Elimination of human rabies in Goa, India through an integrated One Health approach.

Dog-mediated rabies kills tens of thousands of people each year in India, representing one third of the estimated global rabies burden. Whilst the World Health Organization (WHO), World Organization for Animal Health (OIE) and the Food and Agriculture Organization of the United Nations (FAO) have set a target for global dog-mediated human rabies elimination by 2030, examples of large-scale dog vaccination programs demonstrating elimination remain limited in Africa and Asia. We describe the development of a data-driven rabies elimination program from 2013 to 2019 in Goa State, India, culminating in human rabies elimination and a 92% reduction in monthly canine rabies cases. Smartphone technology enabled systematic spatial direction of remote teams to vaccinate over 95,000 dogs at 70% vaccination coverage, and rabies education teams to reach 150,000 children annually. An estimated 2249 disability-adjusted life years (DALYs) were averted over the program period at 526 USD per DALY, making the intervention 'very cost-effective' by WHO definitions. This One Health program demonstrates that human rabies elimination is achievable at the state level in India.

Serological Evidence of Lyssavirus Infection among Bats in Nagaland, a North-Eastern State in India.

Bats are known to be reservoirs of several medically important viruses including lyssaviruses. However, no systematic surveillance for bat rabies has been carried out in India, a canine rabies endemic country with a high burden of human rabies. Surveillance for rabies virus (RABV) infection in bats was therefore carried out in Nagaland, a north-eastern state in India at sites with intense human-bat interfaces during traditional bat harvests. Brain tissues and sera from bats were tested for evidence of infection due to RABV. Brain tissues were subjected to the fluorescent antibody test for detection of viral antigen and real-time reverse transcriptase PCR for presence of viral RNA. Bat sera were tested for the presence of rabies neutralizing antibodies by the rapid fluorescent focus inhibition test. None of the bat brains tested (n = 164) were positive for viral antigen or viral RNA. However, rabies neutralizing antibodies were detected in 4/78 (5·1%) bat sera tested, suggesting prior exposure to RABV or related lyssaviruses. The serological evidence of lyssaviral infection in Indian bats may have important implications in disease transmission and rabies control measures, and warrant extensive bat surveillance to better define the prevalence of lyssaviral infection in bats.

The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma.

Rap1GAP is a critical tumor suppressor gene that is downregulated in multiple aggressive cancers, such as head and neck squamous cell carcinoma, melanoma and pancreatic cancer. However, the mechanistic basis of rap1GAP downregulation in cancers is poorly understood. By employing an integrative approach, we demonstrate polycomb-mediated repression of rap1GAP that involves Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase in head and neck cancers. We further demonstrate that the loss of miR-101 expression correlates with EZH2 upregulation, and the concomitant downregulation of rap1GAP in head and neck cancers. EZH2 represses rap1GAP by facilitating the trimethylation of histone 3 at lysine 27, a mark of gene repression, and also hypermethylation of rap1GAP promoter. These results provide a conceptual framework involving a microRNA-oncogene-tumor suppressor axis to understand head and neck cancer progression.

Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase.

Mammalian polynucleotide kinase (mPNK) is a critical DNA repair enzyme whose 5'-kinase and 3'-phoshatase activities function with poorly understood but striking specificity to restore 5'-phosphate/3'-hydroxyl termini at sites of DNA damage. Here we integrated site-directed mutagenesis and small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of mPNK. The flexible attachment of the FHA domain to the catalytic segment, elucidated by SAXS, enables the interactions of mPNK with diverse DNA substrates and protein partners required for effective orchestration of DNA end repair. Point mutations surrounding the kinase active site identified two substrate recognition surfaces positioned to contact distinct regions on either side of the phosphorylated 5'-hydroxyl. DNA substrates bind across the kinase active site cleft to position the double-stranded portion upstream of the 5'-hydroxyl on one side, and the 3'-overhang on the opposite side. The bipartite DNA-binding surface of the mPNK kinase domain explains its preference for recessed 5'-termini, structures that would be encountered in the course of DNA strand break repair.

Repression of E-cadherin by the polycomb group protein EZH2 in cancer.

