Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

Using a Balloon-Launched Unmanned Glider to Validate Real-Time WRF Modeling.

The use of small unmanned aerial systems (sUAS) for meteorological measurements has expanded significantly in recent years. SUAS are efficient platforms for collecting data with high resolution in both space and time, providing opportunities for enhanced atmospheric sampling. Furthermore, advances in mesoscale weather research and forecasting (WRF) modeling and graphical processing unit (GPU) computing have enabled high resolution weather modeling. In this manuscript, a balloon-launched unmanned glider, complete with a suite of sensors to measure atmospheric temperature, pressure, and relative humidity, is deployed for validation of real-time weather models. This work demonstrates the usefulness of sUAS for validating and improving mesoscale, real-time weather models for advancements toward reliable weather forecasts to enable safe and predictable sUAS missions beyond visual line of sight (BVLOS).

Vascular endothelial cell growth factors promote the in vitro development of rat photoreceptor cells.

We have identified and characterized a novel trophic effect of vascular endothelial cell growth factor (VEGF) on photoreceptor cells. Treatment of retinal cultures, derived from postnatal day 1 (P1) rats, with VEGF-2 resulted in a dose- and time-dependent increase in the level of rhodopsin protein, as determined by ELISA assay. After 7-9 d of treatment the VEGF-1 or VEGF-2, at a concentration of 10 ng/ml, induced a 200-300% increase in rhodopsin protein and a 220% increase in the number of rhodopsin-immunopositive cells. Treatment with VEGF-2 induced a 250% increase in the number of syntaxin-immunopositive cells and a 67% increase in high-affinity GABA uptake, both markers for amacrine cells. In contrast, there was no increase in the non-neuronal cell populations. VEGF-2 induced an approximately 300% increase in the number of bromodeoxyuridine-labeled (BrdU) retinal cells within 48 hr of treatment. After 3 d in culture both the basal and stimulated levels of BrdU incorporation were reduced, suggesting that the proliferative effect of VEGF was restricted developmentally. Furthermore, there was a developmentally dependent increase in the mitogenic response to VEGF-2, with retinal cultures derived from E15, E20, or P1 animals demonstrating a 50, 100, and 300% increase in thymidine incorporation, respectively. However, VEGF treatment resulted in an increase in the number of rhodopsin-immunopositive cells only when the cultures were derived from P1 animals. Therefore, retinal progenitor cells appear to be targets for VEGF, and thus VEGF may be involved in the regulation of the early developmental program of retinal neurogenesis.

The possibility of transplacental transmission of experimental schistosomiasis.

Congenital transmission of Schistosoma mansoni and S. haematobium in mice was studied by experimentally infecting pregnant mice on day three, or nine or 14 of gestation. None of the pups born to mothers infected in early or mid-pregnancy were infected. However, several pregnancies ended in abortion ranging between 10%-40%. On the other hand, eight out of 35 pups born to mice infected with S. mansoni and five out of 30 pups born to mice infected with S. haematobium in late pregnancy were found to harbour schistosomes with granulomas recovered in their livers and intestines. These findings show that congenital infection of mice with S. mansoni and S. haematobium could occur when pregnant mice were infected during late pregnancy. This may have important implications not only for mice, but also for other mammalian hosts of schistosomes, including man.

Computational EST database analysis identifies a novel member of the neuropoietic cytokine family.

A novel member of the neuropoietic cytokine family has been cloned and the protein expressed and characterized. In an effort to identify novel secreted proteins, an algorithm incorporating neural network algorithms was applied to a large EST database. A full-length clone was identified that is 1710 bp in length and has a single open reading frame of 225 amino acids. This new cytokine is most homologous to cardiotrophin-1, having a similarity and an identity of 46 and 29%, respectively, and therefore we have named it cardiotrophin-like cytokine (CLC). Northern hybridization analysis identified a 1.4-kb messenger RNA that is highly expressed in spleen and peripheral leukocytes. Purified recombinant CLC induced the activation of NFkappaB and SRE reporter constructs in the TF-1, U937, and M1 cell lines. Furthermore, the signal transduction pathway for CLC was characterized in the neuroblastoma cell line SK-N-MC and found to involve tyrosine phosphorylation of gp130 and STAT-1.

Non-equivalent roles for the first and second zinc fingers of protein kinase Cdelta. Effect of their mutation on phorbol ester-induced translocation in NIH 3T3 cells.

Classical and novel protein kinase C (PKC) isozymes contain two, so-called cysteine-rich zinc finger domains that represent the binding sites for phorbol esters and the diacylglycerols. X-ray crystallographic, mutational, and modeling studies are providing detailed understanding of the interactions between the phorbol esters and individual PKC zinc fingers. In the present study, we explore the roles of the individual zinc fingers in the context of the intact enzyme. Our approach was to mutate either the first, the second, or both zinc fingers of PKCdelta, to express the mutated enzyme in NIH 3T3 cells, and to monitor the effect of the mutations on the dose-response curve for translocation induced by phorbol 12-myristate 13-acetate. The introduced mutations change into glycine the consensus proline in the phorbol ester binding loop of the zinc finger; in the isolated zinc finger, this mutation causes a 125-fold decrease in phorbol ester binding affinity. We observed that mutation in the first zinc finger caused almost no shift in the dose-response curve for translocation; mutation in the second zinc finger caused a 21-fold shift, whereas mutation in both zinc fingers caused a 138-fold shift. We conclude that the zinc fingers in the intact PKC are not equivalent and that the second zinc finger plays the predominant role in translocation of protein kinase Cdelta in response to phorbol 12-myristate 13-acetate. Our findings have important implications for the understanding and design of PKC inhibitors targeted to the zinc finger domains.

Detection of Trichinella pseudospiralis antigen in the parenteral phase in experimentally infected mice.

Antigen distribution of T. pseudospiralis in muscles was studied at 10 days, 2,3 and 5 weeks post infection. Immunocytolocalization of antigen in the muscle was performed by peroxidase antiperoxidase (PAP) and immunogold silver staining techniques. Weak positive reaction of the stichosome and the cuticle was observed at 10 days post-infection which increased to attain its highest level in the fully infective stage larvae 3 and 5 weeks post infection. The intensity of staining reaction in the cytoplasm and nucleoplasm of infected host cell and surrounding tissue was highest at 5 weeks p.i. The small nuclei of the host cells and the inflammatory cells were unstained.

Detection of Trichinella pseudospiralis antigen in the enteral phase in experimentally infected mice.

The distribution of T. pseudospiralis antigen in tissue sections of the intestine of infected mice was studied at different intervals (10, 21, 35 hours and 10 days post-infection (P.I.). Immunocytolocalization of antigen in the intestine was done by peroxidase antiperoxidase staining technique. Positive staining reaction of the enterocytes was noted in all durations examined. The mononuclear cells of the lamina propria were positively stained at 10 hrs P.I. while patchy staining of muscularis mucosa was observed at 10 days P.I. The present study revealed intense staining reaction in the stichosome and the cuticle of 10 hrs-old larvae L1, the intensity of the reaction decreased throughout the worm development, until it almost disappeared at 10 days P.I. The gut lining and its occupying substance were positive throughout the enteral phase. Intense staining reaction of the hypodermal cells was observed in L2, L3 and adult stage worm. The genital primordium was weakly stained in the larval stages while the male and female reproductive systems were moderately stained at the adult stage.