Cell Replication Rates Research Articles

Insights into ageing rates comparison across tissues from recalibrating cerebellum DNA methylation clock

DNA methylation (DNAm)-based age clocks have been studied extensively as a biomarker of human ageing and a risk factor for age-related diseases. Despite different tissues having vastly different rates of proliferation, it is still largely unknown whether they age at different rates. It was previously reported that the cerebellum ages slowly; however, this claim was drawn from a single clock using a relatively small sample size and so warrants further investigation. We collected the largest cerebellum DNAm dataset (N = 752) to date. We found the respective epigenetic ages are all severely underestimated by six representative DNAm age clocks, with the underestimation effects more pronounced in the four clocks whose training datasets do not include brain-related tissues. We identified 613 age-associated CpGs in the cerebellum, which accounts for only 14.5% of the number found in the middle temporal gyrus from the same population (N = 404). From the 613 cerebellum age-associated CpGs, we built a highly accurate age prediction model for the cerebellum named CerebellumClockspecific (Pearson correlation=0.941, MAD=3.18 years). Ageing rate comparisons based on the two tissue-specific clocks constructed on the 201 overlapping age-associated CpGs support the cerebellum has younger DNAm age. Nevertheless, we built BrainCortexClock to prove a single DNAm clock is able to unbiasedly estimate DNAm ages of both cerebellum and cerebral cortex, when they are adequately and equally represented in the training dataset. Comparing ageing rates across tissues using DNA methylation multi-tissue clocks is flawed. The large underestimation of age prediction for cerebellums by previous clocks mainly reflects the improper usage of these age clocks. There exist strong and consistent ageing effects on the cerebellar methylome, and we suggest the smaller number of age-associated CpG sites in cerebellum is largely attributed to its extremely low average cell replication rates.

Scaly dermatoses: What’s that itch?

Chronic scaly dermatoses include dandruff, seborrheic dermatitis, and psoriasis. These disorders involve the epidermis, the uppermost layer of skin. The primary manifestation is scaling of the skin, with inflammation, erythema, and other changes in appearance of variable degree and severity. Individuals with scaly dermatoses can receive significant relief through focused OTC products and important educational tips from their pharmacist. Dandruff is the less-inflammatory form of scaly dermatoses. It manifests as a more subtle dermatitis with relatively fine scaling confined to the scalp and occasionally includes mild erythema or inflammation. The goals of self-treatment for dandruff are 1) to reduce the epidermal turnover rate of the scalp skin by reducing the number of Malassezia fungi on the scalp, 2) to minimize the cosmetic embarrassment of visible scaling, and 3) to minimize itch. Shampooing with a general-purpose nonmedicated shampoo daily or every other day often is sufficient to control mild to moderate dandruff. Nonprescription medicated antidandruff shampoos, including those with either a cytostatic agent or ketoconazole, are other options. Topical cytostatic agents include pyrithione zinc, selenium sulfide, and coal tar. These agents can decrease the rate of epidermal cell replication and are of limited effectiveness. Contact time is the key to any efficacy, so medicated shampoos should be massaged into the scalp and left on the hair for three to five minutes before rinsing. Use the medicated shampoo daily for one week, then two to three times weekly for two to three weeks, and thereafter once weekly or every other week to control the disorder. Nonprescription ketoconazole antifungal shampoo is active against most pathogenic fungi but is indicated specifically for Malassezia infections. This product is used twice a week for 4 weeks, with at least three days between each treatment. Once the disorder is controlled, apply once weekly to prevent relapse. Seborrheic dermatitis affects the scalp, face, and chest (usually the sternum) and typically features significant inflammation and erythema. The goals of self-treatment for seborrheic dermatitis are 1) to reduce inflammation and the epidermal turnover rate of the scalp skin by reducing the level of Malassezia fungi, 2) to minimize or eliminate visible erythema and scaling, and 3) to minimize itch. Because of the inflammatory nature of seborrheic dermatitis, treatment is similar to, but more aggressive than, that for dandruff. Topical corticosteroids are needed more frequently to treat the more significant inflammation in seborrheic dermatitis. Corticosteroid products may be used to manage seborrheic dermatitis whenever erythema persists after therapy with medicated shampoos. Hydrocortisone ointment should be applied no more than twice daily because of the reservoir effect of the stratum corneum, which slowly releases the corticosteroid over time, and potential greasiness. Treatment should continue until symptoms subside but for no more than 7 consecutive days since seborrheic dermatitis is rarely cured by nonprescription treatment. If the disorder worsens or symptoms persist longer than 7 days, a dermatologist or other knowledgeable health care provider should be consulted. Psoriasis is a highly inflammatory skin disorder characterized by raised plaques and adherent thick, silvery white scales. The goals of self-treatment for psoriasis are 1) to control or eliminate the signs and symptoms (i.e., inflammation, scaling, itching) and 2) to prevent or minimize the likelihood of flares. Mild cases of psoriasis, characterized by the presence of a few localized lesions no larger than a quarter, are amenable to self-treatment with topical agents. Patients with psoriasis should be encouraged to bathe with lubricating bath products two to three times per week using tepid water. Daily lubrication of the skin after a bath or shower is essential. Emollients moisturize, lubricate, and soothe dry and flaky skin as well as reduce fissure formation within plaques and help maintain flexibility of the surrounding skin. Emollients should be applied to the lesions within minutes of bathing. To be effective, these products need to be applied liberally with gentle rubbing, up to 4 times daily. Hydrocortisone ointment 1% is the nonprescription medication of choice for treatment of bright red lesions. In some patients, nonprescription treatment may be ineffective and more aggressive treatments may be needed. Psoriasis can be controlled but not cured, and remissions do occur. Patients should be educated on controlling the disorder to help prevent flare-ups and to better understand this disorder to help reduce associated emotional stress and frequency of psychogenic exacerbations.

