The importance of side branches of glycosylphosphatidylinositol anchors: a molecular dynamics perspective.

Many proteins are anchored to the cell surface of eukaryotes using a unique family of glycolipids called glycosylphosphatidylinositol (GPI) anchors. These glycolipids also exist without a covalently bound protein, in particular on the cell surfaces of protozoan parasites where they are densely populated. GPIs and GPI-anchored proteins participate in multiple cellular processes such as signal transduction, cell adhesion, protein trafficking and pathogenesis of Malaria, Toxoplasmosis, Trypanosomiasis and prion diseases, among others. All GPIs share a common conserved glycan core modified in a cell-dependent manner with additional side glycans or phosphoethanolamine residues. Here, we use atomistic molecular dynamic simulations and perform a systematic study to evaluate the structural properties of GPIs with different side chains inserted in lipid bilayers. Our results show a flop-down orientation of GPIs with respect to the membrane surface and the presentation of the side chain residues to the solvent. This finding agrees well with experiments showing the role of the side residues as active epitopes for recognition of GPIs by macrophages and induction of GPI-glycan-specific immune responses. Protein-GPI interactions were investigated by attaching parasitic GPIs to Green Fluorescent Protein. GPIs are observed to recline on the membrane surface and pull down the attached protein close to the membrane facilitating mutual contacts between protein, GPI and the lipid bilayer. This model is efficient in evaluating the interaction of GPIs and GPI-anchored proteins with membranes and can be extended to study other parasitic GPIs and proteins and develop GPI-based immunoprophylaxis to treat infectious diseases.

Targeting the ABC transporter ABCB5 sensitizes glioblastoma to temozolomide-induced apoptosis through a cell-cycle checkpoint regulation mechanism.

Glioblastoma multiforme (GBM) is a malignant brain tumor with a poor prognosis resulting from tumor resistance to anticancer therapy and a high recurrence rate. Compelling evidence suggests that this is driven by subpopulations of cancer stem cells (CSCs) with tumor-initiating potential. ABC subfamily B member 5 (ABCB5) has been identified as a molecular marker for distinct subsets of chemoresistant tumor-initiating cell populations in diverse human malignancies. In the current study, we examined the potential role of ABCB5 in growth and chemoresistance of GBM. We found that ABCB5 is expressed in primary GBM tumors, in which its expression was significantly correlated with the CSC marker protein CD133 and with overall poor survival. Moreover, ABCB5 was also expressed by CD133-positive CSCs in the established human U-87 MG, LN-18, and LN-229 GBM cell lines. Antibody- or shRNA-mediated functional ABCB5 blockade inhibited proliferation and survival of GBM cells and sensitized them to temozolomide (TMZ)-induced apoptosis in vitro Likewise, in in vivo human GBM xenograft experiments with immunodeficient mice, mAb treatment inhibited growth of mutant TP53, WT PTEN LN-229 tumors, and sensitized LN-229 tumors to TMZ therapy. Mechanistically, we demonstrate that ABCB5 blockade inhibits TMZ-induced G2/M arrest and augments TMZ-mediated cell death. Our results identify ABCB5 as a GBM chemoresistance marker and point to the potential utility of targeting ABCB5 to improve current GBM therapies.

ATP-binding cassette member B5 (ABCB5) promotes tumor cell invasiveness in human colorectal cancer.

ABC member B5 (ABCB5) mediates multidrug resistance (MDR) in diverse malignancies and confers clinically relevant 5-fluorouracil resistance to CD133-expressing cancer stem cells in human colorectal cancer (CRC). Because of its recently identified roles in normal stem cell maintenance, we hypothesized that ABCB5 might also serve MDR-independent functions in CRC. Here, in a prospective clinical study of 142 CRC patients, we found that ABCB5 mRNA transcripts previously reported not to be significantly expressed in healthy peripheral blood mononuclear cells are significantly enriched in patient peripheral blood specimens compared with non-CRC controls and correlate with CRC disease progression. In human-to-mouse CRC tumor xenotransplantation models that exhibited circulating tumor mRNA, we observed that cancer-specific ABCB5 knockdown significantly reduced detection of these transcripts, suggesting that the knockdown inhibited tumor invasiveness. Mechanistically, this effect was associated with inhibition of expression and downstream signaling of AXL receptor tyrosine kinase (AXL), a proinvasive molecule herein shown to be produced by ABCB5-positive CRC cells. Importantly, rescue of AXL expression in ABCB5-knockdown CRC tumor cells restored tumor-specific transcript detection in the peripheral blood of xenograft recipients, indicating that ABCB5 regulates CRC invasiveness, at least in part, by enhancing AXL signaling. Our results implicate ABCB5 as a critical determinant of CRC invasiveness and suggest that ABCB5 blockade might represent a strategy in CRC therapy, even independently of ABCB5's function as an MDR mediator.

