Selective Translocation of Cyclic Sugars through Dynamic Bacterial Transporter.

The selective translocation of molecules through membrane pores is an integral process in cells. We present a bacterial sugar transporter, CymA of unusual structural conformation due to a dynamic N terminus segment in the pore, reducing its diameter. We quantified the translocation kinetics of various cyclic sugars of different charge, size, and symmetry across native and truncated CymA devoid of the N terminus using single-channel recordings. The chemically divergent cyclic hexasaccharides bind to the native and truncated pore with high affinity and translocate effectively. Specifically, these sugars bind and translocate rapidly through truncated CymA compared to native CymA. In contrast, larger cyclic heptasaccharides and octasaccharides do not translocate but bind to native and truncated CymA with distinct binding kinetics highlighting the importance of molecular charge, size and symmetry in translocation consistent with liposome assays. Based on the sugar-binding kinetics, we suggest that the N terminus most likely resides inside the native CymA barrel, regulating the transport rate of cyclic sugars. Finally, we present native CymA as a large nanopore sensor for the simultaneous single-molecule detection of various sugars at high resolution, establishing its functional versatility. This natural pore is expected to have several applications in nanobiotechnology and will help further our understanding of the fundamental mechanism of molecular transport.

Chandipura virus induces cell death in cancer cell lines of human origin and promotes tumor regression in vivo.

Chandipura virus (CHPV) is an emerging human pathogen of great clinical significance. In this study, we have investigated the susceptibility pattern of both normal and cancer cell lines of human origin to wild-type (wt) CHPV in order to explore the possibility of developing CHPV as an oncolytic vector (OV). Marked cytopathic effect along with enhanced virus output was observed in cancer cell lines (HeLa, A549, U-138, PC-3, and HepG2) in comparison to normal human adult dermal fibroblast (HADF) cells. At an MOI of 0.1, cancer cell lines were differentially susceptible to CHPV, with cells like HeLa and U-138 having pronounced cell death, while the PC-3 were comparatively resistant. All cell lines used in the study except U-138 restricted CHPV infection to varying degrees with IFN-ß pre-treatment and supplementation of interferon (IFN) could neither activate the IFN signaling pathway in U-138 cells. Finally, U-138 tumor xenografts established in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice showed significant delay in tumor growth in the CHPV-challenged animals. Thus, targeted cytopathic effect in cancer cells at a very low dose with restricted replication in normal cells offers a rationale to exploit CHPV as an oncolytic vector in the future.

In the Crosshairs: RNA Viruses OR Complement?

Complement, a part of the innate arm of the immune system, is integral to the frontline defense of the host against innumerable pathogens, which includes RNA viruses. Among the major groups of viruses, RNA viruses contribute significantly to the global mortality and morbidity index associated with viral infection. Despite multiple routes of entry adopted by these viruses, facing complement is inevitable. The initial interaction with complement and the nature of this interaction play an important role in determining host resistance versus susceptibility to the viral infection. Many RNA viruses are potent activators of complement, often resulting in virus neutralization. Yet, another facet of virus-induced activation is the exacerbation in pathogenesis contributing to the overall morbidity. The severity in disease and death associated with RNA virus infections shows a tip in the scale favoring viruses. Growing evidence suggest that like their DNA counterparts, RNA viruses have co-evolved to master ingenious strategies to remarkably restrict complement. Modulation of host genes involved in antiviral responses contributed prominently to the adoption of unique strategies to keep complement at bay, which included either down regulation of activation components (C3, C4) or up regulation of complement regulatory proteins. All this hints at a possible "hijacking" of the cross-talk mechanism of the host immune system. Enveloped RNA viruses have a selective advantage of not only modulating the host responses but also recruiting membrane-associated regulators of complement activation (RCAs). This review aims to highlight the significant progress in the understanding of RNA virus-complement interactions.

A Factor I-Like Activity Associated with Chikungunya Virus Contributes to Its Resistance to the Human Complement System.

