Silica-Antibiotic Hybrid Nanoparticles for Targeting Intracellular Pathogens

We investigated the capability of biodegradable silica xerogel as a novel carrier of antibiotic and the efficacy of treatment compared to that with the same dose of free drug against murine salmonellosis. The drug molecules (31%) entrapped in the sol-gel matrix remained in biologically active form, and the bactericidal effect was retained upon drug release. The in vitro drug release profiles of the gentamicin from the xerogel and that from the xerogel-polyethylene glycol (PEG) were distinctly different at pH 7.4. A delayed release of gentamicin was observed from the silica xerogel network (57% in 33 h), and with the addition of 2% PEG, the release rate reached 90% in 33 h. Administration of two doses of the silica xerogel significantly reduced the Salmonella enterica serovar Typhimurium load in the spleens and livers of infected AJ 646 mice. The silica xerogel and xerogel-PEG achieved a 0.45-log and a 0.41-log reduction in the spleens, respectively, while for the free drug there was no reduction. On the other hand, silica xerogel and xerogel-PEG achieved statistically significant 1.13-log and 1.15-log reductions in the livers, respectively, while for the free drug the reduction was a nonsignificant value of 0.07 log. This new approach, which utilizes a room-temperature synthetic route for incorporating therapeutic drugs into the silica matrix, should improve the capability for targeting intracellular pathogens.Targeting intracellular pathogens like Salmonella, Brucella, and Mycobacterium species remains a medical challenge. These intracellular pathogens can cause persistent infection due to their ability to maintain infections even in the presence of inflammation, specific antimicrobial mechanisms, and a robust adaptive immune response of the host (12). Some individuals who are infected with Salmonella enterica serovar Typhi become lifelong carriers, periodically shedding large numbers of bacteria (11). In host-adapted salmonelloses such as typhoid fever, the Salmonella-infected phagocytes gain access to the lymphatics and bloodstream, allowing the bacteria to spread to the liver and the spleen (20). Treatment and eradication are difficult since infections are localized within phagocytic cells, and it is difficult to achieve the optimum relatively high concentration of therapeutics within the infected cells (15). Thus, an optimum strategy to treat these infections should address targeting of active drugs to the intracellular compartment where bacteria replicate and should prolong release of the antibiotics so that the number of doses and associated drug toxicity can be reduced. The associated problems with delivering free antibiotics to the intracellular space have led to the investigations of improved drug carriers for treating intracellular pathogens, including antibiotics loaded into liposomes, microspheres, polymeric carriers, and nanoplexes (15).The success of any approach for drug delivery to intracellular pathogens depends on the ability to construct a biocompatible carrier with a high loading capacity of therapeutic drugs with no or minimum premature release of the cargo before reaching the replicative niche of the bacteria. The recently developed sol-gel technique has shown great promise for biomedical applications by offering new possibilities for embedding antibiotics within silica and for controlling their release from the host matrix into the surrounding medium (7, 16, 19, 22, 23). The composition of silica xerogels that can be tuned and the fabrication method at low temperatures enable them to carry biologically active agents and to be useful as a drug delivery system (8). Sol-gel silica materials have been shown to be biocompatible in vivo as they are readily degradable inside the body (6), which eliminates the problem of accumulation that has remained a major drawback for many other nanoparticle delivery systems. The various glass compositions cause no adverse tissue reactions and are easily eliminated from the body through the kidneys as they degrade into Si(OH)₄ (1, 18, 21).Also, the size, zeta potential, pore structure, and the surface characteristics of silica xerogel make it a suitable carrier for therapeutics to target the replicative niche of intracellular pathogens.Although gentamicin exhibits several characteristics that make it a useful antimicrobial agent in vitro, it does not kill intracellular Salmonella due to the polar nature of the drug and the associated low level of intracellular penetration. Therefore, the usefulness of gentamicin seems to be limited to eradication of the extracellular pathogens. In the present study, we used a mouse model of human typhoid fever to investigate the capability of sol-gel processed silica as a carrier for gentamicin and the efficacy of treatment compared to that with the free form of the same drug against Salmonella enterica serovar Typhimurium, a leading cause of human gastroenteritis (10).     

D–π–A azine based AIEgen with solvent dependent response towards a nerve agent

We developed a D–π–A based unsymmetrical azine molecule 4-((E)-((E)-(4-(dipropylamino)benzylidene)hydrazono)methyl)benzonitrile [DPBN] and studied its optical and aggregation induced emission properties. The DPBN molecule shows good aggregation induced emission (AIE) behaviour with 1157-fold fluorescence enhancement in the aggregated state. In addition to that, both colorimetric as well as fluorometric sensing studies revealed that DPBN selectively detects diethylchlorophosphate (DCP), a potent nerve agent. Interestingly, DPBN shows solvent dependent optical output in the presence of DCPvia two different mechanisms. In the monomer state, it shows red shifted fluorescence enhancement along with color change from colorless to orange color via the formation of a new intramolecular charge transfer state in pure tetrahydrofuran (THF). In the aggregated state, DPBN shows blue shifted emission with fluorescence enhancement in THF–water mixture by protonation at the amine nitrogen centre. Thus, DPBN can be used as a diagnostic measure to selectively detect nerve agents like DCP. This study also paves the way for further development of molecular probes for nerve agents that would represent immense implications in various fields of chemistry and biology.