A molecular dynamics model for glycosylphosphatidyl-inositol anchors: "flop down" or "lollipop"?

We present a computational model of glycosylphosphatidyl-inositol (GPI) anchors for molecular dynamics studies. The model is based on state-of-the-art biomolecular force fields from the AMBER family, employing GLYCAM06 for carbohydrates and Lipid14 to represent fatty acid tails. We construct an adapted glycero-phosphatidyl-inositol unit to establish a seamless transition between the two domains of atom types. This link can readily be extended into a broad variety of GPI variants by applying either domain's building block scheme. As test cases, selected GPI fragments inserted into DMPC and POPC bilayer patches are considered. Our results suggest that the glycan part of the GPI anchor interacts strongly with the lipid head groups, partially embedding the carbohydrate moieties. This behaviour is supported by the conformational preferences of the GPI anchor, which in particular are conveyed by the strong interactions between the proximal amine and phosphate groups. In a similar way we can conclude that the extension of the anchor away from the lipid bilayer surface that could prevent the contact of the membrane with an attached protein ("lollipop picture") is quite unfavorable. Indeed, when attaching green fluorescent protein to the GPI anchor, it is found to reside close to bilayer surface all the time, and the rather flexible phosphoethanolamine linker governs the extent to which the protein directly interacts not only with the head groups, but also with its own GPI core.

Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type.

Klebsiella pneumoniae ST258 is a globally disseminated, extremely drug resistant, nosocomial clone with limited treatment options. We show that the vast majority of ST258 isolates express modified d-galactan-I lipopolysaccharide O-antigen, termed hereinafter as D-galactan-III. The genetic determinant required for galactan-III synthesis was identified as a distinct operon adjacent to the rfb (wb) locus encoding D-galactan-I synthesis. The three genes within the operon encode predicted glycosyltransferases. Testing an isogenic transformant pair revealed that expression of D-galactan-III, in comparison to D-galactan-I, conferred improved survival in the presence of human serum. Eighty-three percent of the more than 200 ST258 draft genome sequences currently available carries the corresponding operon and hence these isolates are predicted to express galactan-III antigens. A D-galactan-III specific monoclonal antibody (mAb) was shown to bind to extracted LPS from a panel of ST258 isolates. The same mAb confirmed accessibility of galactan-III in surface staining of ST258 irrespective of the distinct capsular antigens expressed by both clades described previously. Based on these data, the galactan-III antigen may represent an attractive target for active and passive immunization approaches against K. pneumoniae ST258.

Heme oxygenase-1 promotes survival of renal cancer cells through modulation of apoptosis- and autophagy-regulating molecules.

The cytoprotective enzyme heme oxygenase-1 (HO-1) is often overexpressed in different types of cancers and promotes cancer progression. We have recently shown that the Ras-Raf-ERK pathway induces HO-1 to promote survival of renal cancer cells. Here, we examined the possible mechanisms underlying HO-1-mediated cell survival. Considering the growing evidence about the significance of apoptosis and autophagy in cancer, we tried to investigate how HO-1 controls these events to regulate survival of cancer cells. Rapamycin (RAPA) and sorafenib, two commonly used drugs for renal cancer treatment, were found to induce HO-1 expression in renal cancer cells Caki-1 and 786-O; and the apoptotic effect of these drugs was markedly enhanced upon HO-1 knockdown. Overexpression of HO-1 protected the cells from RAPA- and sorafenib-induced apoptosis and also averted drug-mediated inhibition of cell proliferation. HO-1 induced the expression of anti-apoptotic Bcl-xL and decreased the expression of autophagic proteins Beclin-1 and LC3B-II; while knockdown of HO-1 down-regulated Bcl-xL and markedly increased LC3B-II. Moreover, HO-1 promoted the association of Beclin-1 with Bcl-xL and Rubicon, a novel negative regulator of autophagy. Drug-induced dissociation of Beclin-1 from Rubicon and the induction of autophagy were also inhibited by HO-1. Together, our data signify that HO-1 is up-regulated in renal cancer cells as a survival strategy against chemotherapeutic drugs and promotes growth of tumor cells by inhibiting both apoptosis and autophagy. Thus, application of chemotherapeutic drugs along with HO-1 inhibitor may elevate therapeutic efficiency by reducing the cytoprotective effects of HO-1 and by simultaneous induction of both apoptosis and autophagy.

