Advances in computational approaches to structure determination of alphaviruses and flaviviruses using cryo-electron microscopy.

Advancements in the field of cryo-electron microscopy (cryo-EM) have greatly contributed to our current understanding of virus structures and life cycles. In this review, we discuss the application of single particle cryo-electron microscopy (EM) for the structure elucidation of small enveloped icosahedral viruses, namely, alpha- and flaviviruses. We focus on technical advances in cryo-EM data collection, image processing, three-dimensional reconstruction, and refinement strategies for obtaining high-resolution structures of these viruses. Each of these developments enabled new insights into the alpha- and flavivirus architecture, leading to a better understanding of their biology, pathogenesis, immune response, immunogen design, and therapeutic development.

Recent advances in the preservation of postharvest fruits using edible films and coatings: A comprehensive review.

In recent years, there has been considerable growth in the creation of edible films and coatings, which is predicted to have a major impact on fruit quality in the coming years. Consumers want fresh fruits that are pesticide-free, good quality, high nutritional value, and a long shelf life. The use of edible coatings and films on fruits is an environmentally dependable approach to a creative solution to this problem. The application, recent trends, and views of coatings and edible films, as well as their impact on fruit quality, are presented in this article, along with a knowledge of their key roles and benefits. According to numerous studies, natural polymers are highly suited for use as packaging material for fresh fruits and can often be a viable alternative to synthetic chemicals. Plasticisers, surfactants, cross-linkers, antimicrobial agents, functional additives, nanoparticles, and fruit and vegetable residues can be used to alter the properties of edible coatings.

Migration study of chemical additives from low density polyethylene (LDPE) into dahi.

Dahi is widely used fermented milk product in India. Low Density Polyethylene (LDPE) is the most extensively used packaging material for Dahi in India. The present study was conducted to develop the analytical methods for extraction and migration of chemical additives from LDPE into dahi. Characterization of dahi packaging materials collected from five different firms was done by Fourier Transform Infrared Spectroscopy. Focused ultrasound solid liquid extraction method was observed to be better as compared to solid liquid extraction method as the former extracted maximum additives from the LDPE. Out of total 76 chemical additives extracted from LDPE, only eight (10.52%) matched with the existing positive list of polyolefins prescribed by Bureau of Indian Standads (BIS). The overall migration of chemical additives from all the LDPE samples was below their maximum limit as given by BIS standards. Chemical additives which migrated into the simulants included the antioxidants, fatty acids and their derivatives, unreacted hydrocarbons, plasticizers, lubricants and surfactant etc.

Mycobacterium tuberculosis Endonuclease VIII 2 (Nei2) forms a prereplicative BER complex with DnaN: Identification, characterization, and disruption of complex formation.

Mycobacterium tuberculosis Nei2 (Rv3297) is a BER glycosylase that removes oxidized base lesions from ssDNA and replication fork-mimicking substrates. We show that Endonuclease VIII 2 (Nei2) forms a BER complex with the ß-clamp (DnaN, Rv0002) with a KD of 170 nM. The Nei2-ß-clamp interactions enhance Nei2's activities up to several folds. SEC analysis shows that one molecule of Nei2 binds to a single ß-clamp dimer. Nei2 interacts with subsites I and II of the ß-clamp via a noncanonical 223 QGCRRCGTLIAY239 Clamp Interacting Protein (CIP) motif in the C-terminal zinc-finger domain, which was previously shown by us to be dispensable for intrinsic Nei2 activity. The 12-mer peptide alone exhibited a KD of 10.28 nM, suggesting that the motif is a key mediator of Nei2-ß-clamp interactions. Finally, we identified inhibitors of Nei2-ß-clamp interactions using rational methods, in vitro disruption, and SPR assays after querying a database of natural products. We found that Tubulosine, Fumitremorgin C, Toyocamycin, and Aleuritic acid exhibit IC50 values of 94.47, 83.49, 109.7, and 71.49 µM, respectively. They act by disrupting Nei2-ß-clamp interactions and do not affect intrinsic Nei2 activity. Among other things, the present study gives insights into the role of Nei2 in bacterial prereplicative BER.

Comparative evaluation of the predictive value of cariogram and informal caries risk assessment among school-going children in the age group of 8-9 years of Yamuna Nagar District, Haryana.