Enhancer of zeste homolog 2 (EZH2) is a critical component of the polycomb-repressive complex 2 (PRC2), which is involved in gene silencing and histone H3 lysine 27 methylation. EZH2 has a master regulatory function in controlling such processes as stem cell differentiation, cell proliferation, early embryogenesis and X chromosome inactivation. Although benign epithelial cells express very low levels of EZH2, increased levels of EZH2 have been observed in aggressive solid tumors such as those of the prostate, breast and bladder. The mechanism by which EZH2 mediates tumor aggressiveness is unclear. Here, we demonstrate that EZH2 mediates transcriptional silencing of the tumor suppressor gene E-cadherin by trimethylation of H3 lysine 27. Histone deacetylase inhibitors can prevent EZH2-mediated repression of E-cadherin and attenuate cell invasion, suggesting a possible mechanism that may be useful for the development of therapeutic treatments. Taken together, these observations provide a novel mechanism of E-cadherin regulation and establish a functional link between dysregulation of EZH2 and repression of E-cadherin during cancer progression.

Fluorescence studies of substrate binding to human recombinant deoxycytidine kinase.

Deoxycytidine kinase (dCK), is responsible for the phosphorylation of deoxynucleosides to the corresponding monophosphates using ATP or UTP as phosphate donors. Steady-state intrinsic fluorescence measurements were used to study interaction of dCK with substrates in the absence and presence of phosphate donors. Enzyme fluorescence quenching by its substrates exhibited unimodal quenching when excited at 295 nm. Binding of substrates induced conformational changes in the protein, suggesting that dCK can assume different conformational states with different substrates and may account for the observed differences in their specificity. dCK bound the substrates more tightly in the presence of phosphate donors and UTP is the preferred phosphate donor. Among the substrates tested, the antitumour drugs gemcitabine and cladribine were bound very tightly by dCK, yielding Kd values of 0.75 and 0.8 microM, respectively, in the presence of UTP.

Physical properties of human polynucleotide kinase: hydrodynamic and spectroscopic studies.

Human polynucleotide kinase (hPNK) is a putative DNA repair enzyme in the base excision repair pathway required for processing and rejoining strand-break termini. This study represents the first systematic examination of the physical properties of this enzyme. The protein was produced in Escherichia coli as a His-tagged protein, and the purified recombinant protein exhibited both the kinase and the phosphatase activities. The predicted relative molecular mass (M(r)) of the 521 amino acid polypeptide encoded by the sequenced cDNA for PNK and the additional 21 amino acids of the His tag is 59,538. The M(r) determined by low-speed sedimentation equilibrium under nondenaturing conditions was 59,600 +/- 1000, indicating that the protein exists as a monomer, in contrast to T4 phage PNK, which exists as a homotetramer. The size and shape of hPNK in solution were determined by analytical ultracentrifugation studies. The protein was found to have an intrinsic sedimentation coefficient, s(0)(20,w), of 3.54 S and a Stokes radius, R(s), of 37.5 A. These hydrodynamic data, together with the M(r) of 59 600, suggest that hPNK is a moderately asymmetric protein with an axial ratio of 5.51. Analysis of the secondary structure of hPNK on the basis of circular dichroism spectra, which revealed the presence of two negative dichroic bands located at 218 and 209 nm, with ellipticity values of -7200 +/- 300 and -7800 +/- 300 deg x cm(2) x d(mol(-1), respectively, indicated the presence of approximately 50% beta-structure and 25% alpha-helix. Binding of ATP to the protein induced an increase in beta-structure and perturbed tryptophan, tyrosine, and phenylalanine signals observed by aromatic CD and UV difference spectroscopy.

Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae. Interaction of inhibitors of nucleoside transport with recombinant hENT1 and a glycosylation-defective derivative (hENT1/N48Q).

We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside {6-[(4-nitrobenzyl)thio]-9-beta-d-ribofuranosyl purine; NBMPR} to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means+/-S.D.) of [3H]NBMPR to intact yeast (Kd 1.2+/-0.2 nM; Bmax 5.0+/-0.5 pmol/mg of protein) and membranes (Kd 0.7+/-0.2 nM; Bmax 6.5+/-1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [3H]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130+/-10 and 380+/-20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [3H]NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The Kd for binding of NBMPR to hENT1/N48Q was 10. 5+/-1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR. The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound [3H]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14+/-0.02 and 0. 36+/-0.05 min-1 for koff, and (1.2+/-0.1)x10(8) and (0.40+/-0. 04)x10(8) M-1.min-1 for kon. These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.

