IRF1 Inhibits Antitumor Immunity through the Upregulation of PD-L1 in the Tumor Cell.

Multiple studies have associated the transcription factor IRF1 with tumor-suppressive activities. Here, we report an opposite tumor cell-intrinsic function of IRF1 in promoting tumor growth. IRF1-deficient tumor cells showed reduced tumor growth in MC38 and CT26 colon carcinoma and B16 melanoma mouse models. This reduction in tumor growth was dependent on host CD8+ T cells. Detailed profiling of tumor-infiltrating leukocytes did not show changes in the various T-cell and myeloid cell populations. However, CD8+ T cells that had infiltrated IRF1-deficieint tumors in vivo exhibited enhanced cytotoxicity. IRF1-deficient tumor cells lost the ability to upregulate PD-L1 expression in vitro and in vivo and were more susceptible to T-cell-mediated killing. Induced expression of PD-L1 in IRF1-deficient tumor cells restored tumor growth. These results indicate differential activity of IRF1 in tumor escape.

Synthesis of Excitation Independent Highly Luminescent Graphene Quantum Dots through Perchloric Acid Oxidation.

We demonstrate a facile liquid phase exfoliation method by only using perchloric acid to synthesize graphene quantum dots (GQDs) having excitation independent strong emission with a quantum yield of about 14%. The proposed simplified synthesis strategy can help in overcoming the limitations of existing aqueous routes which produce GQDs with excitation dependent emission and of low quantum efficiency. Photoluminescence (PL) properties of GQDs have been studied in detail to understand the origin of emission. As-synthesized GQDs show excitation independent photoluminesce (PL) which suggests that the synthesized materials do not have any significant defects. Spectral analysis suggests that the PL emission of the well-defined GQDs originates mainly from the peripheral functional groups conjugated with carbon backbone planes. We also demonstrate a relatively longer PL lifetime (average lifetime of about 10 ns) of the synthesized GQDs determined by time correlated single photon counting (TCSPC) measurement and this high lifetime suggests that the synthesized GQDs may be suitable for biomacromolecular probing. In addition, as-synthesized GQDs interestingly show delayed fluorescence and steady state anisotropy, which make the material an appropriate candidate for application in sensing and bioimaging of cells and organisms.

Exploiting the biomimetic and luminescence properties of multivalent dendrimer-semiconductor nanohybrid materials in the ultra-low level determination of folic acid.

In view of the enhanced generation of folate receptors in cancerous cells and diseases linked to the deficiency of folic acid, such as anemia, mental devolution, congenital malformation, etc., the development of a simple method for the ultra-sensitive determination of folic acid remains a long-standing issue for practical applications in medicine and biotechnology. Thus, the proposed luminescence based strategy involving multifunctional poly(amidoamine) (PAMAM) dendrimer encapsulated quantum dots (QDs) as a probe provides a simple, fast and efficient method for the selective determination of folic acid at the nano-molar level. Absorption and Fourier transform infra-red (FTIR) spectroscopy provide evidence of the binding of folic acid with dendrimer amine groups. The emission quenching of dendrimer encapsulated CdS QDs follows a linear Stern-Volmer plot with an exceedingly high value of the Stern-Volmer constant (KSV = 8.4 × 106 M-1) facilitating a higher detection efficiency. Similar quenching analysis with dendrimer-ZnS QDs showed a slightly lower Stern-Volmer constant (KSV = 2.29 × 106 M-1). The lower probing efficiency of the protein or amino acid capping of QDs has been explained through zeta potential measurements. The solvent polarity dependence suggests a charge transfer process responsible for the emission quenching of CdS QDs, which is static in nature as revealed by lifetime measurements. The determination of folic acid at this low level is not affected by possible interfering molecules, such as vitamin C, vitamin B12 and uric acid. Calorimetric measurements showed that the exothermic binding of folic acid with a dendrimer follows enthalpy-entropy compensation. The detailed mechanistic aspect of interactions of folic acid with the QD probe helps in a better understanding of the detection process, which in turn can assist in developing a dendrimer based material for image analysis and drug delivery in folate receptor rich cells.

Aqueous Synthesis of Protein-Encapsulated ZnSe Quantum Dots and Physical Significance of Semiconductor-Induced CuII Ion Sensing.