AIM: This longitudinal, observational study was conducted in the schools of Yamunanagar, Haryana, to evaluate and compare the predictive value of formal type of caries risk assessment using reduced Cariogram software, including only seven factors and informal type among 8-9 years' school-going children. METHODS: A.total of 111 school-going children were included in the study. Risk profile for each child was created using cariogram as well as informal factors. The same children were scheduled for re-examination at an interval of 9 and 18 months. The caries status was recorded again using the Collapsed International Caries Detection and Assessment System (ICDAS) concept. STATISTICAL ANALYSIS: The precoded data were transferred to the computer and analyzed using the SPSS software (version 17.0). Data were analyzed for the identification of children with lesion progression and numbers of lesions progressing using the Transition Scoring System. RESULTS: Cariogram being a multifactorial model gives significant individual weightage to each etiological factor causing dental caries as compared to informal caries risk assessment which though easy to implement yet unstructured unlike cariogram and thus does not guarantee consistent implementation. CONCLUSION: Cariogram is a perfect option for patient motivation and supports the clinician in decision making for planning preventive strategies for the patients. Along with this, a combination of the factors for informal caries risk assessment can help in making a simple yet multifactorial model which can be applied in daily practice.

Leprosy drug clofazimine activates peroxisome proliferator-activated receptor-γ and synergizes with imatinib to inhibit chronic myeloid leukemia cells.

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38- progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.

Mycobacterial protein tyrosine kinase, PtkA phosphorylates PtpA at tyrosine residues and the mechanism is stalled by the novel series of inhibitors.

Phosphorylation and dephosphorylation are the key mechanisms for mycobacterial physiology and play critical roles in mycobacterial survival and in its pathogenesis. Mycobacteria evade host immune mechanism by inhibiting phagosome - lysosome fusion in which mycobacterial protein tyrosine phosphatase A (PtpA;TP) plays an indispensable role. Tyrosine kinase (PtkA;TK) activated by autophosphorylation; phosphorylates TP, which subsequently leads to increase in its phosphatase activity. The phosphorylated TP is secreted in phagosome of macrophage. In the present study, we have shown that the phosphorylation at two sites of TP; Y128 and Y129 are critical for TK-mediated phosphatase activity. The disruption of this interaction between TK and TP inhibits activation of later which further leads to the decrease in intracellular survival of mycobacteria. Furthermore, the proof of concept has been established using benzylbenzofurans and benzofuranamides, which inhibit the growth and intracellular survival of mycobacteria, associate with the functional sites of TP and contend with the TK. This binding was further restated by looking at the anchorage of protein-protein and the protein-inhibitor complexes in the homology-based structure models and by surface plasmon resonance analysis.

Estimation of steviol glycosides in food matrices by high performance liquid chromatography.

Steviol glycosides (SGs) are non-caloric, natural sweetener obtained from plant Stevia rebaudiana and are used as sugar substitute in foods. The level of SGs in foods should not exceed maximum permissible limit defined by regulatory agencies. Thus analytical methods are required for assay of stevioside (Stev) and rebaudioside A (Reb A), which are two major constituents of SGs, in foods. A method for extraction of Stev and Reb A from dairy viz., flavoured milk, flavoured yoghurt and non-dairy foods viz., carbonated water, jam, chewing gum and estimation of these by HPLC has been described. Extraction of SGs from dairy samples was achieved by treating samples with 20% acetonitrile in presence of Carrez solutions while these can be simply extracted with water from non-dairy samples. Separation and estimation of these two glycosides was achieved on C18 column (length: 4.6 × 250 mm, particle size: 5 µm) using isocratic mobile phase prepared by mixing of acetonitrile and 10 mM sodium phosphate buffer (pH 2.6) in ratio of 32:68 (v/v). Recovery of two SGs was quantitative. Separation and estimation of SGs by HPLC was robust. Limit of detection and limit of quantitation for Reb A in different food was in range from 1.057-1.834 to 3.525-6.114 mg kg-1 while that of Stev was from 1.679-2.912 to 5.596-9.707 mg kg-1, respectively. Neotame, an artificial sweetener can be used as internal standard for separation of SGs.

Construction of a lateral flow strip for detection of soymilk in milk.

A lateral flow based detection method for ascertaining the presence of soymilk in whole bovine milk has been described. The method uses commercially available rabbit anti-soy protein antibodies conjugated to gold nanoparticles (AuNPs) wherein soymilk protein in adulterated milk and soymilk protein at test line competes for limited antibodies. At control line, anti-rabbit immunoglobulin was immobilized for ensuring flow properties of antibody-conjugated AuNPs. Absence or diminished intensity of band at test line indicates presence of soymilk in milk. The soymilk detection limit was 1.75% (v/v) in whole bovine milk and results are available in 5 min. Constructed lateral flow device can be used for on-spot examination of soymilk in milk.

Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297).

Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5'/3' fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 µM-92.7 µM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.

Rapid screening test for detection of oxytetracycline residues in milk using lateral flow assay.

A rapid, semi-quantitative lateral flow assay (LFA) was developed to screen the oxytetracycline (OTC) antibiotics residues in milk samples. In this study a competitive immuno-assay format was established. Colloidal gold nano-particles (GNP) were prepared and used as labelling material in LFA. Polyclonal antibodies were generated against OTC molecule (anti-OTC), purified and the quality was assessed by enzyme linked immuno sorbet assay. For the first time membrane components required for LFA in milk system was optimized. GNP and anti-OTC stable conjugate preparation method was standardized, and then these components were placed over the conjugate pad. OTC coupled with carrier protein was placed on test line; species specific secondary antibodies were placed on the control line of the membrane matrix. Assay was validated by spiking OTC to antibiotic free milk samples and results could be accomplished within 5min. without need of any equipment. The visual detection limit was 30ppb.

