Thermostability of Biological Systems: Fundamentals, Challenges, and Quantification

This review examines the fundamentals and challenges in engineering/understanding the thermostability of biological systems over a wide temperature range (from the cryogenic to hyperthermic regimen). Applications of the bio-thermostability engineering to either destroy unwanted or stabilize useful biologicals for the treatment of diseases in modern medicine are first introduced. Studies on the biological responses to cryogenic and hyperthermic temperatures for the various applications are reviewed to understand the mechanism of thermal (both cryo and hyperthermic) injury and its quantification at the molecular, cellular and tissue/organ levels. Methods for quantifying the thermophysical processes of the various applications are then summarized accounting for the effect of blood perfusion, metabolism, water transport across cell plasma membrane, and phase transition (both equilibrium and non-equilibrium such as ice formation and glass transition) of water. The review concludes with a summary of the status quo and future perspectives in engineering the thermostability of biological systems

Vitamin D and VDR in cancer cachexia and muscle regeneration

Low circulating levels of vitamin D were associated with decreased muscle strength and physical performance. Along this line, the present study was aimed to investigate: i) the therapeutic potential of vitamin D in cancer-induced muscle wasting; ii) the mechanisms by which vitamin D affects muscle phenotype in tumor-bearing animals.Rats bearing the AH130 hepatoma showed decreased circulating vitamin D compared to control rats, while muscle vitamin D receptor (VDR) mRNA was up-regulated. Both circulating vitamin D and muscle VDR expression increased after vitamin D administration, without exerting appreciable effects on body weight and muscle mass.The effects of vitamin D on muscle cells were studied in C2C12 myocytes. Vitamin D-treated myoblasts did not differentiate properly, fusing only partially and forming multinucleated structures with aberrant shape and low myosin heavy chain content. Vitamin D treatment resulted in VDR overexpression and myogenin down-regulation. Silencing VDR expression in C2C12 cultures abrogated the inhibition of differentiation exerted by vitamin D treatment.These data suggest that VDR overexpression in tumor-bearing animals contributes to muscle wasting by impairing muscle regenerative program. In this regard, attention should be paid when considering vitamin D supplementation to patients affected by chronic pathologies where muscle regeneration may be involved

Reconfiguration of amino acid biosynthesis in TGF-β1-induced myofibroblasts

At sites of tissue injury, the differentiation of fibroblasts into highly synthetic extracellular matrix (ECM)-producing myofibroblasts is essential for tissue repair and chiefly mediated by the pro-fibrotic cytokine, TGF-β1. In a fibrotic setting, these myofibroblasts fail to respond to pro-apoptotic signals during the resolution phase and relentlessly deposit ECM components, leading to destruction of healthy tissue and, in the case of lung fibrosis, respiratory failure and death. Differentiating myofibroblasts exhibit a reprograming in their metabolic networks which, like cancer cells, is believed to support the biosynthetic and bioenergetic needs of a highly protein-synthesizing cell. Evidence suggests that the changes observed in glucose and glutamine metabolic networks are critical for fibrogenesis. The present study shows that TGF-β1 induces ATF4 protein levels via mTOR to upregulate the serine-glycine biosynthetic axis enzymes (PHGDH, PSAT1, PSPH, SHMT2) which are required for enhanced collagen synthesis. Pharmacological inhibition of PHGDH and siRNA-mediated silencing of PSAT1 both prevent TGF-β1-stimulated pHLFs from synthesizing enhanced levels of collagen protein. Glycine is essential in the growth media for pHLFs to synthesize TGF-β1-enhanced collagen I levels. Glutamine, which supports the serine-glycine biosynthetic axis via its generation of glutamate, is critical for collagen synthesis and compensates for a withdrawal of glucose for pHLFs to synthesize TGF-β1-induced collagen I. TGF-β1 accelerates glutamine consumption and increases intracellular glutamate synthesis, an even facilitated by upregulating GLS1 and downregulating GLUL. GLS1-derived glutamate supports the biosynthetic pathways for alanine and proline, the latter mediated by GPT2 and required for TGF-β1-induced mTORC1 activation via a mechanism which is independent of the amino acid sensing Rag-GTPases. Furthermore, the group of enzymes to which GPT2 and PSAT1 belong, the aminotransferases, prevent TGF-β1-induced collagen synthesis upon pharmacological inhibition, an event rescued by exogenous addition of nonessential amino acids and α-ketoglutarate. Together, this work identifies the aminotransferases as a promising therapeutic target for fibrotic conditions by limiting the capacity for enhanced collagen synthesis in fibroblasts. Further understanding the pro-fibrotic functions of the members of this enzyme family may yield promising results to aid in the development of therapeutic approaches and strategies

