Globospiramine Exhibits Inhibitory and Fungicidal Effects against Candida albicans via Apoptotic Mechanisms.

Candidiasis is considered an emerging public health concern because of the occurrence of drug-resistant Candida strains and the lack of an available structurally diverse antifungal drug armamentarium. The indole alkaloid globospiramine from the anticandidal Philippine medicinal plant Voacanga globosa exhibits a variety of biological activities; however, its antifungal properties remain to be explored. In this study, we report the in vitro anticandidal activities of globospiramine against two clinically relevant Candida species (C. albicans and C. tropicalis) and the exploration of its possible target proteins using in silico methods. Thus, the colony-forming unit (CFU) viability assay revealed time- and concentration-dependent anticandidal effects of the alkaloid along with a decrease in the number of viable CFUs by almost 50% at 60 min after treatment. The results of the MIC and MFC assays indicated inhibitory and fungicidal effects of globospiramine against C. albicans (MIC = 8 µg/mL; MFC = 8 µg/mL) and potential fungistatic effects against C. tropicalis at lower concentrations (MIC = 4 µg/mL; MFC > 64 µg/mL). The FAM-FLICA poly-caspase assay showed metacaspase activation in C. albicans cells at concentrations of 16 and 8 µg/mL, which agreed well with the MIC and MFC values. Molecular docking and molecular dynamics simulation experiments suggested globospiramine to bind strongly with 1,3-ß-glucan synthase and Als3 adhesin-enzymes indirectly involved in apoptosis-driven candidal inhibition.

Globospiramine from Voacanga globosa Exerts Robust Cytotoxic and Antiproliferative Activities on Cancer Cells by Inducing Caspase-Dependent Apoptosis in A549 Cells and Inhibiting MAPK14 (p38α): In Vitro and Computational Investigations.

Bisindole alkaloids are a source of inspiration for the design and discovery of new-generation anticancer agents. In this study, we investigated the cytotoxic and antiproliferative activities of three spirobisindole alkaloids from the traditional anticancer Philippine medicinal plant Voacanga globosa, along with their mechanisms of action. Thus, the alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) showed in vitro cytotoxicity and antiproliferative activities against the tested cell lines (L929, KB3.1, A431, MCF-7, A549, PC-3, and SKOV-3) using MTT and CellTiter-Blue assays. Globospiramine (1) was also screened against a panel of breast cancer cell lines using the sulforhodamine B (SRB) assay and showed moderate cytotoxicity. It also promoted the activation of apoptotic effector caspases 3 and 7 using Caspase-Glo 3/7 and CellEvent-3/7 apoptosis assays. Increased expressions of cleaved caspase 3 and PARP in A549 cells treated with 1 were also observed. Apoptotic activity was also confirmed when globospiramine (1) failed to promote the rapid loss of membrane integrity according to the HeLa cell membrane permeability assay. Network pharmacology analysis, molecular docking, and molecular dynamics simulations identified MAPK14 (p38α), a pharmacological target leading to cancer cell apoptosis, as a putative target. Low toxicity risks and favorable drug-likeness were also predicted for 1. Overall, our study demonstrated the anticancer potentials and apoptotic mechanisms of globospiramine (1), validating the traditional medicinal use of Voacanga globosa.

Yeast oral vaccines against infectious diseases.

Vaccines that are delivered orally have several advantages over their counterparts that are administered via injection. Despite the advantages of oral delivery, however, approved oral vaccines are currently limited either to diseases that affect the gastrointestinal tract or to pathogens that have a crucial life cycle stage in the gut. Moreover, all of the approved oral vaccines for these diseases involve live-attenuated or inactivated pathogens. This mini-review summarizes the potential and challenges of yeast oral vaccine delivery systems for animal and human infectious diseases. These delivery systems utilize whole yeast recombinant cells that are consumed orally to transport candidate antigens to the immune system of the gut. This review begins with a discussion of the challenges associated with oral administration of vaccines and the distinct benefits offered by whole yeast delivery systems over other delivery systems. It then surveys the emerging yeast oral vaccines that have been developed over the past decade to combat animal and human diseases. In recent years, several candidate vaccines have emerged that can elicit the necessary immune response to provide significant protection against challenge by pathogen. They serve as proof of principle to show that yeast oral vaccines hold much promise.

Is Self-Amputation for Survival Morally Justifiable?

Are acts of self-amputation for survival morally justifiable in the Catholic tradition? I propose that a proper understanding of the principle of totality that involves a benefit/risk assessment of the impact of a surgical intervention on the well-being of the embodied person justifies such acts: A bodily part may be sacrificed to save the life of an individual person because all bodily parts are ordered towards the flourishing of that person. As such they can be sacrificed for the good of the whole, because this would be in accord with their natural end.

Interrogating COVID-19 vaccine intent in the Philippines with a nationwide open-access online survey.

To mitigate the unprecedented health, social, and economic damage of COVID-19, the Philippines is undertaking a nationwide vaccination program to mitigate the effects of the global pandemic. In this study, we interrogated COVID-19 vaccine intent in the country by deploying a nationwide open-access online survey, two months before the rollout of the national vaccination program. The Health Belief Model (HBM) posits that people are likely to adopt disease prevention behaviors and to accept medical interventions like vaccines if there is sufficient motivation and cues to action. A majority of our 7,193 respondents (62.5%) indicated that they were willing to be vaccinated against COVID-19. Moreover, multivariable analysis revealed that HBM constructs were associated with vaccination intention in the Philippines. Perceptions of high susceptibility, high severity, and significant benefits were all good predictors for vaccination intent. We also found that external cues to action were important. Large majorities of our respondents would only receive the COVID-19 vaccines after many others had received it (72.8%) or after politicians had received it (68.2%). Finally, our study revealed that most (21%) were willing to pay an amount of PHP 1,000 (USD20) for the COVID-19 vaccines with an average willing-to-pay amount of PHP1,892 (USD38).

Prudential Use of the Morally Controversial COVID-19 Vaccines.

In recent months, there has been a lot of debate surrounding the use of those COVID-19 vaccines that have been either tested or manufactured with cell lines that were isolated from the remains of an aborted fetal child. Most faithful and orthodox Catholic moral theologians, among whom I count myself, have concluded that their use is not intrinsically evil. Therefore, like every other decision that falls into the category of actions that are not intrinsically evil, the decision to be vaccinated with these morally controversial vaccines has to be governed by the virtue of prudence. It is a decision that calls for a wisdom that properly sees this action within the constellation of actions that propels the human agent to the heights of holiness. This is why prayer is so essential for authentic moral judgment. With prayer, we ask the Holy Spirit who is the all-prudent one to guide our actions so that we can choose and act well not only for our only well-being but for the well-being of all. Acts that are not themselves intrinsically evil are deemed virtuous or not within the narrative of the individual person's life.

Sulforaphane alters the acidification of the yeast vacuole.

Sulforaphane (SFN) is a compound [1-isothiocyanato-4-(methylsulfinyl)-butane] found in broccoli and other cruciferous vegetables that is currently of interest because of its potential as a chemopreventive and a chemotherapeutic drug. Recent studies in a diverse range of cellular and animal models have shown that SFN is involved in multiple intracellular pathways that regulate xenobiotic metabolism, inflammation, cell death, cell cycle progression, and epigenetic regulation. In order to better understand the mechanisms of action behind SFN-induced cell death, we undertook an unbiased genome wide screen with the yeast knockout (YKO) library to identify SFN sensitive (SFNS) mutants. The mutants were enriched with knockouts in genes linked to vacuolar function suggesting a link between this organelle and SFN's mechanism of action in yeast. Our subsequent work revealed that SFN increases the vacuolar pH of yeast cells and that varying the vacuolar pH can alter the sensitivity of yeast cells to the drug. In fact, several mutations that lower the vacuolar pH in yeast actually made the cells resistant to SFN (SFNR). Finally, we show that human lung cancer cells with more acidic compartments are also SFNR suggesting that SFN's mechanism of action identified in yeast may carry over to higher eukaryotic cells.

Metabolism-induced oxidative stress and DNA damage selectively trigger genome instability in polyploid fungal cells.

Understanding how cellular activities impact genome stability is critical to multiple biological processes including tumorigenesis and reproductive biology. The fungal pathogen Candida albicans displays striking genome dynamics during its parasexual cycle as tetraploid cells, but not diploid cells, exhibit genome instability and reduce their ploidy when grown on a glucose-rich "pre-sporulation" medium. Here, we reveal that C. albicans tetraploid cells are metabolically hyperactive on this medium with higher rates of fermentation and oxidative respiration relative to diploid cells. This heightened metabolism results in elevated levels of reactive oxygen species (ROS), activation of the ROS-responsive transcription factor Cap1, and the formation of DNA double-strand breaks. Genetic or chemical suppression of ROS levels suppresses each of these phenotypes and also protects against genome instability. These studies reveal how endogenous metabolic processes can generate sufficient ROS to trigger genome instability in polyploid C. albicans cells. We also discuss potential parallels with metabolism-induced instability in cancer cells and speculate that ROS-induced DNA damage could have facilitated ploidy cycling prior to a conventional meiosis in eukaryotes.

