Exploring the differences in metabolic behavior of astrocyte and glioblastoma: a flux balance analysis approach

Brain cancers demonstrate a complex metabolic behavior so as to adapt the external hypoxic environment and internal stress generated by reactive oxygen species. To survive in these stringent conditions, glioblastoma cells develop an antagonistic metabolic phenotype as compared to their predecessors, the astrocytes, thereby quenching the resources expected for nourishing the neurons. The complexity and cumulative effect of the large scale metabolic functioning of glioblastoma is mostly unexplored. In this study, we reconstruct a metabolic network comprising of pathways that are known to be deregulated in glioblastoma cells as compared to the astrocytes. The network, consisted of 147 genes encoding for enzymes performing 247 reactions distributed across five distinct model compartments, was then studied using constrained-based modeling approach by recreating the scenarios for astrocytes and glioblastoma, and validated with available experimental evidences. From our analysis, we predict that glycine requirement of the astrocytes are mostly fulfilled by the internal glycine–serine metabolism, whereas glioblastoma cells demand an external uptake of glycine to utilize it for glutathione production. Also, cystine and glucose were identified to be the major contributors to glioblastoma growth. We also proposed an extensive set of single and double lethal reaction knockouts, which were further perturbed to ascertain their role as probable chemotherapeutic targets. These simulation results suggested that, apart from targeting the reactions of central carbon metabolism, knockout of reactions belonging to the glycine–serine metabolism effectively reduce glioblastoma growth. The combinatorial targeting of glycine transporter with any other reaction belonging to glycine–serine metabolism proved lethal to glioblastoma growth.

Electronic supplementary material

The online version of this article (doi:10.1007/s11693-015-9183-9) contains supplementary material, which is available to authorized users.     

Antimonial treatment of visceral leishmaniasis: are current in vitro susceptibility assays adequate for prognosis of in vivo therapy outcome?

In most of the Indian subcontinent, the first line treatment for visceral leishmaniasis (VL) is sodium stibogluconate (SSG), an antimonial drug, but the efficacy of the drug varies according to region. We aimed to characterize the in vitro antimony susceptibility of clinical isolates of Nepalese VL patients, and to correlate this in vitro parasite phenotype to clinical therapy outcome. Thirty-three clinical isolates of L. donovani were taken from patients with known disease history. These isolates were typed and the susceptibility of intracellular amastigotes to pentavalent (SbV) and trivalent (SbIII) antimonials was determined. We observed (i) 22 SbV-resistant isolates out of 33 tested and (ii) 3 SbIII-resistant isolates out of 12 tested. Amongst the latter, there were three combinations of in vitro phenotypes: (i) parasites sensitive (n=4) or (ii) resistant to both drugs (n=3) and (iii) resistant to SbV only (n=5). There was no geographical clustering in terms of in vitro susceptibility. The relation between the in vitro susceptibility to antimonials and the corresponding in vivo treatment outcome was ambiguous. Our results highlight the need to adjust the currently used Leishmania drug susceptibility assays if they are to be used for prognosis of in vivo SSG treatment outcome.     

Hilar Somatostatin Interneurons Contribute to Synchronized GABA Activity in an In Vitro Epilepsy Model

Epilepsy is a disorder characterized by excessive synchronized neural activity. The hippocampus and surrounding temporal lobe structures appear particularly sensitive to epileptiform activity. Somatostatin (SST)-positive interneurons within the hilar region have been suggested to gate hippocampal activity, and therefore may play a crucial role in the dysregulation of hippocampal activity. In this study, we examined SST interneuron activity in the in vitro 4-aminopyridine (4-AP) model of epilepsy. We employed a multi-disciplinary approach, combining extracellular multi-electrode array (MEA) recordings with patch-clamp recordings and optical imaging using a genetically encoded calcium sensor. We observed that hilar SST interneurons are strongly synchronized during 4-AP-induced local field potentials (LFPs), as assayed by Ca²⁺ imaging as well as juxtacellular or intracellular recording. SST interneurons were particularly responsive to GABA-mediated LFPs that occurred in the absence of ionotropic glutamatergic transmission. Our results present evidence that the extensive synchronized activity of SST-expressing interneurons contribute to the generation of GABAergic LFPs in an in vitro model of temporal lobe seizures.     

