Semi-quantification of antibody-dependent enhancement (ADE) in the uptake of Adenovirus serotype 5 into THP-1 cells.

Replication defective recombinant Ad5 vectors (rAdV5) are extensively explored for its applications in gene therapy and vaccine delivery. Ad5 enter into monocytes and macrophages through CAR independent route as an immune complex termed as antibody-dependent enhancement (ADE). We developed an effective method for estimating the ADE of rAdV5 encoding GFP (rAdV5-GFP) into THP-1 cells, using fluorimetric semi-quantification of GFP. Initially, twenty numbers of human sera samples were screened in HeLa cells for anti-Ad5 antibody titer using neutralization assay. Uptake of rAdV5-GFP in THP-1 cells was observed only after pre-incubation with the serially diluted human sera which are attributed to ADE. The optimal dilution which showed the maximum GFP expression as per the fluorescence microscopic analysis in THP-1 cells was used for further analysis. Fluorimetric analysis of the THP-1 cell lysate showed a maximum GFP intensity of 17058 RFU, which was equivalent to the 0.397 pmoles of Alexa Fluor 488 under the same experimental condition. Similarly, immunoblot analysis of GFP in THP-1 cell lysate and HeLa cell lysate confirmed the entry of rAdV5-GFP into the cells. The assay can serve as a platform for understanding the molecular events involved in ADE for the uptake of viruses into immune cells.

Human skin fibrosis: up-regulation of collagen type III gene transcription in the fibrotic skin nodules of lower limb lymphoedema.

OBJECTIVES: To investigate the cellular and molecular pathophysiology involved in the development of fibrotic skin of grade-3 lymphoedema patients with a focus on collagen types. METHODS: Fibrotic and normal skin biopsy samples obtained from grade-3 lymphoedema patients and normal individuals, respectively, were analysed by histopathology, quantitative real-time PCR and immunohistochemistry to examine collagen gene expression. RESULTS: Histopathologic analysis revealed epidermal changes such as orthokeratosis, hypergranulosis and irregular acanthosis in the skin biopsies. The thickened dermis contained nodules of haphazardly arranged thick collagen bundles. Real-time PCR data showed significant (P-value 0.0003) up-regulation of Collagen type I and type III gene transcripts in the fibrotic skin of patients resulting in 38.94-fold higher transcription of Collagen type III alpha-1 gene than of Collagen type I alpha-1 gene. Semi-quantification of the per cent of haematoxylin-DAB-stained area of immunohistochemistry images also showed significant (P < 0.0001) enhancement of both collagen proteins in the fibrotic skin of patients vs. normal human skin. CONCLUSIONS: Gene transcript analysis revealed significant up-regulation of Collagen type III vs. Collagen type I in fibrotic skin of limb nodules from patient biopsies. Histopathological and immunohistochemical analysis also revealed enhancement of Collagen types I and III in fibrotic vs. normal skin. The findings of this preliminary study indicate the potentially significant involvement of Collagen type III in the development of the fibrotic skin of grade-3 lymphoedema patients.

OBJECTIFS: Etudier la physiopathologie cellulaire et moléculaire impliquée dans le développement de la fibrose cutanée chez les patients atteints de lymphœdème de grade 3 en mettant l'accent sur les types de collagène. MÉTHODES: Des échantillons de biopsie cutanée fibrotique et normale obtenus respectivement de patients atteints de lymphœdème de grade 3 et d'individus normaux ont été analysés par histopathologie, par PCR quantitative en temps réel et par immunohistochimie pour examiner l'expression des gènes de collagène. RÉSULTATS: L'analyse histopathologique a révélé des changements épidermiques tels que l'orthokératose, l'hypergranulose et l'acanthose irrégulière dans les biopsies cutanées. Le derme épaissi contenait des nodules de faisceaux de collagène épais disposés au hasard. Les données de PCR en temps réel ont montré une régulation à la hausse significative (P = 0.0003) des transcrits des gènes de collagène de type I et III dans la peau fibrotique des patients, résultant en une transcription 38,94 fois plus élevée du gène alpha-1 du collagène de type III par rapport à celui du gène alpha-1 du collagène de type I. La semi-quantification du pourcentage de zone colorée à l'hématoxyline-DAB des images d'immunohistochimie a également montré une amélioration significative (P < 0.0001) des deux protéines de collagène dans la peau fibrotique des patients par rapport à la peau humaine normale. CONCLUSIONS: L'analyse de transcription génétique a révélé une régulation à la hausse importante du collagène de type III par rapport à celle du collagène de type I dans la peau fibrotique des nodules des membres provenant de biopsies de patients. L'analyse histopathologique et immunohistochimique a également révélé une amélioration du collagène de types I et III dans la peau fibrotique pa rapport à la peau normale. Les résultats de cette étude préliminaire indiquent l'implication potentiellement significative du collagène de type III dans le développement de la peau fibrotique des patients atteints de lymphœdème de grade 3.