Guidelines and recommendations on yeast cell death nomenclature.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.

A virtue analysis of recreational marijuana use.

Several empirical studies suggest that recreational marijuana is popularly perceived as an essentially harmless rite of passage that ends as young people settle into their careers and their adult intimate relationships. Is this perception accurate? To answer this question, we evaluate the morality of recreational marijuana use from a virtue perspective guided by the theological synthesis of St. Thomas Aquinas. Since the medical data reveals that recreational marijuana use is detrimental to the well-being of the user, we conclude that it is a vicious activity, an instance of the vice of intoxication, and as such would be morally illicit. LAY SUMMARY: In contrast to its medical use, the recreational use of marijuana cannot be justified for at least three reasons. First, as scientists have amply documented, it harms the organic functioning of the human body. Second, it impedes our ability to reason and in so doing does harm to us. Finally, it has lasting detrimental effects on the user and his neighbor, even when it occurs in a casual setting. Intoxication is always contrary to the integral good of the person. Thus, the use of marijuana is never warranted even for good, non-medical reasons.

A philosophical assessment of TK's autopsy report: Implications for the debate over the brain death criteria.

In recent years, there has been increasing evidence that the totally brain-dead patient is able to continue to live and to maintain some integrated functions, albeit with the necessary assistance of mechanical ventilation. Several years ago, the autopsy report of a totally brain-dead patient named TK who was kept on life support for nearly twenty years was published in the Journal of Child Neurology. He remains the individual kept on life support the longest after suffering total brain failure. In this essay, I argue that the clinical data described in the autopsy report demonstrate that TK's long-term survival after total brain failure supports the claim acknowledged by the President's Council on Bioethics that the brain-dead patient retains his bodily integrity. As such, he is not dead. He is still a living, though severely disabled, human organism, a human person made in the image and likeness of God. LAY SUMMARY: Traditionally, the presence or absence of bodily integration has been used to definitively discern the presence or absence of life in the human being where decomposition of the body is the surest sign of death. The autopsy report of a patient named TK who was brain-dead for nearly twenty years demonstrates that brain-dead patients retain their bodily integrity. As such, TK and other brain-dead patients are not dead. They are living, though severely disabled, human organisms, who are human persons made in the image and likeness of God.

Filamentation protects Candida albicans from amphotericin B-induced programmed cell death via a mechanism involving the yeast metacaspase, MCA1.

The budding yeast Candida albicans is one of the most significant fungal pathogens worldwide. It proliferates in two distinct cell types: blastopores and filaments. Only cells that are able to transform from one cell type into the other are virulent in mouse disease models. Programmed cell death is a controlled form of cell suicide that occurs when C. albicans cells are exposed to fungicidal drugs like amphotericin B and caspofungin, and to other stressful conditions. We now provide evidence that suggests that programmed cell death is cell-type specific in yeast: Filamentous C. albicans cells are more resistant to amphotericin B- and caspofungin-induced programmed cell death than their blastospore counterparts. Finally, our genetic data suggests that this phenomenon is mediated by a protective mechanism involving the yeast metacaspase, MCA1.

The Brain Dead Patient Is Still Sentient: A Further Reply to Patrick Lee and Germain Grisez.

Patrick Lee and Germain Grisez have argued that the total brain dead patient is still dead because the integrated entity that remains is not even an animal, not only because he is not sentient but also, and more importantly, because he has lost the radical capacity for sentience. In this essay, written from within and as a contribution to the Catholic philosophical tradition, I respond to Lee and Grisez's argument by proposing that the brain dead patient is still sentient because an animal with an intact but severed spinal cord can still perceive and respond to external stimuli. The brain dead patient is an unconscious sentient organism.

Aging and cell death in the other yeasts, Schizosaccharomyces pombe and Candida albicans.