Preference for daily oral pills over long-acting antiretroviral therapy options among people with HIV.

OBJECTIVE: To examine the characteristics of people with HIV (PWH) who prefer remaining on daily oral antiretroviral therapy (ART), rather than switching to long-acting ART (LA-ART). DESIGN: Building upon a discrete choice experiment (DCE), we examined characteristics of individuals who always selected their current daily oral tablet regimen over either of two hypothetical LA-ART options presented in a series of 17 choice tasks. METHODS: We used LASSO to select sociodemographic, HIV-related, and other health-related predictors of preferring current therapy over LA-ART, and logistic regression to measure the associations with those characteristics. RESULTS: Among 700 PWH in Washington State and Atlanta, Georgia, 11% of participants ( n  = 74) chose their current daily treatment over LA-ART in all DCE choice tasks. We found that people with lower educational attainment, good adherence, more aversion to injections, and who participated from Atlanta to be more likely to prefer their current daily regimen over LA-ART. CONCLUSIONS: Gaps in ART uptake and adherence remain, and emerging LA-ART treatments show promise to address these challenges and help a larger portion of PWH to achieve viral suppression, but preferences for these new treatments are understudied. Our results show that certain drawbacks of LA-ART may help to maintain demand for daily oral tablets, especially for PWH with certain characteristics. Some of these characteristics (lower educational attainment and Atlanta participation) were also associated with a lack of viral suppression. Future research should focus on overcoming barriers that impact preferences for LA-ART among those patients who could benefit most from this innovation.

Hemolysin induces Toll-like receptor (TLR)-independent apoptosis and multiple TLR-associated parallel activation of macrophages.

Vibrio cholerae hemolysin (HlyA) displays bipartite property while supervising macrophages (MΦ). The pore-forming toxin causes profound apoptosis within 3 h of exposure and in parallel supports activation of the defying MΦ. HlyA-induced apoptosis of MΦ remains steady for 24 h, is Toll-like receptor (TLR)-independent, and is driven by caspase-9 and caspase-7, thus involving the mitochondrial or intrinsic pathway. Cell activation is carried forward by time dependent up-regulation of varying TLRs. The promiscuous TLR association of HlyA prompted investigation, which revealed the ß-prism lectin domain of HlyA simulated TLR4 up-regulation by jacalin, a plant lectin homologue besides expressing CD86 and type I cytokines TNF-α and IL-12. However, HlyA cytolytic protein domain up-regulated TLR2, which controlled CD40 for continuity of cell activation. Expression of TOLLIP before TLR2 and TLR6 abrogated TLR4, CD40, and CD86. We show that the transient expression of TOLLIP leading to curbing of activation-associated capabilities is a plausible feedback mechanism of MΦ to deploy TLR2 and prolong activation involving CD40 to encounter the HlyA cytolysin domain.

The heme oxygenase-1 protein is overexpressed in human renal cancer cells following activation of the Ras-Raf-ERK pathway and mediates anti-apoptotic signal.

