Brain Organoids to Evaluate Cellular Therapies.

Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies.

Implementation of a Mindfulness-Based Crisis Intervention for Frontline Healthcare Workers During the COVID-19 Outbreak in a Public General Hospital in Madrid, Spain.

Introduction: The COVID-19 outbreak is having an impact on the well-being of healthcare workers. Mindfulness-based interventions have shown effectiveness in reducing stress and fostering resilience and recovery in healthcare workers. There are no studies examining the feasibility of brief mindfulness-based interventions during the COVID-19 outbreak. Materials and Methods: This is an exploratory study with a post intervention assessment. We describe an on-site brief mindfulness intervention and evaluate its helpfulness, safety, and feasibility. Results: One thousand out of 7,000 (14%) healthcare workers from La Paz University Hospital in Madrid (Spain) participated in at least one session. One hundred and fifty out of 1,000 (15%) participants filled out a self-report questionnaire evaluating the helpfulness of the intervention for on-site stress reduction. Ninety two subjects (61%) participated in more than one session. Most of the participants were women (80%) with a mean age of 38.6 years. Almost half of the sample were nurses (46%). Sessions were perceived as being helpful with a mean rating of 8.4 on a scale from 0 to 10. Only 3 people (2%) reported a minor adverse effect (increased anxiety or dizziness). Discussion: Our data supports the utility, safety and feasibility of an on-site, brief mindfulness-based intervention designed to reduce stress for frontline health workers during a crisis. There is a need to continue testing this type of interventions, and to integrate emotion regulation strategies as an essential part of health workers' general training. Clinical Trial Registration number: NCT04555005.

Fostering Environmental Awareness with Smart IoT Planters in Campuses.

The decrease in the cost of sensors during the last years, and the arrival of the 5th generation of mobile technology will greatly benefit Internet of Things (IoT) innovation. Accordingly, the use of IoT in new agronomic practices might be a vital part for improving soil quality, optimising water usage, or improving the environment. Nonetheless, the implementation of IoT systems to foster environmental awareness in educational settings is still unexplored. This work addresses the educational need to train students on how to design complex sensor-based IoT ecosystems. Hence, a Project-Based-Learning approach is followed to explore multidisciplinary learning processes implementing IoT systems that varied in the sensors, actuators, microcontrollers, plants, soils and irrigation system they used. Three different types of planters were implemented, namely, hydroponic system, vertical garden, and rectangular planters. This work presents three key contributions that might help to improve teaching and learning processes. First, a holistic architecture describing how IoT ecosystems can be implemented in higher education settings is presented. Second, the results of an evaluation exploring teamwork performance in multidisciplinary groups is reported. Third, alternative initiatives to promote environmental awareness in educational contexts (based on the lessons learned) are suggested. The results of the evaluation show that multidisciplinary work including students from different expertise areas is highly beneficial for learning as well as on the perception of quality of the work obtained by the whole group. These conclusions rekindle the need to encourage work in multidisciplinary teams to train engineers for Industry 4.0 in Higher Education.

The inhibitory mechanism of Hal3 on the yeast Ppz1 phosphatase: A mutagenesis analysis.

The Ser/Thr protein phosphatase (PPase) Ppz1 is an enzyme related to the ubiquitous type-1 PPases (PP1c) but found only in fungi. It is regulated by an inhibitory subunit, Hal3, which binds to its catalytic domain. Overexpression of Ppz1 is highly toxic for yeast cells, so its de-regulation has been proposed as a target for novel antifungal therapies. While modulation of PP1c by its many regulatory subunits has been extensively characterized, the manner by which Hal3 controls Ppz1 remains unknown. We have used error-prone PCR mutagenesis to construct a library of Ppz1 variants and developed a functional assay to identify mutations affecting the binding or/and the inhibitory capacity of Hal3. We have characterized diverse Ppz1 mutated versions in vivo and in vitro and found that, although they were clearly refractory to Hal3 inhibition, none of them exhibited significant reduction in Hal3 binding. Mapping the mutations strengthened the notion that Hal3 does not interact with Ppz1 through its RVxF-like motif (found in most PP1c regulators). In contrast, the most relevant mutations mapped to a conserved α-helix region used by mammalian Inhibitor-2 to regulate PP1c. Therefore, modulation of PP1c and Ppz1 by their subunits likely differs, but could share some structural features.

Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1.

The Saccharomyces cerevisiae type 2C protein phosphatase Ptc1 is required for a wide variety of cellular functions, although only a few cellular targets have been identified. A genetic screen in search of mutations in protein kinase-encoding genes able to suppress multiple phenotypic traits caused by the ptc1 deletion yielded a single gene, MKK1, coding for a MAPK kinase (MAPKK) known to activate the cell-wall integrity (CWI) Slt2 MAPK. In contrast, mutation of the MKK1 paralog, MKK2, had a less significant effect. Deletion of MKK1 abolished the increased phosphorylation of Slt2 induced by the absence of Ptc1 both under basal and CWI pathway stimulatory conditions. We demonstrate that Ptc1 acts at the level of the MAPKKs of the CWI pathway, but only the Mkk1 kinase activity is essential for ptc1 mutants to display high Slt2 activation. We also show that Ptc1 is able to dephosphorylate Mkk1 in vitro. Our results reveal the preeminent role of Mkk1 in signaling through the CWI pathway and strongly suggest that hyperactivation of Slt2 caused by upregulation of Mkk1 is at the basis of most of the phenotypic defects associated with lack of Ptc1 function.