Human bronchial epithelial cells from patients with asthma have an altered gene expression profile.

Gene changes observed in asthma bronchial epithelial cells are maintained following repeated culture, presenting with an exaggerated response to viral infection and immune responses as well as having differences in the rate of cell division and replication https://bit.ly/3Cq2xKf

Insights from Leishmania (Viannia) guyanensis in vitro behavior and intercellular communication

BackgroundPentavalent antimonial-based chemotherapy is the first-line approach for leishmaniasis treatment and disease control. Nevertheless antimony-resistant parasites have been reported in some endemic regions. Treatment refractoriness is complex and is associated with patient- and parasite-related variables. Although amastigotes are the parasite stage in the vertebrate host and, thus, exposed to the drug, the stress caused by trivalent antimony in promastigotes has been shown to promote significant modification in expression of several genes involved in various biological processes, which will ultimately affect parasite behavior. Leishmania (Viannia) guyanensis is one of the main etiological agents in the Amazon Basin region, with a high relapse rate (approximately 25%).MethodsHerein, we conducted several in vitro analyses with L. (V.) guyanensis strains derived from cured and refractory patients after treatment with standardized antimonial therapeutic schemes, in addition to a drug-resistant in vitro-selected strain. Drug sensitivity assessed through Sb(III) half-maximal inhibitory concentration (IC50) assays, growth patterns (with and without drug pressure) and metacyclic-like percentages were determined for all strains and compared to treatment outcomes. Finally, co-cultivation without intercellular contact was followed by parasitic density and Sb(III) IC50 measurements.ResultsPoor treatment response was correlated with increased Sb(III) IC50 values. The decrease in drug sensitivity was associated with a reduced cell replication rate, increased in vitro growth ability, and higher metacyclic-like proportion. Additionally, in vitro co-cultivation assays demonstrated that intercellular communication enabled lower drug sensitivity and enhanced in vitro growth ability, regardless of direct cell contact.ConclusionsData concerning drug sensitivity in the Viannia subgenus are emerging, and L. (V.) guyanensis plays a pivotal epidemiological role in Latin America. Therefore, investigating the parasitic features potentially related to relapses is urgent. Altogether, the data presented here indicate that all tested strains of L. (V.) guyanensis displayed an association between treatment outcome and in vitro parameters, especially the drug sensitivity. Remarkably, sharing enhanced growth ability and decreased drug sensitivity, without intercellular communication, were demonstrated.Graphical

Exploring a new approach for regenerative medicine: Ti-doped polycrystalline diamond layers as bioactive platforms for osteoblast-like cells growth