The stress-inducible cytoprotective enzyme heme oxygenase-1 (HO-1) may play a critical role in the growth and metastasis of tumors. We demonstrated that overexpressed HO-1 promotes the survival of renal cancer cells by inhibiting cellular apoptosis; we also showed that the proto-oncogene H-Ras becomes activated in these cells under stress following treatment with immunosuppressive agents. However, it is not known if there is an association between Ras activation and HO-1 overexpression. Here, we examined if the activation of H-Ras pathway could induce HO-1, and promote the survival of renal cancer cells (786-0 and Caki-1). In co-transfection assays, using HO-1 promoter-luciferase construct, we found that the activated H-Ras, H-Ras(12V), promoted HO-1 transcriptional activation. The inhibition of endogenous H-Ras by specific dominant-negative mutant/siRNA markedly ablated the HO-1 promoter activity. Active H-Ras increased HO-1 mRNA and protein expression. Moreover, transfection with effector domain mutant constructs of active H-Ras showed that H-Ras-induced HO-1 overexpression was primarily mediated through the Raf signaling pathway. Using pharmacological inhibitor, we observed that ERK is a critical intermediary molecule for Ras-Raf-induced HO-1 expression. Activation of H-Ras and ERK promoted nuclear translocation of the transcription factor Nrf2 for its binding to the specific sequence of HO-1 promoter. The knockdown of Nrf2 significantly inhibited H-Ras-induced HO-1 transcription. Finally, by FACS analysis using Annexin-V staining, we demonstrated that the H-Ras-ERK-induced and HO-1-mediated pathway could protect renal cancer cells from apoptosis. Thus, targeting the Ras-Raf-ERK pathway for HO-1 overexpression may serve as novel therapeutics for the treatment of renal cancer.

Hydrochemical analysis to evaluate the seawater ingress in a small coral island of India.

The sustainable development of the limited groundwater resources in the tropical island requires a thorough understanding of detail hydrogeological regime including the hydrochemical behavior of groundwater. Detail analysis of chemical data of groundwater helps in assessing the different groundwater zone affected by formation as well as sea water. Groundwater and saline water interaction is better understood using groundwater major ion chemistry over an island aquifer. Multivariate methods to analyze the geochemical data are used to understand geochemical evolution of groundwater. The methods are successfully used to group the data to evaluate influence of various environs in the study area. Various classification methods such as piper, correlation method, and salinity hazard measurements are also employed to critical study of geochemical characteristics of groundwater to identify vulnerable parts of the aquifer. These approaches have been used to successfully evaluate the aquifer zones of a tiny island off the west coast of India. The most part of island is found to be safe for drinking, however some parts of island are identified that are affected by sea water ingress and dissolution of formation minerals. The analysis has successfully leaded to identification of that part of aquifer on the island which needs immediate attention for restoration and avoids further deterioration.

An unusual presentation of Salmonella typhi bacteriuria in a 10 year old girl from North India.

Enteric fever caused mainly by Salmonella typhi (S. typhi), has a high incidence in India. We report a case of enteric fever in a ten year old patient presenting with clinical history of less than one week. Although blood culture, the most important diagnostic modality in early stage of enteric fever was sterile, S. typhi was isolated from culture of urine sample. Serum antibody titres against S. typhi were unusually raised considering that the infection was still in its early stage. Intravenous ceftriaxone therapy was given leading to complete recovery.

Deep care: The COVID-19 pandemic and the work of marginal feminist organizing in India.

In this paper, we adopt a Southern feminist epistemology to critically appraise the ways in which media discourse on gendered organizing during the Indian COVID-19-induced migrant crisis resists or reinforces hegemonic caste hierarchies. To contextualize this work, we briefly historicize scholarship on feminist organizing around land rights, hunger, and violence, while noting the politics of contagion and pollution narratives plaguing the pandemic discourse in India. After conducting a qualitative content analysis (QCA) followed by a critical discourse analysis (CDA) of media discourses across three tiers (international, national, and local), we found that international and national tiers of discourse largely deployed a savarna gaze that worked to 1) Reinforce brahminical and technocratic pandemic narratives and 2) Delegitimize Dalit marginal organizing feminist work and Dalit sensibilities through seven overlapping metrics of erasure. On the other hand, local tier of discourse confronted the savarna gaze, amplified voices of Dalit and Muslim women by centering their narratives of resistance, and tackled the exacerbation of casteist oppression under the pandemic in the service of emancipation. Local discourses also highlight how marginal organizing during the first pandemic lockdown involved provision of essential resources and services (food, medical care, security) for mostly Dalit and Muslim migrant workers, and women intersectionally facing domestic violence and savarna violence. Despite the brahmininal structural oppression, Dalit feminist praxis' emblematic resistance of oppressive structures, during and beyond times of crisis, constitutes what we call the work of deep care.

CXCR3-B can mediate growth-inhibitory signals in human renal cancer cells by down-regulating the expression of heme oxygenase-1.

The chemokine receptor CXCR3 may play a critical role in the growth and metastasis of tumor cells, including renal tumors. It has been shown that CXCR3 has two splice variants with completely opposite functions; CXCR3-A promotes cell proliferation, whereas CXCR3-B inhibits cell growth. We recently demonstrated that the expression of growth-promoting CXCR3-A is up-regulated, and the growth-inhibitory CXCR3-B is markedly down-regulated in human renal cancer tissues; and the overexpression of CXCR3-B in renal cancer cells can significantly inhibit cell proliferation. However, the growth-inhibitory signal(s) through CXCR3-B are not well characterized. Here, we investigated the effector molecule(s) involved in CXCR3-B-mediated signaling events. We found that the overexpression of CXCR3-B in human renal cancer cells (Caki-1) promoted cellular apoptosis as observed by FACS analysis through Annexin-V staining. To examine whether the overexpression of CXCR3-B could alter the expression of any apoptosis-related genes in renal cancer cells, we performed a protein array. We found that CXCR3-B overexpression significantly down-regulated the expression of antiapoptotic heme oxygenase-1 (HO-1). By utilizing a HO-1 promoter-luciferase plasmid, we showed that CXCR3-B-mediated down-regulation of HO-1 was controlled at the transcriptional level as observed by luciferase assay. We also demonstrated that the inhibition of HO-1 expression using siRNA promoted apoptosis of renal cancer cells. Finally, we observed that human renal cancer tissues expressing low amounts of CXCR3-B significantly overexpress HO-1 at both mRNA and protein level. Together, we suggest that the overexpression of CXCR3-B may prevent the growth of renal tumors through the inhibition of antiapoptotic HO-1.

Application of drastic model and GIS: for assessing vulnerability in hard rock granitic aquifer.

Geographic information system (GIS) has become one of the leading tools in the field of hydrogeological science that helps in assessing, monitoring, and conserving groundwater resources. Groundwater is a finite resource, which is being overexploited due to increase in demand over the years leading to decrease in its potentiality. In the present study, DRASTIC model has been used to prepare groundwater vulnerable zone in hard rock aquifer of granitic terrain. The main objective is to determine susceptible zone for groundwater pollution by integrating hydrogeological layers in GIS environment. The layers such as depth of aquifer, recharge, aquifer yield, soil type, topography, vadose zone, and transmissivity are incorporated in the DRASTIC model. The final output of the map shows that around 60% of the area falls under low to no risk of pollution zone. The high risk of pollution zones are mostly present towards the margin of southeastern periphery. The lower part of the basin as well as small area on northern side falls under moderate risk of pollution zone. For the assessment of groundwater pollution zone, 24 groundwater samples have been collected from different vulnerable zones. The chemical analysis of sample shows that the southeastern margin of basin has relatively high concentration of nitrate as compared to other parts of the basin. It is present in high pollution zone as well as moderate pollution zone. The present model can be used for assessment and management of groundwater.

Coarse-Grained Molecular Model for the Glycosylphosphatidylinositol Anchor with and without Protein.

Glycosylphosphatidylinositol (GPI) anchors are a unique class of complex glycolipids that anchor a great variety of proteins to the extracellular leaflet of plasma membranes of eukaryotic cells. These anchors can exist either with or without an attached protein called GPI-anchored protein (GPI-AP) both in vitro and in vivo. Although GPIs are known to participate in a broad range of cellular functions, it is to a large extent unknown how these are related to GPI structure and composition. Their conformational flexibility and microheterogeneity make it difficult to study them experimentally. Simplified atomistic models are amenable to all-atom computer simulations in small lipid bilayer patches but not suitable for studying their partitioning and trafficking in complex and heterogeneous membranes. Here, we present a coarse-grained model of the GPI anchor constructed with a modified version of the MARTINI force field that is suited for modeling carbohydrates, proteins, and lipids in an aqueous environment using MARTINI's polarizable water. The nonbonded interactions for sugars were reparametrized by calculating their partitioning free energies between polar and apolar phases. In addition, sugar-sugar interactions were optimized by adjusting the second virial coefficients of osmotic pressures for solutions of glucose, sucrose, and trehalose to match with experimental data. With respect to the conformational dynamics of GPI-anchored green fluorescent protein, the accessible time scales are now at least an order of magnitude larger than for the all-atom system. This is particularly important for fine-tuning the mutual interactions of lipids, carbohydrates, and amino acids when comparing to experimental results. We discuss the prospective use of the coarse-grained GPI model for studying protein-sorting and trafficking in membrane models.