Selective detection of diethylchlorophosphate using a D–π–A based AIEgen in aqueous as well as non-aqueous environment via different sensing mechanisms.     

ZnO-embedded S-doped g-C3N4 heterojunction: mediator-free Z-scheme mechanism for enhanced charge separation and photocatalytic degradation

The design of UV-visible light active photocatalysts for organic pollutant removal is a challenging task. Herein, we have developed an LED light active ZnO-embedded S-doped g-C₃N₄ (SCN) heterojunction by a facile sol–gel assisted calcination method. The heterojunction between ZnO and SCN nanoparticles generates a Z-scheme photocatalyst, which helps to separate the photo-induced charge carriers in the opposite direction, and is beneficial for more visible light absorption for photocatalytic dye degradation. The composite heterojunction shows better photocatalytic redox in comparison with pristine nanomaterials. The enhanced degradation efficiency is attributed to the high production rate of ˙OH (hydroxyl) radicals during the photocatalysis process, which is analyzed by the TA test and elemental trapping experiment. Hence, we hope that this Z-scheme heterojunction provides a new way to develop UV-visible light active photocatalysts for environmental remediation applications.

We have developed the LED light active ZnO-embedded S-doped g-C₃N₄ (SCN) mediator free Z-scheme heterojunction photocatalyst for effective organic pollutant degradation.     

Synergistic terpene combinations as larvicides against the dengue vector Aedes aegypti Linn.

Most of the essential oils (EO) known for mosquitocidal activity contain terpenes as part of their chemical constituents. In this study, four monoterpenes namely γ-terpinene (T), R-(+)-limonene (L), carvacrol (C), and trans-anethole (A) were screened against late third instar Aedes aegypti Linn. larvae singly and in combination to find out the synergistic interaction. The monoterpenes γ-terpinene, R-(+)-limonene, trans-anethole, and carvacrol showed larvicidal activity with LC₅₀ values of 9.76, 11.88, 50.19, 48.57 ppm and LC₉₀ values of 16.99, 17.78, 65.21, 75.02 ppm, respectively for Ae. aegypti when tested individually. Prepared binary combinations at LC₂₀ values and studied the synergistic interactions based on cotoxicity factor (CTF) and found that all the binary combinations showed synergistic interactions (CTF > 20). Further, the binary (1:1), ternary (1:1:1), and quaternary (1:1:1:1) terpene combinations on equal weight basis were prepared, evaluated against Ae. aegypti larvae and synergistic interactions were studied by Wadley's method. Among the eight combinations two combinations viz. LC (LC₅₀10.42 ppm) and LA (LC₅₀11.86 ppm) were showing synergistic interactions (R > 1.5) and others were exhibiting additive effect (1.5 ≥ R > 0.5) and none of the combinations showed antagonism (R < 0.5). Among the two methods used the Wadley's method found suitable when combinations were made on an equal weight basis. The two binary combinations found in this study are promising synergistic mixtures which has scope in designing mosquito larvicidal EOs by enriching the EOs with terpenes or by producing EO combinations with these terpenes as chemical constituents in the correct proportions.     

Cold preservation mediated renal injury: involvement of mitochondrial oxidative stress

Cold preservation has greatly facilitated the use of cadaveric kidneys for renal transplantation, but, clearly, damage occurs during both the preservation episode and the reperfusion phase (following transplantation). The aims of this study were twofold: to develop an in vivo model that was capable of evaluating renal function at early time points following cold preservation, and to evaluate the extent of renal mitochondrial damage that occurs following short periods of cold preservation in vivo. To accomplish these goals, we developed a novel rat model of in vivo renal cold ischemia followed by warm reperfusion (cold I/R) which avoided the complexity involved with transplantation. Briefly, after a right nephrectomy, cold I/R was initiated via pulsatile perfusion (40 minutes) of the left kidney with a cold University of Wisconsin solution followed by 18 hours of warm reperfusion. Cold I/R resulted in significant renal injury, nitrotyrosine production, and inactivation of the key mitochondrial antioxidant enzyme, manganese superoxide dismutase. Furthermore, the activities of the mitochondrial respiratory complexes were significantly reduced following cold I/R. In conclusion, short-term cold I/R results in inactivation of MnSOD, which may lead to the inhibition of mitochondrial complexes and subsequent renal injury. These data suggest that compounds designed to prevent early mitochondrial injury in kidneys that undergo cold preservation would significantly improve renal function and graft survival following transplantation.     

Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilage of both knees in other rabbits. The cultured allogenic chondrocytes were transplanted into the defect in the right knees and closed with a periosteal flap, while the defects in the left knees served as controls and were closed with a periosteal flap alone, without chondrocytes. Healing of the defects was assessed at 12 weeks by histological studies. Allogenic chondrocyte transplantation significantly increased the amount of newly formed repair tissue (P=0.04) compared with that found in the control knees. The histological quality score of the repair tissue was significantly better (P=0.05), with more hyaline characteristics in the knees treated with allogenic chondrocytes than in the control knees. Articular cartilage defects treated with allogenic chondrocyte transplantation result in better repair tissue formation with hyaline characteristics than those in control knees.     

An effective integrated learning programme in the first year of the medical course

BACKGROUND: An integrated approach to teaching medical subjects is an effective educational strategy. Yet, this has not become popular in medical colleges in India. We describe an integrated learning programme to teach the gastrointestinal system in the first year of the medical course. METHODS: The integrated learning programme was conducted for 3 years (2003-2005). It incorporated elements of problem-based learning, early clinical exposure, lectures and small group laboratory work. Student assessment was formative (for problem-based learning sessions) and summative (using problem-based learning and knowledge tests). Evaluation of the programme was based on feedback from the students and faculty members. RESULTS: Ninety-six per cent of the students obtained more than 60% marks in the problem-based learning test. The mean (SD) score in the knowledge test was 62 (0.89)%. The majority of students received satisfactory and more than satisfactory grades for their performance in the problem-based learning sessions. The feedback from faculty members and students was positive, which highlighted benefits such as integrated learning of the basic sciences, their application to clinical cases and active student learning. The challenges encountered included the higher input required from faculty members. Most of the faculty members and students recommended that the integrated programme should be continued and extended to other parts of the curriculum. CONCLUSION: An integrated learning programme is feasible within a conventional medical curriculum of an Indian medical college.     

Quorum-sensing signals produced by plant-growth promoting Burkholderia strains under in vitro and in planta conditions

The genus Burkholderia is a heterogeneous group with extraordinary nutritional versatility and which occupies a diversity of niches. In recent decades, members of Burkholderia have been shown to be active participants in plant-microbe interactions, imparting beneficial effects as plant-growth-promoting bacteria (PGPB) or as pathogens. The study of quorum sensing and cell-density-dependent gene regulation, which play an important role in host colonization and pathogenesis, is extremely important in such a versatile organism. We report the identification and characterization by thin-layer chromatography (TLC) of N-acyl homoserine lactone (HSL) quorum sensing signal molecules by PGP Burkholderia. The Burkholderia spp. strains CBMB40, CBPB-HOD and CBPB-HIM investigated in this study were isolated from rice and possessed one or more PGP characteristics. Culture extracts of these strains contained detectable levels of hexanoyl (C(6)-), heptanoyl (C(7)-) and octanoyl (C(8)-) HSLs. Burkholderia sp. strain CBMB40 produced an additional molecule that migrated along decanoyl (C(10)-) HSL. Inoculation of HSL-producing Burkholderia strains through seed bacterization to canola stimulated root elongation. Signal molecules produced by Burkholderia strains could also be detected in planta, as determined by plate assays and TLC analysis of plant extracts. This study advances the hypothesis that signaling molecules by PGPB in planta might play a substantial role in increasing the pathogen resistance of plants.     

Anti‐VEGF antibody enhances the antitumor effect of CD40

As its central immunomodulatory effects, CD40 induces interleukin (IL)‐12‐dependent antitumor immune responses; as its local protumor effects, CD40 induces the expression of vascular endothelial growth factor (VEGF) that promotes tumor angiogenesis and growth. Therefore, using a previously established tumor model in mouse, we examined if the antitumor functions of CD40 are self‐limited by VEGF induction. We observed that as the tumor mass grew during day 6 to day 18, VEGF expression in the tumor peaked with concomitant decrease in expressions of CD40 and IL‐12 but not of IL‐10. Among the angiogenic factors, VEGF‐B, VEGFR‐1, VEGFR‐2, angiopoietin‐1 and Tie2 expressions decreased, whereas the expressions of angiopoietin‐2 and angiopoietin‐3 increased with tumor growth. As significant changes in the expressions of these factors were observed on day 6, we treated the tumor‐bearing mice with the agonistic anti‐CD40 antibody or neutralizing anti‐VEGF antibody—alone or in combination—from the fifth day after the injection of tumor cells. The anti‐VEGF antibody significantly enhanced the antitumor effects of the anti‐CD40 antibody, as observed through increased survival of the mice, accompanied by reduced angiogenesis and angiopoietin‐2 expression but higher T‐cell proliferation in response to tumor antigens, increased interferon‐γ production and tumor cell cytotoxicity and higher levels of tumor antigen‐specific serum IgM, IgG1 and IgG2a, indicating B‐cell activation. Thus, our data show for the first time that the combined treatment with an agonistic anti‐CD40 antibody and a neutralizing anti‐VEGF antibody, which increases antitumor immune response or reduces local angiogenesis, respectively, is a novel antitumor strategy.