This study explores the feasibility to use electroconductive Ti-doped polycrystalline diamond layers as scaffolds for tissue engineering. The synthesis of the diamond–based materials is accomplished in a HFCVD reactor where Ti(IV) acetyl acetonate powders are delivered by N2 fluxes to the growing diamond phase. In-depth investigations (Raman spectroscopy, SEM, AFM, XRD, XPS) allowed the characterization of the morphological/structural/compositional features and the properties of charge transport (KPFM, I-V) induced in the diamond layers by the incorporation of Ti-species. The bioactivity of the Ti-doped diamond surface was verified investigating the growth of MG-63 osteoblast-like cells by using MTT assays and confocal microscopy. The study evidenced a net increase of cell replication rate on diamond scaffolds after 4 days of incubation. After 6-days incubation, the cell growth on the Ti-doped diamond scaffolds increased up to 150% compared with the reference polystyrene tissue culture vessel, with a dominant presence of cells in active division.The cell behavior is discussed and related to the structural and functional surface properties of the Ti-diamond systems, acting as bioactive platforms able to offer an extremely beneficial environment for cell proliferation and viability.

Dynamics of Mammalian Cell Infection by Trypanosoma cruzi trypomastigotes

In a recent work we demonstrated that Trypanosoma cruzi trypomastigotes change their motility patterns in the presence of mammalian cells, that the extent of the changes depends on the cell line, and that this extent is positively correlated with the efficiency with which parasites invade the different cell lines. These results open the question of what cellular characteristics are relevant for parasite identification and invasion. In the present work, we tackled such question. We performed infection-kinetics experiments on various cell lines, and developed a mathematical model to simulate the experimental outcomes. An analysis of the cell-parasite mechanisms included in the model, together with the parameter values that allowed it to replicate the experimental results, suggests that a process related to the cell replication rate may strongly influence the parasite invasion efficiency, and the infection dynamics in general.

Mathematical modelling of telomere length dynamics

Telomeres are repetitive DNA sequences located at the ends of chromosomes. During cell division, an incomplete copy of each chromosome’s DNA is made, causing telomeres to shorten on successive generations. When a threshold length is reached replication ceases and the cell becomes ‘senescent’. In this paper, we consider populations of telomeres and, from discrete models, we derive partial differential equations which describe how the distribution of telomere lengths evolves over many generations. We initially consider a population of cells each containing just a single telomere. We use continuum models to compare the effects of various mechanisms of telomere shortening and rates of cell division during normal ageing. For example, the rate (or probability) of cell replication may be fixed or it may decrease as the telomeres shorten. Furthermore, the length of telomere lost on each replication may be constant, or may decrease as the telomeres shorten. Where possible, explicit solutions for the evolution of the distribution of telomere lengths are presented. In other cases, expressions for the mean of the distribution are derived. We extend the models to describe cell populations in which each cell contains a distinct subpopulation of chromosomes. As for the simpler models, constant telomere shortening leads to a linear reduction in telomere length over time, whereas length-dependent shortening results in initially rapid telomere length reduction, slowing at later times. Our analysis also reveals that constant telomere loss leads to a Gaussian (normal) distribution of telomere lengths, whereas length-dependent loss leads to a log-normal distribution. We show that stochastic models, which include a replication probability, also lead to telomere length distributions which are skewed.

Estimation of the cancer risk induced by therapies targeting stem cell replication and treatment recommendations

Rejuvenation of stem cell activity might increase life expectancy by prolonging functionality of organs. Higher stem cell replication rates also bear the risk of cancer. The extent of this risk is not known. While it is difficult to evaluate this cancer risk in experiments, it can be estimated using a mathematical model for tissue homeostasis by stem cell replication and associated cancer risk. The model recapitulates the observation that treatments targeting stem cell replication can induce a substantial delay of organ failure. The model predicts that the cancer risk is minor under particular conditions. It depends on the assumed implications for cell damage repair during treatment. The benefit of rejuvenation therapy and its impact on cancer risk depend on the biological age at the time of treatment and on the overall cell turnover rate of the organs. Different organs have to be considered separately in the planning of systemic treatments. In recent years, the transfer of blood from young to old individuals was shown to bear the potential of rejuvenation of stem cell activity. In this context, the model predicts that the treatment schedule is critical for success and that schedules successful in animal experiments are not transferable to humans. Guidelines for successful protocols are proposed. The model presented here may be used as a guidance for the development of stem cell rejuvenation treatment protocols and the identification of critical parameters for cancer risk.

Targeting metabolic pathways for head and neck cancers therapeutics.