Chikungunya virus (CHIKV) is an emerging pathogen capable of causing explosive outbreaks. Prior studies showed that exacerbation in arthritogenic alphavirus-induced pathogenesis is attributed to its interaction with multiple immune components, including the complement system. Viremia concomitant to CHIKV infection makes exposure of the virus to complement unavoidable, yet very little is known about CHIKV-complement interactions. Here, we show that CHIKV activated serum complement to modest levels in a concentration- and time-dependent manner, but the virus effectively resisted complement-mediated neutralization. Heat-inactivated serum from seropositive donors could actively neutralize CHIKV due to the presence of potent anti-CHIKV antibodies. Deposition of key complement components C3 and C4 did not alter the resistance of CHIKV to complement. Further, we identified a factor I-like activity in CHIKV that limited complement by inactivating C3b into inactive C3b (iC3b), the complement component known to significantly contribute to disease severity in vivo, but this activity had no effect on C4b. Inactivation of C3b by CHIKV was largely dependent on the concentration of the soluble host cofactor factor H and the virus concentration. A factor I function-blocking antibody had only a negligible effect on the factor I-like activity associated with CHIKV, suggesting that this activity is independent of host factor I and could be of viral origin. Thus, our findings suggest a complement modulatory action of CHIKV which not only helps the virus to evade human complement but may also have implications in alphavirus-induced arthritogenic symptoms.IMPORTANCE Chikungunya virus is a vector-borne pathogen of global significance. The morbidity associated with chikungunya virus (CHIKV) infection, neurovirulence and adaptability to Aedes albopictus, necessitates a deeper understanding of the interaction of CHIKV with the host immune system. Here, we demonstrate that CHIKV is resistant to neutralization by one of the potent barriers of the innate immune arm, the complement system. Chikungunya virus showed marked resistance to complement despite activation and deposition of complement proteins. Interestingly the C3 component associated with the virion was found to be inactive C3b (iC3b), a key factor implicated in the pathogenesis and disease severity in the mouse model of Ross River virus infection. CHIKV also had an associated unique factor I-like activity that mediated the inactivation of C3b into iC3b. We have unraveled a smart strategy adopted by CHIKV to limit complement which has serious implications in viral dissemination, pathogenesis, and disease.

Complement-Mediated Neutralization of a Potent Neurotropic Human Pathogen, Chandipura Virus, Is Dependent on C1q.

Among the innate immune sentinels, the complement system is a formidable first line of defense against pathogens, including viruses. Chandipura virus (CHPV), a neurotropic vesiculovirus of the family Rhabdoviridae, is a deadly human pathogen known to cause fatal encephalitis, especially among children. The nature of interaction and the effect of human complement on CHPV are unknown. Here, we report that CHPV is a potent activator of complement and, thus, is highly sensitive to complement proteins in normal human serum (NHS). Utilizing a panel of specific complement component depleted/reconstituted human serum, we have demonstrated that CHPV neutralization is C3, C4, and C1q dependent and independent of factor B, suggesting the importance of the classical pathway in limiting CHPV. Employing a range of biochemical approaches, we showed (i) a direct association of C1q to CHPV, (ii) deposition of complement proteins C3b, C4b, and C1q on CHPV, and (iii) virus aggregation. Depletion of C8, an important component of the pore-forming complex of complement, had no effect on CHPV, further supporting the finding that aggregation and not virolysis is the mechanism of virus neutralization. With no approved vaccines or treatment modalities in place against CHPV, insights into such interactions can be exploited to develop potent vaccines or therapeutics targeting CHPV.IMPORTANCE Chandipura virus is a clinically important human pathogen of the Indian subcontinent. The rapidity of death associated with CHPV infection in addition to the absence of an effective vaccine or therapeutics results in poor clinical prognosis. The biology of the virus and its interaction with the host immune system, including the complement system, are understudied. Our investigation reveals the susceptibility of CHPV to fluid phase complement and also dissects the pathway involved and the mechanism of virus neutralization. Direct binding of C1q, an important upstream component of the classical pathway of complement to CHPV, and the strong dependency on C1q for virus neutralization highlight the significance of identifying such interactions to better understand CHPV pathogenesis and devise strategies to target this deadly pathogen.