Porin-incorporated liposome induces Toll-like receptors 2- and 6-dependent maturation and type 1 response of dendritic cell.

Porin of Shigella dysenteriae was incorporated in liposome (PIL) and presented to mouse splenic dendritic cells (DC). PIL up-regulated Toll-like receptor (TLR) 2 and TLR6 on DC, showing that co-expression of the two TLRs is involved in recognition of porin. Detection of myeloid differentiating factor 88 (MyD88)-TLR2 complex confirmed interaction between the two for triggering the downstream signaling, which ultimately led to TLR2-dependent nuclear translocation of nuclear factor-kappa B. PIL-induced expression of MHC class II (I-Ab), CD40 and CD80 showed maturation of DC, whereas up-regulation of intercellular adhesion molecule-1 and CCR7 implicated the capacity of splenic DC to migrate. Induction of messenger ribonucleic acid for the chemokines, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta and regulated upon activation, normal T cell expressed and secreted indicated a strong bias of PIL for type 1 polarization that was supported by the intracellular expression and release of tumor necrosis factor (TNF)-alpha and IL-12. Along with CD40 and CD80 expression, release of the cytokines of CD11c+ JAWS II cells was inhibited by TLR2 or simultaneous TLR2 and 6 knockdown showing that recognition of PIL by the two TLRs is essential for DC activation and type 1 polarization. The signaling pathway initiated upon recognition of PIL by the TLRs was MyD88 dependent as confirmed by inhibition of IL-6, TNF-alpha and IL-12 release of MyD88-knockdown JAWS II cells. The maturation and polarization of DC induced T(h)1 phenotype, as evident from proliferation, activation and IFN-gamma release of allogeneic CD4+ T cells in response to PIL-stimulated DC, thereby suggesting that the adjuvant activity of PIL can successfully bridge the innate and adaptive immunity.

Priming of CD4+ T cells with porin of Shigella dysenteriae activates the cells toward type 1 polarization.

Macrophages treated with porin of Shigella dysenteriae were potent stimulators of naive T(h) cells since the porin-pulsed macrophages strongly proliferated the T cells and up-regulated the activation molecules CD69 and CD25 on CD4(+) T cells in allogeneic mixed leukocyte reaction. Immunization of C57BL/6 mice with porin selectively induced the intracellular expression and release of IFN-gamma and had no effect on IL-4 expression. In parallel to the predominant release of the T(h)1 cytokine IFN-gamma, up-regulation of CCR5 on the immune CD4(+) T cells and messenger ribonucleic acid (mRNA) expression of the T cell chemokines macrophage inflammatory protein-1 alpha and regulated on activation normal T cell expressed and secreted showed T(h)1 bias. The porin-primed CD4(+) T cells expressed the mRNA for T-box expressed in T cells, a T(h)1-specific transcription factor confirming the adjuvant-induced transition of T cells to polarized effector T(h)1 cells. The immune CD4(+) T cells proliferated and released IL-2 and IFN-gamma profoundly in response to re-challenge with porin-pulsed macrophages but not to BSA-pulsed macrophages in vitro, which demonstrated the presence of porin-specific CD4(+) T cells of T(h)1 phenotype. The study highlights that porin has the capacity of an adjuvant to unfold and maintain the T cell function and thereby to activate adaptive immunity.

Porin of Shigella dysenteriae activates mouse peritoneal macrophage through Toll-like receptors 2 and 6 to induce polarized type I response.

