A versatile yeast model identifies the pesticides cymoxanil and metalaxyl as risk factors for synucleinopathies.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of Lewy bodies, which predominantly consist of aggregated forms of the protein alpha-synuclein (aSyn). While these aggregates are a pathological hallmark of PD, the etiology of most cases remains elusive. Although environmental risk factors have been identified, such as the pesticides dieldrin and MTPT, many others remain to be assessed and their molecular impacts are underexplored. This study aimed to identify pesticides that could enhance aSyn aggregation using a humanized yeast model expressing aSyn fused to GFP as a primary screening platform, which we validated using dieldrin. We found that the pesticides cymoxanil and metalaxyl induce aggregation of aSyn in yeast, which we confirmed also occurs in a model of aSyn inclusion formation using human H4 cells. In conclusion, our approach generated invaluable molecular data on the effect of pesticides, therefore providing insights into mechanisms associated with the onset and progression of PD and other synucleinopathies.

Enhancement of Acetate-Induced Apoptosis of Colorectal Cancer Cells by Cathepsin D Inhibition Depends on Oligomycin A-Sensitive Respiration.

Colorectal cancer (CRC) is a leading cause of death worldwide. Conventional therapies are available with varying effectiveness. Acetate, a short-chain fatty acid produced by human intestinal bacteria, triggers mitochondria-mediated apoptosis preferentially in CRC but not in normal colonocytes, which has spurred an interest in its use for CRC prevention/therapy. We previously uncovered that acetate-induced mitochondrial-mediated apoptosis in CRC cells is significantly enhanced by the inhibition of the lysosomal protease cathepsin D (CatD), which indicates both mitochondria and the lysosome are involved in the regulation of acetate-induced apoptosis. Herein, we sought to determine whether mitochondrial function affects CatD apoptotic function. We found that enhancement of acetate-induced apoptosis by CatD inhibition depends on oligomycin A-sensitive respiration. Mechanistically, the potentiating effect is associated with an increase in cellular and mitochondrial superoxide anion accumulation and mitochondrial mass. Our results provide novel clues into the regulation of CatD function and the effect of tumor heterogeneity in the outcome of combined treatment using acetate and CatD inhibitors.

Acetic acid triggers cytochrome c release in yeast heterologously expressing human Bax.

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3ß. This novel system mimics human Bax α regulation by GSK3ß and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.

The PALliative MUlticenter Study in Intensive Care (PalMuSIC). Results From a Multicenter Study Addressing Frailty and Palliative Care Interventions in Intensive Care Units in Portugal.

Objective: Frailty is a clinically recognizable state of increased vulnerability common in critical medicine. When underrecognized, it may lead to invasive treatments that do not serve the patients' best interest. Our aim was to evaluate the use of both palliative care consultation and invasive interventions in frail patients admitted to Intensive Care Units in Portugal. Methods: This was a prospective, observational study. All consecutive adult patients admitted for more than 24 h, over a 15-day period were enrolled. Twenty-three Portuguese Intensive Care Units were included. Informed consent was obtained from all patients or their surrogate. The doctor and nurse in charge calculated the Clinical Frailty Score as well as the reference family member Results: A total of 335 patients were included in the study (66% male). Mean age was 63.2 ± 16.8 and SAPS II score was 41.8 ± 17.4. Mean Clinical Frailty Score value was 3.5 ± 1.7. Frailty prevalence (mean score ≥ 5) was 20.9%. Frail patients were offered organ support therapy (64,3% invasive mechanical ventilation; 24,3% renal replacement therapy; 67,1% vasopressors) more often than non-frail patients. Nevertheless, limitation of therapeutic effort or a do not resuscitate order (p < 0.001) were more common in frail patients. Mortality rate by 6 months was higher among frail patients (50% vs. 32.3%, p < 0.001). Palliative Care was offered to only 15% of frail patients (3.9% overall). Conclusions: The authors suggest that palliative care should be universally consulted once frailty is identified in critical patients.

Lactoferrin perturbs lipid rafts and requires integrity of Pma1p-lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae.

Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.

Lactate Induces Cisplatin Resistance in S. cerevisiae through a Rad4p-Dependent Process.