Self-assembling peptide nanofibers containing phenylalanine for the controlled release of 5-fluorouracil.

The study shows that RADA-F6 peptide with pH-responsive self-assembling nature can be effectively used as a drug delivery system for the sustained release of a potent anticancer drug 5-fluorouracil (5-FU) at basic pH. As 5-FU contains the aromatic pyrimidine ring, RADA-F6 system is suitable for entrapping an aromatic drug due to effective π-π stacking with phenylalanine and be able to show better controlled release behavior. The stability and controlled release nature of RADA-F6 in different conditions followed by 5-FU entrapment at in silico conditions was confirmed by molecular dynamics simulation taking RADA-16 as control. Cytotoxicity of the drug-loaded RADA-F6 was measured by MTT assay and cellular uptake by confocal microscopy. Physicochemical characterization and further Western blot analysis and flow cytometric studies confirm that RADA-F6 can be successfully used as an efficient vector for pH-sensitive, controlled 5-FU delivery system.

A Randomized Double-Blind Placebo-Controlled Phase IIB Trial of Curcumin in Oral Leukoplakia.

Oral leukoplakia is a potentially malignant lesion of the oral cavity, for which no effective treatment is available. We investigated the effectiveness of curcumin, a potent inhibitor of NF-κB/COX-2, molecules perturbed in oral carcinogenesis, to treat leukoplakia. Subjects with oral leukoplakia (n = 223) were randomized (1:1 ratio) to receive orally, either 3.6 g/day of curcumin (n = 111) or placebo (n = 112), for 6 months. The primary endpoint was clinical response obtained by bi-dimensional measurement of leukoplakia size at recruitment and 6 months. Histologic response, combined clinical and histologic response, durability and effect of long-term therapy for an additional six months in partial responders, safety and compliance were the secondary endpoints. Clinical response was observed in 75 (67.5%) subjects [95% confidence interval (CI), 58.4-75.6] in the curcumin and 62 (55.3%; 95% CI, 46.1-64.2) in placebo arm (P = 0.03). This response was durable, with 16 of the 18 (88.9%; 95% CI, 67.2-96.9) subjects with complete response in curcumin and 7 of 8 subjects (87.5%) in placebo arm, demonstrating no relapse after 6 months follow-up. Difference in histologic response between curcumin and placebo was not significant (HR, 0.88, 95% CI, 0.45-1.71; P = 0.71). Combined clinical and histologic response assessment indicated a significantly better response with curcumin (HR, 0.50; 95% CI, 0.27-0.92; P = 0.02). Continued therapy, in subjects with partial response at 6 months, did not yield additional benefit. The treatment did not raise any safety concerns. Treatment of oral leukoplakia with curcumin (3.6 g for six months), thus was well tolerated and demonstrated significant and durable clinical response for 6 months. Cancer Prev Res; 9(8); 683-91. ©2016 AACR.

5-Fluorouracil-lipid conjugate: potential candidate for drug delivery through encapsulation in hydrophobic polyester-based nanoparticles.

The encapsulation of 5-fluorouracil (5-FU) in hydrophobic polymeric materials is made feasible by a lipid-based prodrug approach. A lipid-5-FU conjugate of 5-FU with palmitic acid was synthesized in two-step process. A synthesized dipalmitoyl derivative (5-FUDIPAL) was characterized using Fourier transform infrared spectroscopy and (1)H-nuclear magnetic resonance. The 5-FUDIPAL was encapsulated in polyester-based polymers by the double emulsion-solvent evaporation method. The nanoparticles were characterized by scanning electron microscopy, transmission electron microscopy and dynamic light scattering. The thermal stability was assessed by differential scanning calorimetry data. In vitro release kinetics measurements of the drug from nanoparticles showed the controlled release pattern over a period of time. Cytotoxicity measurements by MTT assay confirmed that dipalmitoyl derivative in nano formulation successfully inhibited the cell growth. Thus the combined physical and biological evaluation of the different polyester-based nanoparticle containing the modified drug showed a facile approach to delivering 5-FU to the tumour site with enhanced efficacy.