In view of their promising bio-applicability, we have synthesized water-soluble bovine serum albumin (BSA)-encapsulated ZnSe quantum dots (QDs) with visible emission with longer average luminescence lifetimes of approximately 125 ns at ambient conditions. BSA-ZnSe QDs are shown to be efficient selective copper ion probes in the presence of physiologically important metal ions through luminescence quenching with a high Stern-Volmer constant (3.3×105 m-1 ). The mechanism of sensing has been explained in terms of electron transfer processes and the apparent rate of electron transfer (Ket ) from ZnSe QDs to Cu2+ has been calculated to be 2.8×108  s-1 . It is demonstrated that the negative conduction band potential plays a major role in the feasibility of the electron transfer process, which is reflected in the higher efficacy of ZnSe QDs in sensing copper(II) ions over other group II-VI quantum dots, namely, CdSe, ZnS, or CdS. The results observed with cysteine-capped QDs are almost identical to those with BSA-encapsulated QDs and this presumably negates the possible reason of CuII ion induced quenching ascribed to its binding with surface groups or replacement of metal sites as proposed by several groups previously.

A comparative evaluation of the activity modulation of flavo and non-flavo enzymes induced by graphene oxide.

Graphene, and its water soluble derivative graphene oxide, has shown great promise in various biomedical applications, such as cancer therapeutics, drug delivery, etc. and in industrial applications such as enzyme immobilization, etc. Thus, modulation of the activities of different classes of enzymes by graphene materials is an important aspect in the formulation of different biological applications. We have demonstrated here how flavin adenine dinucleotide (FAD) moieties protect the binding site from conformational change in the presence of an inhibitor, graphene oxide, and also explore differences in the mode of interactions between flavo and non-flavo enzymes. It was shown that there was a much greater loss of activity with the non-flavo enzyme, l-lactate dehydrogenase (LDH), of ∼74% compared to that with the flavo-enzyme, glucose oxidase (GOX), of ∼45%, in the presence of equal concentrations of GO. Furthermore, GO acts as an enzyme inhibitor and the mode of inhibition is uncompetitive for GOX and competitive for LDH. Circular dichromism measurements showed a 21% decrease in the α helix of GOX and a 31% decrease in the α helix of LDH in the presence of a given concentration of GO (0.5 mg mL-1). There was a slight change in the average emission lifetime of tryptophan in GOX in the presence of GO from 3.2 to 2.6 ns. In contrast, there was no change in the average emission lifetime of tryptophan in LDH in the presence of GO. The extents of fluorescence quenching for GOX and LDH were 39% and 70% upon addition of a certain amount of GO. The present study provides insight into the development of sensors through the immobilization of enzymes and the possible formulation of a multifunctional protein and graphene composite system for various biomedical applications such as bio-sensing, gene and drug delivery, etc.

SERS Enhancement on the Basis of Temperature-Dependent Chemisorption: Microcalorimetric Evidence.

The mechanism of surface-enhanced Raman spectroscopy (SERS) is not very clear in view of the magnitude of the contribution of electromagnetic factor as well as the chemical mechanism. This report presents the extent of adsorption at different temperatures in terms of signal enhancements in SERS employing silver nanoparticles (AgNPs) of various shapes as substrate and dye molecules, crystal violet or Rhodamine 6G, as model Raman probes. Initially, the SERS signal increases with increasing temperature until a maximum intensity is reached, before it gradually decreases with increasing temperature. This trend is independent of the shape of the Raman substrates and probes. However, the temperature at which maximum intensity is obtained may depend upon the nature of the Raman probe. The energetics involved in the chemisorption process between dye molecules and AgNPs were determined through isothermal titration calorimetry and their implications for the observed SERS signals were assessed. The maximum heat change occurred at the temperature at which the maximum signal enhancement in SERS was obtained and the enhanced interaction at optimum temperature was confirmed by absorption spectroscopy.

Synthesis and spectral measurements of sulphonated graphene: some anomalous observations.

The present report demonstrates how a sulphonation process, a key route for synthesizing water soluble graphene, can influence the optical behavior of precursor graphene oxide, intermediate reaction products and sulphonated graphene. We observed that there is constant emission maximum at 500 nm for graphene oxide in the excitation range of 320-450 nm. During sulphonation, sulphonated reduced graphene oxide (rGO-SO3H) is initially formed which has an emission at 358 nm. However, the reduction of oxygen containing groups in rGO-SO3H with hydrazine hydrate leading to the formation of SG caused a shift in the emission to 430 nm, which has been attributed to the extended delocalization of π-electrons involving the phenyl sulphonate group. In the present investigation, we have identified many existing anomalies in the important spectral features of these materials, such as violation of Kasha's rule on fluorescence and pH dependence emission. Furthermore, it has also been shown that proper care is necessary to be taken in monitoring the fluorescence of sulphonated graphene in view of possible interference from the components produced during sulphonation.