Demonstration of equilibrative nucleoside transporters (hENT1 and hENT2) in nuclear envelopes of cultured human choriocarcinoma (BeWo) cells by functional reconstitution in proteoliposomes.

The equilibrative nucleoside transporters (ENTs) are a newly recognized family of membrane proteins of which hENT1 is the nitrobenzylmercaptopurine ribonucleoside (NBMPR)-sensitive (es) and hENT2 the NBMPR-insensitive (ei) transporter of human cells. BeWo cells exhibit large numbers (>10(7)/cell) of NBMPR-binding sites and high es and ei nucleoside transport activities relative to other cell types. In this work, we have demonstrated that proliferating BeWo cells possess (i) mRNA encoding hENT1 and hENT2 and (ii) hENT1-specific immunoepitopes. We examined NBMPR binding and its inhibition of uridine transport in various BeWo membrane fractions and proteoliposomes derived therefrom to determine if NBMPR binding to intracellular membranes represented interaction with functional es transporters. Unfractionated membranes and fractions enriched 5-fold in plasma membranes relative to postnuclear supernatants exhibited high NBMPR binding activity. Intact nuclei and nuclear envelopes also exhibited abundant quantities of NBMPR-binding sites with affinities similar to those of enriched plasma membranes (Kd = 0.4-0.9 nM). When proteoliposomes were made from octyl glucoside-solubilized membranes, high affinity NBMPR-binding sites were not only observed in crude membrane preparations and plasma membrane-enriched fractions but also in nuclear envelope fractions. Proteoliposomes prepared from either unfractionated membranes or nuclear envelopes exhibited both hENT1-mediated (82-85%) and hENT2-mediated (15-18%) transport of [3H]uridine. These results provided evidence for the presence of functional es and ei transporters in nuclear membranes and endoplasmic reticulum, suggesting that hENT1 and hENT2 may function in the translocation of nucleosides between the cytosol and the luminal compartments of one or both of these membrane types.

Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines.

Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucleoside drug with clinical efficacy in several common epithelial cancers. We have proposed that gemcitabine requires nucleoside transporter (NT) proteins to permeate the plasma membrane and to exhibit pharmacological activity. In humans, there are seven reported distinct NT activities varying in substrate specificity, sodium dependence, and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole. To determine which NTs are required for gemcitabine-dependent growth inhibition, cultures from a panel of 12 cell lines with defined plasma membrane NT activities were incubated with different concentrations of gemcitabine. Cell proliferation was assessed by the sulforhodamine B assay and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC50s). NT activity was a prerequisite for growth inhibition in vitro because: (a) the nucleoside transport-deficient cells were highly resistant to gemcitabine; and (b) treatment of cells that exhibited only equilibrative NT activity with NBMPR or dipyridamole increased resistance to gemcitabine by 39- to 1800-fold. These data suggested that the type of NT activities possessed by a cell may be an important determinant of its sensitivity to gemcitabine and that NT deficiency may confer significant gemcitabine resistance. We analyzed the uptake kinetics of [3H]gemcitabine by each of five human NT activities in cell lines that exhibited a single NT activity in isolation; transient transfection of the cDNAs encoding the human concentrative NT proteins (hCNT1 and hCNT2) was used to study the cit and cif activities, respectively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es approximately = cit > ei > cib >>> cif. The transportability of [3H]gemcitabine was demonstrated by reconstitution of the human es NT in proteoliposomes, confirming that gemcitabine permeation is a protein-mediated process.

Influence of caltropin on the caldesmon induced polymerization of G-actin.

The effect of caltropin (CaT) on the caldesmon (CaD)-G-actin interaction was monitored by viscosity measurements, bioassays measuring the release of inorganic phosphate (Pi) following G-actin polymerization and fluorescence studies using acrylodan labelled G-actin. CaD induced polymerization of G-actin into filaments in the absence of salt was accompanied by an increase in relative viscosity. This effect of CaD was essentially abolished by CaT in the presence of Ca2+. In bioassays the rate of Pi release was reduced significantly in the presence of Ca2+/CaT. Acrylodan labelled G-actin when excited at 375 nm exhibited an emission maximum at 478 nm. Polymerization of G-actin resulted in shifting the emission maximum to 465 nm. When CaD was added to G-actin containing Ca2+/CaT, the rate of G-actin polymerization was reduced considerably, suggesting that CaT interferes in the CaD-G-actin interaction.