Functional interactions between potassium and phosphate homeostasis in Saccharomyces cerevisiae.

Maintenance of ion homeostatic mechanisms is essential for living cells, including the budding yeast Saccharomyces cerevisiae. Whereas the impact of changes in phosphate metabolism on metal ion homeostasis has been recently examined, the inverse effect is still largely unexplored. We show here that depletion of potassium from the medium or alteration of diverse regulatory pathways controlling potassium uptake, such as the Trk potassium transporters or the Pma1 H(+) -ATPase, triggers a response that mimics that of phosphate (Pi) deprivation, exemplified by accumulation of the high-affinity Pi transporter Pho84. This response is mediated by and requires the integrity of the PHO signaling pathway. Removal of potassium from the medium does not alter the amount of total or free intracellular Pi, but is accompanied by decreased ATP and ADP levels and rapid depletion of cellular polyphosphates. Therefore, our data do not support the notion of Pi being the major signaling molecule triggering phosphate-starvation responses. We also observe that cells with compromised potassium uptake cannot grow under limiting Pi conditions. The link between potassium and phosphate homeostasis reported here could explain the invasive phenotype, characteristic of nutrient deprivation, observed in potassium-deficient yeast cells.

Ptc6 is required for proper rapamycin-induced down-regulation of the genes coding for ribosomal and rRNA processing proteins in S. cerevisiae.

Ptc6 is one of the seven components (Ptc1-Ptc7) of the protein phosphatase 2C family in the yeast Saccharomyces cerevisiae. In contrast to other type 2C phosphatases, the cellular role of this isoform is poorly understood. We present here a comprehensive characterization of this gene product. Cells lacking Ptc6 are sensitive to zinc ions, and somewhat tolerant to cell-wall damaging agents and to Li(+). Ptc6 mutants are sensitive to rapamycin, albeit to lesser extent than ptc1 cells. This phenotype is not rescued by overexpression of PTC1 and mutation of ptc6 does not reproduce the characteristic genetic interactions of the ptc1 mutation with components of the TOR pathway, thus suggesting different cellular roles for both isoforms. We show here that the rapamycin-sensitive phenotype of ptc6 cells is unrelated to the reported role of Pt6 in controlling pyruvate dehydrogenase activity. Lack of Ptc6 results in substantial attenuation of the transcriptional response to rapamycin, particularly in the subset of repressed genes encoding ribosomal proteins or involved in rRNA processing. In contrast, repressed genes involved in translation are Ptc6-independent. These effects cannot be attributed to the regulation of the Sch9 kinase, but they could involve modulation of the binding of the Ifh1 co-activator to specific gene promoters.

Molecular analysis of a conditional hal3 vhs3 yeast mutant links potassium homeostasis with flocculation and invasiveness.

Yeast flocculation and invasive growth are processes of great interest in fundamental biology and also relevant in biotechnology and medicine. Hal3 and Vhs3 are moonlighting proteins acting in Saccharomyces cerevisiae both as inhibitors of the Ppz protein phosphatases and as components of a catalytic step in CoA biosynthesis. The double hal3 vhs3 mutant is not viable but, under semi-permissive conditions, the tetO:HAL3 vhs3 strain shows a flocculent phenotype, invasive growth and increased expression of the flocculin-encoding FLO11 gene. We show here that all these effects are caused by hyperactivation of Ppz1 as a result of depletion of its natural inhibitors. The evidence indicates that hyperactivation of Ppz1 would impair potassium transport through the Trk1/Trk2 transporters, thus resulting in a decrease in the intracellular pH and a subsequent increase in the levels of cAMP. Mutation of the TPK2 isoform of protein kinase A blocks the increase in FLO11 expression, and eliminates the flocculent and invasive phenotypes produced by depletion of Hal3 and Vhs3. Interestingly, mutation of RIM101 also significantly decreases FLO11 expression under these conditions. Cells lacking Trk1,2 display an invasive phenotype that is abolished by deletion of FLO8 or by increasing the potassium concentration in the medium. Therefore, our results support a model in which hyperactivation of Ppz phosphatases would result in alteration of potassium transport, activation of Tpk2 and signaling to the FLO11 promoter by means of the Flo8 transcription factor, thus modulating flocculation and invasive growth. This model highlights an unsuspected link between potassium homeostasis and these important morphogenetic events.

Protein phosphatase Z modulates oxidative stress response in fungi.