Apoptosis and Calcification

Calcification in necrosis has long been known. Of the tissue components, the cells are most vulnerable. Nevertheless, little attention has been paid to the role of cell death in calcification. This review attempts to update the mechanism of calcification with an emphasis on the role of apoptosis in calcification. A brief review on the basic sciences relevant to calcification is followed by a discussion of abnormal Ca2+ and Pi homeostasis in cell injury and apoptosis. Concomitant increases in Ca2+ and Pi in blebs (and matrix vesicles) formed by apoptotic and/or necrotic cells are apparently the primary mechanism of calcification. In addition, membranous cellular degradation products (CDP) resulting from cell disintegration in toto frequently serve as the nidus of calcification. Published data on physiological calcification are compared with findings in various dystrophic calcinoses. This led to the conclusion that apoptosis most likely underlies the mechanism of both physiological and pathological calcifications. It is concluded that calcification is an important function of apoptosis. The mechanism of calcification by CDP and morphology of the resultant calcific deposits are complex

Measurement and mathematical modeling of thermally induced injury and heat shock protein expression kinetics in normal and cancerous prostate cells.

Abstract Purpose: Hyperthermia can induce heat shock protein (HSP) expression in tumours, which will cause enhanced tumour viability and increased resistance to additional thermal, chemotherapy, and radiation treatments. The study objective was to determine the relationship of hyperthermia protocols with HSP expression kinetics and cell death and develop corresponding computational predictive models of normal and cancerous prostate cell response. Methods: HSP expression kinetics and cell viability were measured in PC3 prostate cancer and RWPE-1 normal prostate cells subjected to hyperthermia protocols of 44 to 60 C for 1 to 30 min. Hsp27, Hsp60, and Hsp70 expression kinetics were determined by western blotting and visualised with immunofluorescence and confocal microscopy. Based on measured HSP expression data, a mathematical model was developed for predicting thermally induced HSP expression. Cell viability was measured with propidium iodide staining and flow cytometry to quantify the injury parameters necessary for predicting cell death following hyperthermia. Results: Significant Hsp27 and Hsp70 levels were induced in both cell types with maximum HSP expression occurring at 16 h post-heating, and diminishing substantially after 72 h. PC3 cells were slightly more sensitive to thermal stress than RWPE-1 cells. Arrhenius analysis of injury data suggested a transition between injury mechanisms at 54 C. HSP expression and injury models were effective at predicting cellular response to hyperthermia. Conclusion: Measurement of thermally induced HSP expression kinetics and cell viability associated with hyperthermia enabled development of thermal dosimetry guidelines and predictive models for HSP expression and cell injury as a function of thermal stress to investigate and design more effective hyperthermia therapies

The potential utility of stem cells in the treatment of congenital heart disease

Non-ischaemic right ventricular dysfunction and cardiac failure is a source of considerable morbidity in children with congenital heart disease. Despite an increasing body of evidence suggesting that the intrinsic regenerative capacity of the heart can be encouraged by stimulation of resident cardiac stem cells or the transplantation of extracardiac progenitor cells, cell transplantation has not previously been studied in the paediatric setting where enhancing the function of the ventricle in response to supraphysiological workloads might be beneficial. Firstly I studied extra-cellular matrix composition, myocyte homeostasis and gene expression in right ventricular biopsies obtained from patients with Hypoplastic Left Heart Syndrome (HLHS) undergoing neonatal surgical palliation and from patients undergoing neonatal truncus arteriosus repair in order to investigate potential differences in the myocardial substrate which could have implications for adaptive growth potential and haemodynamic performance in HLHS. Simultaneous to these activities, I collected, isolated and analysed umbilical cord blood stem cells from children born with either structurally-normal hearts or HLHS to investigate whether such cell populations might be useful in cardiac augmentation. We then transplanted human cord blood stem cells from normal cord blood donors into an immunosuppressed neonatal sheep model of right ventricular training, taking load-independent functional measurements at baseline and again after one month. Transplanted human cells were detected in the myocardium, spleen, kidney and bone marrow up to 6 weeks after transplantation. Human cells expressed the haematopoietic marker CD45 and in the bone marrow and spleen, also the mature B cell marker CD23. Significant functional improvements were seen in the group receiving human cord blood stem cells compared to placebo. Data demonstrated that lineage negative-enriched cord blood stem cells engraft and adopt traditional haematopoietic cell fates in both myocardium as well as natural niches such as bone marrow and spleen

Ultrasound Thermal Therapy: Patient-Specific Planning for Intraluminal and External Delivery

Thermal therapy can be regarded as one of the promising emerging technologies for cancer treatment. In the current study, proof-of concept patient-specific treatment protocols are formulated for high frequency ultrasound thermal therapy for intramural and external delivery. Propagation of ultrasound waves in biological tissue is first determined using the finite difference method, which generates the temperature histories from the bioheat transfer model. Thermal coagulation contours are determined by thermal dose information. From an analysis of the results for a range of parameters, treatment protocols for prostate and breast cancers are developed for a broad spectrum of patients. Results indicate tumors varying in size and location can be targeted effectively using the designs employed. To further improve simulations, thermal damage data for breast cancer is determined through pilot in vitro experiments. The algorithms developed can help optimize the design of delivery equipment and generate accurate patient-specific US clinical protocols

