Blood transcriptomics mirror regulatory mechanisms during hibernation-a comparative analysis of the Djungarian hamster with other mammalian species.

Hibernation enables many species of the mammalian kingdom to overcome periods of harsh environmental conditions. During this physically inactive state metabolic rate and body temperature are drastically downregulated, thereby reducing energy requirements (torpor) also over shorter time periods. Since blood cells reflect the organism´s current condition, it was suggested that transcriptomic alterations in blood cells mirror the torpor-associated physiological state. Transcriptomics on blood cells of torpid and non-torpid Djungarian hamsters and QIAGEN Ingenuity Pathway Analysis (IPA) revealed key target molecules (TMIPA), which were subjected to a comparative literature analysis on transcriptomic alterations during torpor/hibernation in other mammals. Gene expression similarities were identified in 148 TMIPA during torpor nadir among various organs and phylogenetically different mammalian species. Based on TMIPA, IPA network analyses corresponded with described inhibitions of basic cellular mechanisms and immune system-associated processes in torpid mammals. Moreover, protection against damage to the heart, kidney, and liver was deduced from this gene expression pattern in blood cells. This study shows that blood cell transcriptomics can reflect the general physiological state during torpor nadir. Furthermore, the understanding of molecular processes for torpor initiation and organ preservation may have beneficial implications for humans in extremely challenging environments, such as in medical intensive care units and in space.

European space agency's hibernation (torpor) strategy for deep space missions: Linking biology to engineering.

Long-duration space missions to Mars will impose extreme stresses of physical and psychological nature on the crew, as well as significant logistical and technical challenges for life support and transportation. Main challenges include optimising overall mass and maintaining crew physical and mental health. These key scopes have been taken up as the baseline for a study by the European Space Agency (ESA) using its Concurrent Design Facility (CDF). It focussed on the biology of hibernation in reducing metabolism and hence stress, and its links to the infrastructure and life support. We concluded that torpor of crew members can reduce the payload with respect to oxygen, food and water but will require monitoring and artificial intelligence (AI) assisted monitoring of the crew. These studies additionally offer new potential applications for patient care on Earth. Keywords: Space flight, concurrent design facility, metabolic reduction.

Age-related dysfunction of the autophago-lysosomal pathway in human endothelial cells.

Senescent cells, which are cells in a post-proliferative state, show an increased number of dysfunctional mitochondria and oxidatively damaged and aggregated proteins. The mitochondrial-lysosomal axis theory of aging proposes that the autophago-lysosomal system is unable to cope with the rising amount of damaged organelles and proteins. We used human umbilical vein endothelial cells (HUVEC) as in vitro model system to determine which part/s of the autophago-lysosomal pathway become deficient by aging. Senescent HUVEC contained a much larger population of autophagosomes and lysosomes compared to young cells. Transcriptome analysis comparing young and old cells demonstrated several age-related changes of autophagy gene expression. One reason for the observed increase of autophagosomes was an impairment of the autophagic flux in senescent cells due to reduced V-ATPase activity required for acidification of the lysosomes and thus functionality of lysosomal hydrolases. The hypothesis that reduced mitochondrial ATP production underlies low V-ATPase activity was supported by addition of exogenous ATP. This procedure rescued the lysosomal acidification and restored the autophagic flux. Thus, we propose impaired lysosomal acidification due to ATP shortage which may result from mitochondrial dysfunction as a mechanism underlying the accumulation of dysfunctional cellular constituents during aging.

Assessment of Melanogenesis in a Pigmented Human Tissue-Cultured Skin Equivalent.

