Amphotericin B (Fungizone®) enhancement of nitrogen mustard uptake by human tumor cells

In HT29 human colon carcinoma cells, amphotericin B at doses above 120μg/ml increased nitrogen mustard uptake, and this was due to an increase in the apparent V_max without a change in the apparent K_m. Longer incubations (24 to 48 hr) of ascites fluid human ovarian carcinoma cells or SKMES-1 human epidermoid carcinoma cells with amphotericin B 4μg/ml enhanced the uptake of nitrogen mustard to a greater degree than that observed when cells were incubated for only 30 min. Therefore, amphotericin B can enhance nitrogen mustard by human tumor cell lines and by fresh human tumor cells.     

At Limits of Life: Multidisciplinary Insights Reveal Environmental Constraints on Biotic Diversity in Continental Antarctica

Multitrophic communities that maintain the functionality of the extreme Antarctic terrestrial ecosystems, while the simplest of any natural community, are still challenging our knowledge about the limits to life on earth. In this study, we describe and interpret the linkage between the diversity of different trophic level communities to the geological morphology and soil geochemistry in the remote Transantarctic Mountains (Darwin Mountains, 80°S). We examined the distribution and diversity of biota (bacteria, cyanobacteria, lichens, algae, invertebrates) with respect to elevation, age of glacial drift sheets, and soil physicochemistry. Results showed an abiotic spatial gradient with respect to the diversity of the organisms across different trophic levels. More complex communities, in terms of trophic level diversity, were related to the weakly developed younger drifts (Hatherton and Britannia) with higher soil C/N ratio and lower total soluble salts content (thus lower conductivity). Our results indicate that an increase of ion concentration from younger to older drift regions drives a succession of complex to more simple communities, in terms of number of trophic levels and diversity within each group of organisms analysed. This study revealed that integrating diversity across multi-trophic levels of biotic communities with abiotic spatial heterogeneity and geological history is fundamental to understand environmental constraints influencing biological distribution in Antarctic soil ecosystems.     

Application of a Drug-Induced Apoptosis Assay to Identify Treatment Strategies in Recurrent or Metastatic Breast Cancer

BackgroundA drug-induced apoptosis assay has been developed to determine which chemotherapy drugs or regimens can produce higher cell killing in vitro. This study was done to determine if this assay could be performed in patients with recurrent or metastatic breast cancer patients, to characterize the patterns of drug-induced apoptosis, and to evaluate the clinical utility of the assay. A secondary goal was to correlate assay use with clinical outcomes.MethodsIn a prospective, non-blinded, multi institutional controlled trial, 30 evaluable patients with recurrent or metastatic breast cancer who were treated with chemotherapy had tumor samples submitted for the MiCK drug-induced apoptosis assay. After receiving results within 72 hours after biopsy, physicians could use the test to determine therapy (users), or elect to not use the test (non-users).ResultsThe assay was able to characterize drug-induced apoptosis in tumor specimens from breast cancer patients and identified which drugs or combinations gave highest levels of apoptosis. Patterns of drug activity were also analyzed in triple negative breast cancer. Different drugs from a single class of agents often produced significantly different amounts of apoptosis. Physician frequently (73%) used the assay to help select chemotherapy treatments in patients, Patients whose physicians were users had a higher response (CR+PR) rate compared to non-users (38.1% vs 0%, p = 0.04) and a higher disease control (CR+PR+Stable) rate (81% vs 25%, p<0.01). Time to relapse was longer in users 7.4 mo compared to non-users 2.2 mo (p<0.01).ConclusionsThe MiCK assay can be performed in breast cancer specimens, and results are often used by physicians in breast cancer patients with recurrent or metastatic disease. These results from a good laboratory phase II study can be the basis for a future larger prospective multicenter study to more definitively establish the value of the assay.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00901264","term_id":"NCT00901264"}}NCT00901264     