Design, synthesis, and initial biological evaluation of a steroidal anti-estrogen-doxorubicin bioconjugate for targeting estrogen receptor-positive breast cancer cells

As part of our program to develop breast cancer specific therapeutic agents, we have synthesized a conjugate agent that is a conjugate of the steroidal anti-estrogen and the potent cytotoxin doxorubicin. In this effort, we employed a modular assembly approach to prepare a novel 11β-substituted steroidal anti-estrogen functionalized with an azido-tetraethylene glycol moiety, which could be coupled to a complementary doxorubicin benzoyl hydrazone functionalized with a propargyl tetraethylene glycol moiety. Huisgen [3 + 2] cycloaddition chemistry gave the final hybrid that was evaluated for selective uptake and cytotoxicity in ER(+)-MCF-7 and ER(-)-MDA-MB-231 breast cancer cell lines. The results demonstrated that the presence of the anti-estrogenic component in the hybrid compound was critical for selectivity and cytotoxicity in ER(+)-MCF-7 human breast cancer cells as the hybrid was ~70-fold more potent than doxorubicin in inhibition of cell proliferation and promoting cell death.     

Molecular and functional characterization of cynomolgus monkey IgG subclasses

Studies for vaccine and human therapeutic Ab development in cynomolgus monkeys (cynos) are influenced by immune responses, with Ab responses playing a significant role in efficacy and immunogenicity. Understanding the nature of cyno humoral immune responses and characterizing the predominant cyno IgG types produced and the Fc-FcγR interactions could provide insight into the immunomodulatory effects of vaccines. Anti-drug Ab responses against human IgG therapeutic candidates in cynos may affect efficacy and safety assessments because of the formation of immune complexes. There is, however, limited information on the structure and function of cyno IgG subclasses and how they compare with human IgG subclasses in Fc-dependent effector functions. To analyze the functional nature of cyno IgG subclasses, we cloned four cyno IgG C regions by using their sequence similarity to other primate IgGs. The four clones, cyno (cy)IGG1, cyIGG2, cyIGG3, cyIGG4, were then used to construct chimeric Abs. The sequence features of cyno IgG subclasses were compared with those of rhesus monkey and human IgG. Our data show that rhesus monkey and cyno IgG C regions are generally highly conserved, with differences in the hinge and hinge-proximal CH2 regions. Fc-dependent effector functions of cyno IgG subclasses were assessed in vitro with a variety of binding and functional assays. Our findings demonstrate distinctive functional properties of cyno IgG subclasses. It is notable that human IgG1 was less potent than cyno IgG1 in cyno FcγR binding and effector functions, with the differences emphasizing the need to carefully interpret preclinical data obtained with human IgG1 therapeutics.     

The Voltage-dependent Anion Channel (VDAC) Binds Tissue-type Plasminogen Activator and Promotes Activation of Plasminogen on the Cell Surface

The voltage-dependent anion channel (VDAC), a major pore-forming protein in the outer membrane of mitochondria, is also found in the plasma membrane of a large number of cells where in addition to its role in regulating cellular ATP release and volume control it is important for maintaining redox homeostasis. Cell surface VDAC is a receptor for plasminogen kringle 5, which promotes partial closure of the channel. In this study, we demonstrate that VDAC binds tissue-type plasminogen activator (t-PA) on human neuroblastoma SK-N-SH cells. Binding of t-PA to VDAC induced a decrease in K_m and an increase in the V_max for activation of its substrate, plasminogen (Pg). This resulted in accelerated Pg activation when VDAC, t-PA, and Pg were bound together. VDAC is also a substrate for plasmin; hence, it mimics fibrin activity. Binding of t-PA to VDAC occurs between a t-PA fibronectin type I finger domain located between amino acids Ile⁵ and Asn³⁷ and a VDAC region including amino acids ²⁰GYGFG²⁴. These VDAC residues correspond to a GXXXG repeat motif commonly found in amyloid β peptides that is necessary for aggregation when these peptides form fibrillar deposits on the cell surface. Furthermore, we also show that Pg kringle 5 is a substrate for the NADH-dependent reductase activity of VDAC. This ternary complex is an efficient proteolytic complex that may facilitate removal of amyloid β peptide deposits from the normal brain and cell debris from injured brain tissue.     

Direct observation of protein folding in nanoenvironments using a molecular ruler

We observe folding of horse heart cytochrome c in various environments including nano-compartments (micelles and reverse micelles). Using picosecond-resolved Förster resonance energy transfer (FRET) dynamics of an extrinsic covalently attached probe dansyl (donor) at the surface of the protein to a heme group (acceptor) embedded inside the protein, we measured angstrom-resolved donor–acceptor distances in the environments. The overall structural perturbations of the protein revealed from the FRET experiments are in close agreement with our circular dichroism (CD) and dynamic light scattering (DLS) studies on the protein in a variety of solution conditions. The change of segmental motion of the protein due to imposed restriction in the nano-compartments compared to that in bulk buffer is also revealed by temporal fluorescence anisotropy of the dansyl probe.