Co-immunization with hemagglutinin stem immunogens elicits cross-group neutralizing antibodies and broad protection against influenza A viruses.

Current influenza vaccines predominantly induce immunity to the hypervariable hemagglutinin (HA) head, requiring frequent vaccine reformulation. Conversely, the immunosubdominant yet conserved HA stem harbors a supersite that is targeted by broadly neutralizing antibodies (bnAbs), representing a prime target for universal vaccines. Here, we showed that the co-immunization of two HA stem immunogens derived from group 1 and 2 influenza A viruses elicits cross-group protective immunity and neutralizing antibody responses in mice, ferrets, and nonhuman primates (NHPs). Immunized mice were protected from multiple group 1 and 2 viruses, and all animal models showed broad serum-neutralizing activity. A bnAb isolated from an immunized NHP broadly neutralized and protected against diverse viruses, including H5N1 and H7N9. Genetic and structural analyses revealed strong homology between macaque and human bnAbs, illustrating common biophysical constraints for acquiring cross-group specificity. Vaccine elicitation of stem-directed cross-group-protective immunity represents a step toward the development of broadly protective influenza vaccines.

Infants Infected with Respiratory Syncytial Virus Generate Potent Neutralizing Antibodies that Lack Somatic Hypermutation.

Respiratory syncytial virus (RSV) is a leading cause of infant mortality, and there are currently no licensed vaccines to protect this vulnerable population. A comprehensive understanding of infant antibody responses to natural RSV infection would facilitate vaccine development. Here, we isolated more than 450 RSV fusion glycoprotein (F)-specific antibodies from 7 RSV-infected infants and found that half of the antibodies recognized only two antigenic sites. Antibodies targeting both sites showed convergent sequence features, and structural studies revealed the molecular basis for their recognition of RSV F. A subset of antibodies targeting one of these sites displayed potent neutralizing activity despite lacking somatic mutations, and similar antibodies were detected in RSV-naive B cell repertoires, suggesting that expansion of these B cells in infants may be possible with suitably designed vaccine antigens. Collectively, our results provide fundamental insights into infant antibody responses and a framework for the rational design of age-specific RSV vaccines.

Quadrivalent influenza nanoparticle vaccines induce broad protection.

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines3.

Isolation of lectin from Musa acuminata for its antibiofilm potential against Methicillin-resistant Staphylococcus aureus and its synergistic effect with Enterococcus species.

Methicillin resistant Staphylococcus aureus (MRSA) is an emerging pathogen posing a considerable burden on the healthcare system due to its involvement in skin and soft tissue infections (SSTIs). Lectins are carbohydrate binding proteins found ubiquitously in animals, plants and microorganisms. Extraction and isolation of proteins from Musa acuminata were performed by using Affinity chromatography with Sephadex G 75 to determine antibiofilm activity against MRSA. Enterococcus strains obtained from dairy products, beans and vegetables were also screened for its potential to inhibit growth and biofilm formation of MRSA by using 96 well microtiter plates. Synergistic effect of cell free supernatant of Enterococcus with proteins from ripe banana were also tested. BanLec was successfully isolated and appeared as 15 KDa band after SDS-PAGE (15%) while multiple bands of unbound protein fractions were observed. The unbound fractions showed inhibition of planktonic cells and biofilm but BanLec exhibited no significant effect. The CFS of Enterococcus faecium (LCM002), Enterococcus lactis (LCM003) and Enterococcus durans (LCM004 and LCM005) displayed antagonistic effects against pathogen. The synergistic effect of CFS from E. lactis (LCM003) and unbound proteins showed inhibition of biofilm and pathogenic growth. This study demonstrates the use of Enterococcus species and plant proteins against pathogens and results suggested that it can inhibit the growth of resistant strains of Staphylococcus aureus and their synergistic effect has opened new ways to tackle emerging resistance. Furthermore, after assessment of Enterococcus as probiotics, this could be used in food industries as well as in treatment of severe skin infections.