Mycophenolic acid (MPA) modulates host cellular autophagy progression in sub genomic dengue virus-2 replicon cells.

Cellular autophagy (Macrophagy) is a self-degradative process, executed through the network of autophagy associated genes (ATGs) encoded proteins. Both in vitro and in vivo studies suggest that dengue virus (DENV) induces autophagy and supports the viral genome replication and translation. Therefore, the cellular autophagy induced by dengue virus can be a good target for antiviral drug development. The action of mycophenolic acid (MPA), a specific inhibitor of DENV replication, was investigated in the stable BHK-21/DENV2 replicon cells. The inhibition was mediated by enhanced degradation of autophagic substrates in stable BHK-21/DENV2 replicon cells as evidenced by a decrease in lapidated LC3 (LC3II) and p62 expression in the presence of MPA. In contrast, the results indicated that four gene sets, namely Transmembrane protein 74 (TMEM74), Unc-51-like kinase 2 (ULK2), Cathepsin D (CTSD) and Estrogen receptor 1 (ESR1) were upregulated in stable BHK-21/DENV2 replicon cells, due to the sustained dynamic replication of DENV2 genome. These ATGs involved in the pre-autophagosomal structure (PAS) formation, were suppressed in the presence MPA. Instead, MPA induced the expression of different set of autophagy genes such as ATG4, AKT1, APP, ATG16L1, ATG16L2, B2M and HPRT1. An enzyme involved in the nucleotide salvage pathway, HPRT1, was highly expressed in the presence of MPA. The study shows that DENV2 replication is dependent on PAS formation and is inhibited in the presence of MPA by enhancing the degradation of autophagic substrates and suppression of PAS formation. This study provides impetus in designing MPA analogues to effectively inhibit dengue viral replication.

Proteomic analysis reveals the enhancement of human serum apolipoprotein A-1(APO A-1) in individuals infected with multiple dengue virus serotypes.

OBJECTIVES: Human serum protein profiling of the individual infected with multiple dengue virus serotypes for identifying the potential biomarkers and to investigate the cause for the severity of dengue virus infection. METHODS: Dengue virus NS1-positive serum samples were pooled into two groups (S2 and S3) based on the molecular serotyping and number of heterotypic infections. The pooled serum samples were subjected to two-dimensional gel electrophoresis (2DGE) to identify the differentially expressed proteins. The peptide masses of upregulated protein were detected by matrix-assisted laser desorption-ionisation time-of-flight MALDI-TOF mass spectrometry and analysed by MASCOT search engine. The results were compared with the control group (S1). The commonly upregulated protein was validated by quantitative ELISA and compared with control as well as single serotypic infected samples. RESULTS: Based on 2DGE, total thirteen proteins were differentially upregulated in S2 and S3 groups as compared to control. Some of the upregulated proteins were involved in mediating the complement activation of immune response. The apolipoprotein A-1 (APO A-1) was upregulated in S2 and S3 groups. Upon validation, APO A-1 levels were increased in line with the number of heterotypic infection of dengue viruses. CONCLUSION: Heterotypic infection of dengue viruses upregulate the serum proteins involved in the complement pathway in the early phase of infection. There was a significant increase in the level of APO A-1 in three different serotypic infections of dengue virus as compared to control. Further, the role of APO-A1 can be explored in elucidating the mechanism of dengue pathogenesis.

