treat more diverse pathologies and to improve overall health outcomes.

Akt Is S-Palmitoylated: A New Layer of Regulation for Akt.

The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and 3H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation in vitro. These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death.

Cytoplasmic Expression of the ALS/FTD-Related Protein TDP-43 Decreases Global Translation Both in vitro and in vivo.

TDP-43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of TDP-43 in mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models overexpressing a cytoplasmic form of human TDP-43 (TDP-43-ΔNLS), recapitulating ALS/FTD features. We applied in HEK293 cells a method for labeling de novo translation, surface sensing of translation (SUnSET), based on puromycin (PURO) incorporation. While control cells displayed robust puromycilation, TDP-43-ΔNLS transfected cells exhibited reduced ongoing protein synthesis. Next, by using a transgenic mouse overexpressing cytoplasmic TDP-43 in the forebrain (TDP-43-ΔNLS mice) we assessed whether cytoplasmic TDP-43 regulates global translation in vivo. Polysome profiling of brain cortices from transgenic mice showed a shift toward non-polysomal fractions as compared to wild-type littermates, indicating a decrease in global translation. Lastly, cellular level translational assessment by SUNSET was performed in TDP-43-ΔNLS mice brain slices. Control mice slices incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP-43 staining. Neurons in TDP-43-ΔNLS mice slices incubated with PURO exhibited high cytoplasmic expression of TDP-43 and reduced puromycilation respect to control mice. These in vitro and in vivo results indicate that cytoplasmic TDP-43 decreases global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. Our study provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP-43 in the regulation of global mRNA translation, with implications for TDP-43 proteinopathies.

A new tool to sense pH changes at the neuromuscular junction synaptic cleft.

Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxin derived from the snake Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.

Relationship between activity and stability: Design and characterization of stable variants of human frataxin.

The relationships between conformational dynamics, stability and protein function are not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex dedicated to the iron-sulfur (Fe-S) cluster assembly within the mitochondrial matrix. In humans, the loss of FXN expression or a decrease in its functionality results in Friedreich's Ataxia, a cardio-neurodegenerative disease. Recently, the way in which FXN interacts with the rest of the subunits of the supercomplex was uncovered. This opens a window to explore relationships between structural dynamics and function. In this study, we prepared a set of FXN variants spanning a broad range of conformational stabilities. Variants S160I, S160M and A204R were more stable than the wild-type and showed similar biological activity. Additionally, we prepared SILCAR, a variant that combines S160I, L203C and A204R mutations. SILCAR was 2.4 kcal mol-1 more stable and equally active. Some of the variants were significantly more resistant to proteolysis than the wild-type FXN. SILCAR showed the highest resistance, suggesting a more rigid structure. It was corroborated by means of molecular dynamics simulations. Relaxation dispersion NMR experiments comparing SILCAR and wild-type variants suggested similar internal motions in the microsecond to millisecond timescale. Instead, variant S157I showed higher denaturation resistance but a significant lower function, similarly to that observed for the FRDA variant N146K. We concluded that the contribution of particular side chains to the conformational stability of FXN might be highly subordinated to their impact on both the protein function and the stability of the functional supercomplex.

Yeast GPCR signaling reflects the fraction of occupied receptors, not the number.

According to receptor theory, the effect of a ligand depends on the amount of agonist-receptor complex. Therefore, changes in receptor abundance should have quantitative effects. However, the response to pheromone in Saccharomyces cerevisiae is robust (unaltered) to increases or reductions in the abundance of the G-protein-coupled receptor (GPCR), Ste2, responding instead to the fraction of occupied receptor. We found experimentally that this robustness originates during G-protein activation. We developed a complete mathematical model of this step, which suggested the ability to compute fractional occupancy depends on the physical interaction between the inhibitory regulator of G-protein signaling (RGS), Sst2, and the receptor. Accordingly, replacing Sst2 by the heterologous hsRGS4, incapable of interacting with the receptor, abolished robustness. Conversely, forcing hsRGS4:Ste2 interaction restored robustness. Taken together with other results of our work, we conclude that this GPCR pathway computes fractional occupancy because ligand-bound GPCR-RGS complexes stimulate signaling while unoccupied complexes actively inhibit it. In eukaryotes, many RGSs bind to specific GPCRs, suggesting these complexes with opposing activities also detect fraction occupancy by a ratiometric measurement. Such complexes operate as push-pull devices, which we have recently described.

Akt/PKB: one kinase, many modifications.

Akt/PKB, a serine/threonine kinase member of the AGC family of proteins, is involved in the regulation of a plethora of cellular processes triggered by a wide diversity of extracellular signals and is thus considered a key signalling molecule in higher eukaryotes. Deregulation of Akt signalling is associated with a variety of human diseases, revealing Akt-dependent pathways as an attractive target for therapeutic intervention. Since its discovery in the early 1990s, a large body of work has focused on Akt phosphorylation of two residues, Thr308 and Ser473, and modification of these two sites has been established as being equivalent to Akt activation. More recently, Akt has been identified as a substrate for many different post-translational modifications, including not only phosphorylation of other residues, but also acetylation, glycosylation, oxidation, ubiquitination and SUMOylation. These modifications could provide additional regulatory steps for fine-tuning Akt function, Akt trafficking within the cell and/or for determining the substrate specificity of this signalling molecule. In the present review, we provide an overview of these different post-translational modifications identified for Akt, focusing on their consequences for this kinase activity.

Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells.

The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative splicing in an Argonaute-1 (AGO1)-dependent manner. Here we used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to investigate the genome-wide distribution of AGO1 nuclear targets. Unexpectedly, we found that about 80% of AGO1 clusters are associated with cell-type-specific transcriptional enhancers, most of them (73%) overlapping active enhancers. This association seems to be mediated by long, rather than short, enhancer RNAs and to be more prominent in intragenic, rather than intergenic, enhancers. Paradoxically, crossing ChIP-seq with RNA-seq data upon AGO1 depletion revealed that enhancer-bound AGO1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing, which was confirmed by an individual gene analysis explaining how AGO1 controls inclusion levels of the cassette exon 107 in the SYNE2 gene.

Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle.

Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G1/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase.

Modulation of the Akt pathway reveals a novel link with PERK/eIF2α, which is relevant during hypoxia.

The unfolded protein response (UPR) and the Akt signaling pathway share several regulatory functions and have the capacity to determine cell outcome under specific conditions. However, both pathways have largely been studied independently. Here, we asked whether the Akt pathway regulates the UPR. To this end, we used a series of chemical compounds that modulate PI3K/Akt pathway and monitored the activity of the three UPR branches: PERK, IRE1 and ATF6. The antiproliferative and antiviral drug Akt-IV strongly and persistently activated all three branches of the UPR. We present evidence that activation of PERK/eIF2α requires Akt and that PERK is a direct Akt target. Chemical activation of this novel Akt/PERK pathway by Akt-IV leads to cell death, which was largely dependent on the presence of PERK and IRE1. Finally, we show that hypoxia-induced activation of eIF2α requires Akt, providing a physiologically relevant condition for the interaction between Akt and the PERK branch of the UPR. These data suggest the UPR and the Akt pathway signal to one another as a means of controlling cell fate.

Involvement of hnRNP A1 in the matrix metalloprotease-3-dependent regulation of Rac1 pre-mRNA splicing.

Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57-nucleotide exon 3b. Rac1b was originally identified through its over-expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signaling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase-3 (MMP-3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP-3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP-3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer.