Improved Cryopreservation of Human Induced Pluripotent Stem Cell (iPSC) and iPSC-derived Neurons Using Ice-Recrystallization Inhibitors.

Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iPSC-Ns) represent a differentiated modality toward developing novel cell-based therapies for regenerative medicine. However, the successful application of iPSC-Ns in cell-replacement therapies relies on effective cryopreservation. In this study, we investigated the role of ice recrystallization inhibitors (IRIs) as novel cryoprotectants for iPSCs and terminally differentiated iPSC-Ns. We found that one class of IRIs, N-aryl-D-aldonamides (specifically 2FA), increased iPSC post-thaw viability and recovery with no adverse effect on iPSC pluripotency. While 2FA supplementation did not significantly improve iPSC-N cell post-thaw viability, we observed that 2FA cryopreserved iPSC-Ns re-established robust neuronal network activity and synaptic function much earlier compared to CS10 cryopreserved controls. The 2FA cryopreserved iPSC-Ns retained expression of key neuronal specific and terminally differentiated markers and displayed functional electrophysiological and neuropharmacological responses following treatment with neuroactive agonists and antagonists. We demonstrate how optimizing cryopreservation media formulations with IRIs represents a promising strategy to improve functional cryopreservation of iPSCs and post-mitotic iPSC-Ns, the latter of which have been challenging to achieve. Developing IRI enabling technologies to support an effective cryopreservation and an efficiently managed cryo-chain is fundamental to support the delivery of successful iPSC-derived therapies to the clinic.

Cross protection to SARS-CoV-2 variants in hamsters with naturally-acquired immunity.

Since SARS-CoV-2 was first reported in late 2019, multiple variations of the original virus have emerged. Each variant harbors accumulations of mutations, particularly within the spike glycoprotein, that are associated with increased viral transmissibility and escape immunity. The different mutations in the spike protein of different variants shape the subsequent antibody and T cell responses, such that exposure to different spike proteins can result in reduced or enhanced responses to heterologous variants further down the line. Globally, people have been exposed and re-exposed to multiple variations of the Ancestral strain, including the five variants of concerns. Studies have shown that the protective immune response of an individual is influenced by which strain or combination of strains they are exposed to. The initial exposure to a specific strain may also shape their subsequent immune patterns and response to later infections with a heterologous virus. Most immunological observations were carried out early during the pandemic when the Ancestral strain was circulating. However, SARS-CoV-2 variants exhibit varying patterns of disease severity, waning immunity, immune evasion and sensitivity to therapeutics. Here we investigated the cross-protection in hamsters previously infected with a variant of concern (VOC) and subsequently re-infected with a heterologous variant. We also determined if cross-protection and immunity were dependent on the specific virus to which the hamster was first exposed. We further profiled the host cytokine response induced by each SARS-CoV-2 variants as well as subsequent to re-infection. A comparative analysis of the three VOCs revealed that Alpha variant was the most pathogenic VOC to emerge. We showed that naturally acquired immunity protected hamsters from subsequent re-infection with heterologous SARS-CoV-2 variant, regardless which variant the animal was first exposed to. Our study supports observations that heterologous infection of different SARS-CoV-2 variants do not exacerbate disease in subsequent re-infections. The continual emergence of new SARS-CoV-2 variants mandates a better understanding of cross-protection and immune imprinting in infected individuals. Such information is essential to guide vaccine strategy and public policy to emerging SARS-CoV-2 VOCs and future novel pandemic coronaviruses.

A Canadian perspective on severe acute respiratory syndrome coronavirus 2 infection and treatment: how prevalent underlying inflammatory disease contributes to pathogenesis.

The coronavirus disease 2019 (COVID-19), a serious respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a global pandemic. Canada reported its first case of COVID-19 on the 25th January 2020. By March 2020, the virus had spread within Canadian communities reaching the most frail and vulnerable elderly population in long-term care facilities. The majority of cases were reported in the provinces of Quebec, Ontario, Alberta, and British Columbia, and the highest mortality was seen among individuals aged 65 years or older. Canada has the highest prevalence and incidence rates of several chronic inflammatory diseases, such as multiple sclerosis, inflammatory bowel disease, and Parkinson's disease. Many elderly Canadians also live with comorbid medical illnesses, such as hypertension, diabetes, cardiovascular disease, and chronic lung disease, and are more likely to suffer from severe COVID-19 with a poor prognosis. It is becoming increasingly evident that underlying inflammatory disease contributes to the pathogenesis of SARS-CoV-2. Here, we review the mechanisms behind SARS-CoV-2 infection, and the host inflammatory responses that lead to resolution or progression to severe COVID-19 disease. Furthermore, we discuss the landscape of COVID-19 therapeutics that are currently in development in Canada.

Decoding Mast Cell-Microglia Communication in Neurodegenerative Diseases.

Microglia, resident immune cells of the central nervous system (CNS), play a pivotal role in immune surveillance and maintenance of neuronal health. Mast cells are also important resident immune cells of the CNS but they are underappreciated and understudied. Both microglia and mast cells are endowed with an array of signaling receptors that recognize microbes and cellular damage. As cellular sensors and effectors in the CNS, they respond to many CNS perturbations and have been implicated in neuroinflammation and neurodegeneration. Mast cells contain numerous secretory granules packaged with a plethora of readily available and newly synthesized compounds known as 'mast cell mediators'. Mast cells act as 'first responders' to a pathogenic stimuli and respond by degranulation and releasing these mediators into the extracellular milieu. They alert other glial cells, including microglia to initiate neuroinflammatory processes that culminate in the resolution of injury. However, failure to resolve the pathogenic process can lead to persistent activation, release of pro-inflammatory mediators and amplification of neuroinflammatory responses, in turn, resulting in neuronal dysfunction and demise. This review discusses the current understanding of the molecular conversation between mast cells and microglia in orchestrating immune responses during two of the most prevalent neurodegenerative diseases, namely Alzheimer's disease and Parkinson's disease. Here we also survey the potential emerging therapeutic approaches targeting common pathways in mast cells and microglia to extinguish the fire of inflammation.

Aberrant NLRP3 Inflammasome Activation Ignites the Fire of Inflammation in Neuromuscular Diseases.

Inflammasomes are molecular hubs that are assembled and activated by a host in response to various microbial and non-microbial stimuli and play a pivotal role in maintaining tissue homeostasis. The NLRP3 is a highly promiscuous inflammasome that is activated by a wide variety of sterile triggers, including misfolded protein aggregates, and drives chronic inflammation via caspase-1-mediated proteolytic cleavage and secretion of proinflammatory cytokines, interleukin-1ß and interleukin-18. These cytokines further amplify inflammatory responses by activating various signaling cascades, leading to the recruitment of immune cells and overproduction of proinflammatory cytokines and chemokines, resulting in a vicious cycle of chronic inflammation and tissue damage. Neuromuscular diseases are a heterogeneous group of muscle disorders that involve injury or dysfunction of peripheral nerves, neuromuscular junctions and muscles. A growing body of evidence suggests that dysregulation, impairment or aberrant NLRP3 inflammasome signaling leads to the initiation and exacerbation of pathological processes associated with neuromuscular diseases. In this review, we summarize the available knowledge about the NLRP3 inflammasome in neuromuscular diseases that affect the peripheral nervous system and amyotrophic lateral sclerosis, which affects the central nervous system. In addition, we also examine whether therapeutic targeting of the NLRP3 inflammasome components is a viable approach to alleviating the detrimental phenotype of neuromuscular diseases and improving clinical outcomes.

Orally delivered water soluble Coenzyme Q10 (Ubisol-Q10) blocks on-going neurodegeneration in rats exposed to paraquat: potential for therapeutic application in Parkinson's disease.

BACKGROUND: Paraquat, still used as an herbicide in some parts of the world, is now regarded as a dangerous environmental neurotoxin and is linked to the development Parkinson's disease (PD). Paraquat interacts with cellular redox systems and causes mitochondrial dysfunction and the formation of reactive oxygen species, which in turn, plays a crucial role in the pathophysiology of PD. Various antioxidant therapies have been explored with the expectations that they deliver health benefits to the PD patients, however, no such therapies were effective. Here we have tested the neuroprotective efficacy of a novel water-soluble CoQ10 (Ubisol-Q10), in a rat model of paraquat-induced neurodegeneration in order to evaluate its potential application in the management of PD. RESULTS: We have developed a rat model of progressive nigrostriatal degeneration by giving rats five intraperitoneal injections of paraquat (10 mg/kg/injection), once every five days. Neuronal death occurred over a period of 8 weeks with close to 50% reduction in the number of tyrosine hydroxylase-positive cells. Ubisol-Q10, at 6 mg CoQ10/kg body weight/day, was delivered as a supplement in drinking water. The intervention begun after the completion of paraquat injections when the neurodegenerative process had already began and about 20% of TH-positive neurons were lost. Ubisol-Q10 treatment halted the progression of neurodegeneration and remaining neurons were protected. The outcomes were evaluated based on the number of surviving tyrosine hydroxylase-positive neurons in the substantia nigra region and improved motor skills in response to the Ubisol-Q10 intervention. To maintain this neuroprotection, however, continuous Ubisol- Q10 supplementation was required, if withdrawn, the neuronal death pathway resumed, suggesting that the presence of CoQ10 was essential for blocking the pathway. CONCLUSION: The CoQ10, given orally as Ubisol-Q10 in drinking solution, was effective in blocking the progression of neurodegeneration when administered therapeutically (post-toxin injection), at a much lower concentration than other previously tested oil soluble formulations and well within the acceptable daily intake of 12 mg/kg/day. Such unprecedented neuroprotection has never been reported before. These results are very encouraging and suggest that Ubisol-Q10 should be further tested and developed as a therapy for halting the progression of PD.

Effectiveness of VSV vectored SARS-CoV-2 spike when administered through intranasal, intramuscular or a combination of both.

A critical feature of the VSV vector platform is the ability to pseudotype the virus with different glycoproteins from other viruses, thus altering cellular tropism of the recombinant virus. The route of administration is critical in triggering local and systemic immune response and protection. Most of the vaccine platforms used at the forefront are administered by intramuscular injection. However, it is not known at what level ACE2 is expressed on the surface of skeletal muscle cells, which will have a significant impact on the efficiency of a VSV-SARS-CoV-2 spike vaccine to mount a protective immune response when administered intramuscularly. In this study, we investigate the immunogenicity and efficacy of a prime-boost immunization regimen administered intranasally (IN), intramuscularly (IM), or combinations of the two. We determined that the prime-boost combinations of IM followed by IN immunization (IM + IN) or IN followed by IN immunization (IN + IN) exhibited strong spike-specific IgG, IgA and T cell response in vaccinated K18 knock-in mice. Hamsters vaccinated with two doses of VSV expressing SARS-CoV-2 spike, both delivered by IN or IM + IN, showed strong protection against SARS-CoV-2 variants of concern Alpha and Delta. This protection was also observed in aged hamsters. Our study underscores the highly crucial role immunization routes have with the VSV vector platform to elicit a strong and protective immune response.

The SARS-CoV-2 spike glycoprotein interacts with MAO-B and impairs mitochondrial energetics.

SARS-CoV-2 infection is associated with both acute and post-acute neurological symptoms. Emerging evidence suggests that SARS-CoV-2 can alter mitochondrial metabolism, suggesting that changes in brain metabolism may contribute to the development of acute and post-acute neurological complications. Monoamine oxidase B (MAO-B) is a flavoenzyme located on the outer mitochondrial membrane that catalyzes the oxidative deamination of monoamine neurotransmitters. Computational analyses have revealed high similarity between the SARS-CoV-2 spike glycoprotein receptor binding domain on the ACE2 receptor and MAO-B, leading to the hypothesis that SARS-CoV-2 spike glycoprotein may alter neurotransmitter metabolism by interacting with MAO-B. Our results empirically establish that the SARS-CoV-2 spike glycoprotein interacts with MAO-B, leading to increased MAO-B activity in SH-SY5Y neuron-like cells. Common to neurodegenerative disease pathophysiological mechanisms, we also demonstrate that the spike glycoprotein impairs mitochondrial bioenergetics, induces oxidative stress, and perturbs the degradation of depolarized aberrant mitochondria through mitophagy. Our findings also demonstrate that SH-SY5Y neuron-like cells expressing the SARS-CoV-2 spike protein were more susceptible to MPTP-induced necrosis, likely necroptosis. Together, these results reveal novel mechanisms that may contribute to SARS-CoV-2-induced neurodegeneration.

Involvement of NOS3 in RA-Induced neural differentiation of human NT2/D1 cells.

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2-derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5' regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter (-734 to -989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2-N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3. This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region.

Effects of Tolerance-Induced Preconditioning on Mitochondrial Biogenesis in Undifferentiated and Differentiated Neuronal Cells.