How do cells age and die? For the past 20 years, the budding yeast, Saccharomyces cerevisiae, has been used as a model organism to uncover the genes that regulate lifespan and cell death. More recently, investigators have begun to interrogate the other yeasts, the fission yeast, Schizosaccharomyces pombe, and the human fungal pathogen, Candida albicans, to determine if similar longevity and cell death pathways exist in these organisms. After summarizing the longevity and cell death phenotypes in S. cerevisiae, this mini-review surveys the progress made in the study of both aging and programed cell death (PCD) in the yeast models, with a focus on the biology of S. pombe and C. albicans. Particular emphasis is placed on the similarities and differences between the two types of aging, replicative aging, and chronological aging, and between the three types of cell death, intrinsic apoptosis, autophagic cell death, and regulated necrosis, found in these yeasts. The development of the additional microbial models for aging and PCD in the other yeasts may help further elucidate the mechanisms of longevity and cell death regulation in eukaryotes.

Deletion of AIF1 but not of YCA1/MCA1 protects Saccharomyces cerevisiae and Candida albicans cells from caspofungin-induced programmed cell death.

Caspofungin was the first member of a new class of antifungals called echinocandins to be approved by a drug regulatory authority. Like the other echinocandins, caspofungin blocks the synthesis of ß(1,3)-D-glucan of the fungal cell wall by inhibiting the enzyme, ß(1,3)-D-glucan synthase. Loss of ß(1,3)-D-glucan leads to osmotic instability and cell death. However, the precise mechanism of cell death associated with the cytotoxicity of caspofungin was unclear. We now provide evidence that Saccharomyces cerevisiae cells cultured in media containing caspofungin manifest the classical hallmarks of programmed cell death (PCD) in yeast, including the generation of reactive oxygen species (ROS), the fragmentation of mitochondria, and the production of DNA strand breaks. Our data also suggests that deleting AIF1 but not YCA1/MCA1 protects S. cerevisiae and Candida albicans from caspofungin-induced cell death. This is not only the first time that AIF1 has been specifically tied to cell death in Candida but also the first time that caspofungin resistance has been linked to the cell death machinery in yeast.

S-Adenosyl-L-methionine protects the probiotic yeast, Saccharomyces boulardii, from acid-induced cell death.

BACKGROUND: Saccharomyces boulardii is a probiotic yeast routinely used to prevent and to treat gastrointestinal disorders, including the antibiotic-associated diarrhea caused by Clostridium difficile infections. However, only 1-3% of the yeast administered orally is recovered alive in the feces suggesting that this yeast is unable to survive the acidic environment of the gastrointestinal tract. RESULTS: We provide evidence that suggests that S. boulardii undergoes programmed cell death (PCD) in acidic environments, which is accompanied by the generation of reactive oxygen species and the appearance of caspase-like activity. To better understand the mechanism of cell death at the molecular level, we generated microarray gene expression profiles of S. boulardii cells cultured in an acidic environment. Significantly, functional annotation revealed that the up-regulated genes were significantly over-represented in cell death pathways Finally, we show that S-adenosyl-L-methionine (AdoMet), a commercially available, FDA-approved dietary supplement, enhances the viability of S. boulardii in acidic environments, most likely by preventing programmed cell death. CONCLUSIONS: In toto, given the observation that many of the proven health benefits of S. boulardii are dependent on cell viability, our data suggests that taking S. boulardii and AdoMet together may be a more effective treatment for gastrointestinal disorders than taking the probiotic yeast alone.

Endoplasmic reticulum involvement in yeast cell death.

Yeast cells undergo programed cell death (PCD) with characteristic markers associated with apoptosis in mammalian cells including chromatin breakage, nuclear fragmentation, reactive oxygen species generation, and metacaspase activation. Though significant research has focused on mitochondrial involvement in this phenomenon, more recent work with both Saccharomyces cerevisiae and Schizosaccharomyces pombe has also implicated the endoplasmic reticulum (ER) in yeast PCD. This minireview provides an overview of ER stress-associated cell death (ER-SAD) in yeast. It begins with a description of ER structure and function in yeast before moving to a discussion of ER-SAD in both mammalian and yeast cells. Three examples of yeast cell death associated with the ER will be highlighted here including inositol starvation, lipid toxicity, and the inhibition of N-glycosylation. It closes by suggesting ways to further examine the involvement of the ER in yeast cell death.

Yeast Bax inhibitor, Bxi1p, is an ER-localized protein that links the unfolded protein response and programmed cell death in Saccharomyces cerevisiae.

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that Δbxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death.