Cancer cells have distinctive energy metabolism pathways that support their rapid cell division. The preference for anaerobic glycolysis under the normal oxygen condition is known as the Warburg effect and has been observed in head and neck cancers. These metabolic changes are controlled by cancer-related transcription factors, such as tumor suppressor gene and hypoxia inducible factor 1α. In addition, various metabolic enzymes also actively regulate cancer-specific metabolism including the switch between aerobic and anaerobic glycolysis. For a long time, these metabolic changes in cancer cells have been considered a consequence of transformation required to maintain the high rate of tumor cell replication. However, recent studies indicate that alteration of metabolism is sufficient to initiate tumor transformation. Indeed, oncogenic mutations in the metabolic enzymes, isocitrate dehydrogenase and succinate dehydrogenase, have been increasingly found in various cancers, including head and neck cancers. In the present review, we introduce recent findings regarding the cancer metabolism, including the molecular mechanisms of how they affect cancer pathogenesis and maintenance. We also discuss the current and future perspectives on therapeutics that target metabolic pathways, with an emphasis on head and neck cancer.

Colorectal polyps: cancer risk and classification

Colorectal polyps are histologically classified as inflammatory, hamartomatous, hyperplastic or neoplastic (adenomas). Adenomas can develop into malignant adenocarcinomas, which result in 95% of colorectal cancers. Polyps can be detected and examined by endoscopy, where the size, pit pattern, shape (sessile or pedunculated) and elevation (protruded, superficially elevated, flat or depressed) of a polyp can be indicative of its type and cancer risk. Chromoendoscopy and the lifting sign technique can help in diagnosis. Histological examination of the shape (serrated or not), structure (tubular, villous or tubulovillous) and cell replication rates of polyp tissue can be effective at identifying dysplasia and cancer risk. Should a polyp develop into a carcinoma, the extent of its invasion through the colon wall can be classified by the Haggit and Kikuchi scales to inform prognosis and appropriate interventions. Endoscopy nurses have an important role in detecting and diagnosing polyps and thus preventing the development of colorectal cancers.

Differences in Flower Transcriptome between Grapevine Clones Are Related to Their Cluster Compactness, Fruitfulness, and Berry Size.

Grapevine cluster compactness has a clear impact on fruit quality and health status, as clusters with greater compactness are more susceptible to pests and diseases and ripen more asynchronously. Different parameters related to inflorescence and cluster architecture (length, width, branching, etc.), fruitfulness (number of berries, number of seeds) and berry size (length, width) contribute to the final level of compactness. From a collection of 501 clones of cultivar Garnacha Tinta, two compact and two loose clones with stable differences for cluster compactness-related traits were selected and phenotyped. Key organs and developmental stages were selected for sampling and transcriptomic analyses. Comparison of global gene expression patterns in flowers at the end of bloom allowed identification of potential gene networks with a role in determining the final berry number, berry size and ultimately cluster compactness. A large portion of the differentially expressed genes were found in networks related to cell division (carbohydrates uptake, cell wall metabolism, cell cycle, nucleic acids metabolism, cell division, DNA repair). Their greater expression level in flowers of compact clones indicated that the number of berries and the berry size at ripening appear related to the rate of cell replication in flowers during the early growth stages after pollination. In addition, fluctuations in auxin and gibberellin signaling and transport related gene expression support that they play a central role in fruit set and impact berry number and size. Other hormones, such as ethylene and jasmonate may differentially regulate indirect effects, such as defense mechanisms activation or polyphenols production. This is the first transcriptomic based analysis focused on the discovery of the underlying gene networks involved in grapevine traits of grapevine cluster compactness, berry number and berry size.

Telomeres, NAFLD and Chronic Liver Disease

Telomeres consist of repeat DNA sequences located at the terminal portion of chromosomes that shorten during mitosis, protecting the tips of chromosomes. During chronic degenerative conditions associated with high cell replication rate, progressive telomere attrition is accentuated, favoring senescence and genomic instability. Several lines of evidence suggest that this process is involved in liver disease progression: (a) telomere shortening and alterations in the expression of proteins protecting the telomere are associated with cirrhosis and hepatocellular carcinoma; (b) advanced liver damage is a feature of a spectrum of genetic diseases impairing telomere function, and inactivating germline mutations in the telomerase complex (including human Telomerase Reverse Transcriptase (hTERT) and human Telomerase RNA Component (hTERC)) are enriched in cirrhotic patients independently of the etiology; and (c) experimental models suggest that telomerase protects from liver fibrosis progression. Conversely, reactivation of telomerase occurs during hepatocarcinogenesis, allowing the immortalization of the neoplastic clone. The role of telomere attrition may be particularly relevant in the progression of nonalcoholic fatty liver, an emerging cause of advanced liver disease. Modulation of telomerase or shelterins may be exploited to prevent liver disease progression, and to define specific treatments for different stages of liver disease.