RNA Interference in the Age of CRISPR: Will CRISPR Interfere with RNAi?

The recent emergence of multiple technologies for modifying gene structure has revolutionized mammalian biomedical research and enhanced the promises of gene therapy. Over the past decade, RNA interference (RNAi) based technologies widely dominated various research applications involving experimental modulation of gene expression at the post-transcriptional level. Recently, a new gene editing technology, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system, has received unprecedented acceptance in the scientific community for a variety of genetic applications. Unlike RNAi, the CRISPR/Cas9 system is bestowed with the ability to introduce heritable precision insertions and deletions in the eukaryotic genome. The combination of popularity and superior capabilities of CRISPR/Cas9 system raises the possibility that this technology may occupy the roles currently served by RNAi and may even make RNAi obsolete. We performed a comparative analysis of the technical aspects and applications of the CRISPR/Cas9 system and RNAi in mammalian systems, with the purpose of charting out a predictive picture on whether the CRISPR/Cas9 system will eclipse the existence and future of RNAi. The conclusion drawn from this analysis is that RNAi will still occupy specific domains of biomedical research and clinical applications, under the current state of development of these technologies. However, further improvements in CRISPR/Cas9 based technology may ultimately enable it to dominate RNAi in the long term.

The bioactive lipid 4-hydroxyphenyl retinamide inhibits flavivirus replication.

Dengue virus (DENV), a member of the Flaviviridae family, is a mosquito-borne pathogen and the cause of dengue fever. The increasing prevalence of DENV worldwide heightens the need for an effective vaccine and specific antivirals. Due to the dependence of DENV upon the lipid biosynthetic machinery of the host cell, lipid signaling and metabolism present unique opportunities for inhibiting viral replication. We screened a library of bioactive lipids and modulators of lipid metabolism and identified 4-hydroxyphenyl retinamide (4-HPR) (fenretinide) as an inhibitor of DENV in cell culture. 4-HPR inhibits the steady-state accumulation of viral genomic RNA and reduces viremia when orally administered in a murine model of DENV infection. The molecular target responsible for this antiviral activity is distinct from other known inhibitors of DENV but appears to affect other members of the Flaviviridae, including the West Nile, Modoc, and hepatitis C viruses. Although long-chain ceramides have been implicated in DENV replication, we demonstrate that DENV is insensitive to the perturbation of long-chain ceramides in mammalian cell culture and that the effect of 4-HPR on dihydroceramide homeostasis is separable from its antiviral activity. Likewise, the induction of reactive oxygen species by 4-HPR is not required for the inhibition of DENV. The inhibition of DENV in vivo by 4-HPR, combined with its well-established safety and tolerability in humans, suggests that it may be repurposed as a pan-Flaviviridae antiviral agent. This work also illustrates the utility of bioactive lipid screens for identifying critical interactions of DENV and other viral pathogens with host lipid biosynthesis, metabolism, and signal transduction.

Biochemical characterization of P-type copper ATPases.

Copper ATPases, in analogy with other members of the P-ATPase superfamily, contain a catalytic headpiece including an aspartate residue reacting with ATP to form a phosphoenzyme intermediate, and transmembrane helices containing cation-binding sites [TMBS (transmembrane metal-binding sites)] for catalytic activation and cation translocation. Following phosphoenzyme formation by utilization of ATP, bound copper undergoes displacement from the TMBS to the lumenal membrane surface, with no H+ exchange. Although PII-type ATPases sustain active transport of alkali/alkali-earth ions (i.e. Na+, Ca2+) against electrochemical gradients across defined membranes, PIB-type ATPases transfer transition metal ions (i.e. Cu+) from delivery to acceptor proteins and, prominently in mammalian cells, undergo trafficking from/to various membrane compartments. A specific component of copper ATPases is the NMBD (N-terminal metal-binding domain), containing up to six copper-binding sites in mammalian (ATP7A and ATP7B) enzymes. Copper occupancy of NMBD sites and interaction with the ATPase headpiece are required for catalytic activation. Furthermore, in the presence of copper, the NMBD allows interaction with protein kinase D, yielding phosphorylation of serine residues, ATP7B trafficking and protection from proteasome degradation. A specific feature of ATP7A is glycosylation and stabilization on plasma membranes. Cisplatin, a platinum-containing anti-cancer drug, binds to copper sites of ATP7A and ATP7B, and undergoes vectorial displacement in analogy with copper.