Porin of Shigella dysenteriae type 1 coexpressed Toll-like receptor (TLR) 2 and TLR6 on peritoneal cavity (PerC) macrophages (MPhi) of C57BL/6 mice implicating that both the TLRs are essential as a combinatorial repertoire to recognize the protein. Besides TLRs, mRNA for MyD88 and TRAF6, and nuclear translocation of NF-kappaB were enhanced that indicate their involvement in tandem in the activity of porin. The protein selectively up-regulated CD80 on the activated MPhi together with MHC class II molecule and CD40, and had no effect on CD86 expression. The porin-induced profile of MIP-1alpha, MIP-1beta and RANTES showed strong bias for chemokines correlated with M1 polarization. Intracellular expression and release of TNF-alpha and IL-12 in presence of porin was found to be TLR2 and NF-kappaB dependent. Induction of TNF-alpha and IL-12 along with the chemokine profile suggests type I polarization of the MPhi that would influence Th1-type response.

Coarse-Grained Molecular Dynamics Force-Field for Polyacrylamide in Infinite Dilution Derived from Iterative Boltzmann Inversion and MARTINI Force-Field.

We present a mesoscale model of aqueous polyacrylamide in the infinitely dilute concentration regime, by combining an extant coarse-grained (CG) force-field, MARTINI, and the Iterative Boltzmann Inversion protocol (IBI). MARTINI force-field was used to retain the thermodynamics of solvation of the polymer in water, whereas the structural properties and intrapolymer interactions were optimized by IBI. Atomistic molecular dynamics simulations of polymer in water were performed to benchmark the mesoscale simulations. Our results from the CG model show excellent agreement in structure with the atomistic system. We also studied the dynamical behavior of our CG system by computing the shear viscosity and compared it with the standard IBI model. The viscosity trends of our model were similar to the atomistic system, whereas the standard IBI model was highly dissimilar as expected. In summary, our hybrid CG model sufficiently mimics an infinitely dilute system, and is superior to both MARTINI and IBI in representing the structure and thermodynamics of the atomistic system, respectively. Our hybrid coarse-graining strategy promises applicability in large-scale simulations of polymeric/biological systems where the structure needs to be replicated accurately while preserving the thermodynamics of a smoother surrounding.

Expression of Cell-Surface Marker ABCB5 Causes Characteristic Modifications of Glucose, Amino Acid and Phospholipid Metabolism in the G3361 Melanoma-Initiating Cell Line.

We present a pilot study aimed at determining the effects of expression of ATP-binding cassette member B5 (ABCB5), a previously described marker for melanoma-initiating cells, on cellular metabolism. Metabolic profiles for two groups of human G3361 melanoma cells were compared, i.e. wildtype melanoma cells with intact ABCB5 expression (ABCB5-WT) and corresponding melanoma cell variants with inhibited ABCB5 expression, through shRNA-mediated gene knockdown (ABCB5-KD). A comprehensive metabolomic analysis was performed by using proton and phosphorus NMR spectroscopy of cell extracts to examine water-soluble metabolites and lipids. Parametric and non-parametric statistical analysis of absolute and relative metabolite levels yielded significant differences for compounds involved in glucose, amino acid and phospholipid (PL) metabolism. By contrast, energy metabolism was virtually unaffected by ABCB5 expression. The sum of water-soluble metabolites per total protein was 17% higher in ABCB5-WT vs. ABCB5-KD G3361 variants, but no difference was found for the sum of PLs. Enhanced abundance was particularly pronounced for lactate (+ 23%) and alanine (+ 26%), suggesting an increase in glycolysis and potentially glutaminolysis. Increases in PL degradation products, glycerophosphocholine and glycerophosphoethanolamine (+ 85 and 123%, respectively), and redistributions within the PL pool suggested enhanced membrane PL turnover as a consequence of ABCB5 expression. The possibility of glycolysis modulation by an ABCB5-dependent IL1ß-mediated mechanism was supported by functional studies employing monoclonal antibody (mAb)-dependent ABCB5 protein inhibition in wildtype G3361 melanoma cells. Our metabolomic results suggest that the underlying biochemical pathways may offer targets for melanoma therapy, potentially in combination with other treatment forms.