Reductant Control on Particle Size, Size Distribution and Morphology in the Process of Surface Enhanced Raman Spectroscopy Active Silver Colloid Synthesis.

The present study demonstrates how reducing agents play an important role in synthesis of silver nanoparticles (AgNPs) in colloidal phase. It is apparent from the observed results that borohydride, one of the most widely used reductants, induces reduction leading to the formation of spherical particles with narrowest size distribution. In contrast, ascorbic or citrate mediated reduction leads to formation of anisotropic silver nanoparticles, indicating the role of anionic carboxylate in template driving process. In view of recent green chemistry approach for synthesizing silver nanoparticles involving glucose as reductant and starch as capping groups, we have followed in detail the dependence of glucose-induced reduction process on different synthesis parameters, such as concentration, temperature and time of reactions. The phase of the synthesized particles was found to be face centred cubic (fcc), which was independent of the reductants employed. Further, we have endeavored to look into the Surface enhanced Raman spectroscopy (SERS) of crystal violet and rhodamine 6G in the presence of AgNPs substrate synthesized by using the reducing agents in question without involving any other structural modulating additive, such as ionic salt, etc. Here, the observed results provide a guideline on the selection of reducing agents and appropriate conditions for application specific synthesis of silver nanoparticles.

Evolutionary Dynamics of Tat in HIV-1 Subtypes B and C.

Evolutionary characteristics of HIV-1 have mostly studied focusing its structural genes, Gag, Pol and Env. However, regarding the process of HIV-1's evolution, few studies emphasize on genetic changes in regulatory proteins. Here we investigate the evolutionary dynamics of HIV-1, targeting one of its important regulatory proteins, Tat. We performed a phylogenetic analysis and employed a Bayesian coalescent-based approach using the BEAST package to investigate the evolutionary changes in Tat over time in the process of HIV-1 evolution. HIV-1 sequences of subtypes B and C from different parts of the world were obtained from the Los Alamos database. The mean estimated nucleotide substitution rates for Tat in HIV-1 subtypes B and C were 1.53 x 10(-3) (95% highest probability density- HPD Interval: 1.09 x10-3 to 2.08 x 10(-3)) and 2.14 x 10(-3) (95% HPD Interval: 1.35 x 10(-3) to 2.91 x 10(-3)) per site per year, respectively, which is relatively low compared to structural proteins. The median times of the most recent common ancestors (tMRCA) were estimated to be around 1933 (95% HPD, 1907-1952) and 1956 (95% HPD, 1934-1970) for subtypes B and C, respectively. Our analysis shows that subtype C appeared in the global population two decades after the introduction of subtype B. A Gaussian Markov random field (GMRF) skyride coalescent analysis demonstrates that the early expansion rate of subtype B was quite high, rapidly progressing during the 1960s and 1970s to the early 1990s, after which the rate increased up to the 2010s. In contrast, HIV-1 subtype C exhibited a relatively slow occurrence rate until the late 1980s when there was a sharp increase up to the end of 1990s; thereafter, the rate of occurrence gradually slowed. Our study highlights the importance of examining the internal/regulatory genes of HIV-1 to understand its complete evolutionary dynamics. The study results will therefore contribute to better understanding of HIV-1 evolution.

Molecular characterization of full-length Tat in HIV-1 subtypes B and C.

HIV-1Tat (trans-acting activator of transcription) plays essential roles in the replication through viral mRNA and genome transcription from the HIV-1 LTR promoter. However, Tat undergoes continuous amino acid substitutions. As a consequence, the virus escapes from host immunity indicating that genetic diversity of Tat protein in major HIV-1 subtypes is required to be continuously monitored. We analyzed available full-length HIV-1 sequences of subtypes B (n=493) and C (n=280) strains circulating worldwide. We observed 81% and 84% nucleotide sequence identities of HIV-1 Tat for subtypes B and C, respectively. Based on phylogenetic and mutation analyses, global diversity of subtype B was apparently higher compared to that of subtype C. Positively selected sites, such as positions Ser68 and Ser70 in both subtypes, were located in the Tat-transactivation responsive RNA (TAR) interaction domain. We also found positively selected sites in exon 2, such as positions Ser75, Pro77, Asp80, Pro81 and Ser87 for both subtypes. Our study provides useful information on the full-length HIV-1 Tat sequences in globally circulating strains.