Serotype-specific clinical features and spatial distribution of dengue in northern Kerala, India.

Background: Collection and compilation of spatial, meteorological, entomological, and virological data are critical in mitigating climate-sensitive emerging infections like dengue. This study was a holistic attempt to understand the dengue situation in the Kasaragod district of Kerala, India. Methods: This cross-sectional study was conducted in 13 health institutions from June to July 2021. Adult patients presenting with fever and testing positive for NS1 ELISA were subjected to Dengue RT-PCR and serotyping. The spatial and clinical features of the RT-PCR-positive patients, the district's meteorological data, and the vector indices were studied. Results: The pre-epidemic months were marked by intermittent rainfall, peak ambient temperature and high larval indices. Among the 136 dengue RT-PCR patients studied, 41.2% had DENV2 followed by DENV1 (22.8%), DENV3 (5.9%) and DENV4 (4.4%); with 25% mixed infections. DENV1 showed a higher risk of gastrointestinal manifestations (80.6%, p=0.019) and musculoskeletal symptoms (77.4%, p=0.026) compared with other serotypes. Conclusions: In the context of dengue hyperendemicity, the possibility of an emerging serotype's dominance coupled with the mixing up of strains should warn the health system regarding future outbreaks. Furthermore, the study emphasizes the importance of monitoring larval indices and the window of opportunity to intervene between environmental predictors and dengue outbreaks.

Distinctive features of catalytic and transport mechanisms in mammalian sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases.

Ca(2+) (sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA)) and Cu(+) (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca(2+), demonstrated by the addition of ATP and Ca(2+) to SERCA deprived of Ca(2+) (E2) as compared with ATP to Ca(2+)-activated enzyme (E1·2Ca(2+)). Activation by Ca(2+) is slower at low pH (2H(+)·E2 to E1·2Ca(2+)) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca(2+) translocation. A "H(+)-gated pathway," demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca(2+) release by H(+)/Ca(2+) exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu(+)/H(+) exchange. As opposed to SERCA after Ca(2+) chelation, ATP7A/B does not undergo reverse phosphorylation with P(i) after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.

Development of PEGylated Cu nanoclusters: A nontoxic, multifunctional colloidal system for bioimaging and peroxide sensing.

This study introduces the development of blue-emitting colloidal Cu NCs through a novel and easy PEGylation method using different functional groups, including -SH and -COOH. The surface functionalization controls the size, cellular toxicity, and emission properties of Cu NCs. The combination of PEG, thiol, and carboxylic groups protects the particle surface from aggregation and oxidation. Among the samples, CAGP (Surface modified Cu NCs with -SH-COOH-PEG combination) emerges as an amazing candidate with the lowest toxicity and enhanced blue emission properties. The bright blue fluorescence emission from Hela cells after treatment with CAGP demonstrated this property. It also has excellent peroxide sensing potential, with a detection limit of 1.4 µM. Because of their excellent bioimaging and peroxide sensing properties, these Cu NCs could be a promising candidate for cellular oxidative stress sensing applications with high clinical relevance.

Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase).

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.

Development of a paper printed colorimetric sensor based on Cu-Curcumin nanoparticles for evolving point-of-care clinical diagnosis of sodium.