BACKGROUND: Organotypic tissue-cultured skin equivalents are used for a broad range of applications either as possible substitute for animal tests or for transplantation in patient-centered care. AIMS: In this study, we implemented melanocytes in a tissue-cultured full-thickness skin equivalent, consisting of epidermis and dermis. The versatility of this skin-like model with respect to pigmentation and morphological criteria was tested. MATERIALS AND METHODS: Pigmented skin equivalents were morphologically characterized, and melanogenesis was evaluated after treatment with kojic acid - a tyrosinase inhibitor and forskolin - a well-known activator of the cyclic adenosine 3,5-monophosphate pathway. Pigmentation was measured either by determination of the extinction at 400 nm after melanin extraction with KOH correlated to a melanin standard curve or by reflectance colorimetric analysis, monitoring reflectance of 660 nm and 880 nm emitting diodes. RESULTS: The morphological analysis revealed characteristic epidermal stratification with melanocytes located at the basal layer. Stimulation with forskolin increased the pigmentation, whereas treatment with kojic acid caused bleaching. CONCLUSION: The present study demonstrates that the herein-introduced organotypic tissue-cultured skin equivalent is comparable to the normal human skin and its versatility in tests regarding skin pigmentation. Therefore, this model might help understand diseases with dysfunctional pigmentation such as melasma, vitiligo, and postinflammatory hyperpigmentation.

Influence of the HDAC Inhibitor Valproic Acid on the Growth and Proliferation of Temsirolimus-Resistant Prostate Cancer Cells In Vitro.

The mechanistic target of rapamycin (mTOR) is elevated in prostate cancer, making this protein attractive for tumor treatment. Unfortunately, resistance towards mTOR inhibitors develops and the tumor becomes reactivated. We determined whether epigenetic modulation by the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), may counteract non-responsiveness to the mTOR inhibitor, temsirolimus, in prostate cancer (PCa) cells. Prostate cancer cells, sensitive (parental) and resistant to temsirolimus, were exposed to VPA, and tumor cell growth behavior compared. Temsirolimus resistance enhanced the number of tumor cells in the G2/M-phase, correlating with elevated cell proliferation and clonal growth. The cell cycling proteins cdk1 and cyclin B, along with Akt-mTOR signaling increased, whereas p19, p21 and p27 decreased, compared to the parental cells. VPA significantly reduced cell growth and up-regulated the acetylated histones H3 and H4. Cdk1 and cyclin B decreased, as did phosphorylated mTOR and the mTOR sub-complex Raptor. The mTOR sub-member Rictor and phosphorylated Akt increased under VPA. Knockdown of cdk1, cyclin B, or Raptor led to significant cell growth reduction. HDAC inhibition through VPA counteracts temsirolimus resistance, probably by down-regulating cdk1, cyclin B and Raptor. Enhanced Rictor and Akt, however, may represent an undesired feedback loop, which should be considered when designing future therapeutic regimens.

Hibernating astronauts-science or fiction?

For long-duration manned space missions to Mars and beyond, reduction of astronaut metabolism by torpor, the metabolic state during hibernation of animals, would be a game changer: Water and food intake could be reduced by up to 75% and thus reducing payload of the spacecraft. Metabolic rate reduction in natural torpor is linked to profound changes in biochemical processes, i.e., shift from glycolysis to lipolysis and ketone utilization, intensive but reversible alterations in organs like the brain and kidney, and in heart rate control via Ca2+. This state would prevent degenerative processes due to organ disuse and increase resistance against radiation defects. Neuro-endocrine factors have been identified as main targets to induce torpor although the exact mechanisms are not known yet. The widespread occurrence of torpor in mammals and examples of human hypometabolic states support the idea of human torpor and its beneficial applications in medicine and space exploration.

Scanning Acoustic Microscopy-A Novel Noninvasive Method to Determine Tumor Interstitial Fluid Pressure in a Xenograft Tumor Model.

