The Chd1 Chromatin Remodeler Shifts Nucleosomal DNA Bidirectionally as a Monomer.

Chromatin remodelers catalyze dynamic packaging of the genome by carrying out nucleosome assembly/disassembly, histone exchange, and nucleosome repositioning. Remodeling results in evenly spaced nucleosomes, which requires probing both sides of the nucleosome, yet the way remodelers organize sliding activity to achieve this task is not understood. Here, we show that the monomeric Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome. During sliding, Chd1 generates unstable remodeling intermediates that spontaneously relax to a pre-remodeled position. We demonstrate that nucleosome sliding is tightly controlled by two regulatory domains: the DNA-binding domain, which interferes with sliding when its range is limited by a truncated linking segment, and the chromodomains, which play a key role in substrate discrimination. We propose that active interplay of the ATPase motor with the regulatory domains may promote dynamic nucleosome structures uniquely suited for histone exchange and chromatin reorganization during transcription.

Interdomain Communication of the Chd1 Chromatin Remodeler across the DNA Gyres of the Nucleosome.

Chromatin remodelers use a helicase-like ATPase motor to reposition and reorganize nucleosomes along genomic DNA. Yet, how the ATPase motor communicates with other remodeler domains in the context of the nucleosome has so far been elusive. Here, we report for the Chd1 remodeler a unique organization of domains on the nucleosome that reveals direct domain-domain communication. Site-specific cross-linking shows that the chromodomains and ATPase motor bind to adjacent SHL1 and SHL2 sites, respectively, on nucleosomal DNA and pack against the DNA-binding domain on DNA exiting the nucleosome. This domain arrangement spans the two DNA gyres of the nucleosome and bridges both ends of a wrapped, ∼90-bp nucleosomal loop of DNA, suggesting a means for nucleosome assembly. This architecture illustrates how Chd1 senses DNA outside the nucleosome core and provides a basis for nucleosome spacing and directional sliding away from transcription factor barriers.

Role of nuclear localization signals in the DNA delivery function of Chikungunya virus capsid protein.

Capsids of several RNA viruses are reported to have unconventional roles attributed to their subcellular trafficking property. The capsid of CHIKV is also found to localize in the nucleus, but the rationale is not yet clear. To understand the role of the nuclear-localized capsid, we examined the nucleic acid binding and cargo delivery activity of the CHIKV capsid. We used bacterially purified capsid protein to probe the binding affinity with CHIKV genome-specific and non-specific nucleic acids. We found that the capsid was able to bind non-specifically to different forms of nucleic acids. The successful transfection of GFP-tagged plasmid DNA by CHIKV capsid protein shows the DNA delivery ability of the protein. Further, we selected and investigated the DNA binding and cargo delivery activity of commercially synthesized Nuclear Localization Signal sequences (NLS 1 and NLS2) of capsid protein. Both peptides showed comparable DNA binding affinity, however, only the NLS1 peptide was capable of delivering plasmid DNA inside the cell. Furthermore, the cellular uptake study using the FITC-labelled NLS1 peptide was performed to highlight the membrane penetrating ability. Structural analysis was performed using circular dichroism and NMR spectroscopy to elucidate the transfection ability of the NLS1 peptides. Our findings suggest that the capsid of CHIKV might influence cellular trafficking in the infected cell via non-specific interactions. Our study also indicates the significance of NLS sequences in the multifunctionality of CHIKV capsid protein.

Epidemiology and molecular characterization of chikungunya virus from human cases in North India, 2016.

Chikungunya virus (CHIKV), an arthropod-borne Alphavirus is responsible for chikungunya disease. Arthralgia and arthritis are the major symptom. Some patients recover early while others for a very long time. This study provides, epidemiology and molecular characterization of three whole-genome sequences of CHIKV and assessed phylogenetic analysis, physiological properties, antigenicity, and B-cell epitope prediction by in silico. We report the clinical epidemiology of 325 suspected patients. Of these, 118 (36.30%) were confirmed CHIKV positive by either PCR or ELISA. Clinical analysis showed joint pain, joint swelling and headache were frequent and significant features. Phylogenie analysis showed the currently circulating strain is in close clustring to Africa, Uganda, and Singapore CHIKV strains. Molecular characterization by WGS was done. Thirty eight amino acid changes in the nonstructural proteins were found with respect to the S27 (ECSA) strain. Of these five located in nsP2. Similarly, 34 amino acid changes in structural proteins were observed. The major change was notice; in E3 protein hydropathicity -0.281 to -0.362, in E2 isoelectric point (pI) 8.24 to 8.37, instability index 66.08 to 71.062, aliphatic index varied from 74.69 to 68.59 and E3 75.79 to 70.05. In nsP1 protein pI varies from 6.62 to 8.04, while no other change was observed in structural and nonstructural protein. The linear B-cell epitopes, position, and number varied with the mutation. The molecular characterizations of WGS demonstrate the observation of protein, antigenicity with respect to the mutation.