The homeostatic control of Sodium (Na+) ion in the human body assumes paramount relevance owing to its physiological importance. Any deviation from the normal level causes serious health problems like hypernatremia, hyponatremia, stroke, kidney problems etc. Therefore, quantification of Na+ levels in body fluids has significant diagnostic and prognostic importance. However, interfering ions like Potassium ion (K+) is the major hurdle in sodium detection. In this work, we synthesized the clusters of 3-9 nm-sized highly stable and pure Copper nanoparticles surface functionalised with curcumin, through chemical reduction method. Each cluster of particles is encapsulated in a curcumin layer which is clearly visible in TEM images. The results show that these curcumin functionalized Cu NPs (CuC) are highly selective to the colorimetric detection of Na+. The ions like K+, Mg2+ and Zn2+ did not interfere with the Na+ in this sensing technique. Low-cost paper-based sensor strips are fabricated and calibrated for the sensing of sodium in the physiological range and shade cards were developed as a calorimetric guide for estimation of Na+ which makes them ideal point of care diagnostic platform. We demonstrate that the proposed CuC paper strip can be used for detecting Na+ concentration within the whole physiological range in both blood serum and urine.

Sensitization of cancer cells towards Cisplatin and Carboplatin by protein kinase D inhibitors through modulation of ATP7A/B (copper transport ATPases).

Drug resistance of cancer cells is a significant impediment to effective chemotherapy. One primary reason for this is copper exporters - ATPase copper transporting alpha (ATP7A) and ATPase copper transporting beta (ATP7B). These molecular pumps belong to P-type ATPases and dispose off the Platinum (Pt) based anticancer drugs from cancer cells, causing resistance in them. For the disposal of Pt-drugs, copper exporters require phosphorylation mediated by protein kinase D (PKD) for their activation and trafficking. Even though various research works are underway to overcome resistance to anticancer drugs, the role of PKD is mainly ignored. In this study, we have found a significant upregulation of ATP7A and ATP7B in cervical cancer cells (HeLa) and Liver Hepatocellular Carcinoma cells (HepG2) in the presence of Cisplatin or Carboplatin; both at transcriptional as well as translational levels. Interestingly, the expression of ATP7A and ATP7B were significantly downregulated in the presence of a PKD inhibitor (CID2011756), resulting in the reduction of PKD mediated phosphorylation of ATP7A/7B. This causes enhancement of proteasome-mediated degradation of ATP7A/7B and thereby sensitizes the cells towards Cisplatin and Carboplatin. Similarly, the treatment of Cisplatin resistant HepG2 cells with PKD inhibitor causes enhanced sensitivity towards Cisplatin drug. However, the presence of proteasome inhibitor (MG132) reversed the effect of the PKD inhibitor on the expression level of ATP7A/7B, indicating the necessity of phosphorylation for its stability. Hence, we conclude that the combinatorial usage of Cisplatin with drugs targeting PKD can be developed as an effective chemotherapeutic approach to overcome drug resistance.

Green Synthesis of Engineered CdS Nanoparticles with Reduced Cytotoxicity for Enhanced Bioimaging Application.

The modern epoch of semiconductor nanotechnology focuses on its application in biology, especially in medical sciences, to fetch direct benefits to human life. Fabrication of devices for biosensing and bioimaging is a vibrant research topic nowadays. Luminescent quantum dots are the best option to move with, but most of them are toxic to living organisms and hence cannot be utilized for biological applications. Recent publications demonstrate that surface treatment on the nanoparticles leads to enhanced luminescence properties with a drastic reduction in toxicity. The current work introduces surface-modified CdS, prepared via a simple green chemical route with different medicinal leaf extracts as the reaction media. Lower toxicity and multiple emissions in the visible region, observed for the CdS-O.tenuiflorum hybrid structures, make them a better option for future biological applications. Furthermore, the hybrid structure showed enhanced electrical properties, which promises its use in modifying the current optoelectronic devices.

Evaluation of diagnostic accuracy of developed rapid SARS-COV-2 IgG antibody test kit using novel diluent system.