Attempting Well-Tempered Funnel Metadynamics Simulations for the Evaluation of the Binding Kinetics of Methionine Aminopeptidase-II Inhibitors.

Understanding the unbinding kinetics of protein-ligand complexes is considered a significant approach for the design of ligands with desired specificity and safety. In recent years, enhanced sampling methods have emerged as effective tools for studying the unbinding kinetics of protein-ligand complexes at the atomistic level. MetAP-II is a target for the treatment of cancer for which not a single effective drug is available yet. The identification of the dissociation rate of ligands from the complexes often serves as a better predictor for in vivo efficacy than the ligands' binding affinity. Here, funnel-based restraint well-tempered metadynamics simulations were applied to predict the residence time of two ligands bound to MetAP-II, along with the ligand association and dissociation mechanism involving the identification of the binding hotspot during ligand egress. The ligand-egressing route revealed by metadynamics simulations also correlated with the identified pathways from the CAVER analysis and by the enhanced sampling simulation using PLUMED. Ligand 1 formed a strong H-bond interaction with GLU364 estimating a higher residence time of 28.22 ± 5.29 ns in contrast to ligand 2 with a residence time of 19.05 ± 3.58 ns, which easily dissociated from the binding pocket of MetAP-II. The results obtained from the simulations were consistent to reveal ligand 1 being superior to ligand 2; however, the experimental data related to residence time were close for both ligands, and no kinetic data were available for ligand 2. The current study could be considered the first attempt to apply an enhanced sampling method for the evaluation of the binding kinetics and thermodynamics of two different classes of ligands to a binuclear metalloprotein.

Dynamic Profiling of ß-Coronavirus 3CL Mpro Protease Ligand-Binding Sites.

ß-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensable role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV Mpro. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across ß-CoV homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.

Iron coordination to pyochelin siderophore influences dynamics of FptA receptor from Pseudomonas aeruginosa: a molecular dynamics simulation study.

FptA is a TonB-dependent transporter that permits the high affinity binding and transport of Fe(III)-pyochelin complex across the outer membrane of Pseudomonas aeruginosa. Molecular dynamics simulations were employed to FptA receptor and its complexes with pyochelin, and co-crystallized Fe(III)-pyochelin-ethanediol and Fe(III)-pyochelin-water embedded in dilauroyl phosphatidyl choline bilayer for the evaluation of their structural and dynamical properties. The evaluation of properties of the receptor bound to pyochelin molecule and Fe(III)-pyochelin complexes helped to figure out the iron coordination effect on the receptor properties. Moreover, comparison of these four simulation systems revealed further information on the dynamical changes occurred in extracellular loops, in particular loop-7 corresponding to the missing amino acid residues including the close-by loop-8 that was largely affected by the metal coordination to pyochelin. The binding of iron to pyochelin molecule affected the overall structure of the receptor therefore, evaluation fo the gyration radii and hydrogen bonding were evaluated as well as analysis of the pore size were also carried out to understand the effect of metal coordination on the dynamics of the helices which form a kind of translocation channel to transport the siderophore across the FptA protein into the periplasmic space. The properties of each component of the molecular systems were therefore observed to be perturbed by the incorporation of iron to the pyochelin molecule thus demonstrating that the bacteria use its receptor to abstract and transport iron from extracellular environment for its survival and that was made possible to understand at the molecular level through successful implementation of molecular dynamics simulations.

Solvation of cholesterol in different solvents: a molecular dynamics simulation study.