The genome of the filamentous fungus Aspergillus nidulans harbors the gene ppzA that codes for the catalytic subunit of protein phosphatase Z (PPZ), and the closely related opportunistic pathogen Aspergillus fumigatus encompasses a highly similar PPZ gene (phzA). When PpzA and PhzA were expressed in Saccharomyces cerevisiae or Schizosaccharomyces pombe they partially complemented the deleted phosphatases in the ppz1 or the pzh1 mutants, and they also mimicked the effect of Ppz1 overexpression in slt2 MAP kinase deficient S. cerevisiae cells. Although ppzA acted as the functional equivalent of the known PPZ enzymes its disruption in A. nidulans did not result in the expected phenotypes since it failed to affect salt tolerance or cell wall integrity. However, the inactivation of ppzA resulted in increased sensitivity to oxidizing agents like tert-butylhydroperoxide, menadione, and diamide. To demonstrate the general validity of our observations we showed that the deletion of the orthologous PPZ genes in other model organisms, such as S. cerevisiae (PPZ1) or Candida albicans (CaPPZ1) also caused oxidative stress sensitivity. Thus, our work reveals a novel function of the PPZ enzyme in A. nidulans that is conserved in very distantly related fungi.

The role of the Snf1 kinase in the adaptive response of Saccharomyces cerevisiae to alkaline pH stress.

Alkaline pH stress invokes a potent and fast transcriptional response in Saccharomyces cerevisiae that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and -response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. In the present study we show that the addition of glucose to the medium improves the growth of wild-type cells at high pH, fully abolishes the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr(210). Lack of Elm1, one of the three upstream Snf1 kinases (Tos3, Elm1 and Sak1), markedly increases alkali sensitivity, whereas the phenotype of the triple mutant tos3 elm1 sak1 is even more pronounced than that of snf1 cells and is poorly rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of the genes induced in the short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those encoding high-affinity hexose transporters and phosphorylating enzymes. The induction of genes encoding enzymes involved in glycogen, but not trehalose, metabolism is largely dependent of the presence of Snf1. Therefore the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium resulted in an additive effect and allows near-normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment.

Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans.

The opportunistic pathogen Candida albicans has a single protein phosphatase Z (PPZ) candidate gene termed CaPPZ1, which shows significant allele variability. We demonstrate here that bacterially expressed CaPpz1 protein exhibits phosphatase activity which can be inhibited by recombinant Hal3, a known inhibitor of Saccharomyces cerevisiae Ppz1. Site-directed mutagenesis experiments based on natural polymorphisms allowed the identification of three amino acid residues that affect enzyme activity or stability. The expression of CaPPZ1 in ppz1 S. cerevisiae and pzh1 Schizosaccharomyces pombe cells partially rescued the salt and caffeine phenotypes of the deletion mutants. CaPpz1 also complemented the slt2 S. cerevisiae mutant, which is crippled in the mitogen-activated protein (MAP) kinase that mediates the cell wall integrity signalling pathway. Collectively, our results suggest that the orthologous PPZ enzymes have similar but not identical functions in different fungi. The deletion of the CaPPZ1 gene in C. albicans resulted in a mutant that was sensitive to salts such as LiCl and KCl, to caffeine, and to agents that affect cell wall biogenesis such as Calcofluor White and Congo red, but was tolerant to spermine and hygromycin B. Reintegration of the CaPPZ1 gene into the deletion mutant alleviated all of the mutant phenotypes tested. Thus CaPpz1 is involved in cation homeostasis, cell wall integrity and the regulation of the membrane potential of C. albicans. In addition, the germ tube growth rate, and virulence in the BALB/c mouse model, were reduced in the null mutant, suggesting a novel function for CaPpz1 in the yeast to hypha transition that may have medical relevance.

The role of the protein kinase A pathway in the response to alkaline pH stress in yeast.

Exposure of Saccharomyces cerevisiae to alkaline pH provokes a stress condition that generates a compensatory reaction. In the present study we examined a possible role for the PKA (protein kinase A) pathway in this response. Phenotypic analysis revealed that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation enhances tolerance to this stress. We observed that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding with STRE (stress response element) sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alkaline stress. A msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 min of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated genome expression remodelling. However, the relevance of attenuation of PKA in high pH tolerance is probably not restricted to regulation of Msn2 function.

Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase.

The phosphatase calcineurin and the kinases Hal4/Hal5 regulate high-affinity potassium uptake in Saccharomyces cerevisiae through the Trk1 transporter. We demonstrate that calcineurin is necessary for high-affinity potassium uptake even in the absence of Na(+) stress. HAL5 expression is induced in response to stress in a calcineurin-dependent manner through a newly identified functional CDRE (nt -195/-189). Lack of calcineurin decreases Hal5 protein levels, although with little effect on Trk1 amounts. However, the growth defect of cnb1 cells at K(+)-limiting conditions can be rescued in part by overexpression of HAL5, and this mutation further aggravates the potassium requirements of a hal4 strain. This suggests that the control exerted by calcineurin on Hal5 expression may be biologically relevant for Trk1 regulation.