Immunochromatographic assay kits are used in primary diagnostics which is based on the principle of antigen and antibody interaction. These kits play pivotal role in rapid surveillance of infectious diseases at early stages as well as for the surveillance of the contagious diseases. The immunochromatographic test kits lacks sensitivity and specificity with certain diseases. In this study, our intention was to develop a rapid test kit for SARS-COV-2 with a novel diluent system to enhance the efficacy of antigen-antibody binding and thereby the improvement in the sensitivity outlined. Finally, IgG antibodies against SARS-COV-2 virus peptides were analyzed using 25 positive and 25 negative confirmed clinical samples. The sensitivity of the clinical studies showed 91% sensitivity and 100% specificity. Therefore, the authors propose that this assay will be a potential tool for efficient community or sentinel surveillance of SARS-COV-2 infection and additionally, for effective monitoring of convalescent sera therapy.

Comparative features of copper ATPases ATP7A and ATP7B heterologously expressed in COS-1 cells.

ATP7A and ATP7B are P-type ATPases required for copper homeostasis and involved in the etiology of Menkes and Wilson diseases. We used heterologous expression of ATP7A or ATP7B in COS-1 cells infected with adenovirus vectors to characterize differential features pertinent to each protein expressed in the same mammalian cell type, rather than to extrinsic factors related to different cells sustaining expression. Electrophoretic analysis of the expressed protein, before and after purification, prior or subsequent to treatment with endoglycosidase, and evidenced by protein or glycoprotein staining as well as Western blotting, indicates that the ATP7A protein is glycosylated while ATP7B is not. This is consistent with the prevalence of glycosylation motifs in the ATP7A sequence, and not in ATP7B. ATP7A and ATP7B undergo copper-dependent phosphorylation by utilization of ATP, forming equal levels of an "alkali labile" phosphoenzyme intermediate that undergoes similar catalytic (P-type ATPase) turnover in both enzymes. In addition, incubation with ATP yields an "alkali stable" phosphoprotein fraction, attributed to phosphorylation of serines. Alkali stable phosphorylation occurs at lower levels in ATP7A, consistent with a different distribution of serines in the amino acid sequence. Immunostaining of COS-1 cells sustaining heterologous expression shows initial association of both ATP7A and ATP7B with Golgi and the trans-Golgi network. However, in the presence of added copper, ATP7A undergoes prevalent association with the plasma membrane while ATP7B exhibits intense trafficking with cytosolic vesicles. Glycosylation of ATP7A and phosphorylation of ATP7B apparently contribute to their different trafficking and membrane association when expressed in the same cell type.

The prevalence of antibodies to adenovirus serotype 5 in an adult Indian population and implications for adenovirus vector vaccines.

In vivo gene delivery using human adenovirus serotype 5 (AdV5) vectors is being explored for vaccination purposes. The presence of anti-AdV5 antibodies in human serum arising from natural exposure to AdV5 can interfere potentially with and compromise the efficacy of rAdV5-based vaccine vectors. In this report, a collection of 114 sera from healthy adult Indian blood donors was analyzed for the presence of anti-AdV5 antibodies, using an AdV5 vector encoding the green fluorescent protein (GFP) to monitor the presence of anti-AdV5 neutralizing antibodies in human sera based on their ability to block virus entry into HeLa cells which express the Coxsackievirus-and-Adenovirus Receptor (CAR). In this assay all samples tested were positive for anti-AdV5 antibodies, with titers varying over a very wide range. It was also observed that these antibodies facilitated the uptake of the reporter AdV5 vector into the monocytic cell line U937 which does not express CAR, but expresses Fc receptors (FcRs) instead. These observations have implications for rAdV5-based vaccine development. J. Med. Virol. 82:407-414, 2010. (c) 2010 Wiley-Liss, Inc.

Label Free, Nontoxic Cu-GSH NCs as a Nanoplatform for Cancer Cell Imaging and Subcellular pH Monitoring Modulated by a Specific Inhibitor: Bafilomycin A1.