Dual drug delivery of 5-fluorouracil (5-FU) and methotrexate (MTX) through random copolymeric nanomicelles of PLGA and polyethylenimine demonstrating enhanced cell uptake and cytotoxicity.

We now report the synthesis of a random copolymer of poly-lactic-co-glycolic acid (PLGA) grafted branched polyethylenimine (BPEI) and the use of it as a multi drug delivery system (DDS). The methotrexate (MTX) was conjugated to BPEI through DCC/NHS chemistry. The copolymer-drug conjugate (PBP-MTX) was characterised by FT-IR and (1)H NMR spectroscopy. The PBP-MTX was converted into nanomicelles with entrapped 5-fluorouracil (5-FU) through nanoprecipitation technique. The size, shape, morphology and surface charge of the nanomicelles were confirmed using different techniques. The thermal behaviour and distribution of both conjugated and entrapped drug through the polymeric matrix were assessed by differential scanning calorimetry (DSC) and powder X-ray diffraction analysis (PXRD). In vitro drug release pattern of the nanomicelles was examined to ascertain the release pattern of two drugs namely 5-FU and MTX. The cellular uptake studies demonstrated higher uptake of the nanomicelles in colon cancer cell line HCT 116. Further the cytotoxicity evaluation of nanomicelles illustrated promising action which confirms the use of the system as a potential DDS to colon cancer.

Methacrylic-based nanogels for the pH-sensitive delivery of 5-fluorouracil in the colon.

Methacrylic-based copolymers in drug-delivery systems demonstrate a pH-sensitive drug-releasing behavior in the colon. In this study, copolymers of methacrylic acid and 2-ethyl hexyl acrylate were prepared using a microemulsion polymerization technique. The purified copolymer was characterized by Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. 5-Fluorouracil (5-FU) was entrapped within methacrylic-based copolymers by a solvent evaporation method. The size of the nanogels formed was characterized by transmission electron microscopy and atomic force microscopy. In vitro drug-release studies using phosphate-buffered saline at different pH levels demonstrated the sustained release of 5-FU and its pH dependence. Cell proliferation assay of a human colon tumor colon cancer cell line (HCT-116) was performed and showed that the nanogels containing 5-FU exhibited considerable cytotoxicity in comparison with free 5-FU. Cell uptake of the nanogels was also monitored using confocal microscopy. Western blot analysis and flow cytometry studies confirmed that the nanogels could be successfully used as an efficient vector for pH-sensitive and controlled delivery of drugs specifically targeted to the colon.

Poly (D,L-lactic-co-glycolide) nanoparticles for the improved therapeutic efficacy of all-trans-retinoic acid: a study of acute myeloid leukemia (AML) cell differentiation in vitro.

All-trans-retinoic acid reverses malignant cell growth and induces cell differentiation and apoptosis. Poor aqueous solubility and uncertain bioavailability are the limiting factors for using all-trans-retinoic acid for tumor therapy. The objective of present study was to encapsulate the hydrophobic drug all-trans-retinoic acid in the polymer poly (lactide-coglycolide). The encapsulation was expected to improve the bioavailability and solubility of the drug. Oil in water single emulsion solvent evaporation technique used for the preparation efficiently encapsulated about 60% of the drug. The drug release profile showed a biphasic pattern with 70% of the drug being released in first 48 hrs and the residual drug showing a slow controlled release reaching up to 8 days. The particle size of 150-200 nm as determined with TEM was ideal for tumor targeting. All-trans-retinoic acid loaded nanoparticles were efficient to induce differentiation and blocked the proliferation of HL-60 cells invitro. These studies also revealed that the dosage of drug required for the therapeutic effects have been reduced efficiently. Our studies thereby demonstrate that Poly (lactide-co-glycolide) based nanoparticles may be efficient for parenteral administration of the drug.