BACKGROUND: Mitochondrial biogenesis occurs in response to chronic stresses as an adaptation to the increased energy demands and often renders cells more refractive to subsequent injuries which is referred to as preconditioning. This phenomenon is observed in several non-neuronal cell types, but it is not yet fully established in neurons, although it is fundamentally important for neuroprotection and could be exploited for therapeutic purposes. METHODS: This study was designed to examine whether the preconditioning treatment with hypoxia or nitric oxide could trigger biogenesis in undifferentiated and differentiated neuronal cells (rat PC12 and human NT2 cells) as well as in primary mouse cortical neurons. RESULTS: The results showed that both preconditioning paradigms induced mitochondrial biogenesis in undifferentiated cell lines, as indicated by an increase of mitochondrial mass (measured by flow cytometry of NAO fluorescence) and increased expression of genes required for mitochondrial biogenesis (Nrf1, Nrf2, Tfam, Nfκb1) and function (Cox3, Hk1). All these changes translated into an increase in the organelle copy number from an average of 20-40 to 40-60 mitochondria per cell. The preconditioning treatments also rendered the cells significantly less sensitive to the subsequent oxidative stress challenge brought about by oxygen/glucose deprivation, consistent with their improved cellular energy status. Mitochondrial biogenesis was abolished when preconditioning treatments were performed in the presence of antioxidants (vitamin E or CoQ10), indicating clearly that ROS-signaling pathway(s) played a critical role in the induction of this phenomenon in undifferentiated cells. However, mitochondrial biogenesis could not be re-initiated by preconditioning treatments in any of the post-mitotic neuronal cells tested, i.e., neither rat PC12 cells differentiated with NGF, human NT2 cells differentiated with retinoic acid nor mouse primary cortical neurons. Instead, differentiated neurons had a much higher organelle copy number per cell than their undifferentiated counterparts (100-130 mitochondria per neuron vs. 20-40 in proliferating cells), and this feature was not altered by preconditioning. CONCLUSIONS: Our study demonstrates that mitochondrial biogenesis occurred during the differentiation process resulting in more beneficial energy status and improved tolerance to oxidative stress in neurons, putting in doubt whether additional enhancement of this phenomenon could be achieved and successfully exploited as a way for better neuroprotection.

Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor.

The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood-brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB. Surface plasmon resonance binding analyses demonstrated the species cross-reactivity of IGF1R5 toward IGF1R from multiple species. To overcome the short serum half-life of sdAbs, we fused IGF1R5 to the human (hFc) or mouse Fc domain (mFc). IGF1R5 in both N- and C-terminal mFc fusion showed enhanced transmigration across a rat BBB model (SV-ARBEC) in vitro. Increased levels of hFc-IGF1R5 in the cerebrospinal fluid and vessel-depleted brain parenchyma fractions further confirmed the ability of IGF1R5 to cross the BBB in vivo. We next tested whether this carrier was able to ferry a pharmacologically active payload across the BBB by measuring the hypothermic and analgesic properties of neurotensin and galanin, respectively. The fusion of IGF1R5-hFc to neurotensin induced a dose-dependent reduction in the core temperature. The reversal of hyperalgesia by galanin that was chemically linked to IGF1R5-mFc was demonstrated using the Hargreaves model of inflammatory pain. Taken together, our results provided a proof of concept that appropriate antibodies, such as IGF1R5 against IGF1R, are suitable as RMT carriers for the delivery of therapeutic cargos for CNS applications.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models.

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

Grabody B, an IGF1 receptor-based shuttle, mediates efficient delivery of biologics across the blood-brain barrier.

Effective delivery of therapeutics to the brain is challenging. Molecular shuttles use receptors expressed on brain endothelial cells to deliver therapeutics. Antibodies targeting transferrin receptor (TfR) have been widely developed as molecular shuttles. However, the TfR-based approach raises concerns about safety and developmental burden. Here, we report insulin-like growth factor 1 receptor (IGF1R) as an ideal target for the molecular shuttle. We also describe Grabody B, an antibody against IGF1R, as a molecular shuttle. Grabody B has broad cross-species reactivity and does not interfere with IGF1R-mediated signaling. We demonstrate that administration of Grabody B-fused anti-alpha-synuclein (α-Syn) antibody induces better improvement in neuropathology and behavior in a Parkinson's disease animal model than the therapeutic antibody alone due to its superior serum pharmacokinetics and enhanced brain exposure. The results indicate that IGF1R is an ideal shuttle target and Grabody B is a safe and efficient molecular shuttle.

