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.

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.

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.

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.

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.

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.

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.

Decreased expression of Drp1 and Fis1 mediates mitochondrial elongation in senescent cells and enhances resistance to oxidative stress through PINK1.

Mitochondria display different morphologies, depending on cell type and physiological situation. In many senescent cell types, an extensive elongation of mitochondria occurs, implying that the increase of mitochondrial length in senescence could have a functional role. To test this hypothesis, human endothelial cells (HUVECs) were aged in vitro. Young HUVECs had tubular mitochondria, whereas senescent cells were characterized by long interconnected mitochondria. The change in mitochondrial morphology was caused by downregulation of the expression of Fis1 and Drp1, two proteins regulating mitochondrial fission. Targeted photodamage of mitochondria induced the formation of reactive oxygen species (ROS), which triggered mitochondrial fragmentation and loss of membrane potential in young cells, whereas senescent cells proved to be resistant. Alterations of the Fis1 and Drp1 expression levels also influenced the expression of the putative serine-threonine kinase PINK1, which is associated with the PARK6 variant of Parkinson's disease. Downregulation of PINK1 or overexpression of a PINK1 mutant (G309D) increased the sensitivity against ROS in young cells. These results indicate that there is a Drp1- and Fis1-induced, and PINK1-mediated protection mechanism in senescent cells, which, when compromised, could contribute to the age-related progression of Parkinson's disease and arteriosclerosis.

During epithelial differentiation of human adipose-derived stromal/stem cells, expression of zonula occludens protein-1 is induced by a combination of retinoic acid, activin-A and bone morphogenetic protein-7.

BACKGROUND AIMS: Adipose-derived stromal/stem cells (ASC) possess a multilineage differentiation potential, can be used from an autologous origin, and are, therefore, attractive candidates for clinical applications to repair or regenerate damaged tissues and organs. Beside their well-known differentiation into cells of mesodermal origin, ASC are able to differentiate into cells of ecto- and endodermal origin. METHODS: Previous studies have shown that all trans retinoic acid (ATRA) induces the expression of cytokeratin 18 (CK18), indicating the beginning of differentiation into the epithelial lineage. Nevertheless, ATRA does not induce the expression of other epithelial markers. Therefore, we tested the additional influence of two growth factors on the onset of epithelial differentiation of ASC. The cells were cultured with ATRA, Activin A (ActA) and bone morphogenetic protein-7 (BMP-7), either alone or in combination. Differentiation into the epithelial lineage was assessed by the expression of the characteristic epithelial markers CK18 and zonula occludens protein 1 (ZO-1) using Western blot, immunofluorescence staining and polymerase chain reaction (PCR) analysis. RESULTS: The mixture of all three factors induced epithelial differentiation of ASC without enhancing cell proliferation. Upon induction, the ASC showed phenotypic changes consistent with an epithelial phenotype. The addition of the growth factors ActA and BMP-7 enhanced the inductive effect of ATRA, as shown by the de novo expression of ZO-1 in addition to CK18 expression. CONCLUSIONS: Our study highlights the onset of the epithelial differentiation of ASC induced by culture with a combination of ATRA, ActA and BMP-7.

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.

Regulation of the cardiovascular function by CO2 laser stimulation in anesthetized rats.

Physical stimulation of body surface points is known to affect various organ functions. In traditional Chinese medicine, so-called acupoints were defined. These points can be physically stimulated to effectively treat various diseases. Here we describe for the first time the effect of CO(2) laser stimulation at the acupoints Neiguan (PC-6), Quchi (LI-11), Zusanli (ST-36), and Taichong (LR-3) on heart rate and mean arterial blood pressure in anesthetized rats. CO(2) laser stimulation increased the skin surface temperature to 54°C. Our results revealed that the laser stimulation at the left or right PC-6 and LR-3 increased heart rate and mean arterial pressure. There was no response of heart rate and mean arterial pressure during and after stimulation of the left LI-11, but laser stimulation at the right LI-11 slightly increased heart rate and mean arterial pressure. On the other hand, laser stimulation at the left and right ST-36 decreased heart rate and mean arterial pressure. The effects on mean arterial pressure were more pronounced than those on heart rate. After full spinal cord transection, all heart-rate and mean-arterial-pressure responses were attenuated or completely abolished. These results suggest that CO(2) laser stimulation at either the left or right PC-6, ST-36, and LR-3, as well as at the right LI-11 can modulate the cardiovascular functions in anesthetized rats, and its modulatory site might be supraspinal.