Cisplatin is a widely used antineoplastic agent that has DNA as the main target, though cellular resistance hampers its therapeutic efficacy. An emerging hallmark of cancer cells is their altered metabolism, characterized by increased glycolysis even under aerobic conditions, with increased lactate production (known as the Warburg effect). Although this altered metabolism often results in increased resistance to chemotherapy, it also provides an opportunity for targeted therapeutic intervention. It has been suggested that cisplatin cytotoxicity can be affected by tumor metabolism, though with varying effects. We therefore sought to better characterize how lactate affects cisplatin sensitivity in the simplified Saccharomyces cerevisiae model. We show that lactate renders yeast cells resistant to cisplatin, independently of growth rate or respiration ability. We further show that histone acetylation is not affected, but histone phosphorylation is decreased in lactate-containing media. Finally, we show that Rad4p, essential for nucleotide excision repair, is required for the observed phenotype and thus likely underlies the mechanism responsible for lactate-mediated resistance to cisplatin. Overall, understanding how lactate modulates cisplatin sensitivity will aid in the development of new strategies to overcome drug resistance.

The Role of Diet Related Short-Chain Fatty Acids in Colorectal Cancer Metabolism and Survival: Prevention and Therapeutic Implications.

Colorectal Cancer (CRC) is a major cause of cancer-related death worldwide. CRC increased risk has been associated with alterations in the intestinal microbiota, with decreased production of Short Chain Fatty Acids (SCFAs). SCFAs produced in the human colon are the major products of bacterial fermentation of undigested dietary fiber and starch. While colonocytes use the three major SCFAs, namely acetate, propionate and butyrate, as energy sources, transformed CRC cells primarily undergo aerobic glycolysis. Compared to normal colonocytes, CRC cells exhibit increased sensitivity to SCFAs, thus indicating they play an important role in cell homeostasis. Manipulation of SCFA levels in the intestine, through changes in microbiota, has therefore emerged as a potential preventive/therapeutic strategy for CRC. Interest in understanding SCFAs mechanism of action in CRC cells has increased in the last years. Several SCFA transporters like SMCT-1, MCT-1 and aquaporins have been identified as the main transmembrane transporters in intestinal cells. Recently, it was shown that acetate promotes plasma membrane re-localization of MCT-1 and triggers changes in the glucose metabolism. SCFAs induce apoptotic cell death in CRC cells, and further mechanisms have been discovered, including the involvement of lysosomal membrane permeabilization, associated with mitochondria dysfunction and degradation. In this review, we will discuss the current knowledge on the transport of SCFAs by CRC cells and their effects on CRC metabolism and survival. The impact of increasing SCFA production by manipulation of colon microbiota on the prevention/therapy of CRC will also be addressed.

Causal attributions of potentially traumatic life events in fibromyalgia patients.

AIM: In common clinical practice, potentially traumatic life events and the possible attribution of such events to fibromyalgia often go undetected. Having demonstrated in a previous study that hypnosis facilitates the exploration of such events, we now aim to explore and classify the content of these events and to determine whether there is an association between the type of event, the state of consciousness in which the patient verbalized it, and possible causal attributions of the disorder. METHOD: Thirty-two women (age 52 ± 7 years) suffering from fibromyalgia and receiving care in a primary healthcare center participated in the same semi-structured interview twice: once in a wakeful state and once under hypnosis. They answered two questions: what other events in your life coincided with the onset of fibromyalgia? Are those events related to your fibromyalgia in any way? To counteract biases related to order or recall, the order of the two interviews was randomized with a 3-month period in between. RESULTS: The patients verbalized 23 different types of events, which we classified into eight categories, mostly psychosocial events. Although the relationship between the three variables was not statistically significant, two of the bivariate associations predicted (type of event and state of consciousness; and state of consciousness and causal attribution) were confirmed (χ2 , P values of .01 and <.001 respectively). CONCLUSION: Potentially traumatic life events could remain undetected in primary healthcare consultations and hypnosis could prove a useful tool for their detection.

Proteasome inhibition prevents cell death induced by the chemotherapeutic agent cisplatin downstream of DNA damage.

Cisplatin is a highly effective chemotherapeutic drug acting as a DNA-damaging agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance still occurs. Mutations in p53 in a large fraction of tumor cells contribute to defects in apoptotic pathways and drug resistance. To uncover new strategies to eliminate tumors through a p53-independent pathway, we established a simplified model devoid of p53 to study cisplatin-induced regulated cell death, using the yeast Saccharomyces cerevisiae. We previously showed that cisplatin induces an active form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction. We further demonstrated that proteasome inhibition, either with MG132 or genetically, increased resistance to cisplatin. In this study, we sought to determine how proteasome inhibition is important for cisplatin resistance by analyzing how it affects several phenotypes associated with the DNA damage response. We found MG132 does not seem to affect the activation of the DNA damage response or increase damage tolerance. Moreover, central modulators of the DNA damage response are not required for cisplatin resistance imparted by MG132. These results suggest the proteasome is involved in modulation of cisplatin toxicity downstream of DNA damage. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.