Modelling and optimization of process parameters for production of desiccated Chhana-murki (Indian cottage cheese-based dessert).

In the present study, process parameters were optimized for the production of desiccated chhana-murki (Indian cottage cheese-based dessert). Response Surface Methodology (RSM) was employed to explore the mutual effects of coagulation temperature (CT) of milk (70-90 °C), % fat level in milk (3.5%-5.5%), and sugar-to-paneer cube (SP) ratio (0.6-0.9) on instrumental hardness (N), water activity (aw), yield (%), sensory sweetness and overall acceptability (on 100-point intensity scale) of chhana-murki. The resulted responses were evaluated by analysis of variance (ANOVA), and the second-order polynomial response surface equations were fitted using multiple regression analysis. Determination coefficients (R 2) were equal to 80% or higher for individual responses stated that the developed models were well fitted to the experimental results. The optimized product was prepared using CT 79.22 °C, milk fat 4.8%, and SP ratio 0.7. Confirmatory experiment values for instrument hardness, water activity (aw), yield (%), sensory sweetness and overall acceptability were 105.05 N, 0.85, 115.2%, 61.2 and 78.8, respectively.

Differential expression of structural and functional proteins during bean common mosaic virus-host plant interaction.

Bean common mosaic virus (BCMV), the most common seed-borne pathogen in Phaseolus vulgaris L. is known to cause severe loss in productivity across the globe. In the present study, proteomic analyses were performed for leaf samples from control (healthy) and susceptible BCMV infected plants. The differential expression of proteins was evaluated using two-dimensional gel electrophoresis (2-DE). Approximately, 1098 proteins were spotted, amongst which 107 proteins were observed to be statistically significant with differential expression. The functional categorization of the differential proteins illustrated that they were involved in biotic/abiotic stress (18%), energy and carbon metabolism (11%), photosynthesis (46%), protein biosynthesis (10%), chaperoning (5%), chlorophyll (5%) and polyunsaturated fatty acid biosynthesis (5%). This is the first report on the comparative proteome study of compatible plant-BCMV interactions in P. vulgaris which contributes largely to the understanding of protein-mediated disease resistance/susceptible mechanisms.

Advances in our understanding of the structure and functionality of edible fats and fat mimetics.

The reasons for the increased world-wide incidence of obesity, type-2 diabetes, and cardiovascular disease include sedentary lifestyles and poor food choices. Regulatory agencies in several countries now require companies to add unattractive front of package labels to their products where salt, sugar and fat (or saturated fat) levels are prominently displayed. After the demise of partially hydrogenated fats, saturated fat has become the new target. Consumption of saturated fat over polyunsaturated oil has been clearly shown to increase cholesterol levels in humans. However, saturated fats provide the functionality required in many food products. To complicate matters, concerns over sustainability, veganism, genetically modified organisms, animal welfare, as well as religious beliefs, severely limit our sources of saturated fat. In this review we will discuss recent advances in our understanding of the nano and mesoscale structure of fats, responsible for their physical functionality and contrast it to that of fat mimetics. Fat mimetics include polymeric networks of ethylcellulose, emulsion-templated networks of proteins and polysaccharides, colloidal and self-assembled fibrillar networks of polar lipid crystals, as well as solid o/w emulsions of oil trapped within crystallized lamellar mesophases. Clean label and economic considerations will also be touched upon.

Evaluation of medicinal herbs as a potential therapeutic option against SARS-CoV-2 targeting its main protease.

The COVID-19 disease caused by the SARS-CoV-2 has emerged as a worldwide pandemic and caused huge damage to the lives and economy of more than hundred countries. As on May 10, 2020, more than 4,153,300 people stand infected from the virus due to an unprecedented rate of transmission and 282,700 have lost their lives because of the disease. In this context, medicinal plants may provide a way to treat the disease by targeting specific essential proteins of the virus. We screened about 51 medicinal plants and found that Tea (Camellia sinensis) and Haritaki (Terminalia chebula) has potential against SARS-COV-2 3CLpro , with an IC50 for Green Tea as 8.9 ± 0.5 µg/ml and Haritaki 8.8 ± 0.5 µg/ml. The in-silico studies suggested that Tea component Thearubigins binds to the cysteine 145 of protease active site and could be a pharmacoactive molecule. We predict that the inhibition in protease activity may be able to halt the SARS-CoV-2 replication cycle and therefore, we propose Green Tea, Black Tea, and Haritaki plant extracts as potential therapeutic candidates for SARS-CoV-2 infection. Further investigation on role of bioactive constituents of extracts is needed to establish the molecular basis of inhibition and towards expedited drug discovery.