To the best of our knowledge, molecular dynamics simulations of an isolated cholesterol immersed in four different solvents of varying polarity, such as water, methanol, dimethyl sulfoxide and benzene, were reported for the first time to gain insights into the structural and dynamical properties. The study was mainly focused on the evaluation of solvation of cholesterol with respect to its hydrophilic and hydrophobic structural components in the form of respective functional groups interacting with the solvents. Structural evaluations suggested that both hydrophilic and hydrophobic groups of cholesterol were interacting with the solvents, in particular methanol and dimethyl sulfoxide, which presented both types of interactions that are polar and non-polar. On the other hand, the highly polar water and non-polar benzene demonstrated extreme solvation behavior, since water was involved only in hydrogen bonding to the solute hydroxyl group and non-polar benzene formed strong van der Waals interactions only. Furthermore, the hydrophobic effect of cholesterol was also analyzed mainly in polar solvents, as the effect was more pronounced in the polar environment thereby preventing the solvent mobility in the solvation layer(s). The dynamical properties in terms of lateral diffusion and hydrogen bond dynamics as well as free energies of solvation also corroborated the findings based on the structural data and the hydrophobic character of cholesterol was later quantified by the computation of the averaged solvent accessible surface area. The polarity effect of the solvents on the aggregation property of cholesterol was further investigated, which is of big concern from the clinical point of view due to its major role in cardiovascular ailments. It was another major finding of the present study that aggregation was shown to be facilitated by highly polar solvents like water.

Causes and incidence of community-acquired serious infections among young children in south Asia (ANISA): an observational cohort study.

BACKGROUND: More than 500 000 neonatal deaths per year result from possible serious bacterial infections (pSBIs), but the causes are largely unknown. We investigated the incidence of community-acquired infections caused by specific organisms among neonates in south Asia. METHODS: From 2011 to 2014, we identified babies through population-based pregnancy surveillance at five sites in Bangladesh, India, and Pakistan. Babies were visited at home by community health workers up to ten times from age 0 to 59 days. Illness meeting the WHO definition of pSBI and randomly selected healthy babies were referred to study physicians. The primary objective was to estimate proportions of specific infectious causes by blood culture and Custom TaqMan Array Cards molecular assay (Thermo Fisher, Bartlesville, OK, USA) of blood and respiratory samples. FINDINGS: 6022 pSBI episodes were identified among 63 114 babies (95·4 per 1000 livebirths). Causes were attributed in 28% of episodes (16% bacterial and 12% viral). Mean incidence of bacterial infections was 13·2 (95% credible interval [CrI] 11·2-15·6) per 1000 livebirths and of viral infections was 10·1 (9·4-11·6) per 1000 livebirths. The leading pathogen was respiratory syncytial virus (5·4, 95% CrI 4·8-6·3 episodes per 1000 livebirths), followed by Ureaplasma spp (2·4, 1·6-3·2 episodes per 1000 livebirths). Among babies who died, causes were attributed to 46% of pSBI episodes, among which 92% were bacterial. 85 (83%) of 102 blood culture isolates were susceptible to penicillin, ampicillin, gentamicin, or a combination of these drugs. INTERPRETATION: Non-attribution of a cause in a high proportion of patients suggests that a substantial proportion of pSBI episodes might not have been due to infection. The predominance of bacterial causes among babies who died, however, indicates that appropriate prevention measures and management could substantially affect neonatal mortality. Susceptibility of bacterial isolates to first-line antibiotics emphasises the need for prudent and limited use of newer-generation antibiotics. Furthermore, the predominance of atypical bacteria we found and high incidence of respiratory syncytial virus indicated that changes in management strategies for treatment and prevention are needed. Given the burden of disease, prevention of respiratory syncytial virus would have a notable effect on the overall health system and achievement of Sustainable Development Goal. FUNDING: Bill & Melinda Gates Foundation.

Highly selective enrichment of phosphopeptides using poly(dibenzo-18-crown-6) as a solid-phase extraction material.

A poly(dibenzo-18-crown-6) was used as a new solid-phase extraction material for the selective enrichment of phosphopeptides. Isolation of phosphopeptides was achieved based on specific ionic interactions between poly(dibenzo-18-crown-6) and the phosphate group of phosphopeptides. Thus, a method was developed and optimized, including loading, washing and elution steps, for the selective enrichment of phosphopeptides. To assess this potential, tryptic digest of three proteins (α- casein, ß-casein and ovalbumin) was applied on poly(dibenzo-18-crown-6). The nonspecific products were removed by centrifugation and washing. The spectrometric analysis was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Highly selective enrichment of both mono- and multiphosphorylated peptides was achieved using poly(dibenzo-18-crown-6) as solid-phase extraction material with minimum interference from nonspecific compounds. Furthermore, evaluation of the efficiency of the poly(dibenzo-18-crown-6) was performed by applying the digest of egg white. Finally, quantum mechanical calculations were performed to calculate the binding energies to predict the affinity between poly(dibenzo-18-crown-6) and various ligands. The newly identified solid-phase extraction material was found to be a highly efficient tool for phosphopeptide recovery from tryptic digest of proteins.

Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate.

Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo, and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation.IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site Ø binding and greater protection against RSV challenge.

Hydration facilitates oxygenation of hemocyanin: perspectives from molecular dynamics simulations.

Molecular dynamics simulations were applied to deoxy- and oxy-hemocyanins using newly developed force field parameters for the dicopper site to evaluate their structural and dynamical properties. Data obtained from the simulations provided information of the oxygenation effect on the active site and overall topology of the protein that was analyzed by root-mean-square deviations, b-factors, and dicopper coordination geometries. Domain I of the protein was found to demonstrate higher flexibility with respect to domain II because of the interfacial rotation between domain I and II that was further endorsed by computing correlative domain movements for both forms of the protein. The oxygenation effect on the overall structure of the protein or polypeptide subunit was further explored via gyration radii evaluated for the metal-binding domain and for the whole subunit. The evaluation of hydration dynamics was carried out to understand the water mediated role of amino acid residues of the solvent tunnel facilitating the entry of oxygen molecule to the dicopper site of hemocyanin.

A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats.

UNLABELLED: Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants. However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (ΔSH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-ΔSH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10 total PFU and 2.7 log10 PFU/g of tissue, respectively, compared to those in unvaccinated animals (P < 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity. IMPORTANCE: RSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codon-deoptimized nonstructural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate, DB1, was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats.

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector.

Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced "high-quality" RSV-neutralizing antibodies, defined as antibodies that neutralize RSV in vitro without added complement (B. Liang et al., J Virol 89:9499-9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials. IMPORTANCE: Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia worldwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) strain expressing the RSV fusion (F) protein was previously shown to be well tolerated by seronegative children but was insufficiently immunogenic for RSV F. In the present study, the RSV F protein was engineered to be packaged efficiently into vaccine virus particles. This resulted in a significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and nonhuman primates. In nonhuman primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this well-tolerated vaccine candidate and should be applicable to other vectored vaccines.

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein.

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.

Hydration of iron-porphyrins: ab initio quantum mechanical charge field molecular dynamics simulation study.

The ab initio quantum mechanical charge field molecular dynamics (QMCF-MD) simulation approach was successfully applied to Fe2+-P and Fe3+-P in water to evaluate their structural, dynamical and energetic properties. Based on the structural data, it was found that Fe2+-P accommodates one water molecule in the first coordination sphere of the Fe2+ ion including the four nitrogen atoms of the porphyrin system coordinating with central metal species. On the other hand, two water molecules were coordinated to Fe3+-P, thus forming a hexa-coordinated species. Comparison of dynamical properties such as the vibrational power spectrum and ligand mean residence times to other metal-free porphyrin systems demonstrate the ions' influence on the hydration structure, enabling a characterisation of the strong interaction of the ions which greatly reduces the hydrogen bonding potential of the complex. The association of water molecules with the metal ions in both solutes was quantified by computing the free energy of binding obtained via the potential of mean force. This further confirmed the strong association of water to the metal ions which was conversely weak as inferred from the energetic data for the Fe2+-P system.

Syntheses of 4,6-dihydroxypyrimidine diones, their urease inhibition, in vitro, in silico, and kinetic studies.

A library of 4,6-dihydroxypyrimidine diones (1-35) were synthesized and evaluated for their urease inhibitory activity. Structure-activity relationships, and mechanism of inhibition were also studied. All compounds were found to be active with IC50 values between 22.6±1.14-117.4±0.73µM, in comparison to standard, thiourea (IC50=21.2±1.3µM). Kinetics studies on the most active compounds 2-7, 16, 17, 28, and 33 were performed to investigate their modes of inhibition, and dissociation constants Ki. Compounds 2, 3, 7, 16, 28, and 33 were found to be mixed-type of inhibitors with Ki values in the range of 7.91±0.024-13.03±0.013µM, whereas, compounds 4-6, and 17 were found to be non-competitive inhibitors with Ki values in the range of 9.28±0.019-13.05±0.023µM. In silico study was also performed, and a good correlation was observed between experimental and docking studies. This study is continuation of our previously reported urease inhibitory activity of pyrimidine diones, representing potential leads for further research as possible treatment of diseases caused by ureolytic bacteria.

Histatin peptides: Pharmacological functions and their applications in dentistry.

There are many human oral antimicrobial peptides responsible for playing important roles including maintenance, repairing of oral tissues (hard or soft) and defense against oral microbes. In this review we have highlighted the biochemistry, physiology and proteomics of human oral histatin peptides, secreted from parotid and submandibular salivary glands in human. The significance of these peptides includes capability for ionic binding that can kill fungal Candida albicans. They have histidine rich amino acid sequences (7-12 family members; corresponding to residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respectively) for Histatin-3. However, Histatin-3 can be synthesized proteolytically from histatin 5 or 6. Due to their fungicidal response and high biocompatibility (little or no toxicity), these peptides can be considered as therapeutic agents with most probable applications for example, artificial saliva for denture wearers and salivary gland dysfunction conditions. The objectives of current article are to explore the human histatin peptides for its types, chemical and biological aspects. In addition, the potential for therapeutic bio-dental applications has been elaborated.

Synthesis and in vitro α-chymotrypsin inhibitory activity of 6-chlorobenzimidazole derivatives.

A library of benzimidazole derivatives 1-20 were synthesized, and studied for their α-chymotrypsin (α-CT) inhibitory activity in vitro. Kinetics and molecular docking studies were performed to identify the type of inhibition. Compound 1 was found to be a good inhibitor of α-chymotrypsin enzyme (IC50=14.8±0.1µM, Ki=16.4µM), when compared with standard chymostatin (IC50=5.7±0.13µM). Compounds 2-8, 15, 17, and 18 showed significant inhibitory activities. All the inhibitors were found to be competitive inhibitors, except compound 17, which was a mixed type inhibitor. The substituents (R) in para and ortho positions of phenyl ring B, apparently played a key role in the inhibitory potential of the series. Compounds 1-20 were also studied for their cytotoxicity profile by using 3T3 mouse fibroblast cells and compounds 3, 5, 6, 8, 12-14, 16, 17, 19, and 20 were found to be cytotoxic. Molecular docking was performed on the most active members of the series in comparison to the standard compound, chymostatin, to identify the most likely binding modes. The compounds reported here can serve as templates for further studies for new inhibitors of α-chymotrypsin and other chymotrypsin-like serine proteases enzymes.

2-Arylquinazolin-4(3H)-ones: A new class of α-glucosidase inhibitors.

Twenty-five derivatives of 2-arylquinazolin-4(3H)-ones (1-25) were evaluated for their yeast (Saccharomyces cerevisiae) α-glucosidase inhibitory activities. All synthetic compounds, except 1 and 6, were found to be several hundred fold more active (IC50 values in the range of 0.3±0.01-117.9±1.76µM), than the standard drug, acarbose (IC50=840±1.73µM). The enzyme kinetic studies on the most active compounds 12, 4, 19, and 13 were performed for the determination of their modes of inhibition and dissociation constants Ki. Study of the modes of inhibition of compounds 12, and 4 were also performed using molecular modeling techniques. In brief, current study identifies a novel class of α-glucosidase inhibitors which can be further studied for the treatment of hyperglycemia and obesity.