Elevated tumor interstitial fluid pressure (TIFP) is a prominent feature of solid tumors and hampers the transmigration of therapeutic macromolecules, for example, large monoclonal antibodies, from tumor-supplying vessels into the tumor interstitium. TIFP values of up to 40 mm Hg have been measured in experimental solid tumors using two conventional invasive techniques: the wick-in-needle and the micropuncture technique. We propose a novel noninvasive method of determining TIFP via ultrasonic investigation with scanning acoustic microscopy at 30-MHz frequency. In our experimental setup, we observed for the impedance fluctuations in the outer tumor hull of A431-vulva carcinoma-derived tumor xenograft mice. The gain dependence of signal strength was quantified, and the relaxation of tissue was calibrated with simultaneous hydrostatic pressure measurements. Signal patterns from the acoustical images were translated into TIFP curves, and a putative saturation effect was found for tumor pressures larger than 3 mm Hg. This is the first noninvasive approach to determine TIFP values in tumors. This technique can provide a potentially promising noninvasive assessment of TIFP and, therefore, can be used to determine the TIFP before treatment approach as well to measure therapeutic efficacy highlighted by lowered TFP values.

Cyclic three-dimensional wall motion of the human ascending and abdominal aorta characterized by time-resolved three-dimensional ultrasound speckle tracking.

The aim of this study was to measure, characterize, and compare the time-resolved three-dimensional wall kinematics of the ascending and the abdominal aorta. Comprehensive description of aortic wall kinematics is an important issue for understanding its physiological functioning and early detection of adverse changes. Data on the three-dimensional, dynamic cyclic deformation of the aorta in vivo are scarce. Either most imaging techniques available are too slow to capture aortic wall motion (CT, MRI) or they do not provide three-dimensional geometry data. Three-dimensional volume data sets of ascending and abdominal aortae of male healthy subjects (25.5 [24.5, 27.5] years) were acquired by use of a commercial echocardiography system with a temporal resolution of 11-25 Hz. Longitudinal and circumferential strain, twist, and relative volume change were determined by use of a commercial speckle tracking algorithm and in-house software. The kinematics of the abdominal aorta is characterized by diameter change, almost constant length and unidirectional, either clockwise or counter clockwise twist. In contrast, the ascending aorta undergoes a complex deformation with alternating clockwise and counterclockwise twist. Length and diameter changes were in the same order of magnitude with a phase shift between both. Longitudinal strain and its phase shift to circumferential strain contribute to the proximal aorta's Windkessel function. Complex cyclic deformations are known to be highly fatiguing. This may account for increased degradation of components of the aortic wall and therefore promote aortic dissection or aneurysm formation.

Impact of Different Spa Waters on Inflammation Parameters in Human Keratinocyte HaCaT Cells.

BACKGROUND: The treatment of different skin conditions with spa waters is a long tradition dating back to at least late Hellenism. Interestingly, independent scientific examinations studying the effect of spa waters are scarce. OBJECTIVE: In the present in vitro study, we compared the effect of culture media supplemented with (a) thermal spa waters (La Roche-Posay, Avène) and (b) two natural mineral drinking waters (Heppinger, Adelholzener) on physiological parameters in HaCaT keratinocytes. METHODS: The different medium preparations were investigated with regard to cell proliferation and cell damage. Moreover, the impact on inflammation parameters with and without ultraviolet B (UVB) irradiation was examined. RESULTS: Two popular thermal spring waters were found to suppress cell proliferation and cell damage. Moreover, these waters reversed the induction of interleukin-6, as measured using enzyme-linked immunosorbent assay and promoter transactivation, and the formation of reactive oxygen species after UVB stimulation. Of note, the two natural mineral waters, which are distributed as drinking waters, had some effect on the above-mentioned parameters but to a lesser extent. CONCLUSION: In summary, our results show that spa waters, and particularly those derived from thermal springs, reduce parameters associated with inflammation. It seems likely that trace elements such as selenium and zinc are critical for the observed effects.

Cellular responses to egg-oil (charismon©).