Up-regulation of vitamin B1 homeostasis genes in breast cancer

An increased carbon flux and exploitation of metabolic pathways for the rapid generation of biosynthetic precursors is a common phenotype observed in breast cancer. To support this metabolic phenotype, cancer cells adaptively regulate the expression of glycolytic enzymes and nutrient transporters. However, activity of several enzymes involved in glucose metabolism requires an adequate supply of cofactors. In particular, vitamin B1 (thiamine) is utilized as an essential cofactor for metabolic enzymes that intersect at critical junctions within the glycolytic network. Intracellular availability of thiamine is facilitated by the activity of thiamine transporters and thiamine pyrophosphokinase-1 (TPK-1). Therefore, the objective of this study was to establish if the cellular determinants regulating thiamine homeostasis differ between breast cancer and normal breast epithelia. Employing cDNA arrays of breast cancer and normal breast epithelial tissues, SLC19A2, SLC25A19 and TPK-1 were found to be significantly up-regulated. Similarly, up-regulation was also observed in breast cancer cell lines compared to human mammary epithelial cells. Thiamine transport assays and quantitation of intracellular thiamine and thiamine pyrophosphate established a significantly greater extent of thiamine transport and free thiamine levels in breast cancer cell lines compared to human mammary epithelial cells. Overall, these findings demonstrate an adaptive response by breast cancer cells to increase cellular availability of thiamine.     

Hypolithic microbial communities of quartz rocks from Miers Valley, McMurdo Dry Valleys, Antarctica

The McMurdo Dry Valleys region of eastern Antarctica is a cold desert that presents extreme challenges to life. Hypolithic microbial colonisation of the subsoil surfaces of translucent quartz rocks represent a significant source of terrestrial biomass and productivity in this region. Previous studies have described hypoliths as dominated by cyanobacteria. However, hypoliths that occur in the lower Dry Valleys such as the Miers, Garwood and Marshall Valleys are unusual as they are not necessarily cyanobacteria-dominated. These hypoliths support significant eukaryal colonisation by fungi and mosses in addition to cyanobacteria-dominated bacterial assemblages and so have considerable ecological value in this barren landscape. Here, we characterise these novel hypoliths by analysis of environmental rRNA gene sequences. The hypolithic community was demonstrated to be distinct from the surrounding soil and non-translucent rocks. Hypoliths supported cyanobacterial signatures from the Oscillatoriales and Nostocales. Other heterotrophic bacterial signatures were also recovered, and these were phylogenetically diverse and spanned 8 other bacterial phyla. Archaeal phylotypes recovered were phylogenetically affiliated with the large group of unclassified, uncultured Crenarcheota. Eukaryal phylotypes indicated that free-living ascomycetous fungi, chlorophytes and mosses (Bryum sp.) were all supported by these hypoliths, and these are thought to be responsible for the extensive eukaryotic biomass that develops around quartz rocks.     

Measurement of in vivo anterior cruciate ligament strain during dynamic jump landing

Despite recent attention in the literature, anterior cruciate ligament (ACL) injury mechanisms are controversial and incidence rates remain high. One explanation is limited data on in vivo ACL strain during high-risk, dynamic movements. The objective of this study was to quantify ACL strain during jump landing. Marker-based motion analysis techniques were integrated with fluoroscopic and magnetic resonance (MR) imaging techniques to measure dynamic ACL strain non-invasively. First, eight subjects' knees were imaged using MR. From these images, the cortical bone and ACL attachment sites of the tibia and femur were outlined to create 3D models. Subjects underwent motion analysis while jump landing using reflective markers placed directly on the skin around the knee. Next, biplanar fluoroscopic images were taken with the markers in place so that the relative positions of each marker to the underlying bone could be quantified. Numerical optimization allowed jumping kinematics to be superimposed on the knee model, thus reproducing the dynamic in vivo joint motion. ACL length, knee flexion, and ground reaction force were measured. During jump landing, average ACL strain peaked 55±14 ms (mean and 95% confidence interval) prior to ground impact, when knee flexion angles were lowest. The peak ACL strain, measured relative to its length during MR imaging, was 12±7%. The observed trends were consistent with previously described neuromuscular patterns. Unrestricted by field of view or low sampling rate, this novel approach provides a means to measure kinematic patterns that elevate ACL strains and that provide new insights into ACL injury mechanisms.     

Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP_1,2, to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP_1,2, ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP_1,2-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP_1,2, as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP_1,2-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.     

Oncolytic vesicular stomatitis virus induces apoptosis in U87 glioblastoma cells by a type II death receptor mechanism and induces cell death and tumor clearance in vivo

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.