Determination of protein mobility in mitochondrial membranes of living cells.

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F(1)F(0) ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D=0.7µm(2)/s and 0.6µm(2)/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F(1)F(0) ATPase: 0.4µm(2)/s and 0.0005µm(2)/s; COX: 0.3µm(2)/s and 0.007µm(2)/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.

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.

Curcumin in combination with visible light inhibits tumor growth in a xenograft tumor model.

It is known that curcumin, a dietary pigment from the plant Curcuma longa, inhibits cell proliferation and induces apoptosis in different cell lines; however, the therapeutic benefit is hampered by very low absorption after transdermal or oral application. Recent studies from our laboratory have demonstrated that curcumin at low concentrations (0.2-1 microg/ml) offered the described effects only when applied with UVA or visible light. Nevertheless, the in vivo efficacy of this combination is lacking. In the present study, we used a xenograft tumor model with human epithelial carcinoma A431 cells to test the effect of curcumin and visible light on tumor growth. It was found that tumor growth was significantly inhibited in mice that were i.p. injected with curcumin and consecutively irradiated with visible light. Furthermore, immunohistochemistry showed a reduction of Ki 67 expression, indicating a decrease of cycling cells and induction of apoptotic bodies. The effect on apoptosis was further confirmed by Western blot analysis showing enhanced activation of caspases-9. Vice versa inhibition of extracellular regulated kinases (ERK) 1/2 and epidermal growth factor receptor (EGF-R) was observed which may aid inhibition of proliferation and induction of apoptosis. In summary, the present findings suggest a combination of curcumin and light as a new therapeutic concept to increase the efficacy of curcumin in the treatment of cancer.

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.

How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells.

Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions tau(2) and tau(3) increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of pi-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.

Short- and long-term alterations of mitochondrial morphology, dynamics and mtDNA after transient oxidative stress.

Cells are exposed during their life span to fluctuating levels of reactive oxygen species (ROS). To investigate the effects of a single ROS boost in vitro, human endothelial cells (HUVEC) were treated with one short-term dose of hydrogen peroxide. This treatment resulted in a short, dose-dependent ROS peak that caused transient changes in the mitochondrial morphology and fine structure, in the frequency of mitochondrial fission and fusion and in the mRNA levels of distinct fission and fusion factors. Treatment with a higher dose induced prolonged mtDNA damage; these cells exhibited a significantly shortened replicative lifespan, indicating dose-dependent effects of oxidative stress on mitochondria.

Evaluation of beneficial and adverse effects of glucocorticoids on a newly developed full-thickness skin model.

Glucocorticoids (GCs) are highly effective compounds widely used in the treatment of inflammatory diseases; however, they offer distinct adverse effects such as skin thinning in response to long-term topical treatment. Nevertheless it is difficult to deduce the safety of a newly synthesized compound from its structural formula. Efficient assay systems that measure beneficial and adverse effects are needed. In the present study the applicability of a three-dimensional full-thickness skin model (FTSM) is tested to display GC-induced effects regarding anti-inflammation and atrophy. It is shown that topical application of a commercial GC ointment suppresses the ultraviolet (UV)B induced induction of interleukin (IL)-6 and IL-8. Addition of purified betamethasone-17-valerate, prednicarbate and clobetasol-17-propionate to the culture medium for 14 days caused a reduction in the number of epidermal cell-layers corresponding to the atrophic risk found in vivo. Similarly, repeated topical application of five GC creams induced epidermal thinning. Evidence is given that the inhibitory effect on keratinocyte proliferation contributes to this effect. Furthermore, dermal thinning was monitored by measuring type I collagen synthesis; a decreased collagen synthesis similar to the in vivo situation is shown. The present study demonstrates the versatility of this FTSM in the validation of effectiveness and safety of GCs.