COMPARATIVE BIOLOGY OF AGING

1. Introduction and end of life pathologies Norman Wolf and Steven Austad 2. Animal size, metabolic rate, and survival, among and within species Steven Austad 3. Hormonal Influences on Aging and Lifespan Adam Spong and Andrzej Bartke 4. Exploring mechanisms of aging retardation by caloric restriction: studies in model organisms and mammals. Rozalyn Anderson, Ricki Colman & Richard Weindruch 5. Cell Replication Rates In Vivo and In Vitro and Wound Healing as Affected by Animal Age, Diet, and Species. Norman Wolf 6. The Comparative Biology of Sirtuin Function in Longevity Daniel L. Smith, Jr., and Jeffrey S. Smith 7. The Role of TOR signaling in Aging. Matt Kaeberlein and Lara S. Shamieh 8. Mitochondria, Oxidative Damage and Longevity: What Can Comparative Biology Teach Us? Yun Shi, Rochelle Buffenstein and Holly Van Remmen 9. Comparative genomics of aging. Jan Vijg, Ana Maria Garcia, Brent Calder and Martijn Dolle 10. Changes in lysosomes and their autophagic function in aging. The comparative biology of lysosomal function. Samantha J. Orenstein and Ana Maria Cuervo 11. Telomeres and telomerase. Inter-species comparisons of genetic, mechanistic and functional aging changes. N.M.V. Gomes, J.W. Shay, W.E. Wright, 12. Cardiac Aging. Dao-Fu Dai, Robert J. Wessells, Rolf Bodmer, Peter S. Rabinovitch 13. Comparative Skeletal Muscle Aging. David J. Marcinek, Jonathan Wanagat, Jason J. Villarin 14. Aging of the Nervous System. Catherine A. Wolkow, Sige Zou and Mark P. Mattson 15. Aging of the Immune System across Different Species. Janko Nikolich-Zugich and Luka Cicin-Sain

Peto's paradox and human cancers.

Peto's paradox is the lack of the expected trend in cancer incidence as a function of body size and lifespan across species. The leading hypothesis to explain this pattern is natural selection for differential cancer prevention in larger, longer lived species. We evaluate whether a similar effect exists within species, specifically humans. We begin by reanalysing a recently published dataset to separate the effects of stem cell number and replication rate, and show that each has an independent effect on cancer risk. When considering the lifetime number of stem cell divisions in an extended dataset, and removing cases associated with other diseases or carcinogens, we find that lifetime cancer risk per tissue saturates at approximately 0.3-1.3% for the types considered. We further demonstrate that grouping by anatomical site explains most of the remaining variation. Our results indicate that cancer risk depends not only on the number of stem cell divisions but varies enormously (approx. 10 000 times) depending on anatomical site. We conclude that variation in risk of human cancer types is analogous to the paradoxical lack of variation in cancer incidence among animal species and may likewise be understood as a result of evolution by natural selection.

A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.

Types 1 and 2 diabetes affect some 380 million people worldwide. Both result ultimately from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with peak beta cell labeling indices achieving approximately 2% in first year of life1-4. In embryonic life and after early childhood, beta cell replication rates are very low. While beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1-8. Hence, there remains an urgent need for diabetes therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, we report the results of a high-throughput small molecule screen (HTS) revealing a novel class of human beta cell mitogenic compounds, analogues of the small molecule, harmine. We also define dual specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine, and the Nuclear Factors of activated T-cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation as well as beta cell differentiation. These observations suggest that harmine analogues (“harmalogs”) may have unique therapeutic promise for human diabetes therapy. Enhancing potency and beta cell specificity are important future challenges.

Tailored silica-antibiotic nanoparticles: overcoming bacterial resistance with low cytotoxicity.