In vivo effect of two different dietary fiber blends on the milk calcium bioavailability.

Milk is a valuable source of dietary calcium and it becomes important to establish whether incorporation of dietary fiber (DF), a health promoting food constituent, would lead to any undesirable impact on the bioavailability of milk calcium or not. The DF fortified spray dried partly skimmed milk powder with prestandardized fiber Blend-I (psyllium husk, oat fiber, MCC, inulin) and fiber Blend-II (psyllium husk, oat bran, wheat fiber and inulin) was subjected to rat-feeding studies to examine the possible effects on the bioavailability of milk calcium. The differences for calcium absorption and retention among diets containing DF Blend-I, DF Blend-II and cellulose (control) were found to be non-significant. It was evident that the milk calcium bioavailability of the diets containing two fiber formulations tested (at the levels studied) was at par with that of control standard diet containing only cellulose as DF. Therefore, it is reasonable to incorporate these DF blends into dairy products, and thereby add value.

The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome.

Chromatin remodelers are essential for establishing and maintaining the placement of nucleosomes along genomic DNA. Yet how chromatin remodelers recognize and respond to distinct chromatin environments surrounding nucleosomes is poorly understood. Here, we use Lac repressor as a tool to probe how a DNA-bound factor influences action of the Chd1 remodeler. We show that Chd1 preferentially shifts nucleosomes away from Lac repressor, demonstrating that a DNA-bound factor defines a barrier for nucleosome positioning. Rather than an absolute block in sliding, the barrier effect was achieved by altered rates of nucleosome sliding that biased redistribution of nucleosomes away from the bound Lac repressor site. Remarkably, in addition to slower sliding toward the LacO site, the presence of Lac repressor also stimulated sliding in the opposite direction. These experiments therefore demonstrate that Chd1 responds to the presence of a bound protein on both entry and exit sides of the nucleosome. This sensitivity to both sides of the nucleosome allows for a faster and sharper response than would be possible by responding to only the entry side, and we speculate that dual entry/exit sensitivity is also important for regularly spaced nucleosome arrays generated by Chd1 and the related ISWI remodelers.

An improved micropropagation system, ex vitro rooting and validation of genetic homogeneity in wild female Momordica dioica: an underutilized nutraceutical vegetable crop.

Momordica dioica Roxb. ex Willd., is a perennial and dioecious (2n = 28) plant of family Cucurbitaceae. Conventional methods of propagation through seeds, stem cuttings and rhizomatous/tuberous roots are inadequate for its mass cultivation as a vegetable crop. This paper reports an improved and efficient micropropagation method for wild female M. dioica using nodal explants. Shoot amplification was achieved using subculturing of in vitro raised shoots on MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid (IAA). The maximum number of shoots (45.30 ± 3.83) with an average length 6.52 ± 0.89 cm were differentiated on MS medium containing 0.5 mg L-1 BAP, 0.1 mg L-1 IAA and additives (50 mg L-1 ascorbic acid, 25 mg L-1 each of adenine sulphate, citric acid and l-arginine). The cloned shoots were rooted ex vitro. Each shoot treated with 250 mg L-1 IBA for 5 min produced 12.3 ± 1.33 with a mean length 5.4 ± 0.73 cm. More than 85% (46 plants) of ex vitro rooted plantlets were successfully hardened in a greenhouse with normal growth characteristics. In order to evaluate the genetic stability of micropropagated plants, the two PCR-based techniques, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were used. The amplification patterns of the micropropagated and mother plant were monomorphic thus depicting genetic stability of the micropropagation system. This protocol could be effectively employed for the mass multiplication of wild female M. dioica, a popular summer vegetable crop.

The Contribution of Modern Margarine and Fat Spreads to Dietary Fat Intake.

The study of dietary fat consumption and its resultant effects on human health has been one of the most investigated topics in the field of human nutrition. Based on the results obtained from such studies, specific dietary recommendations on fat intake (both in terms of quantity and quality) have been established by health organizations around the globe. Among the various food industry sectors, the margarine manufacturers have also responded to these guidelines and now offer improved formulations with a desirable balance of fat contents and fat types. The main aim of this article is to provide an overview on how these modern margarines can contribute towards reaching the dietary guidelines relating to fat intake. In particular, the dietary recommendations with respect to the specific fatty acid types are comprehensively detailed along with an emphasis on the role of modern margarines in providing balanced fat types (more polyunsaturated fats, less saturated fats and a near-complete absence of trans fats) in the daily diet.

Production of a protein-rich extruded snack base using tapioca starch, sorghum flour and casein.

A protein-rich puffed snack was produced using a twin screw extruder and the effects of varying levels of tapioca starch (11 to 40 parts), rennet casein (6 to 20 parts) and sorghum flour (25 to 75 parts) on physico-chemical properties and sensory attributes of the product studied. An increasing level of sorghum flour resulted in a decreasing whiteness (Hunter L* value) of the snack. Although the starch also generally tended to make the product increasingly darker, both starch and casein showed redness parameter (a* value) was not significantly influenced by the ingredients levels, the yellow hue (b* value) generally declined with the increasing sorghum level. Tapioca starch significantly increased the expansion ratio and decreased the bulk density and hardness value of the snack, whereas the opposite effects seen in case of sorghum flour. While the water solubility index was enhanced by starch, water absorption index was appreciably improved by sorghum. Incorporation of casein (up to 25 %) improved the sensory color and texture scores, and so also the overall acceptability rating of the product. Sorghum flour had an adverse impact on all the sensory attributes whereas starch only on the color score. The casein or starch level had no perceivable effect on the product's flavor score. The response surface data enabled optimization of the snack-base formulation for the desired protein level or desired sensory characteristics.

Biopolymer-based structuring of liquid oil into soft solids and oleogels using water-continuous emulsions as templates.

Physical trapping of a hydrophobic liquid oil in a matrix of water-soluble biopolymers was achieved using a facile two-step process by first formulating a surfactant-free oil-in-water emulsion stabilized by biopolymers (a protein and a polysaccharide) followed by complete removal of the water phase (by either high- or low-temperature drying of the emulsion) resulting in structured solid systems containing a high concentration of liquid oil (above 97 wt %). The microstructure of these systems was revealed by confocal and cryo-scanning electron microscopy, and the effect of biopolymer concentrations on the consistency of emulsions as well as the dried product was evaluated using a combination of small-amplitude oscillatory shear rheometry and large deformation fracture studies. The oleogel prepared by shearing the dried product showed a high gel strength as well as a certain degree of thixotropic recovery even at high temperatures. Moreover, the reversibility of the process was demonstrated by shearing the dried product in the presence of water to obtain reconstituted emulsions with rheological properties comparable to those of the fresh emulsion.

Decoupling nucleosome recognition from DNA binding dramatically alters the properties of the Chd1 chromatin remodeler.

Chromatin remodelers can either organize or disrupt nucleosomal arrays, yet the mechanisms specifying these opposing actions are not clear. Here, we show that the outcome of nucleosome sliding by Chd1 changes dramatically depending on how the chromatin remodeler is targeted to nucleosomes. Using a Chd1-streptavidin fusion remodeler, we found that targeting via biotinylated DNA resulted in directional sliding towards the recruitment site, whereas targeting via biotinylated histones produced a distribution of nucleosome positions. Remarkably, the fusion remodeler shifted nucleosomes with biotinylated histones up to 50 bp off the ends of DNA and was capable of reducing negative supercoiling of plasmids containing biotinylated chromatin, similar to remodelling characteristics observed for SWI/SNF-type remodelers. These data suggest that forming a stable attachment to nucleosomes via histones, and thus lacking sensitivity to extranucleosomal DNA, seems to be sufficient for allowing a chromatin remodeler to possess SWI/SNF-like disruptive properties.

A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub.