Mitigating Long-Term COVID-19 Consequences on Brain Health.

COVID-19 is increasingly being linked to brain health impacts. The emerging situation is consistent with evidence of immunological injury to the brain, which has been described as a resulting "brain fog." The situation need not be medicalized but rather clinically managed in terms of improving resilience for an over-stressed nervous system. Pre-existing comparisons include managing post-concussion syndromes and/or brain fog. The objective evaluation of changes in cognitive functioning will be an important clinical starting point, which is being accelerated through pandemic digital health innovations. Pre-morbid brain health can significantly optimize risk factors and existing clinical frameworks provide useful guidance in managing over-stressed COVID-19 nervous systems.

Ubisol-Q10, a Nanomicellar and Water-Dispersible Formulation of Coenzyme-Q10 as a Potential Treatment for Alzheimer's and Parkinson's Disease.

The world continues a desperate search for therapies that could bring hope and relief to millions suffering from progressive neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's (PD). With oxidative stress thought to be a core stressor, interests have long been focused on applying redox therapies including coenzyme-Q10. Therapeutic use has failed to show efficacy in human clinical trials due to poor bioavailability of this lipophilic compound. A nanomicellar, water-dispersible formulation of coenzyme-Q10, Ubisol-Q10, has been developed by combining coenzyme-Q10 with an amphiphilic, self-emulsifying molecule of polyoxyethanyl α-tocopheryl sebacate (derivatized vitamin E). This discovery made possible, for the first time, a proper assessment of the true therapeutic value of coenzyme-Q10. Micromolar concentrations of Ubisol-Q10 show unprecedented neuroprotection against neurotoxin exposure in in vitro and in vivo models of neurodegeneration and was extremely effective when delivered either prior to, at the time of, and most significantly, post-neurotoxin exposure. These findings indicate a possible way forward for clinical development due to effective doses well within Federal Drug Administration guidelines. Ubisol-Q10 is a potent mobilizer of astroglia, antioxidant, senescence preventer, autophagy activator, anti-inflammatory, and mitochondrial stabilizer. Here we summarize the work with oil-soluble coenzyme-Q10, its limitations, and focus mainly on efficacy of water-soluble coenzyme-Q10 in neurodegeneration.

IFN-alpha receptor deficiency enhances host resistance to oral Salmonella enterica serovar Typhimurium infection during murine pregnancy.

PROBLEM: Maternal tolerance during pregnancy increases the risk of infection with certain intracellular pathogens. Systemic Salmonella enterica serovar Typhimurium (S.Tm) infection during pregnancy in normally resistant 129X1/SvJ mice leads to severe placental infection, as well as fetal and maternal deaths. However, the effect of oral infection with S.Tm in pregnant mice and the roles of infection-induced inflammation and cell death pathways in contributing to susceptibility to infection are unclear. METHOD OF STUDY: Non-pregnant and pregnant C57BL/6J wild-type (WT) and cell death pathway-altered mice (IFNAR1-/- , Caspase-1, 11-/- , RIP3-/- ) were infected orally with S.Tm. Host survival and fetal resorption were determined. Bacterial burden in mesenteric lymph nodes (MLNs), spleen, liver, and placentas was enumerated at various time points post-infection. Serum cytokine expression was measured through cytometric bead array. RESULTS: Oral infection of WT mice with S.Tm on days 9-10 of gestation resulted in systemic dissemination of the bacteria, substantial placental colonization, and fetal loss 5 days post-infection. Histopathological examination of the placentas indicated that infection-induced widespread focal necrosis and neutrophil infiltration throughout the spongiotrophoblast (SpT) layer. In the non-pregnant state, IFNAR1-/- mice exhibited increased survival following oral S.Tm infection relative to Caspase-1, 11-/- , RIP3-/- , and WT mice. The increased resistance to S.Tm infection in IFNAR1-/- mice was seen during pregnancy as well, with decreased bacterial burden within MLNs, spleen, and placenta, which correlated with the decreased resorptions relative to WT and Caspase-1, 11-/- mice. CONCLUSION: Oral S.Tm exposure leads to placental infection, inflammation, and resorption, whereas IFNAR1 deficiency enhances host resistance both in the non-pregnant and pregnant states.

Salmonella enterica serovar Typhimurium-induced placental inflammation and not bacterial burden correlates with pathology and fatal maternal disease.