New and more aggressive antibiotic resistant bacteria arise at an alarming rate and represent an ever-growing challenge to global health care systems. Consequently, the development of new antimicrobial agents is required to overcome the inefficiency of conventional antibiotics and bypass treatment limitations related to these pathologies. In this study, we present a synthesis protocol, which was able to entrap tetracycline antibiotic into silica nanospheres. Bactericidal efficacy of these structures was tested against bacteria that were susceptible and resistant to antibiotics. For nonresistant bacteria, our composite had bactericidal efficiency comparable to that of free-tetracycline. On the other hand, the synthesized composites were able to avoid bacterial growth of resistant bacteria while free-tetracycline has shown no significant bactericidal effect. Finally, we have investigated the cytotoxicity of these nanoparticles against mammalian cells to check any possible poisoning effect. It was found that these nanospheres are not apoptosis-inducers and only a reduction on the cell replication rate was seen when compared to the control without nanoparticles.

Picoplankton community structure before, during and after convection event in the offshore waters of the Southern Adriatic Sea

Abstract. This paper documents the picoplankton community's response to changes in oceanographic conditions in the period between October 2011 and September 2012 at two stations belonging to the South Adriatic Pit (SAP). The recorded data include the community's abundance, composition, prokaryotic production rates and bacterial metabolic capacity. The sampling period included an intense sea cooling with formation of exceptional, record-breaking dense water. We documented an especially intense winter convection episode that completely diluted the core of Levantine intermediate waters (LIW) in a large area encompassing the SAP's center and its margin. During this convection event the whole picoplankton community had significantly higher abundances with a recorded picoeukaryotic peak at the SAP margin. In the post-convection phase in March, prokaryotic heterotrophic production strongly increased in the entire SAP area (up to 50 times; 456.8 nM C day−1). An autotrophic biomass increase (up to 5 times; 4.86 μg L−1) and a disruption of a close correspondence between prokaryotic heterotrophic biomass production and cell replication rates were observed only in the center of the SAP, which was not under the influence of LIW. At the SAP's margin such an effect was attenuated by LIW, since the waters affected by LIW were characterized by decreased concentrations of dissolved inorganic nitrogen, decreased autotrophic biomasses, and by increased bacterial biomass production balanced with cell replication rates as well as by the domination of Synechococcus among autotrophic picoplankton. The metabolic capacity was lowest in spring when autotrophic biomass largely increased, while the highest levels found in the pre-convection phase (October 2011) suggest that the system was more oligotrophic before than after the convection event. Furthermore, we showed that metabolic capacity is a trait of bacterial community independent of environmental conditions and tightly linked to cell replication and substrate availability. In contrast, the bacterial community composition appears to be strongly influenced by physico-chemical characteristics of waters (e.g., temperature and nutrients) and environmental forcing (e.g., convection and LIW). Our results showed that the two oceanographic phenomena of the Southern Adriatic, strongly relevant for the total production of the Adriatic Sea, winter convection and LIW intrusion, regulate the changes in picoplankton community structure and activities.

Killing cancer cells with nanotechnology: the effect of CaS nanostructures on the cell replication rate and survival of human mammary adenocarcinoma cell lines (780.9)

Calcium sulfide (CaS) nanostructures have the potential to play an important role as cadmium‐free nanoscaled semiconductors with applications in bioimaging, in vivo labeling, and sensing, and for drug delivery systems. The objective of this work is to establish the effect of CaS nanostructures on cell replication rate and survival of human mammary adenocarcinoma cell lines. Human adenocarcinoma cell lines are grown in eagle’s minimum essential medium (EMEM). Single doses of dispersions containing traces of CaS nanostructures were added to human mammary adenocarcinoma cell lines with a 70 % confluence. After a 24 hour period, 200,000 cancer cells were subculture in T‐75 treated flasks. The number of dead cells in the cell culture media supernatant was determined in intervals of 24 hours using a Biorad T10 cell counter. The number of dead cells in the supernatant is found to increase from 2,000 in control cell cultures to over 70,000 in cancer cell cultures exposed to a single dose of the CaS dispersion in the first 24 hours: this represents 35 % of the initial cell seeding. The number of dead cells remains in the range of 60,000 to 30,000 in measurements performed between 48 and 72 hours following the single dose of the dispersion containing the CaS nanostructures. The number of replicating cells is found to decrease in the first 72 hours and to increase thereafter. We conclude that the CaS nanostructures affect the replication rate and survival of human adenocarcinoma cells and demonstrate the role CaS nanostructures may play to treat localized cancers.Grant Funding Source: NIH NIGMS‐R25GM088023 and NIGMS‐R25GM096955