Metal nanoparticles-based sensors invoked much research attention in the biomedical field, especially in applications involving live cell imaging and monitoring. Here, a simple cost-effective method is adopted to synthesize glutathione coated copper nanoclusters (Cu-GSH NCs) with strong bright red fluorescence (625 nm). The clusters were found to be containing five Cu(0) atoms complexed with one molecule of glutathione (GSH) as evidenced by MALDI-TOF MS analysis. The synthesized Cu-GSH NCs system responds linearly to the pH in the acidic and alkaline ranges with a high degree of in vitro pH reversibility, projecting its potential as a real time pH sensor. Higher intensity emission observed in acidic conditions can be exploited for its employability as cellular organelle markers. The imaging and sensing potential of Cu-GSH NCs in the live human adenocarcinoma cell line, the HeLa cells, was tested. The treatment of HeLa cells for 48 h imparted deep red fluorescence, owing to the lower level of intracellular pH in cancer cells. In contrast, the imaging using normal cell lines (L-132, lung epithelial cell line) showed significantly lower fluorescence intensity as compared to that of HeLa cells. The subcellular pH-dependent fluorescence emission of Cu-GSH NCs was further assessed by treating HeLa cells with proton pump (V-ATPase) inhibitor Bafilomycin A1, which increases the vesicular pH. Interestingly, the fluorescent intensity of HeLa cells decreases with increasing concentration of Bafilomycin A1 in the presence of Cu-GSH NCs, as evidenced by the fluorescence microscopic images and quantitative fluorescent output. Accordingly, the developed Cu-GSH NCs system can be employed as an efficient pH-based bioimaging probe for the detection of cancer cells with an implied potential for the label free subcellular organelle tracking and marking. Importantly, the Cu-GSH NCs can be used for live cell pH imaging owing to their high degree of reversibility in sensing of pH variation.

A slow, efficient and safe nanoplatform of tailored ZnS QD-mycophenolic acid conjugates for in vitro drug delivery against dengue virus 2 genome replication.

Dengue is a major health concern causing significant mortality, morbidity and economic loss. The development of anti-dengue viral drugs is challenging due to high toxicity, as well as off-target/side effects. We engineered size tuned ZnS QDs as a platform for the efficient delivery of mycophenolic acid (MPA) against dengue virus serotype 2 (DENV2) to evaluate the drug efficacy and toxicity using the DENV2 sub-genomic replicon system in BHK21 cells. The results indicate that the Selectivity Index 50 (SI50) of the ZnS QD-MPA conjugate was two orders higher than that of free MPA with lower cytotoxicity. The effect is attributed to the sustained release of MPA from ZnS QD-MPA. The conjugated MPA caused significant inhibition of the virus at the level of replication and viral protein translation. The study underpins the efficiency of the ZnS QD for the delivery of antiviral drugs against DENV2 with negligible toxicity and side effects.

Fabrication of a Greener TiO2@Gum Arabic-Carbon Paste Electrode for the Electrochemical Detection of Pb2+ Ions in Plastic Toys.

A novel greener methodology is reported for the synthesis of titanium dioxide (TiO2) nanoparticles (NPs) using gum Arabic (Acacia senegal) and the characterization of the ensuing TiO2 NPs by various techniques such as X-ray diffraction (XRD), Fourier transform infrared, Raman spectroscopy, scanning electron microscopy-energy dispersive X-ray, transmission electron microscopy (TEM), high resolution-TEM, and UV-visible spectroscopy. The XRD analysis confirmed the formation of TiO2 NPs in the anatase phase with high crystal purity, while TEM confirmed the size to be 8.9 ± 1.5 nm with a spherical morphology. The electrode for the electrochemical detection of Pb2+ ions was modified by a carbon paste fabricated using the synthesized TiO2 NPs. Compared to the bare electrode, the fabricated electrode exhibited improved electro-catalytic activity toward the reduction of Pb2+ ions. The detection limit, quantification limit, and the sensitivity of the developed electrode were observed by using differential pulse voltammetry to be 506 ppb, 1.68 ppm, and 0.52 ± 0.01 µA µM-1, respectively. The constructed electrode was tested for the detection of lead content in plastic toys.