Evaluation of a Psychological Intervention for Patients with Chronic Pain in Primary Care.

According to evidence from recent decades, multicomponent programs of psychological intervention in people with chronic pain have reached the highest levels of efficacy. However, there are still many questions left to answer since efficacy has mainly been shown among upper-middle class patients in English-speaking countries and in controlled studies, with expert professionals guiding the intervention and with a limited number of domains of painful experience evaluated. For this study, a program of multicomponent psychological intervention was implemented: (a) based on techniques with empirical evidence, but developed in Spain; (b) at a public primary care center; (c) among patients with limited financial resources and lower education; (d) by a novice psychologist; and (e) evaluating all domains of painful experience using the instruments recommended by the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT). The aim of this study was to evaluate this program. We selected a consecutive sample of 40 patients treated for chronic non-cancer pain at a primary care center in Utrera (Seville, Spain), adults who were not in any employment dispute, not suffering from psychopathology, and not receiving psychological treatment. The patients participated in 10 psychological intervention sessions, one per week, in groups of 13-14 people, which addressed psychoeducation for pain; breathing and relaxation; attention management; cognitive restructuring; problem-solving; emotional management; social skills; life values and goal setting; time organization and behavioral activation; physical exercise promotion; postural and sleep hygiene; and relapse prevention. In addition to the initial assessment, measures were taken after the intervention and at a 6-month follow-up. We assessed the program throughout the process: before, during and after the implementation. Results were analyzed statistically (significance and effect size) and from a clinical perspective (clinical significance according to IMMPACT standards). According to this analysis, the intervention was successful, although improvement tended to decline at follow-up, and the detailed design gave the program assessment a high degree of standardization and specification. Finally, suggestions for improvement are presented for upcoming applications of the program.

Colorectal cancer-related mutant KRAS alleles function as positive regulators of autophagy.

The recent interest to modulate autophagy in cancer therapy has been hampered by the dual roles of this conserved catabolic process in cancer, highlighting the need for tailored approaches. Since RAS isoforms have been implicated in autophagy regulation and mutation of the KRAS oncogene is highly frequent in colorectal cancer (CRC), we questioned whether/how mutant KRAS alleles regulate autophagy in CRC and its implications. We established two original models, KRAS-humanized yeast and KRAS-non-cancer colon cells and showed that expression of mutated KRAS up-regulates starvation-induced autophagy in both. Accordingly, KRAS down-regulation inhibited autophagy in CRC-derived cells harboring KRAS mutations. We further show that KRAS-induced autophagy proceeds via up-regulation of the MEK/ERK pathway in both colon models and that KRAS and autophagy contribute to CRC cell survival during starvation. Since KRAS inhibitors have proven difficult to develop, our results suggest using autophagy inhibitors as a combined/alternative therapeutic approach in CRCs with mutant KRAS.

RAF-1 promotes survival of thyroid cancer cells harboring RET/PTC1 rearrangement independently of ERK activation.

Thyroid cancer (TC) is frequently associated with BRAF or RAS oncogenic mutations and RET/PTC rearrangements, with aberrant RAF-MEK-ERK and/or PI3K pathway activation. BRAF underlies ERK activation in most TC cells, but not in TPC-1 cells with RET/PTC1 rearrangement. Here, we show that depletion of RAF-1, a RAF family member with a poorly defined role in TC, decreases proliferation and increases apoptosis in TPC-1 cells and, less significantly, in cells harboring a BRAF(V600E) or HRAS(G13R) mutations, but without affecting ERK activation. We further demonstrate that constitutive activation of ERKs in TPC-1 cells is not caused by mutations in 50 oncogenes and tumor suppressors prone to activate the ERK pathway, or affected by inhibition of BRAF, MEK1/2 or PI3K. Our data indicate that RAF-1 is important for the survival of TPC-1 cells independently of the classical MEK1/2-ERK activation, offering new perspectives on RET/PTC signaling and for the therapy of thyroid cancers.

Cell wall dynamics modulate acetic acid-induced apoptotic cell death of Saccharomyces cerevisiae.