New strategies to slow down the photoaging of the human skin

Previous reports indicate that inflammatory and proliferating skin conditions like acne and psoriasis are also associated with decreased ATP and cyclic nucleotides (cAMP) in blood and epidermal cells. In the late 80’s we noticed that psoriatic skin lesions significantly respond to supplementation with energy generating compounds and melanin promoters. In clinical studies, topically applied energy generating compounds like AMP, ADP and NAD normalized the cell replication rate in psoriatic skin, (CELL ENERGY) diminished the acne pustulae and induced a significant improvement of skin structure and wrinkles (anti-aging-effect). The photoaging process of the human skin in the presence of natural sunlight or artificial UV-sources is closelyy related to a strong generation of free radicals, lipid peroxidation, collagenase activation, glycation/ oxidation of proteins (AGE products), activation of p53 transcription factors, low DNA repair capacity and cumulative DNA mutations. Clinically, the adverse effects of natural sunlight and other UV-sources on normal human skin may vary from sunburn with erythema, oedema and DNA damage (12-24 hrs. after UV-exposure) to polymorphic light reaction (eczema solare), solar actinic elastosis and actinic hyperkeratosis (as common precancerous condition), up to different skin cancer forms like basal cell carcinoma (BCC), squamous cell carcinoma (SCC) or malignant melanoma (MM). The study of the lipid, protein and DNA oxidative damage triggered by the free radical attack subsequent to sunlight exposure has conducted to appropriate strategies to slow down or block these reactions making possible the design of innovative skin care formulations. In this respect, a new German photoaging defence formula (SOLARIS SPF 25) combines for the first time the double UVA + UVB protection (SPF 25) with melanin promoting aminoacids directly enhancing the natural tanning process. Free radical blocking agents like Vit. E, carrot oil and metal chelators are preventing the sun exposure side-effects together with immune stimulating plant extracts (β-glucans) with anti-herpes virus activity. To slow down the photoaging related wrinkle formation efficiently, a new collagen synthase stimulating formula offers a synergistic anti-aging combination of UV-light blockers, free radical quenchers (Vit. E, Coenzyme Q10) and collagen/ elastin synthesis promoters like hydroxyprolin and plant bioflavonoids (ENERGO Repair)). The active ingredients are incorporated in liposomes containing skin identical phospholipids and ceramides by means of the patented DMS® nanoparticle technology. A rapid uptake in the epidermis cells is thus granted. The use of the described hypoallergenic topical products results in a significant improvement of the skin structure and appearance within 30 days, as documented with the standardized Surface Evaluation of Living Skin (SELS) methodology in a group of 35 women aged 40 to 63 years. Our recent research shows that appropriate combinations of hypoallergenic protein hydrolysates with sugar alcohols and omega-3 polyunsaturated fatty acids (EQUIDERM PLUS) are showing a remarkable free radical quenching and anti-inflammatory activity leading to a significant symptom alleviation in skin and geriatric patients. Finally, with the emergence of affordable microarray gene expression profiling methods we had the opportunity to test several oral and i.v. nutraceutical combinations (NUTRIGEN PA, NUTRIGEN PS ) for their silencing ability on inflammatory genes (nutrigenomic evaluation) and correlate the “ex in vivo” results in blood cells with their “ in vivo” clinical efficacy.

Histone Deacetylase Inhibition Activates Transgene Expression from Integration-Defective Lentiviral Vectors in Dividing and Non-Dividing Cells

Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed in both basic and preclinical gene transfer settings. Often, however, the IDLV transgene expression profile is muted when compared to that of their integration-proficient counterparts. We hypothesized that the episomal nature of IDLVs turns them into preferential targets for epigenetic silencing involving chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array of cis-acting elements and transcriptional unit components were assembled with the aid of packaging constructs encoding either the wild-type or the class I mutant D116N integrase moieties. The transduction levels and transgene-product yields provided by each vector class were assessed in the presence and absence of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A. To investigate the role of the target cell replication status, we performed experiments in growth-arrested human mesenchymal stem cells and in post-mitotic syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless of their genetic makeup or target cell replication rate. Interestingly, the magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a vector backbone- and cell type-dependent manner. Finally, investigation of histone modifications by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV genomes when compared to those measured on isogenic integration-competent vector templates. These findings support the view that IDLVs constitute preferential targets for epigenetic silencing involving histone deacetylation, which contributes to dampening their full transcriptional potential. Our data provide leads on how to most optimally titrate and deploy these promising episomal gene delivery vehicles.