Iron (II)-chelating activity of buffalo αS-casein hydrolysed by corolase PP, alcalase and flavourzyme.

Iron is a vital substance for human health which participates in many biochemical reactions. It also act as initiator for many harmful oxidative process. Buffalo αS-casein enriched fraction (80 %) was hydrolysed independently by corolase PP (H1), alcalase (H2), flavourzyme (H3) and sequentially by alcalase-flavourzyme (H4). After ultrafiltration (10 and 3 kDa) hydrolysates were analysed for their iron chelation activity using ferrozine. For H1 group of hydrolysates highest iron (II)-chelation activity (265.58 µM Fe(2+/)mg protein) was found after 8 h of hydrolysis for H2 (267.56 µM Fe(2+/)mg protein) and H3 group of hydrolysates (380.68 µM Fe(2+/)mg protein) after 6 h of hydrolysis. Sequential hydrolysis was not effective for iron (II)-chelation activity. 3 kDa fractions show higher iron (II)-chelation activity than 10 kDa fraction. Flavourzyme was more effective for generation of iron (II)-chelating peptides from buffalo αS-casein.

Synthesis and application of cephalexin imprinted polymer for solid phase extraction in milk.

Molecular imprinted polymer (MIP) against cephalexin was synthesized by co-polymerization of functional monomer, cross-linker, radical initiator, along with target molecule (cephalexin) in a porogenic material. Binding of cephalexin towards prepared MIP was studied in different solvents (water, methanol, 1M NaCl, acetone and acetonitrile) and best binding was observed in methanol. Partition coefficient and selectivity of prepared imprint and non-imprint was also studied. Cross reactivity in terms of binding efficiency was also assessed with other antibiotics. Chromatographic study of MIP was carried out by packing prepared imprint into glass column. MIP was used as matrix in solid phase extraction (SPE) for recovery of cephalexin from spiked milk samples for further estimation by high performance liquid chromatography. No interference was observed from milk components after elution of cephalexin from MIP, indicating selectivity and affinity of MIP. On the other hand, interference was observed in eluate obtained from C18 SPE column.

Biofilm in endodontics: A review.

Endodontic disease is a biofilm-mediated infection, and primary aim in the management of endodontic disease is the elimination of bacterial biofilm from the root canal system. The most common endodontic infection is caused by the surface-associated growth of microorganisms. It is important to apply the biofilm concept to endodontic microbiology to understand the pathogenic potential of the root canal microbiota as well as to form the basis for new approaches for disinfection. It is foremost to understand how the biofilm formed by root canal bacteria resists endodontic treatment measures. Bacterial etiology has been confirmed for common oral diseases such as caries and periodontal and endodontic infections. Bacteria causing these diseases are organized in biofilm structures, which are complex microbial communities composed of a great variety of bacteria with different ecological requirements and pathogenic potential. The biofilm community not only gives bacteria effective protection against the host's defense system but also makes them more resistant to a variety of disinfecting agents used as oral hygiene products or in the treatment of infections. Successful treatment of these diseases depends on biofilm removal as well as effective killing of biofilm bacteria. So, the fundamental to maintain oral health and prevent dental caries, gingivitis, and periodontitis is to control the oral biofilms. From these aspects, the formation of biofilms carries particular clinical significance because not only host defense mechanisms but also therapeutic efforts including chemical and mechanical antimicrobial treatment measures have the most difficult task of dealing with organisms that are gathered in a biofilm. The aim of this article was to review the mechanisms of biofilms' formation, their roles in pulpal and periapical pathosis, the different types of biofilms, the factors influencing biofilm formation, the mechanisms of their antimicrobial resistance, techniques to identify biofilms.

Plaque and salivary pH changes after consumption of fresh fruit juices.

The concept of health has prevailed for centuries and the dietary habits are apparently changing with modernization. "Healthy eating" is now perceived to be important. The desirability of a healthful lifestyle has led to an increased consumption of juices. Drinking large amount of fruit juice is frequently practiced these days and the consumption of these juices is further modified with behavioral habits such as swishing and frothing the drinks around the mouth, sucking frozen fruit juices, use of feeder cups at bed time etc. Hence this study was conducted to find the acidogenic potential of the commonly consumed fresh fruit juices (Grapes, orange, and pineapple) and the juices stored at various temperatures (room temperature, refrigerator and freezer) on the plaque and saliva at various intervals. It was observed that grape juice was more acidogenic compared to orange and pineapple juice. Frozen fruit juices caused a greater drop in plaque and salivary pH followed by the refrigerated juice.