Egg-oil (Charismon©) is known for its beneficial action in wound healing and other skin irritancies and its antibacterial activity. The physiological basis for these actions has been investigated using cells in culture: HaCaT-cells (immortalized human keratinocytes), human endothelial cells in culture (HUVEC), peripheral blood mononuclear lymphocytes (PBML) and a full thickness human skin model (FTSM). Emphasis was on the influence of egg-oil on cell migration and IL-8 production in HaCaT cells, respiration, mitochondrial membrane potential, reactive oxygen (ROS) production and proliferation in HUVEC and HaCaT cells, cytokine and interleukin production in PBML and UV-light induced damage of FTSM. IL-8 production by HaCaT cells is stimulated by egg-oil whilst in phythemagglutin in-activated PBMLs production of the interleukins IL-2, IL-6, IL-10 and IFN-γ and TFN-α is reduced. ROS-production after H(2)O(2) stimulation first is enhanced but later on reduced. Respiration becomes activated due to partial uncoupling of the mitochondrial respiratory chain and proliferation of HaCaT and HUVEC is reduced. Recovery of human epidermis cells in FTSM after UV-irradiation is strongly supported by egg-oil. These results support the view that egg-oil acts through reduction of inflammatory processes and ROS production. Both these processes are equally important in cellular aging as in healing of chronic wounds.

Clinical application of a tissue-cultured skin autograft: an alternative for the treatment of non-healing or slowly healing wounds?

BACKGROUND: The treatment regime of non-healing or slowly healing wounds is constantly improving. One aspect is surgical defect coverage whereby mesh grafts and keratinocyte suspension are applied. OBJECTIVE: Tissue-cultured skin autografts may be an alternative for the treatment of full-thickness wounds and wounds that cover large areas of the body surface. METHODS: Autologous epidermal and dermal cells were isolated, expanded in vitro and seeded on collagen-elastin scaffolds. The developed autograft was immunohistochemically characterized and subsequently transplanted onto a facial chronic ulceration of a 71-year-old patient with vulnerable atrophic skin. RESULTS: Characterization of the skin equivalent revealed comparability to healthy human skin due to the epidermal strata, differentiation and proliferation markers. Within 138 days, the skin structure at the transplantation site closely correlated with the adjacent undisturbed skin. CONCLUSION: The present study demonstrates the comparability of the developed organotypic skin equivalent to healthy human skin and the versatility for clinical applications.

Mitochondrial dynamics in aging and disease.

Mitochondria are self-replicating organelles but nevertheless strongly depend on supply coded in nuclear genes. They serve many physiological demands in living cells. Supply of the cytoplasm with ATP and engagement in Ca(2+) regulation belong to the main functions of mitochondria. In large eukaryotic cells, in particular in neurons, with their long dendrites and axons, mitochondria have to move to the sites of their action. This trafficking involves several motor molecules and mechanisms to sense the sites of requirements of mitochondria. With aging and as a consequence of some diseases, mitochondrial components may be rendered dysfunctional, and mtDNA mutations arise during the course of replication and by the action of reactive oxygen species. Mutants in motor molecules engaged in trafficking and in the machinery of fusion and fission are causing severe deficiencies on the cellular level; they support neurodegeneration and, thus, cause many diseases. Frequent fusion and fission events mediate the elimination of impaired parts from mitochondria which finally will be degraded by autophagosomes. Extensive fusion provides a basis for functional complementation. Mobility of proteins and small molecules within the mitochondria is necessary to reach the functional goals of fusion and fission, although cristae and a large fraction of proteins of the respiratory complexes proved to be stable for hours after fusion and perform slow exchange of material.

Topically applied glycyrrhizic acid causes hair removal in rats.

CONTEXT: Anecdotic reports from Turkmenistan suggest an epilatory effect of sweet licorice extract after topical application. OBJECTIVE: This study examines hair removal after topical application of glycyrrhizic acid, the main compound of sweet licorice. MATERIALS AND METHODS: An aqueous solution containing 15% of the ammonium salt of glycyrrhizic acid, 10% urea, and 20% ethanol was topically applied two times per day on the neck areas of Wistar rats using a toothbrush. RESULTS: After 3 d, 20-30% of the treated areas were free of hair. After treatment for 6-12 d, 90-95% of the hair was gone. Clinical as well as immunohistological examinations showed no signs of inflammation even after long-term treatment for more than 9 months. Interestingly, long-term treatment reduced the regrowth of hair of about 20%. Examination by scanning electron microscopy showed a smoothed hair cuticle that might facilitate detachment of the hair shaft from the follicular wall. DISCUSSION AND CONCLUSION: Our findings suggest glycyrrhizic acid as an interesting molecule for treating hypertrichosis in humans.