Food-borne infections caused by Salmonella enterica species are increasing globally, and pregnancy poses a high risk. Pregnant mice rapidly succumb to S. enterica serovar Typhimurium infection. To determine the mechanisms involved, we addressed the role of inflammation and bacterial burden in causing placental and systemic disease. In vitro, choriocarcinoma cells were a highly conducive niche for intracellular S. Typhimurium proliferation. While infection of mice with S. Typhimurium wild-type (WT) and mutant (Delta aroA and Delta invA) strains led to profound pathogen proliferation and massive burden within placental cells, only the virulent WT S. Typhimurium infection evoked total fetal loss and adverse host outcome. This correlated with substantial placental expression of granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) and increased serum inflammatory cytokines/chemokines, such as G-CSF, IL-6, CCL1, and KC, evoked by WT S. Typhimurium infection. In contrast, infection with high doses of S. Typhimurium Delta aroA, despite causing massive placental infection, resulted in reduced inflammatory cellular and cytokine response. While S. Typhimurium WT bacteria were dispersed in large numbers across all regions of the placenta, including the deeper labyrinth trophoblast, S. Typhimurium Delta aroA bacteria localized primarily to the decidua. This correlated with the widespread placental necrosis accompanied by neutrophil infiltration evoked by the S. Typhimurium WT bacteria. Thus, the ability of Salmonella to localize to deeper layers of the placenta and the nature of inflammation triggered by the pathogen, rather than bacterial burden, profoundly influenced placental integrity and host survival.

Role of Glutathione in Cancer: From Mechanisms to Therapies.

Glutathione (GSH) is the most abundant non-protein thiol present at millimolar concentrations in mammalian tissues. As an important intracellular antioxidant, it acts as a regulator of cellular redox state protecting cells from damage caused by lipid peroxides, reactive oxygen and nitrogen species, and xenobiotics. Recent studies have highlighted the importance of GSH in key signal transduction reactions as a controller of cell differentiation, proliferation, apoptosis, ferroptosis and immune function. Molecular changes in the GSH antioxidant system and disturbances in GSH homeostasis have been implicated in tumor initiation, progression, and treatment response. Hence, GSH has both protective and pathogenic roles. Although in healthy cells it is crucial for the removal and detoxification of carcinogens, elevated GSH levels in tumor cells are associated with tumor progression and increased resistance to chemotherapeutic drugs. Recently, several novel therapies have been developed to target the GSH antioxidant system in tumors as a means for increased response and decreased drug resistance. In this comprehensive review we explore mechanisms of GSH functionalities and different therapeutic approaches that either target GSH directly, indirectly or use GSH-based prodrugs. Consideration is also given to the computational methods used to describe GSH related processes for in silico testing of treatment effects.

Analysis and Simulation of Glioblastoma Cell Lines-Derived Extracellular Vesicles Metabolome.

Glioblastoma (GBM) is one of the most aggressive cancers of the central nervous system. Despite current advances in non-invasive imaging and the advent of novel therapeutic modalities, patient survival remains very low. There is a critical need for the development of effective biomarkers for GBM diagnosis and therapeutic monitoring. Extracellular vesicles (EVs) produced by GBM tumors have been shown to play an important role in cellular communication and modulation of the tumor microenvironment. As GBM-derived EVs contain specific "molecular signatures" of their parental cells and are able to transmigrate across the blood-brain barrier into biofluids such as the blood and cerebrospinal fluid (CSF), they are considered as a valuable source of potential diagnostic biomarkers. Given the relatively harsh extracellular environment of blood and CSF, EVs have to endure and adapt to different conditions. The ability of EVs to adjust and function depends on their lipid bilayer, metabolic content and enzymes and transport proteins. The knowledge of EVs metabolic characteristics and adaptability is essential for their utilization as diagnostic and therapeutic tools. The main aim of this study was to determine the metabolome of small EVs or exosomes derived from different GBM cells and compare to the metabolic profile of their parental cells using NMR spectroscopy. In addition, a possible flux of metabolic processes in GBM-derived EVs was simulated using constraint-based modeling from published proteomics information. Our results showed a clear difference between the metabolic profiles of GBM cells, EVs and media. Machine learning analysis of EV metabolomics, as well as flux simulation, supports the notion of active metabolism within EVs, including enzymatic reactions and the transfer of metabolites through the EV membrane. These results are discussed in the context of novel GBM diagnostics and therapeutic monitoring.