Acetic acid triggers apoptotic cell death in Saccharomyces cerevisiae, similar to mammalian apoptosis. To uncover novel regulators of this process, we analyzed whether impairing MAPK signaling affected acetic acid-induced apoptosis and found the mating-pheromone response and, especially, the cell wall integrity pathways were the major mediators, especially the latter, which we characterized further. Screening downstream effectors of this pathway, namely targets of the transcription factor Rlm1p, highlighted decreased cell wall remodeling as particularly important for acetic acid resistance. Modulation of cell surface dynamics therefore emerges as a powerful strategy to increase acetic acid resistance, with potential application in industrial fermentations using yeast, and in biomedicine to exploit the higher sensitivity of colorectal carcinoma cells to apoptosis induced by acetate produced by intestinal propionibacteria.

Genome-wide identification of genes involved in the positive and negative regulation of acetic acid-induced programmed cell death in Saccharomyces cerevisiae.

BACKGROUND: Acetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional analysis of a yeast Euroscarf knock-out mutant collection. RESULTS: The screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function, transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were found, validating the approach developed. Metabolism stood out as a main regulator of this process, since impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved developmental role of programmed cell death in unicellular eukaryotes. CONCLUSIONS: This work advanced the characterization of acetic acid-induced PCD, providing a wealth of new information on putative molecular targets for its control with impact both in biotechnology and biomedicine.

C2-phytoceramide perturbs lipid rafts and cell integrity in Saccharomyces cerevisiae in a sterol-dependent manner.

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.

Cisplatin-induced cell death in Saccharomyces cerevisiae is programmed and rescued by proteasome inhibition.

Cisplatin is a highly effective chemotherapeutic drug used in the treatment of several tumors. It is a DNA-damaging agent that induces apoptosis of rapidly proliferating cells, an important factor underlying its therapeutic efficacy. Unfortunately, cellular resistance occurs often. A large fraction of tumor cells harbor mutations in p53, contributing to defects in apoptotic pathways and drug resistance. However, cisplatin-induced apoptosis can also occur in p53 deficient cells; thus, elucidation of the molecular mechanism involved will potentially yield new strategies to eliminate tumors that have defects in the p53 pathway. Most of the studies in this field have been conducted in cultured mammalian cells, not amenable to systematic genetic manipulation. Therefore, we aimed to establish a simplified model devoid of a p53 ortholog to study cisplatin-induced programmed cell death (PCD), using the yeast Saccharomyces cerevisiae. Our results indicate cisplatin induces an active form of cell death in yeast, as this process was partially dependent on de novo protein synthesis and did not lead to loss of membrane integrity. Cisplatin also increased DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction other than partial fragmentation. Co-incubation with the proteasome inhibitor MG132 increased resistance to cisplatin and, accordingly, yeast strains deficient in proteasome activity were more resistant to cisplatin than wild-type strains. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Our results indicate inhibition of the proteasome protects budding yeast from cisplatin-induced cell death and validate yeast as a model to study the role of the proteasome in cisplatin-induced PCD. Elucidation of this mechanism will aid in the development of new strategies to increase the efficacy of chemotherapy.

Cyclin-dependent kinase-associated proteins Cks1 and Cks2 are essential during early embryogenesis and for cell cycle progression in somatic cells.

Cks proteins associate with cyclin-dependent kinases and have therefore been assumed to play a direct role in cell cycle regulation. Mammals have two paralogs, Cks1 and Cks2, and individually deleting the gene encoding either in the mouse has previously been shown not to impact viability. In this study we show that simultaneously disrupting CKS1 and CKS2 leads to embryonic lethality, with embryos dying at or before the morula stage after only two to four cell division cycles. RNA interference (RNAi)-mediated silencing of CKS genes in mouse embryonic fibroblasts (MEFs) or HeLa cells causes cessation of proliferation. In MEFs CKS silencing leads to cell cycle arrest in G(2), followed by rereplication and polyploidy. This phenotype can be attributed to impaired transcription of the CCNB1, CCNA2, and CDK1 genes, encoding cyclin B1, cyclin A, and Cdk1, respectively. Restoration of cyclin B1 expression rescues the cell cycle arrest phenotype conferred by RNAi-mediated Cks protein depletion. Consistent with a direct role in transcription, Cks2 is recruited to chromatin in general and to the promoter regions and open reading frames of genes requiring Cks function with a cell cycle periodicity that correlates with their transcription.