Development and Characterization of an Engraftable Tissue-Cultured Skin Autograft: Alternative Treatment for Severe Electrical Injuries.

BACKGROUND/AIMS: Optimizing the treatment regimens of extensive or nonhealing defects is a constant challenge. Tissue-cultured skin autografts may be an alternative to mesh grafts and keratinocyte suspensions that are applied during surgical defect coverage. METHODS: Autologous epidermal and dermal cells were isolated, in vitro expanded and seeded on collagen-elastin scaffolds. The developed autograft was immunohistochemically and electron microscopically characterized. Subsequently, it was transplanted onto lesions of a severely burned patient. RESULTS: Comparability of the skin equivalent to healthy human skin could be shown due to the epidermal strata, differentiation, proliferation markers and development of characteristics of a functional basal lamina. Approximately 2 weeks after skin equivalent transplantation the emerging new skin correlated closely to the adjacent normal skin. CONCLUSION: The present study demonstrates the comparability of the developed organotypic skin equivalent to healthy human skin and its versatility for clinical applications.

Do we age because we have mitochondria?

The process of aging remains a great riddle. Production of reactive oxygen species (ROS) by mitochondria is an inevitable by-product of respiration, which has led to a hypothesis proposing the oxidative impairment of mitochondrial components (e.g., mtDNA, proteins, lipids) that initiates a vicious cycle of dysfunctional respiratory complexes producing more ROS, which again impairs function. This does not exclude other processes acting in parallel or targets for ROS action in other organelles than mitochondria. Given that aging is defined as the process leading to death, the role of mitochondria-based impairments in those organ systems responsible for human death (e.g., the cardiovascular system, cerebral dysfunction, and cancer) is described within the context of "garbage" accumulation and increasing insulin resistance, type 2 diabetes, and glycation of proteins. Mitochondrial mass, fusion, and fission are important factors in coping with impaired function. Both biogenesis of mitochondria and their degradation are important regulatory mechanisms stimulated by physical exercise and contribute to healthy aging. The hypothesis of mitochondria-related aging should be revised to account for the limitations of the degradative capacity of the lysosomal system. The processes involved in mitochondria-based impairments are very similar across a large range of organisms. Therefore, studies on model organisms from yeast, fungi, nematodes, flies to vertebrates, and from cells to organisms also add considerably to the understanding of human aging.

Vascular endothelial growth factor C-induced lymphangiogenesis decreases tumor interstitial fluid pressure and tumor.

Characteristically, most solid tumors exhibit an increased tumor interstitial fluid pressure (TIFP) that directly contributes to the lowered uptake of macromolecular therapeutics into the tumor interstitium. Abnormalities in the tumor-associated lymph vessels are a central brick in the development and prolonged sustaining of an increased TIFP. In the current study, vascular endothelial growth factor C (VEGF-C) was used to enhance tumor-associated lymphangiogenesis as a new mechanism to actively reduce the TIFP by increased lymphatic drainage of the tumor tissue. Human A431 epidermoid vulva carcinoma cells were inoculated in NMRI nu/nu mice to generate a xenograft mouse model. Seven days after tumor cell injection, VEGF-C was peritumorally injected to induce lymphangiogenesis. Tumor growth and TIFP was lowered significantly over time in VEGF-C-treated tumors in comparison to control or VEGF-A-treated animals. These data demonstrate for the first time that actively induced lymphangiogenesis can lower the TIFP in a xenograft tumor model and apparently reduce tumor growth. This model represents a novel approach to modulate biomechanical properties of the tumor interstitium enabling a lowering of TIFP in vivo.