Design considerations of benchtop fluid flow bioreactors for bio-engineered tissue equivalents in vitro

One of the major aims of bio-engineering tissue equivalents in vitro is to create physiologically relevant culture conditions to accurately recreate the cellular microenvironment. This often includes incorporation of factors such as the extracellular matrix, co-culture of multiple cell types and three-dimensional culture techniques. These advanced techniques can recapitulate some of the properties of tissue in vivo, however fluid flow is a key aspect that is often absent. Fluid flow can be introduced into cell and tissue culture using bioreactors, which are becoming increasingly common as we seek to produce increasingly accurate tissue models. Bespoke technology is continuously being developed to tailor systems for specific applications and to allow compatibility with a range of culture techniques. For effective perfusion of a tissue culture many parameters can be controlled, ranging from impacts of the fluid flow such as increased shear stress and mass transport, to potentially unwanted side effects such as temperature fluctuations. A thorough understanding of these properties and their implications on the culture model can aid with a more accurate interpretation of results. Improved and more complete characterisation of bioreactor properties will also lead to greater accuracy when reporting culture conditions in protocols, aiding experimental reproducibility, and allowing more precise comparison of results between different systems. In this review we provide an analysis of the different factors involved in the development of benchtop flow bioreactors and their potential biological impacts across a range of applications

Perinatal Nicotine Exposure and Programming of HIE Sensitive Phenotype in Neonatal Rat Brains

Large epidemiological and animal studies have revealed a clear association of adverse intrauterine environment with the increased risk of metabolic, cardiovascular and neurological diseases. Maternal smoking is the single most widespread perinatal insult in the world and has been associated with adverse pregnancy outcomes for mother, fetus and the newborn. Our study aims to test the hypothesis that perinatal nicotine exposure induces reprogramming of susceptibility to hypoxic ischemic brain injury in the immature brain, focusing on the roles of AT1R and/or AT2R and trying to reveal the underlying epigenetic mechanisms. Therefore, we established two rat models: perinatal nicotine exposure model in time-dated pregnant rats; hypoxic-ischemic encephalopathy (HIE) in 10-day-old rat pups. In the first part of our study, we demonstrated that nicotine exposure induces aberrant brain development in P10 pups, downregulating AT2R expression in male but upregulating AT2R in female pup brain, enhancing brain vulnerability to HIE in a sex-specific manner. In addition, we observed both AT1R and AT2R are implicated in the pathogenesis of neonatal HIE and confers neuroprotective property; AT2R plays the pivotal and causal role in nicotine induced sex-dependent alteration of vulnerability to HIE in the developing rat brain. Our further study focused on the epigenetic mechanisms involved in nicotine exposure mediated pathological effects in HIE. We demonstrated that perinatal nicotine exposure causes heightened methylation status of a single CpG adjacent to TATA-box at AT2R promoter, inhibiting TBP and recruiting MeCP2 binding, repressing AT2R gene expression, contributing to the enhanced vulnerability to HIE brain injury in male rat pups. All of pathological effects are reversed by administration of 5-Aza, a well-known DNA methylase inhibitor. These findings provide new insights in understanding of the pathogenesis of HIE in newborns and may suggest potential targets for the prevention and treatment of HIE, one of the most common causes of brain damage with severe mortality and long-lasing morbidity in infants and children

The potential utility of stem cells in the treatment of congenital heart disease

Non-ischaemic right ventricular dysfunction and cardiac failure is a source of considerable morbidity in children with congenital heart disease. Despite an increasing body of evidence suggesting that the intrinsic regenerative capacity of the heart can be encouraged by stimulation of resident cardiac stem cells or the transplantation of extracardiac progenitor cells, cell transplantation has not previously been studied in the paediatric setting where enhancing the function of the ventricle in response to supraphysiological workloads might be beneficial. Firstly I studied extra-cellular matrix composition, myocyte homeostasis and gene expression in right ventricular biopsies obtained from patients with Hypoplastic Left Heart Syndrome (HLHS) undergoing neonatal surgical palliation and from patients undergoing neonatal truncus arteriosus repair in order to investigate potential differences in the myocardial substrate which could have implications for adaptive growth potential and haemodynamic performance in HLHS. Simultaneous to these activities, I collected, isolated and analysed umbilical cord blood stem cells from children born with either structurally-normal hearts or HLHS to investigate whether such cell populations might be useful in cardiac augmentation. We then transplanted human cord blood stem cells from normal cord blood donors into an immunosuppressed neonatal sheep model of right ventricular training, taking load-independent functional measurements at baseline and again after one month. Transplanted human cells were detected in the myocardium, spleen, kidney and bone marrow up to 6 weeks after transplantation. Human cells expressed the haematopoietic marker CD45 and in the bone marrow and spleen, also the mature B cell marker CD23. Significant functional improvements were seen in the group receiving human cord blood stem cells compared to placebo. Data demonstrated that lineage negative-enriched cord blood stem cells engraft and adopt traditional haematopoietic cell fates in both myocardium as well as natural niches such as bone marrow and spleen