An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments of mature plant were used as explants for culture establishment. Multiple shoots were optimally differentiated from the nodal stem explants through bud breaking on Murashige and Skoog (1962) medium containing 3.0 mg l(-1) benzyladenine (BA). The effect of different plant growth regulators and minerals were studied on different stages of micropropagation procedure (i.e., explant establishment, shoot multiplication/growth and ex vitro rooting). Additionally, for enhancing shoot multiplication during subculture, MS medium was modified (MMS) with higher levels of magnesium, potassium and sulphate ions. Out of these, MMS3 medium containing 0.25 mg l(-1) each of BA and Kin (N6-furfuryladenine), with 0.1 mg l(-1) NAA (α-naphthalene acetic acid) was found the best for shoot multiplication (42.45 ± 3.82 per culture vessel). The in vitro regenerated shoots were rooted under ex vitro conditions on treating the shoot base with 500 mg l(-1) of IBA (indole-3 butyric acid) for 3 min on sterile Soilrite®. The ex vitro rooted plants were hardened in the greenhouse and transferred to the field with ≈85 % survival rate. There were not any visual differences between wild and micropropagated plants in the field, although the later underwent significant changes during acclimatization. Micromorphological changes on leaf surface characters from in vitro to acclimatized plantlets were studied in terms of development of glandular trichomes, changes in vein spacing and vein structure in order to understand the nature of plant responses towards environmental conditions. The method developed and defined can be applied for commercial cultivation, which may be important for extraction of bioactive compounds and may facilitate conservation of this multipurpose endangered medicinal shrub.

Optimization of instant dalia dessert pre-mix production by using response surface methodology.

Dalia, a wheat-based, particulate containing dairy dessert is popularly consumed as a breakfast food and is also considered as a health food. Though popular throughout Northern parts of the country, its limited shelf-life even under refrigeration imposes severe restrictions on its organized manufacture and marketing. In order to promote dalia dessert as a marketable product, in the present study, a process was developed for manufacture of instant dalia pre-mix, as a dry product with long shelf-life, which could be attractively packaged and easily reconstituted for consumption. During the investigation, the effect of different levels of milk solids and wheat solids was studied on dalia pre-mix quality by employing a central composite rotatable design (CCRD). The suggested formulation had 17.82 % milk solids and 2.87 % wheat solids. This formulation was found to be most appropriate for manufacture of instant dalia pre-mix with predicted sensory scores (Max. 100) of 85.35, 41.98 and 67.27 for mouthfeel, consistency and flavor, respectively; the viscosity of the product was 941.0 cp.

Polysaccharide-based oleogels prepared with an emulsion-templated approach.

The preparation and characterization of oleogels structured by using a combination of a surface-active and a non-surface-active polysaccharide through an emulsion-templated approach is reported. Specifically, the oleogels were prepared by first formulating a concentrated oil-in-water emulsion, stabilized with a combination of cellulose derivatives and xanthan gum, followed by the selective evaporation of the continuous water phase to drive the network formation, resulting in an oleogel with a unique microstructure and interesting rheological properties, including a high gel strength, G'>4000 Pa, shear sensitivity, good thixotropic recovery, and good thermostability.

The basic linker of macroH2A stabilizes DNA at the entry/exit site of the nucleosome.

MacroH2A is a histone H2A variant that is typically found in heterochromatic regions of the genome. A positively charged linker that connects the histone-fold with the macro-domain was suggested to have DNA-binding properties, and has been shown to promote oligomerization of chromatin fibers. Here we examine the influence of this basic linker on DNA of mononucleosomes. We find that the macro-linker reduces accessibility to extranucleosomal DNA, and appears to increase compaction of the nucleosome. These properties arise from interactions between the H1-like basic linker region and DNA around the entry/exit site, which increases protection of nucleosomal DNA from exonuclease III digestion by ∼10 bp. By stabilizing the wrapping of DNA around the histone core, this basic linker of macroH2A may alter the distribution of nucleosome-associated factors, and potentially contribute to the more compacted nature of heterochromatin.

Transcriptome analysis of Huh7 cells upon Chikungunya virus infection and capsid transfection reveals regulation of distinct cellular and metabolic pathways.

Chikungunya virus (CHIKV) causes persistent arthritis and neurological problems imposing a huge burden globally. The present study aims to understand the interaction mechanism of Chikungunya virus and CHIKV-capsid in Huh7 cells. The RNA-sequencing and qRT-PCR method was used for the transcript and gene profiles of CHIKV virus and CHIKV capsid alone. Transcriptional analysis showed capsid induced 1114 and 956 differentially expressed genes (DEGs) to be upregulated and downregulated respectively, while in virus, 933 genes were upregulated and 956 were downregulated. Total 202 DEGs were common in both capsid and virus; and nine were validated using qRT-PCR. Identified DEGs were found to be associated with metabolic pathways such as Diabetes, cardiac disease, and visual impairment. Further, knock-down study on one of the DEGs (MafA) responsible for insulin regulation showed low viral proteins expression suggesting a reduction in virus-infection. Thus, the study provides insight into the interplay of the virus-host factors assisting virus replication.