Intersubtype Genetic Variation of HIV-1 Tat Exon 1.

HIV-1 Tat is a regulatory protein that plays a pivotal role in viral transcription and replication. Our study aims to investigate the genetic variation of Tat exon 1 in all subtypes of HIV-1: A, B, C, D, F, G, H, J, and K. We performed phylogenetic, mutation, and selection pressure analyses on a total of 1,179 sequences of different subtypes of HIV-1 Tat obtained from the Los Alamos National Laboratory (LANL). The mean nucleotide divergences (%) among the analyzed sequences of subtypes A, B, C, D, F, G, H, J, and K were 88, 89, 90, 88, 86, 89, 88, 97, and 97, respectively. We revealed that subtype B evolved relatively faster than other subtypes. The second and fifth domains were found comparatively more variable among all subtypes. Site-by-site tests of positive selection revealed that several positions in all subtypes were under significant positive selection. Positively selected sites were found in the acidic domain at positions 3, 4, and 19, in the cysteine-rich domains at positions 24, 29, 32, and 36, in the core domain at position 40, and in the basic domain for the rest of the positions for all subtypes. Positions 58 and 68 in the basic domain were positively selected in subtypes A, B, C and B, C, F, respectively. We also observed high variability within positively selected sites in amino acid positions. Our study findings on HIV-1 Tat genetic variability may contribute to a better understanding of HIV-1 evolution as well as to the development of effective Tat-targeted therapeutics and vaccines.

Detection of enterovirus 68 in serum from pediatric patients with pneumonia and their clinical outcomes.

Enterovirus 68 (EV68) infection occasionally manifests with fatal outcomes. However, detection of EV68 in serum and its clinical outcomes are yet to be determined. In this study, we retrospectively tested stored serum samples collected from pediatric pneumonia patients whose nasopharyngeal specimens were positive for EV68. Of total 28 nasopharyngeal sample-positive patients, EV68 was detected in serum samples among 12 (43%) patients aged between 1 and 4 years. Our results suggest that EV68 can cause viremia by which the virus may exhibit systemic manifestations.

Antigenic and receptor binding properties of enterovirus 68.

UNLABELLED: Increased detection of enterovirus 68 (EV68) among patients with acute respiratory infections has been reported from different parts of the world in the late 2000s since its first detection in pediatric patients with lower-respiratory-tract infections in 1962. However, the underlying molecular mechanisms for this trend are still unknown. We therefore aimed to study the antigenicity and receptor binding properties of EV68 detected in recent years in comparison to the prototype strain of EV68, the Fermon strain. We first performed neutralization (NT) and hemagglutination inhibition (HI) tests using antisera generated for EV68 strains detected in recent years. We found that the Fermon strain had lower HI and NT titers than recently detected EV68 strains. The HI and NT titers were also significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis. In glycan array analysis, all tested EV68 strains showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study demonstrates that emergence of strains with different antigenicity is the possible reason for the increased detection of EV68 in recent years. Additionally, we found that EV68 preferably binds to α2-6 SAs, which suggests that EV68 might have affinity for the upper respiratory tract. IMPORTANCE: Numbers of cases of enterovirus 68 (EV68) infection in different parts of the world increased significantly in the late 2000s. We studied the antigenicity and receptor binding properties of recently detected EV68 strains in comparison to the prototype strain of EV68, Fermon. The hemagglutination inhibition (HI) and neutralization (NT) titers were significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis, which showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study suggested that the emergence of strains with different antigenicities was the possible reason for the increased detections of EV68 in recent years. Additionally, we revealed that EV68 preferably binds to α2-6 SAs. This is the first report describing the properties of EV68 receptor binding to the specific types of sialic acids.

Evolution of biofunctional semiconductor nanocrystals: a calorimetric investigation.

Semiconductor nanomaterials have found numerous applications in optoelectronic device fabrication and in platforms for drug delivery and hyperthermia cancer treatment, and in various other biomedical fields because of their high photochemical stability and size-tunable photoluminescence (PL). However, little attention has been paid to exploring the energetics of formation of these semiconductor nanoparticles. We demonstrate that formation of nanocrystals with biofunctionalization supported by widely used groups, BSA and cysteine, is an exothermic spontaneous process driven by enthalpy. The whole energetics of the reaction shows that formation of smaller particles is favored with lower synthesis temperature. Further, it is shown that the thermodynamics of nanoparticle formation is strongly influenced by the conformation of the protein matrix. We also demonstrate that protein supported formation of nanocrystals is thermodynamically more favorable compared to that involving smaller organic thiol groups. The favorable enthalpy of formation compensates unfavorable entropy, resulting in favorable Gibbs free energy. Thus, this study can open up new avenues for establishing a thermodynamic basis for the design of nanosystems with new and tunable properties.