Interaction between caltropin and the C-terminal region of smooth muscle caldesmon.

Caltropin (CaT) binds caldesmon (CaD) in a Ca(2+)-dependent manner with an affinity higher than that of calmodulin (CaM). Photo-crosslinking between CaT and a benzophenone-labeled C-terminal CaD fragment (27K) results in a 35-kDa protein that corresponds to the 1:1 adduct between CaT and 27K. In the absence of Ca2+, no crosslinking is obtained. This result is similar to that obtained with CaM and 27K. The apparent affinity of CaM for GS17C, a CaM-binding peptide of CaD, is weakened by CaT, suggesting CaT competes with CaM for the peptide. In contrast to CaM, CaT does not induce changes in the tryptophan fluorescence of GS17C. Thus although the two Ca(2+)-binding proteins behave similarly, there are differences in their interactions with CaD.

Effect of caltropin on caldesmon-actin interaction.

The binding of chicken gizzard caldesmon to actin was studied both in the presence and the absence of caltropin using Airfuge centrifugation experiments, disulfide cross-linking studies, and the fluorescent probe acrylodan (6-acryloyl-2-(dimethylamino)naphthalene). In co-sedimentation studies most of the caldesmon pelleted along with actin. However, when caldesmon in the presence of caltropin was mixed with actin, caldesmon did not pellet along with actin following high speed centrifugation, suggesting that caltropin has significantly weakened its binding to actin. The caltropin effect was noticed even when tropomyosin was included in the reaction mixture. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 515 +/- 2 nm. The addition of actin produced a nearly 70% increase in fluorescent intensity, accompanied by a blue shift in the emission maximum (i.e. lambda em (max) = 505 +/- 2 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of labeled caldesmon with actin indicated a strong affinity (K alpha = approximately 6 x 10(7) M-1). When actin was titrated with labeled caldesmon in the presence of caltropin in a 0.2 mM Ca2+ medium, its affinity for caldesmon was lowered (K alpha = approximately 2 x 10(7) M-1). Caltropin, which is very effective in reversing caldesmon's inhibition of the actin-activated myosin ATPase (Mani, R. S., McCubbin, W. D., and Kay, C. M. (1992) Biochemistry 31, 11896-11901), is shown in the present study to have a pronounced effect on its binding to actin, suggesting a major role for caltropin in regulating caldesmon in smooth muscle.

Calcium-dependent regulation of the caldesmon-heavy meromyosin interaction by caltropin.

The binding of chicken gizzard caldesmon to smooth muscle heavy meromyosin (HMM) was studied using caldesmon-Sepharose 4B affinity chromatography, far-ultraviolet circular dichroism (CD), and the fluorescent probe acrylodan. When HMM was applied to a caldesmon-Sepharose column in the presence of 40 mM NaCl, most of the protein was retained on the column, and HMM could be eluted by increasing the NaCl level to 0.5 M; this interaction was not Ca(2+)-dependent. Far-UV CD studies indicated an interaction between caldesmon and HMM since the experimentally observed ellipticity values at 222 and 207 nm deviated from the theoretical values for the complex, and this interaction was also not Ca(2+)-sensitive. Addition of HMM to a caldesmon-caltropin complex induced a conformational change suggesting the formation of a ternary complex for which Ca2+ was essential. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 515 +/- 2 nm. Addition of HMM resulted in a nearly 20% decrease in fluorescence intensity with little or no shift in the emission maximum. Titration of HMM with labeled caldesmon indicated a strong affinity for HMM [K(a) was on the order of (4.5 +/- 0.5) x 10(7) M-1], and this interaction was observed both in the presence and in the absence of calcium. When HMM was titrated with labeled caldesmon in the presence of caltropin in a 0.2 mM Ca2+ medium, its affinity for caldesmon was lowered nearly 3-fold [K(a) approximately (1.50 +/- 0.5) x 10(7) M-1].(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-dependent regulation of caldesmon by an 11-kDa smooth muscle calcium-binding protein, caltropin.

Caldesmon from chicken gizzard muscle has been examined for its ability to interact with caltropin using affinity chromatography and the fluorescent probe acrylodan. The action of caltropin on the inhibitory effect of caldesmon on actomyosin ATPase was also studied. Like calmodulin, caltropin could release the inhibitory effect of caldesmon in the presence of Ca2+. Complete reversal was obtained when 1 mol of caltropin was added per mol of caldesmon. When caldesmon was applied to caltropin-Sepharose in the presence of Ca2+, most of the caldesmon was bound to the column and could be eluted with EGTA, indicating that there is a direct interaction between caldesmon and caltropin. Acrylodan-labeled caldesmon, when excited at 375 nm, had an emission maximum at 504 nm. Addition of caltropin in the presence of Ca2+ resulted in a nearly 50% increase in fluorescence intensity, and this was accompanied by a blue shift in the emission maximum (i.e., lambda em,max 492 nm), suggesting that the probe now occupies a more nonpolar environment. Titration of caltropin with labeled caldesmon indicated a strong affinity for this protein (Kd was in the order of 8 x 10(-8)-2 x 10(-7) M). However, when caltropin was added to labeled caldesmon in the presence of EGTA, there was no indication of any interaction. Caltropin was at least as potent as calmodulin, if not better, in reversing the inhibitory effect of caldesmon in the presence of calcium, making it a potential Ca2+ factor in regulating caldesmon in smooth muscle.

Purification and characterization of a novel 12,000-Da calcium binding protein from smooth muscle.

A new low molecular weight calcium binding protein, designated 12-kDa CaBP, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous and has a molecular weight of 12,000 based on sodium dodecyl sulfate-gel electrophoresis. The amino acid composition of this protein is similar to but distinct from other known low molecular weight Ca2+ binding proteins. Ca2+ binding assays using Arsenazo III (Sigma) indicated that the protein binds 1 mol of Ca2+/mol of protein. The 12-kDa CaBP underwent a conformational change upon binding Ca2+, as revealed by uv difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. Addition of Ca2+ to the 12-kDa CaBP labeled with 2-p-toluidinylnaphthalene-6-sulfonate (TNS) resulted in a sevenfold increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 463 to 445 nm. Hence, the probe in the presence of Ca2+ moves to a more nonpolar microenvironment. Like calmodulin and other related Ca2+ binding proteins, this protein also exposes a hydrophobic site upon binding calcium. Fluorescence titration with Ca2+ using TNS-labeled protein revealed the presence of a single high affinity calcium binding site (kd approximately 1 x 10(-6) M).

Conformation and dynamics of bovine brain S-100a protein determined by fluorescence spectroscopy.

We have used time-resolved laser fluorescence spectroscopy to investigate the intensity and anisotropy decays of the single tryptophan residue in bovine brain S-100a (alpha beta) protein. The steady-state and acrylamide quenching results indicated that the Trp 90 of the alpha-subunit was partially buried in a relatively nonpolar environment at pH 7.5. Both Ca2+ and pH 8.5 slightly enhanced the exposure of the residue to the solvent, but the residue remained partially buried in the calcium complex at both pH values. The best representation of the intensity decays was a linear combination of three exponential terms, regardless of solvent condition and temperature. The three lifetimes (tau i) were in the range of 0.4-5 ns and insensitive to emission wavelength, but their fractional amplitudes (alpha i) shifted in favor of the shortest component (alpha 1) when the decays were measured at the blue end of the emission spectrum. These results suggest that an excited-state interaction between the indole ring and the side chain of an adjacent residue may be responsible for the observed shortest lifetime. In the presence of Ca2+, the three lifetimes remained relatively unaltered, but the values of alpha 1 decreased by a factor of 2.3 at pH 7.2 and a factor of 1.8 at pH 8.2. This Ca(2+)-induced decrease may be attributed to disruption of the putative excited-state interaction resulting from reorientations of the alpha-helical segments flanking a Ca(2+)-binding loop (residues 62-73). At both pH 7.2 and 8.4, the anisotropy decays of the apoprotein followed a biexponential decay law.(ABSTRACT TRUNCATED AT 250 WORDS)