Visualization of localized elastic properties in human tooth and jawbone as revealed by scanning acoustic microscopy.

The elastic properties of human canine and supporting alveolar bone are measured by the distribution of localized speed of sound using scanning acoustic microscopy. Methods for the dynamic, non-destructive diagnostics of dental hard tissues can have a key role in the early detection of demineralization processes and carious lesions, and they are supposed to open the possibility of early dental restorations. The localized distribution of the ultrasound velocity in canine tooth and alveolar bone was obtained using scanning acoustic microscopy with a 5- and 30-MHz transducer. An acoustic material signature curve signifies the interference of the waves and quantitatively maps the localized speed of sound in alveolar bone and the canine tooth. Seven samples, consisting of alveolar jawbone and tooth sliced along the coronally apical axis, were investigated. The average speed of sound was determined along three independent cross sections at enamel, dentin and cortical bone. The average speed of sound in enamel, bone and dentin was SD 3460 ± 193 m/s, 3232 ± 113 m/s and 2928 ± 106 m/s. The distribution of sound wave propagation reveals a decrease in sound speed from the peripheral parts within the enamel and dentin layers toward the proximal zones. These results prove the possibility of linking the elastic properties to different areas within the osseous and dental hard tissues and visualize them in an extremely high local resolution. The results serve as a basis for further study and substantiate the enormous potential of ultrasound based analysis in the field of dento-alveolar diagnosis.

Epithelial cells in culture: injured or differentiated cells?

Isolation of epithelial cells for cell culture is based on destruction of epithelial integrity. The consequences are manifold: cell polarity and specific cell functions are lost; cells acquire non-epithelial characteristics and start to proliferate. This situation may also occur in situ when parts of the epithelium are lost, either by apoptosis or necrosis by organ or tissue injury. During recovery from this injury, surviving epithelial cells proliferate and may restore epithelial integrity and finally re-differentiate into functional epithelial cells. In vitro, this re-differentiation is mostly not complete due to sub-optimal culture conditions. Therefore cultured epithelial cells resemble wounded or injured epithelia rather than healthy and well differentiated epithelia. The value of an in vitro cell model is the extent to which it helps to understand the function of the cells in situ. A variety of parameters influence the state of differentiation of cultured cells in vitro. Although each of these parameters had been studied, the picture how they co-ordinately influence the state of differentiation of epithelial cells in vitro is incomplete. Therefore we discuss the influence of the isolation method and cell culture on epithelial cells, and outline strategies to achieve highly differentiated epithelial cells for the use as an in vitro model.

Autophagy proteins LC3B, ATG5 and ATG12 participate in quality control after mitochondrial damage and influence lifespan.

Mitochondrial health is maintained by the quality control mechanisms of mitochondrial dynamics (fission and fusion) and mitophagy. Decline of these processes is thought to contribute to aging and neurodegenerative diseases. To investigate the role of mitochondrial quality control in aging on the cellular level, human umbilical vein endothelial cells (HUVEC) were subjected to mitochondria-targeted damage by combining staining of mitochondria and irradiation. This treatment induced a short boost of reactive oxygen species, which resulted in transient fragmentation of mitochondria followed by mitophagy, while mitochondrial dynamics were impaired. Furthermore, targeted mitochondrial damage upregulated autophagy factors LC3B, ATG5 and ATG12. Consequently these proteins were overexpressed in HUVEC as an in vitro aging model, which significantly enhanced the replicative life span up to 150% and the number of population doublings up to 200%, whereas overexpression of LAMP-1 did not alter the life span. Overexpression of LC3B, ATG5 and ATG12 resulted in an improved mitochondrial membrane potential, enhanced ATP production and generated anti-apoptotic effects, while ROS levels remained unchanged and the amount of oxidized proteins increased. Taken together, these data relate LC3B, ATG5 and ATG12 to mitochondrial quality control after oxidative damage, and to cellular longevity.