Molecular evolution of enterovirus 68 detected in the Philippines.

BACKGROUND: Detection of Enterovirus 68 (EV68) has recently been increased. However, underlying evolutionary mechanism of this increasing trend is not fully understood. METHODS: Nasopharyngeal swabs were collected from 5,240 patients with acute respiratory infections in the Philippines from June 2009 to December 2011. EV68 was detected by polymerase chain reaction (PCR) targeting for 5' untranslated region (5'UTR), viral protein 1 (VP1), and VP4/VP2. Phylogenetic trees were generated using the obtained sequences. RESULTS: Of the 5,240 tested samples, 12 EV68 positive cases were detected between August and December in 2011 (detection rate, 0.23%). The detection rate was higher among inpatients than outpatients (p<0.0001). Among VP1 sequences detected from 7 patients in 2011, 5 in lineage 2 were diverged from those detected in the Philippines in 2008, however, 2 in lineage 3 were not diverged from strains detected in the Philippines in 2008 but closely associated with strains detected in the United States. Combined with our previous report, EV68 occurrences were observed twice in the Philippines within the last four years. CONCLUSIONS: EV68 detections might be occurring in cyclic patterns, and viruses might have been maintained in the community while some strains might have been newly introduced.

Single quantum dot tracking reveals that an individual multivalent HIV-1 Tat protein transduction domain can activate machinery for lateral transport and endocytosis.

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

Genetic characterization of human respiratory syncytial virus detected in hospitalized children in the Philippines from 2008 to 2012.

BACKGROUND: Human respiratory syncytial virus (HRSV) is the leading cause of acute lower respiratory tract infection in infants and young children. However, molecular characteristic of HRSV is still unknown in the Philippines. OBJECTIVE: To describe the molecular epidemiology of circulating HRSV detected in the Philippines. STUDY DESIGN: From May 2008 to April 2012, nasopharyngeal swabs were collected from infants and children aged between 7 days and 14 years who were hospitalized with severe pneumonia. HRSV was detected by nested PCR targeting M2 gene, and C-terminus of the G gene was sequenced for phylogenetic analysis. RESULT: Out of total 2150 samples, 19.3% (n = 415) were positive for HRSV, and 65.0% of them (n = 270) were identified as HRSV-A and 35.0% (n = 145) as HRSV-B. There were two major HRSV outbreaks: between June 2008 and February 2009, and between June and March 2012. Majority of HRSV strains detected during the former outbreak were HRSV-A (97.5%, 203/208) whereas during the later outbreak, both HRSV-A (54/158, 34.2%) and HRSV-B (104/158, 65.8%) were detected. All HRSV-A strains were classified as genotype NA1 and all HRSV-B as genotype BA, which had 60-nucleotide duplication in secondary hypervariable region of the G gene. Among HRSV-B positive samples, there were 2 distinct clusters with unique amino acid changes and low homology in compared to other strains in BA, suggesting emergence of new variant of HRSV-B. CONCLUSION: The study provides an overview of the genetic variation in circulating HRSV viruses in the Philippines along with identification of possibly a novel variant of HRSV-B.

Single particle tracking confirms that multivalent Tat protein transduction domain-induced heparan sulfate proteoglycan cross-linkage activates Rac1 for internalization.

The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulfate (HS) chains of HS proteoglycans (HSPGs) for TatP binding and intracellular transport was demonstrated by the enzymatic removal of HS and simultaneous observation of two individual particles. Multivalent TatP induces HSPG cross-linking, recruiting activated Rac1 to adjacent lipid rafts and thereby enhancing the recruitment of TatP/HSPG to actin-associated microdomains and its internalization by macropinocytosis. These findings clarify the initial binding mechanism of TatP to the cell surface and demonstrate the importance of TatP valence for strong surface binding and signal transduction. Our data also shed light on the ability of TatP to exploit the machinery of living cells, using HSPG signaling to activate Rac1 and alter TatP mobility and internalization. This work should guide the future design of TatP-based peptides as therapeutic nanocarriers with efficient transduction.