Patient-specific finite element analysis of heart failure and the impact of surgical intervention in pulmonary hypertension secondary to mitral valve disease.

Pulmonary hypertension (PH), a chronic and complex medical condition affecting 1% of the global population, requires clinical evaluation of right ventricular maladaptation patterns under various conditions. A particular challenge for clinicians is a proper quantitative assessment of the right ventricle (RV) owing to its intimate coupling to the left ventricle (LV). We, thus, proposed a patient-specific computational approach to simulate PH caused by left heart disease and its main adverse functional and structural effects on the whole heart. Information obtained from both prospective and retrospective studies of two patients with severe PH, a 72-year-old female and a 61-year-old male, is used to present patient-specific versions of the Living Heart Human Model (LHHM) for the pre-operative and post-operative cardiac surgery. Our findings suggest that before mitral and tricuspid valve repair, the patients were at risk of right ventricular dilatation which may progress to right ventricular failure secondary to their mitral valve disease and left ventricular dysfunction. Our analysis provides detailed evidence that mitral valve replacement and subsequent chamber pressure unloading are associated with a significant decrease in failure risk post-operatively in the context of pulmonary hypertension. In particular, right-sided strain markers, such as tricuspid annular plane systolic excursion (TAPSE) and circumferential and longitudinal strains, indicate a transition from a range representative of disease to within typical values after surgery. Furthermore, the wall stresses across the RV and the interventricular septum showed a notable decrease during the systolic phase after surgery, lessening the drive for further RV maladaptation and significantly reducing the risk of RV failure.

Sex as an important factor in nanomedicine.

Nanomedicine has demonstrated substantial potential to improve the quality and efficacy of healthcare systems. Although the promise of nanomedicine to transform conventional medicine is evident, significant numbers of therapeutic nanomedicine products have failed in clinical trials. Most studies in nanomedicine have overlooked several important factors, including the significance of sex differences at various physiological levels. This report attempts to highlight the importance of sex in nanomedicine at cellular and molecular level. A more thorough consideration of sex physiology, among other critical variations (e.g., health status of individuals), would enable researchers to design and develop safer and more-efficient sex-specific diagnostic and therapeutic nanomedicine products.

NKTR-255, a novel polymer-conjugated rhIL-15 with potent antitumor efficacy.

BACKGROUND: NKTR-255 is a novel polyethylene glycol-conjugate of recombinant human interleukin-15 (rhIL-15), which was designed to retain all known receptor binding interactions of the IL-15 molecule. We explored the biologic and pharmacologic differences between endogenous IL-15 receptor α (IL-15Rα)-dependent (NKTR-255 and rhIL-15) and IL-15Rα-independent (precomplexed rhIL-15/IL-15Rα) cytokines. METHODS: In vitro pharmacological properties of rhIL-15, NKTR-255 and precomplex cytokines (rhIL-15/IL-15Rα and rhIL-15 N72D/IL-15Rα Fc) were investigated in receptor binding, signaling and cell function. In vivo pharmacokinetic (PK) and pharmacodynamic profile of the cytokines were evaluated in normal mice. Finally, immunomodulatory effect and antitumor activity were assessed in a Daudi lymphoma model. RESULTS: NKTR-255 and rhIL-15 exhibited similar in vitro properties in receptor affinity, signaling and leukocyte degranulation, which collectively differed from precomplexed cytokines. Notably, NKTR-255 and rhIL-15 stimulated greater granzyme B secretion in human peripheral blood mononuclear cells versus precomplexed cytokines. In vivo, NKTR-255 exhibited a PK profile with reduced clearance and a longer half-life relative to rhIL-15 and demonstrated prolonged IL-15R engagement in lymphocytes compared with only transient engagement observed for rhIL-15 and precomplexed rhIL-15 N72D/IL-15Rα Fc. As a consequent, NKTR-255 provided a durable and sustained proliferation and activation of natural killer (NK) and CD8+ T cells. Importantly, NKTR-255 is more effective than the precomplexed cytokine at inducing functionally competent, cytotoxic NK cells in the tumor microenvironment and the properties of NKTR-255 translated into superior antitumor activity in a B-cell lymphoma model versus the precomplexed cytokine. CONCLUSIONS: Our results show that the novel immunotherapeutic, NKTR-255, retains the full spectrum of IL-15 biology, but with improved PK properties, over rhIL-15. These findings support the ongoing phase 1 first-in-human trial (NCT04136756) of NKTR-255 in participants with relapsed or refractory hematologic malignancies, potentially advancing rhIL-15-based immunotherapies for the treatment of cancer.

Nanoscale characterization of the biomolecular corona by cryo-electron microscopy, cryo-electron tomography, and image simulation.

The biological identity of nanoparticles (NPs) is established by their interactions with a wide range of biomolecules around their surfaces after exposure to biological media. Understanding the true nature of the biomolecular corona (BC) in its native state is, therefore, essential for its safe and efficient application in clinical settings. The fundamental challenge is to visualize the biomolecules within the corona and their relationship/association to the surface of the NPs. Using a synergistic application of cryo-electron microscopy, cryo-electron tomography, and three-dimensional reconstruction, we revealed the unique morphological details of the biomolecules and their distribution/association with the surface of polystyrene NPs at a nanoscale resolution. The analysis of the BC at a single NP level and its variability among NPs in the same sample, and the discovery of the presence of nonspecific biomolecules in plasma residues, enable more precise characterization of NPs, improving predictions of their safety and efficacies.

Gold Nano/Micro-Islands Overcome the Molecularly Imprinted Polymer Limitations to Achieve Ultrasensitive Protein Detection.

Here, we report on an electrochemical biosensor based on core-shell structure of gold nano/micro-islands (NMIs) and electropolymerized imprinted ortho-phenylenediamine (o-PD) for detection of heart-fatty acid binding protein (H-FABP). The shape and distribution of NMIs (the core) were tuned by controlled electrodeposition of gold on a thin layer of electrochemically reduced graphene oxide (ERGO). NMIs feature a large active surface area to achieve a low detection limit (2.29 fg mL-1, a sensitivity of 1.34 × 1013 µA mM-1) and a wide linear range of detection (1 fg mL-1 to 100 ng mL-1) in PBS. Facile template H-FABP removal from the layer (the shell) in less than 1 min, high specificity against interference from myoglobin and troponin T, great stability at ambient temperature, and rapidity in detection of H-FABP (approximately 30 s) are other advantages of this biomimetic biosensor. The electrochemical measurements in human serum, human plasma, and bovine serum showed acceptable recovery (between 91.1 ± 1.7 and 112.9 ± 2.1%) in comparison with the ELISA method. Moreover, the performance of the biosensor in clinical serum showed lower detection time and limit of detection against lateral flow assay (LFA) rapid test kits, as a reference method. Ultimately, the proposed biosensor based on the core-shell structure of gold NMIs and MIP opens interesting avenues in the detection of proteins with low cost, high sensitivity and significantstability for clinical applications.

Contribution of osteocalcin-mimetic peptide enhances osteogenic activity and extracellular matrix mineralization of human osteoblast-like cells.

A natural peptide motif in the first helix of osteocalcin (OCN) is used to promote nucleation and crystallization of hydroxyapatite (HA) in hard tissue. The capability of osteocalcin mimetic peptides to induce osteogenic activity of osteoblast cells leading to in-vitro mineralization is demonstrated. An osteocalcin-derived peptide consisting of thirteen amino acids is synthesized in both acidic (OSC) and amidic (OSN) forms and added into the human osteoblast-like cells (MG63) culture. The viability, proliferation, alkaline phosphatase activity, HA deposition and osteogenic gene expression by osteoblast cells are evaluated. It is revealed that the addition of 100 µg/ml of peptides enhances the proliferation rate and total protein content of osteoblast cells. Alkaline phosphatase activity is significantly higher in the presence of peptides which in turn stimulated RNA expression of collagen type I and osteopontin in a phosphate-dependent manner. Alizarin red staining and calcium content measurement show that mineral deposition is considerably increased. Ultrastructural characterization of MG63 cultures confirms the crystalline nature and chemical composition of HA mineral formation in the presence of peptides. It is confirmed that the osteocalcin-derived peptide, particularly in amidic form (OSN), is able to act as a bioactive inducer of mineralization process and hence accelerating bone tissue regeneration.

Effect of Cell Sex on Uptake of Nanoparticles: The Overlooked Factor at the Nanobio Interface.

Cellular uptake of nanoparticles (NPs) depends on the nature of the nanobio system including the solid nanocomponents ( e. g., physicochemical properties of NPs), nanobio interfaces ( e. g., protein corona composition), and the cellular characteristics ( e. g., cell type). In this study, we document the role of sex in cellular uptake of NPs as an "overlooked" factor in nanobio interface investigations. We demonstrate that cell sex leads to differences in NP uptake between male and female human amniotic stem cells (hAMSCs), with greater uptake by female cells. hAMSCs are one of the earliest sources of somatic stem cells. The experiments were replicated with primary fibroblasts isolated from the salivary gland of adult male and female donors of similar ages, and again the extent of NP uptake was altered by cell sex. However, in contrast to hAMSCs, uptake was greater in male cells. We also found out that female versus male amniotic stem cells exhibited different responses to reprogramming into induced pluripotent stem cells (iPSCs) by the Yamanaka factors. Thus, future studies should consider the effect of sex on the nanobio interactions to optimize clinical translation of NPs and iPSC biology and to help researchers to better design and produce safe and efficient therapeutic sex-specific NPs.

Sensing of Alzheimer's Disease and Multiple Sclerosis Using Nano-Bio Interfaces.

It is well understood that patients with different diseases may have a variety of specific proteins (e.g., type, amount, and configuration) in their plasmas. When nanoparticles (NPs) are exposed to these plasmas, the resulting coronas may incorporate some of the disease-specific proteins. Using gold (Au) NPs with different surface properties and corona composition, we have developed a technology for the discrimination and detection of two neurodegenerative diseases, Alzheimer's disease (AD) and multiple sclerosis (MS). Applying a variety of techniques, including UV-visible spectra, colorimetric response analyses and liquid chromatography-tandem mass spectrometry, we found the corona-NP complexes, obtained from different human serums, had distinct protein composition, including some specific proteins that are known as AD and MS biomarkers. The colorimetric responses, analyzed by chemometrics and statistical methods, demonstrate promising capabilities of the technology to unambiguously identify and discriminate AD and MS. The developed colorimetric technology might enable a simple, inexpensive and rapid detection/discrimination of neurodegenerative diseases.

A Flow Perfusion Bioreactor System for Vocal Fold Tissue Engineering Applications.

The human vocal folds (VFs) undergo complex biomechanical stimulation during phonation. The aim of the present study was to develop and validate a phono-mimetic VF flow perfusion bioreactor, which mimics the mechanical microenvironment of the human VFs in vitro. The bioreactor uses airflow-induced self-oscillations, which have been shown to produce mechanical loading and contact forces that are representative of human phonation. The bioreactor consisted of two synthetic VF replicas within a silicone body. A cell-scaffold mixture (CSM) consisting of human VF fibroblasts, hyaluronic acid, gelatin, and a polyethylene glycol cross-linker was injected into cavities within the replicas. Cell culture medium (CCM) was perfused through the scaffold by using a customized secondary flow loop. After the injection, the bioreactor was operated with no stimulation over a 3-day period to allow for cell adaptation. Phonation was subsequently induced by using a variable speed centrifugal blower for 2 h each day over a period of 4 days. A similar bioreactor without biomechanical stimulation was used as the nonphonatory control. The CSM was harvested from both VF replicas 7 days after the injection. The results confirmed that the phono-mimetic bioreactor supports cell viability and extracellular matrix proteins synthesis, as expected. Many scaffold materials were found to degrade because of challenges from phonation-induced biomechanical stimulation as well as due to biochemical reactions with the CCM. The bioreactor concept enables future investigations of the effects of different phonatory characteristics, that is, voice regimes, on the behavior of the human VF cells. It will also help study the long-term functional outcomes of the VF-specific biomaterials before animal and clinical studies.

Investigation of the Viability, Adhesion, and Migration of Human Fibroblasts in a Hyaluronic Acid/Gelatin Microgel-Reinforced Composite Hydrogel for Vocal Fold Tissue Regeneration.

The potential use of a novel scaffold biomaterial consisting of cross-linked hyaluronic acid (HA)-gelatin (Ge) composite microgels is investigated for use in treating vocal fold injury and scarring. Cell adhesion integrins and kinematics of cell motion are investigated in 2D and 3D culture conditions, respectively. Human vocal fold fibroblast (hVFF) cells are seeded on HA-Ge microgels attached to a HA hydrogel thin film. The results show that hVFF cells establish effective adhesion to HA-Ge microgels through the ubiquitous expression of ß1 integrin in the cell membrane. The microgels are then encapsulated in a 3D HA hydrogel for the study of cell migration. The cells within the HA-Ge microgel-reinforced composite hydrogel (MRCH) scaffold have an average motility speed of 0.24 ± 0.08 µm min(-1) . The recorded microscopic images reveal features that are presumably associated with lobopodial and lamellipodial cell migration modes within the MRCH scaffold. Average cell speed during lobopodial migration is greater than that during lamellipodial migration. The cells move faster in the MRCH than in the HA-Ge gel without microgels. These findings support the hypothesis that HA-Ge MRCH promotes cell adhesion and migration; thereby they constitute a promising biomaterial for vocal fold repair.

Inhibition of stress mediated cell death by human lactate dehydrogenase B in yeast.

We report the identification of human L- lactate dehydrogenase B (LDHB) as a novel Bax suppressor. Yeast heterologously expressing LDHB is also resistant to the lethal effects of copper indicating that it is a general suppressor of stress mediated cell death. To identify potential LDHB targets, LDHB was expressed in yeast mutants defective in apoptosis, necrosis and autophagy. The absence of functional PCD regulators including MCA1, YBH3, cyclophilin (CPR3) and VMA3, as well as the absence of the pro-survival autophagic pathway (ATG1,7) did not interfere with the LDHB mediated protection against copper indicating that LDHB functions independently of known PCD regulators or by simply blocking or stimulating a common PCD promoting or inhibitory pathway. Measurements of lactate levels revealed that short-term copper stress (1.6 mM, 4 h), does not increase intracellular levels of lactate, instead a three-fold increase in extracellular lactate was observed. Thus, yeast cells resemble mammalian cells where different stresses are known to lead to increased lactate production leading to lactic acidosis. In agreement with this, we found that the addition of exogenous lactic acid to growth media was sufficient to induce cell death that could be inhibited by the expression of LDHB. Taken together our results suggest that lactate dehydrogenase is a general suppressor of PCD in yeast.

Crucial role of the protein corona for the specific targeting of nanoparticles.

AIMS: We aimed to investigate the physicochemical effects of superparamagnetic iron oxide nanoparticles (SPIONs) on the composition of the protein corona and their correspondence toxicological issues. MATERIALS & METHODS: SPIONs of different sizes and surface charges were exposed to fetal bovine serum. The structure/composition and biological effects of the protein corona-SPION complexes were probed. RESULTS & DISCUSSION: The affinity and level of adsorption of specific proteins is strongly dependent on the size and surface charge of the SPIONs. In vivo experiments on the mouse blood-brain barrier model revealed that nontargeted SPIONs containing specific proteins will enter the brain endothelial barrier cells. CONCLUSION: Some commercially available nanoparticles used for target-specific applications may have unintended uptake in the body (e.g., brain tissue) with potential cytotoxity.

Human Thyroid Cancer-1 (TC-1) is a vertebrate specific oncogenic protein that protects against copper and pro-apoptotic genes in yeast.

The human Thyroid Cancer-1 (hTC-1) protein, also known as C8orf4 was initially identified as a gene that was up-regulated in human thyroid cancer. Here we show that hTC-1 is a peptide that prevents the effects of over-expressing Bax in yeast. Analysis of the 106 residues of hTC-1 in available protein databases revealed direct orthologues in jawed-vertebrates, including mammals, frogs, fish and sharks. No TC-1 orthologue was detected in lower organisms, including yeast. Here we show that TC-1 is a general pro-survival peptide since it prevents the growth- and cell death-inducing effects of copper in yeast. Human TC-1 also prevented the deleterious effects that occur due to the over-expression of a number of key pro-apoptotic peptides, including YCA1, YBH3, NUC1, and AIF1. Even though the protective effects were more pronounced with the over-expression of YBH3 and YCA1, hTC-1 could still protect yeast mutants lacking YBH3 and YCA1 from the effects of copper sulfate. This suggests that the protective effects of TC-1 are not limited to specific pathways or processes. Taken together, our results indicate that hTC-1 is a pro-survival protein that retains its function when heterologously expressed in yeast. Thus yeast is a useful model to characterize the potential roles in cell death and survival of cancer related genes.

Macromitophagy, neutral lipids synthesis, and peroxisomal fatty acid oxidation protect yeast from "liponecrosis", a previously unknown form of programmed cell death.

We identified a form of cell death called "liponecrosis." It can be elicited by an exposure of the yeast Saccharomyces cerevisiae to exogenous palmitoleic acid (POA). Our data imply that liponecrosis is: (1) a programmed, regulated form of cell death rather than an accidental, unregulated cellular process and (2) an age-related form of cell death. Cells committed to liponecrotic death: (1) do not exhibit features characteristic of apoptotic cell death; (2) do not display plasma membrane rupture, a hallmark of programmed necrotic cell death; (3) akin to cells committed to necrotic cell death, exhibit an increased permeability of the plasma membrane for propidium iodide; (4) do not display excessive cytoplasmic vacuolization, a hallmark of autophagic cell death; (5) akin to cells committed to autophagic death, exhibit a non-selective en masse degradation of cellular organelles and require the cytosolic serine/threonine protein kinase Atg1p for executing the death program; and (6) display a hallmark feature that has not been reported for any of the currently known cell death modalities-namely, an excessive accumulation of lipid droplets where non-esterified fatty acids (including POA) are deposited in the form of neutral lipids. We therefore concluded that liponecrotic cell death subroutine differs from the currently known subroutines of programmed cell death. Our data suggest a hypothesis that liponecrosis is a cell death module dynamically integrated into a so-called programmed cell death network, which also includes the apoptotic, necrotic, and autophagic modules of programmed cell death. Based on our findings, we propose a mechanism underlying liponecrosis.

Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival.

The human septin7 and the yeast CDC10 septin prevent Bax and copper mediated cell death in yeast.

The mechanisms of programmed cell death activate genetically encoded intracellular programs in a controlled manner, the most common form being apoptosis. Apoptosis is carried out through a cascade of caspase mediated proteolytic cleavages initiated by the oligomerization of Bax, a cardinal regulator of mitochondrial-mediated apoptosis. Heterologous expression of Bax in yeast causes cell death that shares a number of similarities to processes that occur in mammalian apoptosis. A screen of a cardiac cDNA library for suppressors of Bax-mediated apoptosis identified human septin7, a protein that belongs to the septin superfamily of conserved GTP-binding proteins that share a conserved cdc/septin domain. Analysis of the amino acid sequence deduced from the septin7 clone as well as the corresponding human septin7 gene revealed that a novel alternatively spliced transcript called septin7 variant4 (v4) was uncovered. Yeast cells overexpressing the human septin7 v4 cDNA were also capable of resisting copper-mediated cell death suggesting that it is not only a Bax suppressor but also an anti-apoptotic sequence. Analysis of septin7 function in a MCA1Δ yeast strain suggests that septin7 inhibits apoptosis in a caspase independent pathway. Overexpression of the yeast septin7 ortholog CDC10 also conferred resistance to the negative effects of copper as well as protecting cells from the overexpression of Bax. In contrast, septin7 was unable to prevent the increase in cell size associated with mutants lacking the endogenous yeast CDC10 gene. Taken together, our analysis suggests that anti-apoptosis is a novel yet evolutionarily conserved property of the septin7 sub-family of septins.

Macromitophagy is a longevity assurance process that in chronologically aging yeast limited in calorie supply sustains functional mitochondria and maintains cellular lipid homeostasis.

Macromitophagy controls mitochondrial quality and quantity. It involves the sequestration of dysfunctional or excessive mitochondria within double-membrane autophagosomes, which then fuse with the vacuole/lysosome to deliver these mitochondria for degradation. To investigate a physiological role of macromitophagy in yeast, we examined how theatg32Δ-dependent mutational block of this process influences the chronological lifespan of cells grown in a nutrient-rich medium containing low (0.2%) concentration of glucose. Under these longevity-extending conditions of caloric restriction (CR) yeast cells are not starving. We also assessed a role of macromitophagy in lifespan extension by lithocholic acid (LCA), a bile acid that prolongs yeast longevity under CR conditions. Our findings imply that macromitophagy is a longevity assurance process underlying the synergistic beneficial effects of CR and LCA on yeast lifespan. Our analysis of how the atg32Δ mutation influences mitochondrial morphology, composition and function revealed that macromitophagy is required to maintain a network of healthy mitochondria. Our comparative analysis of the membrane lipidomes of organelles purified from wild-type and atg32Δ cells revealed that macromitophagy is required for maintaining cellular lipid homeostasis. We concluded that macromitophagy defines yeast longevity by modulating vital cellular processes inside and outside of mitochondria.

Influence of the physiochemical properties of superparamagnetic iron oxide nanoparticles on amyloid ß protein fibrillation in solution.

Superparamagnetic iron oxide nanoparticles (SPIONs) are recognized as promising nanodiagnostic materials due to their biocompatibility, unique magnetic properties, and their application as multimodal contrast agents. As coated SPIONs have potential use in the diagnosis and treatment of various brain diseases such as Alzheimer's, a comprehensive understanding of their interactions with Aß and other amyloidogenic proteins is essential prior to their clinical application. Here we demonstrate the effect of thickness and surface charge of the coating layer of SPIONs on the kinetics of fibrillation of Aß in aqueous solution. A size and surface area dependent "dual" effect on Aß fibrillation was observed. While lower concentrations of SPIONs inhibited fibrillation, higher concentrations increased the rate of Aß fibrillation. With respect to coating charge, it is evident that the positively charged SPIONs are capable of promoting fibrillation at significantly lower particle concentrations compared with negatively charged or uncharged SPIONs. This suggests that in addition to the presence of particles, which affect the concentration of monomeric protein in solution (and thereby the nucleation time), there are also effects of binding on the protein conformation.

Cell "vision": complementary factor of protein corona in nanotoxicology.

Engineered nanoparticles are increasingly being considered for use as biosensors, imaging agents and drug delivery vehicles. Their versatility in design and applications make them an attractive proposition for new biological and biomedical approaches. Despite the remarkable speed of development in nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and the presentation of the proteins on their surface could lead to a different in vivo response than an uncoated particle. Here, in addition to protein adsorption, we are going to introduce concept of cell "vision", which would be recognized as another crucial factor that should be considered for the safe design of any type of nanoparticles that will be used in specific biomedical applications. The impact of exactly the same nanoparticles on various cells is significantly different and could not be assumed for other cells; the possible mechanisms that justify this cellular response relate to the numerous detoxification strategies that any particular cell can utilize in response to nanoparticles. The uptake and defence mechanism could be considerably different according to the cell type. Thus, what the cell "sees", when it is faced with nanoparticles, is most likely dependent on the cell type.

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells.

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms.