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Earlier, we have found that the enteropathogenic Yersinia enterocolitica have evolved the survival mechanisms that regulate the expression of laccase-encoding genes in the gut. The present study aims to characterize the purified recombinant laccase from Y. enterocolitica strain 8081 biovar 1B and understand its effect on the midgut of cotton bollworm, Helicoverpa armigera (Hubner) larvae. The recombinant laccase protein showed high purity fold and low molecular mass (~ 43 kDa). H. armigera larvae fed with laccase protein showed a significant decrease in body weight and damage in the midgut. Further, transmission electron microscopy (TEM) studies revealed the negative effect of laccase protein on trachea, malpighian tubules, and villi of the insect. The proteome comparison between control and laccase-fed larvae of cotton bollworm showed significant expression of proteolytic enzymes, oxidoreductases, cytoskeletal proteins, ribosomal proteins; and proteins for citrate (TCA cycle) cycle, glycolysis, stress response, cell redox homeostasis, xenobiotic degradation, and insect defence. Moreover, it also resulted in the reduction of antioxidants, increased melanization (insect innate immune response), and enhanced free radical generation. All these data collectively suggest that H. armigera (Hubner) larvae can be used to study the effect of microbes and their metabolites on the host physiology, anatomy, and survival.

Bioethanol production using paper wastes seems a promising approach towards sustainable energy, but mainly hindered by hazardous ink. Therefore, in our experimental plan various methods were applied for the deinking of waste newspapers, laser printed papers and examination papers. A newly designed paper pulper reduced the pulping time from 6 h to 2 h. Cellulase (15 FPU/g) from Aspergillus oryzae MDU-4 was found effective for the deinking of newspapers, whereas laccase isozymes (150 U/g) from Ganoderma lucidum MDU-7 along with 2 mM HOBt was preferred for the ink removal and degradation from the examination papers. Ozonation in the presence of Tween-80 was found to be efficient in the removal of toxic toners used in laser printing papers. The biologically and physically deinked papers, studied with the help of SEM, TEM, FTIR, and XRD analysis revealed significant changes in the chemical and surface structure. Moreover, the saccharification of deinked papers with the help of an enzymatic consortium of Trichoderma citrinoviride MDU-1 resulted in 305 mg/g, 377 mg/g, and 409 mg/g release of sugars from the newspaper, examination paper, and laser printed paper, respectively. Finally, the enzymatic hydrolysates fermented with Saccharomyces cerevisiae NCIM-3640 produced 3.35 g/L ethanol, with 40.85% ethanol yield.

Smaller oligomeric chaperones of α-crystallins (αA- and αB-) have received increasing attention due to their improved therapeutic potential in preventing protein aggregating diseases. Our previous study suggested that deleting 54–61 residues from the N-terminal domain (NTD) of αB-crystallin (αBΔ54–61) decreases the oligomer size and increases the chaperone function. Several studies have also suggested that NTD plays a significant role in protein oligomerization and chaperone function. The current study was undertaken to assess the effect of deleting conserved 21–28 residues from the activated αBΔ54–61 (to get αBΔ21–28, Δ54–61) on the structure–function of recombinant αBΔ21–28, Δ54–61. The αBΔ21–28, Δ54–61 mutant shows an 80% reduction in oligomer size and 3- to 25-fold increases in chaperone activity against model substrates when compared to αB-WT. Additionally, the αB∆21–28, ∆54–61 was found to prevent β-amyloid (Aβ1–42) fibril formation in vitro and suppressed Aβ1–42-induced cytotoxicity in ARPE-19 cells in a more effective manner than seen with αB-WT or αB∆54–61. Cytotoxicity and reactive oxygen species (ROS) detection studies with sodium iodate (SI) showed that the double mutant protein has higher anti-apoptotic and anti-oxidative activities than the wild-type or αB∆54–61 in oxidatively stressed cells. Our study shows that the residues 21–28 and 54–61 in αB-crystallin contribute to the oligomerization and modulate chaperone function. The deletion of conserved 21–28 residues further potentiates the activated αBΔ54–61. We propose that increased substrate affinity, altered subunit structure, and assembly leading to smaller oligomers could be the causative factors for the increased chaperone activity of αBΔ21–28, Δ54–61.

Fluorinated analogues of the fluorophore pyronin B were synthesized as a new class of amine-reactive drug-like small molecules. In water, 2,7-difluoropyronin B was found to reversibly react with primary amines to form covalent adducts. When this fluorinated analogue is added to proteins, these adducts undergo additional oxidation to yield fluorescent 9-aminopyronins. Irradiation with visible blue light enhances this oxidation step, providing a photochemical method to modify the biological properties of reactive amines. In living HeLa cells, 2,7-difluoropyronin B becomes localized in mitochondria, where it is partially transformed into fluorescent aminopyronins, as detected by spectral profiling confocal microscopy. Further excitation of these cells with the blue laser of a confocal microscope can depolarize mitochondria within seconds. This biological activity was only observed with 2,7-difluoropyronin B and was not detected with analogues such as pyronin B or 9-methyl-2,7-difluoropyronin B. This irradiation with blue light enhances the cellular production of reactive oxygen species (ROS), suggesting that increased ROS in mitochondria promotes the formation of aminopyronins that inactivate biomolecules critical for maintenance of mitochondrial membrane potential. The unique reactivity of 2,7-difluoropyronin B offers a novel tool for photochemical control of mitochondrial biology.

μ-Opioid receptor agonists provide potent and effective acute analgesia; however, their therapeutic window narrows considerably upon repeated administration, such as required for treating chronic pain. In contrast, bifunctional μ/δ opioid agonists, such as the endogenous enkephalins, have potential for treating both acute and chronic pain. However, enkephalins recruit β-arrestins, which correlate with certain adverse effects at μ- and δ-opioid receptors. Herein, we identify the C-terminus of Tyr-ψ[(Z)CF[double bond, length as m-dash]CH]-Gly-Leu-enkephalin, a stable enkephalin derivative, as a key site to regulate bias of both δ- and μ-opioid receptors. Using in vitro assays, substitution of the Leu(5) carboxylate with amides (NHEt, NMe(2), N(Cy)Pr) reduced β-arrestin recruitment efficacy through both the δ-opioid and μ-opioid, while retaining affinity and cAMP potency. For this series, computational studies suggest key ligand–receptor interactions that might influence bias. These findings should enable the discovery of a range of tool compounds with previously unexplored biased μ/δ opioid agonist pharmacological profiles.

The C-terminus fragment (Val-Val-Ile-Ala) of amyloid-β is reported to inhibit the aggregation of the parent peptide. In an attempt to investigate the effect of sequential amino-acid scan and C-terminus amidation on the biological profile of the lead sequence, a series of tetrapeptides were synthesized using MW-SPPS. Peptide D-Phe-Val-Ile-Ala-NH2 (12c) exhibited high protection against β-amyloid-mediated-neurotoxicity by inhibiting Aβ aggregation in the MTT cell viability and ThT-fluorescence assay. Circular dichroism studies illustrate the inability of Aβ42 to form β-sheet in the presence of 12c, further confirmed by the absence of Aβ42 fibrils in electron microscopy experiments. The peptide exhibits enhanced BBB permeation, no cytotoxicity along with prolonged proteolytic stability. In silico studies show that the peptide interacts with the key amino acids in Aβ, which potentiate its fibrillation, thereby arresting aggregation propensity. This structural class of designed scaffolds provides impetus towards the rational development of peptide-based-therapeutics for Alzheimer's disease (AD).

Trametes ljubarskyi produces multiple laccase isozymes under various physicochemical conditions. During co-cultivation condition Rhodotorula mucilaginosa showed inter-specific interactions with T. ljubarskyi and hypersecretion of laccases; however, the underlying molecular mechanism is less-known. The analysis of proteomics data of co-cultivated cultures revealed the mechanism of metabolic coupling during fungal-yeast interactions. The results suggested high score GO terms related to stimulus-response, protein binding, membrane components, transport channels, oxidoreductases, and antioxidants. The SEM studies confirmed the cellular communication and their inter-specific interactions. This study allows us to deepen and refine our understanding of fungal-yeast symbiotic interaction; further, it also establishes a mutual relation by metabolic coupling for 10-fold higher laccase isozyme secretion (6532 U/ml). The purified laccase isozymes showed acidic pH optima (pH 3–4), higher thermo-stability (60 °C), and broad enzyme kinetics (Km) values. Our study also provides an in-depth understanding of laccase isozymes and their potential to degrade synthetic dyes, which may help the fungi to survive in an adverse environment.

Novel derivatives were synthesized using natural scaffold, like phenylpropanoids C(6)–C(3) backbone to reduce unfavorable browning of food due to tyrosinase and oxidative spoilage. Most of the compounds displayed mushroom tyrosinase inhibition better than kojic acid. Compound CE48 exhibited better anti-tyrosinase (IC(50)-29.64 μM) and antioxidant (EC(50)-12.67 μM) activity than the reference compounds, kojic acid (IC(50)-50.30 μM) and ascorbic acid (EC(50)-14.55 μM), respectively. Compounds SAM30, SE78, 11F, and CE48 showed better anti-B. subtilis, anti-S. aureus, and anti-A. niger activity, respectively, compared to their parents. Molecular docking studies between inhibitors and mushroom tyrosinase corroborated the experimental reports, except SAM30 (glide score − 8.117) and SE78 (glide score − 6.151). In silico absorption, distribution, metabolism, excretion/toxicity (ADME/T) and toxicological studies of these newly synthesized compounds exhibited acceptable pharmacokinetic and safety profiles, like good aqueous solubility (− 3.34 to − 7.57), low human oral absorption (e.g., SAM30, SE78, FAM34), low gut–blood barrier permeability [36.67–209.88 nm/s in Cancer coli-2 (Caco-2) cells] and [19.45–91.51 nm/s in Madin-Darby Canine Kidney (MDCK) cells], low blood–brain barrier penetration, non-mutagenicity, and non-carcinogenicity. Interestingly, the synthesized compounds also possessed multifunctional properties, like microbial growth inhibitor, free radicals scavenger, and it also prevented browning of raw fruits and vegetables by inhibiting tyrosinase enzyme. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02636-0.

Laccases or benzenediol oxygen oxidoreductases (EC 1.10.3.2) are polyphenol multicopper oxidases that are known for their structural and functional diversity in various life forms. In the present study, the molecular and physico-chemical properties (redox-potential and secondary structures) of fungal laccase isozymes (FLIs) isolated from a medicinal mushroom Ganoderma lucidum were analyzed and compared with those of the recombinant bacterial laccases (rLac) obtained from different Yersinia enterocolitica strains. It was revealed that the FLIs contained His-Cys-His as the most conserved residue in its domain I Cu site, while the fourth and fifth residues were variable (Ile, Leu, or Phe). Evidently, the cyclic voltammetric measurements of Glac L2 at Type 1 Cu site revealed greater E° for ABTS/ABTS+ (0.312 V) and ABTS+/ABTS2+ (0.773 V) compared to the E° of rLac. Furthermore, circular dichroism-based conformational analysis revealed structural stability of the FLIs at acidic pH (3.0) and low temperature ( 70 °C). The zymographic studies further confirmed the functional inactivation of FLIs at high temperatures (≥70 °C), predominantly due to domain unfolding. These findings provide novel insight into the evolution of the catalytic efficiency and redox properties of the FLIs, contributing to the existing knowledge regarding stress responses, metabolite production, and the biotechnological utilization of metabolites.

The bio-engineering of nanoparticles for the transportation of various therapeutic agents specifically to brain has sparked a rapidly growing interest in the field of material chemistry. In this study, L-pGlu-(1-benzyl)–L-His–L-ProNH2 (NP-355) and L-pGlu-(2-propyl)–L-His–L-ProNH2 (NP-647) were synthesized and encapsulated in biodegradable poly-lactide-co-glycolide (PLGA) nanoparticles which were further decorated with mucoadhesive surface coating of chitosan. NP-355 and NP-647 which are analogues of thyrotropin releasing hormone, have been reported to demonstrate potential antiepileptic properties against various animal models of seizures. However, their applicability is limited by their short lives due to rapid metabolism and blood brain barrier. The treatment of disorders associated with central nervous system such as epilepsy has been a major challenge for several decades due to difficulty in delivery of drug molecules and imaging agents to brain. To overcome these issues, development of sustained release delivery system for these neuropeptides is proposed which can be administered directly from nose to brain utilizing olfactory nerve channels. The synthesized nanoparticles were evaluated for their physicochemical properties, sustained release properties, toxicity and antiepileptic potential following intranasal administration. The ability of these nanoparticles to reach the brain was evaluated by utilizing quantum dots as fluorescent probes. Our results proved that the polymeric nanoparticles can be used for successful delivery of neurological drugs to the brain.

Opioid peptides are key regulators in cellular and intercellular physiological responses, and could be therapeutically useful for modulating several pathological conditions. Unfortunately, the use of peptide-based agonists to target centrally located opioid receptors is limited by poor physicochemical (PC), distribution, metabolic, and pharmacokinetic (DMPK) properties that restrict penetration across the blood-brain-barrier via passive diffusion. To address these problems, the present manuscript exploits fluorinated peptidomimetics to simultaneously modify PC and DMPK properties, thus facilitating entry into the central nervous system. As an initial example, the present manuscript exploited the Tyr(1)-ψ[(Z)CF=CH]-Gly(2) peptidomimetic to improve PC drug-like characteristics (computational), plasma and microsomal degradation, and systemic and CNS distribution of Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu). Thus, the fluoroalkene replacement transformed an instable in vitro tool compound into a stable and centrally-distributed in vivo probe. In contrast, the Tyr(1)-ψ[CF(3)CH(2)–NH]-Gly(2) peptidomimetic decreased stability by accelerating proteolysis at the Gly(3)–Phe(4) position.

Non-steroidal anti-inflammatory drugs (NSAIDs) can induce small-intestinal injuries through inhibition of prostaglandin synthesis. Gut has an important role in building and maintaining the barriers to avoid the luminal gut microbiota from invading the host, and cytoskeleton plays a crucial role in the maintenance of cellular barrier. The recent advances suggest a bi-directional interaction between the drugs and gut microbiota, where gut microbes can metabolize the drugs, and in response drugs can alter the composition of gut microbiota. In the present study, we evaluated the effect of diclofenac on rat gut, when co-administrated with either Yersinia enterocolitica strain 8081 (an enteropathogen) or Lactobacillus fermentum strain 9338 (a probiotic). The LC-MS/MS based label-free quantitation of rat gut proteins revealed 51.38% up-regulated, 48.62% down-regulated in diclofenac-Y. enterocolitica strain 8081 (D*Y), and 74.31% up-regulated, 25.69% down-regulated in diclofenac-L. fermentum strain 9338 (D*L) experiments. The identified proteins belonged to cytoskeleton, metabolism, heme biosynthesis and binding, stress response, apoptosis and redox homeostasis, immune and inflammatory response, and detoxification and antioxidant defence. Further, the histopathological and biochemical analysis indicated more pronounced histological alterations and oxidative stress (enhanced malonaldehyde and altered antioxidant levels) in D*Y rats than D*L rats, compared to control rats. Elevated plus maze (EPM) test performed to determine the behavioral changes, suggested increased anxiety in D*Y rats than D*L rats, compared to control rats. These results together suggest the differential role of either bacterium in biotransformation of diclofenac, and inflammatory and cellular redox response.

The new structural classes of ultrashort peptides that exhibit potent microbicidal action have potential as future drugs. Herein, we report that C-2 arylated histidines containing tripeptides His(2-Ar)-Trp-His(2-Ar) exhibit potent antifungal activity against Cryptococcus neoformans with high selectivity. The most potent peptide 12f [His(2-biphenyl)-Trp-His(2-biphenyl)] displayed high in vitro activity against C. neoformans (IC50 = 0.35 μg/mL, MIC = MFC = 0.63 μg/mL) with a selectivity index of >28 and 2 times higher potency compared to amphotericin B. Peptide 12f exhibited proteolytic stability, with no apparent hemolytic activity. The mechanism of action study of 12f by confocal laser scanning microscopy and electron microscopy indicates nuclear fragmentation and membrane disruption of C. neoformans cells. Combinations of 12f with fluconazole and amphotericin B at subinhibitory concentration were synergistic against C. neoformans. This study suggests that 12f is a new structural class of amphiphilic pept...

The growing incidents of cryptococcosis in immuno-compromised patients have created a need for novel drug therapies capable of eradicating the disease. The peptide-based drug therapy offers many advantages over the traditional therapeutic agents, which has been exploited in the present study by synthesizing a series of hexapeptides that exhibits promising activity against a panel of Gram-negative and Gram-positive bacteria and various pathogenic fungal strains; the most exemplary activity was observed against Cryptococcus neoformans. The peptides 3, 24, 32 and 36 displayed potent anticryptococcal activity (IC50 = 0.4–0.46 μg/mL, MIC = 0.63–1.25 μg/mL, MFC = 0.63–1.25 μg/mL), and stability under proteolytic conditions. Besides this, several other peptides displayed promising inhibition of pathogenic bacteria. The prominent ones include peptides 18–20, and 26 that exhibited IC50 values ranged between 2.1 and 3.6 μg/mL, MICs of 5–20 μg/mL and MBCs of 10–20 μg/mL against Staphylococcus aureus and methicillin-resistant S. aureus. The detailed mechanistic study on selected peptides demonstrated absolute selectivity towards the bacterial membranes and fungal cells by causing perturbations in the cell membranes, confirmed by the scanning electron microscopy and transmission electron microscopy studies.

We report copper(I)-catalyzed synthesis of 1,2-diaryl histidines via N(1)-arylation of protected 2-aryl histidines in the presence of 8-hydroxyquinoline as ligand and aryl iodides as electrophilic coupling partners, under microwave irradiation at 140 oC. This methodology provides regioselective access to previously inaccessible multi-substituted histidines with intact chirality in high yields. The synthesized 1,2-diarylated histidines are a hitherto unknown class of derivatives with untapped potential in modified amino acids-based peptides and peptidomimetics drug discovery.

As tool compounds to study cardiac ischemia, the endogenous &delta, opioid receptors (&delta, OR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the &delta, OR&rsquo, s protective role during ischemic events. Thus, a need remains for &delta, OR peptides with improved selectivity and unique signaling properties for investigating the specific roles for &delta, OR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to &delta, ORs and &micro, opioid receptors (&micro, ORs) and potency to inhibit cAMP signaling and to recruit &beta, arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the &delta, OR and &micro, OR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

Phenylpropanoids and their derivatives are plant secondary metabolites widely present in fruits, vegetables, cereal grains, beverages, spices and herbs. They are known to have multifaceted effects which include antimicrobial, antioxidant, anti-inflammatory, antidiabetic, anticancer activities and as well as exhibits renoprotective, neuroprotective, cardioprotective and hepatoprotective effects. Owing to their antioxidant, antimicrobial and photoprotective properties, these compounds have wide application in the food (preservation, packaging films and edible coating), pharmaceutical, cosmetic and other industries such as textile (colorant), biofuel (antioxidant additive) and sensors (sensing biologically relevant molecules). Phenylpropanoids are present in commercially available dietary supplements and skin care products. In this review, we have presented the current knowledge on the biosynthesis, occurrence, biological activities of phenylpropanoids and their derivatives, along with the mechanism of action and their potential applications in various industries.

Although, Karanj (Pongamia glabra) cake is an important proteinaceous feed resource for livestock but the presence of toxic principles such as karanjin, pongamol and trypsin inhibitors in raw solvent extracted karanj cake restricts its comprehensive use as safe animal feed resources. In the present investigation solvent extracted raw karanj cake was subjected to in vitro studies by incorporation in the concentrate mixture at four graded levels replacing soy bean meal on protein equivalent basis (25, 50, 75 and 100%) in order to understand the rumen fermentation pattern. The inclusion of graded levels of solvent extracted raw karanj cake in the concentrate mixture replacing protein in SBM even at 100% resulted no significant difference in rumen in vitro fermentation pattern in terms of substrate degradation and gas volume produced in 24 h, truly degradable organic matter in rumen (TDOMR; mg/ 200mg), TDOMR%, microbial biomass production (MBP; mg/200mg), efficiency of microbial biomass production (MBP/100mg TDOMR) and partitioning factor (PF).

Synthetic dyes are toxic and carcinogenic in nature, which also causes environmental pollution. The present study was aimed to decolorize various commercial dyes using purified recombinant bacterial laccases. Laccase gene from Yersinia enterocolitica strain 8081 (yacK), Y. enterocolitica strain 7 (yacK) and Bacillus pumilus DSKK1 was cloned in vector pET28a and overproduced in host Escherichia coli BL21. The high yield of recombinant laccase protein resulted in the formation of inclusion bodies, which were further solubilized, refolded, and purified. The purified recombinant laccases were alkali-tolerant and thermostable, with pH optima at 7–8, temperature optima at 60–70 °C and low redox potential. For in silico studies, laccase protein models of B. pumilus DSKK1, Y. enterocolitica strain 7 and Y. enterocolitica strain 8081 were docked with commercial dyes. This is the first and foremost study where the stability of docked complexes of pathogenic and non-pathogenic microorganism has been explored via molecular dynamics (MD) simulations using Gromacs version 4.5.5 with the gromos96 43a force field. Finally, the in silico results were validated experimentally and it was found that purified laccases from B. pumilus DSKK1 and Y. enterocolitica strain 7 efficiently decolorized rose bengal (90.4%), malachite green (77.7%), and congo red (74.5%) dyes.

The inhibition of amyloid-β (Aβ) aggregation is a promising approach towards therapeutic intervention for Alzheimer’s disease (AD). Thirty eight tetrapeptides based upon Aβ39-42 C-terminus fragment of the parent Aβ peptide were synthesized. The sequential replacement/modification employing unnatural amino acids imparted scaffold diversity, augmented activity, enhanced blood brain barrier permeability and offered proteolytic stability to the synthetic peptides. Several peptides exhibited promising protection against Aβ aggregation-mediated-neurotoxicity in PC-12 cells at doses ranged between 10 μM and 0.1 μM, further confirmed by the thioflavin-T fluorescence assay. CD study illustrate that these peptides restrict the β-sheet formation, and the non-appearance of Aβ42 fibrillar structures in the electron microscopy confirm the inhibition of Aβ42 aggregation. HRMS and ANS fluorescence spectroscopic analysis provided additional mechanistic insights. Two selected lead peptides 5 and 16 depicted enhanced blood-brain penetration and stability against serum and proteolytic enzyme. Structural insights into ligand-Aβ interactions on the monomeric and proto-fibrillar units of Aβ were computationally studied. Promising inhibitory potential and short sequence of the lead peptides offers new avenues for the advancement of peptide-derived therapeutics for AD.

Thyrotropin-Releasing Hormone (TRH), a tripeptide amide with molecular formula L-pGlu-L-His-L- Pro-NH2, is used in the treatment of brain/spinal injury and certain central nervous system (CNS) disorders, including schizophrenia, Alzheimer's disease, epilepsy, depression, shock and ischemia due to its profound effects on the CNS. However, TRH's therapeutic activity is severely hampered because of instability and hydrophilicity owing to its peptidic nature which results into ineffective penetration into the blood brain barrier. In the present study, we report the synthesis and stability studies of novel chitosan engineered TRH encapsulated poly(lactide-co-glycolide) (PLGA) based nanoformulation. The aim of such an encapsulation is to allow effective delivery of TRH in biological systems as the peptidase degrade naked TRH. The synthesis of TRH was carried out manually in solution phase followed by its encapsulation using PLGA to form polymeric nanoparticles (NPs) via nanoprecipitation technique. Different parameters such as type of organic phase, concentration of stabilizer, ratio of organic phase and aqueous phase, rate of addition of organic phase were optimized, tested and evaluated for particle size, encapsulation efficiency, and stability of NPs. The TRH-PLGA NPs were then surface modified with chitosan to achieve positive surface charge rendering them potential membrane penetrating agents. PLGA, PLGA-TRH, Chitosan-PLGA and Chitosan-PLGA-TRH NPs were characterized and analyzed using Dynamic Light Scattering (DLS), Transmissiom Electron Microscopy (TEM) and Infra-red spectroscopic techniques.

Sixty fungal cultures were isolated from agricultural soil, industrial soil, forest canopy soil having decomposed leaf litter and compost samples collected from different regions of India. Fifteen fungal cultures were selected qualitatively for the production of xylanase and cellulases and were identified employing ITS, NS and MNS primers. The enzyme cocktail consisting of 3811 IU g(-1) of xylanase and 9.9 IU g(-1) of cellulase from Trichoderma longibrachiatum MDU-6 was selected quantitatively for the deinking of diverse paper wastes. The enzyme production increased two fold when produced at tray level in comparison with flasks. The enzyme cocktail was effective in the deinking of old newspaper samples with significant removal of chromophores, phenolics and hydrophobic compounds and less sugar loss. While in case of examination papers and laser printed papers, ink removal was not very significant. Moreover, the sugar loss was significantly high in case of examination papers. The deinking results were further confirmed with FTIR analysis. Deinked newspaper pulp sample shows brightness of 52%, which was 9.6% high than its control sample. The ERIC value for deinked newspaper pulp was found to be 655.9 ppm. Thereafter, the deinked newspaper pulp was examined under light microscope after differential staining with safranin and malachite green and also examined under scanning and transmission electron microscope, which revealed fibrillation and perforation.

Due to the negative health and environmental consequences of fossil fuels, lignocellulosic ethanol has garnered a great interest as a clean and potent renewable energy resource. The presence of excess amount of water hinders the application of ethanol produced, necessitating the implication of robust recovery and purification techniques. Typically, ethanol recovery starts with a conventional distillation process yielding azeotropic ethanol. Further, dehydration and purification steps are employed to achieve fuel-grade ethanol, which consumes tremendous amount of energy and incur high operational cost; eventually limits the economic feasibility of lignocellulosic ethanol on commercial scale. Therefore, to overcome such obstacles, the synergetic application of distillation or adsorption with membrane-mediated processes seems to be a promising approach in terms of energy conservation, cost-effectiveness, and environmental security. In this review article, we have made an appraisal of the past and current purification and recovery technologies for first generation ethanol, which can be a learning trend for the recovery of lignocellulosic ethanol from the fermented broth, with desired purity.

Ganoderma lucidum is an important medicinal fungus that possesses exceedingly high lignocellulose degrading ability. Evidently, Cu2+ has decisive roles on the mycelial growth and enzyme production. To reveal the effect of Cu2+ on G. lucidum transcriptome, predominantly associated with lignocellulolytic progression, we conducted comparative NGS based de novo transcriptome assembly using Illumina Hi SeqTM sequencing platform. We obtained 26,083,372 and 35,713,076 high-quality reads from induced and uninduced cultures. For wood degrading activity, 194 transcript coding for oxidoreductases and 402 transcripts for CAZymes were predicted. Further, secretome studies revealed high score GO terms related to oxidoreductases, glycosyl hydrolases, and chitinases from Cu-induced mycelia. The increased Cu2+ concentrations showed higher secretion of lignocellulases such as laccases, cellulases, and xylanases along with increased production of phenolics and antioxidants. Several differences in the transcriptomic and proteomic signatures for lignocellulolytic enzymes provide vital clues about Cu2+ mediated gene regulation and metabolic pathways in basidiomycetous fungi.

An environmentally benign, mild, cost-effective and gram-scalable copper-catalyzed method for the N-(hetero)arylation of zwitterionic natural and unnatural amino acids using 2-isobutyrylcyclohexanone as a β-diketone ligand and aryl bromides as coupling partners in water for 50 min at 90 °C under microwave irradiation is reported. Electronically and sterically diverse aryl coupling partners were inserted efficiently, including challenging heteroaryl electrophilic partners in high yields without affecting the enantiopurity of the product.

The YacK gene from Yersinia enterocolitica strain 7, cloned in pET28a vector and expressed in Escherichia coli BL21 (DE3), showed laccase activity when oxidized with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and guaiacol. The recombinant laccase protein was purified and characterized biochemically with a molecular mass of ≈58 KDa on SDS-PAGE and showed positive zymogram with ABTS. The protein was highly robust with optimum pH 9.0 and stable at 70 °C upto 12 h with residual activity of 70%. Kinetic constants, Km values, for ABTS and guaiacol were 675 μM and 2070 μM, respectively, with corresponding Vmax values of 0.125 μmol/ml/min and 6500 μmol/ml/min. It also possess antioxidative property against BSA and Cu(2+)/H2O2 model system. Constant pH MD simulation studies at different protonation states of the system showed ABTS to be most stable at acidic pH, whereas, diclofenac at neutral pH. Interestingly, aspirin drifted out of the binding pocket at acidic and neutral pH, but showed stable binding at alkaline pH. The biotransformation of diclofenac and aspirin by laccase also corroborated the in silico results. This is the first report on biotransformation of non-steroidal anti-inflammatory drugs (NSAIDs) using recombinant laccase from gut bacteria, supported by in silico simulation studies.

Strain of Ganoderma lucidum MDU-7 produce multiple extracellular isoforms of laccase in submerged culture condition using malt extract as a carbon source and copper sulfate as an inducer. SDS–PAGE followed by MALDI–TOF peptide fingerprinting confirmed laccase isozyme with molecular mass of 24–66 kDa. Two laccase isozymes (Glac H1 and Glac L1) were purified from native-PAGE protein purification method and a comparative catalytic and antioxidant study has been performed. Both of the laccase isozymes have optimum temperature and pH at 50 °C and 4.0, respectively. Glac L1 has higher stability in comparison to Glac H1, over wide range of temperature, pH, divalent metal ions and surfactants. The Km values of Glac L1 and Glac H1 determined for guaiacol, ABTS and O-tolidine were 98 μM, 26 μM, 320 μM and 281 μM, 29 μM, 338 μM, respectively. Glac H1, irrespective to its laccase activity and stability, acts as a better antioxidant than Glac L1.

Abiotic stress impacts on plants are inevitable, and are mainly the result of impairments in cellular redox homeostasis. In fact, abiotic stress-provoked elevations in reactive oxygen species (ROS) are the main driver of cellular redox homeostasis. In addition to reviewing ROS, their chemistry, and sites of production; this chapter highlights the dual roles of ROS in separate sections, and discusses in detail the role and modulation of major enzymatic and nonenzymatic components of antioxidant defense systems involved in cellular redox regulation in abiotic-stressed plants.

A mild and efficient method for the N-arylation of zwitterionic amino acids, amino acid esters and peptides is described. The procedure provides the first room temperature synthesis of N-arylated amino acids and peptides using CuI as a catalyst, diketone as a ligand, and aryl iodides as coupling partners. The method is equally applicable for using relatively inexpensive aryl bromides as coupling partners at 80 °C. Using this procedure, electronically and sterically diverse aryl halides, containing reactive functional groups were efficiently coupled in good to excellent yields.

Direct, regioselective and metal-free synthesis of fused N-heterocyclic iodides is reported. This regioselective C-H functionalization is mediated by tert-butylhydroperoxide (TBHP), via dual activation of molecular iodine and a heterocyclic substrate, resulting in the in situ generation of electrophilic iodine species (I(+)), and free radical(s) (t)BuO˙ or (t)BuOO˙, driving the iodination reaction.

Laccase in Cryptococcus neoformans is covalently linked to the carbohydrate moiety of the cell wall, which allows it to get access to the different substrates for catalyzing their oxidation and therefore plays a vital role in the virulence. The laccase gene (3.0 kb) from C. neoformans serotype D was amplified, cloned and sequenced for protein modeling, docking and simulation studies. The three dimensional homology models of laccase protein from C. neoformans and other pathogenic gut bacteria were docked with selected biomolecules like prostaglandins (PG), membrane phospholipids, neurotransmitters (serotonin) using GOLD software. The GOLDscore values of laccase from C. neoformans docked with prostaglandinH2 (59.76), prostaglandinG2 (59.45), prostaglandinE2 (60.99), phosphatidylinositol (54.95), phosphatidylcholine (46.26), phosphatidylserine (55.26), arachidonic acid (53.08) and serotonin (46.22) were similar to the laccase from enteropathogenic bacteria but showed a better binding affinity as compared to that of the non-pathogenic bacteria (e.g. Bacillus safensis, Bacillus pumilus and Bacillus subtilis). The RMSD of MD simulation study done for 25 ns using laccase protein from C. neoformans complexed with phosphatidylcholine was found to be highly stable, followed by the laccase-PGE2 and laccase-serotonin complexes. Furthermore, the binding free energy results were found to support the docking and MD simulation results. The present study implies that few candidate ligands can be intermediate substrate in the catalysis of microbial laccases, which can further play some crucial role in the cell signaling and pathogenesis of enteropathogenic gut micro flora and C. neoformans.

Basidiomycetous fungi, Ganoderma lucidum MDU-7 and Ganoderma sp. kk-02 secreted multiple laccase isozymes under diverse growth condition. Aromatic compounds and metal salts were also found to regulate the differential expression of laccase isozymes from both the Ganoderma sp. Laccase isozymes induced in the presence of copper from G. lucidum MDU-7 were purified by gel-based (native-PAGE) purification method. The purity of laccase isozymes was checked by zymogram and SDS-PAGE. The SDS-PAGE of purified proteins confirmed the multimeric nature of laccase isozymes. The molecular mass of isozymes was found to be in the range of 40 kDa to 66 kDa. Further, the purified laccase isozymes and their peptides were confirmed with the help of MALDI-TOF peptide fingerprinting. The biochemical characterization of laccase isozymes viz. Glac L2, Glac L3, Glac L4 and Glac L5 has shown the optimum temperature in the range of 30 oC to 45 oC and pH 3.0. The Km values of all the laccase isozymes determined for guaiacol were (96 µM - 281 µM), ABTS (15 µM - 83 µM) and O-tolidine (78 µM - 724 µM). Further, laccase isozymes from G. lucidum whole genome were studied using bioinformatics tools. The molecular modeling and docking of laccase isozymes with different substrates showed a significant binding affinity, which further validates our experimental results. Interestingly, copper induced laccase of 40 U/ml in culture medium was found to significantly induce cotton callogenesis. Interestingly, all the laccase isozymes were found to have an antioxidative role and therefore capable in free radicals scavenging during callogenesis. This is the first detailed study on the biochemical characterization of all the laccase isozymes purified by a gel-based novel method.

Despite immense advances in the treatment strategies, management of neuropathic pain remains unsatisfactory. Piracetam is a prototype of nootropic drugs, used to improve cognitive impairment. The present study was designed to investigate the effect of piracetam on peripheral neuropathic pain in rats. Neuropathic pain was induced by the chronic constriction injury of the sciatic nerve. Following this, piracetam was intraperitoneally administered for 2 weeks in doses of 50, 100 and 200 mg/kg, and pain was assessed by employing the behavioural tests for thermal hyperalgesia (hot plate and tail flick tests) and cold allodynia (acetone test). After the induction of neuropathic pain, significant development of thermal hyperalgesia and cold allodynia was observed. The administration of piracetam (50 mg/kg) did not have any significant effect on all the behavioural tests. Further, piracetam (100 mg/kg) also had no effect on the hot plate and tail flick tests; however it significantly decreased the paw withdrawal duration in the acetone test. Piracetam in a dose of 200 mg/kg significantly modulated neuropathic pain as observed from the increased hot plate and tail flick latencies, and decreased paw withdrawal duration (in acetone test). Therefore, the present study suggests the potential use of piracetam in the treatment of neuropathic pain, which merits further clinical investigation.

Gold (Au) catalysts have been rarely investigated for the oxidation of glucose in the absence of a base. These conditions are critical, however, to enable the sequential one-pot combination of cellulose hydrolysis and glucoseoxidation. Here we evaluate the catalytic performance and stability of Au nanoparticles supported on metal oxides for the oxidation of glucose to gluconic acid under unadjusted pH and acidic conditions. The study provides insights into the deactivation of the catalysts caused by leaching and hydrothermal sintering of Au nanoparticles, as well as by adsorption of reactive species. We found that lowering the surface density of Au on metal oxides decreases the sintering rate of the Au nanoparticles and hence enhances the stability and activity of the catalysts.

In this communication, we report the design, synthesis and in vitro antimicrobial activity of ultra short peptidomimetics. Besides producing promising antibacterial activities against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), the dipeptidomimetics exhibited high antifungal activity against C. neoformans with IC50 values in the range of 0.16–19 μg mL−1. The most potent analogs exhibited 4-fold higher activity than the currently used drug amphotericin B, with no apparent cytotoxicity in a panel of mammalian cell lines.

Effects of varied dietary energy densities on immune response and performance of Muzzafarnagari lambs were ascertained in a 180-d study. Animals (n = 24), in three groups, were fed diets providing 100% (100E), 80% (80E) or 70% (70E) of their metabolizable energy requirement. Mean nutrient digestibilities varied significantly among treatments. Nitrogen intake was lower (p

We report regioselective N(1)-arylation of protected histidine using copper(I) iodide as a catalyst, trans-N,N′-dimethylcyclohexane-1,2-diamine as a ligand and readily available aryl iodides as coupling partners under microwave irradiation at 130 °C for 40 min. The reaction provides rapid access to electron-donating, electron-withdrawing and bulky group substituted N-arylated histidines in high yields, including previously inaccessible N-heteroaryl histidines. These N(1)-(hetero)aryl histidines are promising building blocks in peptide-based drug design and discovery.

Rapid microwave-assisted solution-phase peptide synthesis protocol has been developed. The reaction temperature of 50 °C, 40 W and short coupling time of 28 min gave racemization-free synthesis of peptides in 50–80% yield. The method is applicable with equal ease in coupling both side-chain protected and unprotected amino acids indicating reactive-functional group tolerance and high atom-economy. In the case of dipeptides, DIC/HONB-mediated coupling rendered the best results. In the case of tri- and tetrapeptides, HATU/HOAt/DIEA emerged as the best coupling combination.

A new, improved and cost effective protocol for extraction of pure, high molecular weight metagenomic DNA from soil samples using ligninolytic enzymes has been developed. DNA yield of 17 μg g−1 of soil from termite nest soil and 25 μg g−1 from forest soil and size of 15–20 kb was achieved by the optimized protocol. More than 90% of DNA was recovered after purification and there was 81% reduction in humic acid content. The strategy allowed processing of several samples at a given time and yielded purified DNA. The isolated metagenomic DNA sample could be readily processed for restriction digestion and PCR amplification. The quantitative PCR revealed CP value of 21 for the treated samples whereas the untreated samples could not cross the threshold fluorescence even after 45 cycles. This clearly indicated that the isolated metagenomic DNA is amenable for molecular manipulations.

Propoxur (a carbamate pesticide) has been shown to adversely affect memory and induce oxidative stress on both acute and chronic exposure. This study was designed to explore the modulation of the effects of propoxur over cognitive function by melatonin (MEL). Cognitive function was assessed using step-down latency (SDL) on a passive avoidance apparatus, and transfer latency (TL) on an elevated plus maze. Oxidative stress was assessed by examining brain malondialdehyde (MDA) and reduced glutathione (GSH) levels and catalase (CAT) activity. A significant reduction in SDL and prolongation of TL was observed for the propoxur (10 mg/kg/d; p.o.) treated group at weeks 6 and 7 when compared with control. One week treatment with MEL (50 mg/kg/d; i.p.) antagonized the effect of propoxur on SDL, as well as TL. Propoxur produced a statistically significant increase in the brain MDA levels and decrease in the brain GSH levels and CAT activity. Treatment with MEL attenuated the effect of propoxur on oxidative stress. The results of the present study thus show that MEL has the potential to attenuate cognitive dysfunction and oxidative stress induced by toxicants like propoxur in the brain.

We report on the synthesis and catalytic properties of a class of hybrid organic–inorganic mesoporous bifunctional heterogeneous catalysts for efficient catalysis of two-step tandem reactions in one pot. With a solvent-assisted grafting method, two different catalytic groups, that is, an organoamine and a palladium–organodiamine complex, were immobilized onto high-surface-area mesoporous silica sequentially, by using their corresponding organosilanes in 2-propanol or toluene as the solvent in the first step and toluene as the solvent in the second step. Both MCM-41 and SBA-15 mesoporous silicas were used as support materials for the two catalytic groups, and the effect of different sequential grafting of the two organosilanes in 2-propanol or toluene on the structures and the catalytic properties of the resulting bifunctional catalysts were investigated. By using the resulting amine/PdII–diamine bifunctional mesoporous material as catalyst, the occurrence of two very important C–C bond forming reactions, that is, the Sonogashira and the Henry reactions, was demonstrated in one pot for the first time. Yields of approximately 100 % in 2.5 h for the Sonogashira reaction and approximately 100 % in 45 min for the Henry reaction were obtained in the presence of the bifunctional catalyst when the reactions were run individually. When the bifunctional catalyst was used to catalyze the Sonogashira–Henry reactions in tandem in one pot, a yield of up to approximately 60 % of the Sonogashira–Henry product in 5 h was obtained. The synthesis and use of such bifunctional catalysts for efficient catalysis of the tandem reaction is of importance in three significant ways: it prevents the unnecessary use of solvents and other chemicals required for the purification of the intermediate products of either individual reaction, eliminates some of the work-up procedures, and lowers the cost of synthesis of the final product.

Epilepsy is a chronic neurological disorder affecting 1% population worldwide. A number of experimental studies have reported anticonvulsant, neuroprotective and antioxidant activity of certain natural products like curcumin, an active ingredient of turmeric. The present study was designed to explore the effect of acute administration of curcumin at doses 50, 100 and 200 mg/kg, orally (p.o.) pentylenetetrazole-induced kindling in mice. Further two oxidative stress markers viz., malondialdehyde (MDA) and glutathione were estimated in brain tissues of rodents. Curcumin (50, 100 and 200 mg/kg, p.o.) dose dependently suppressed the progression of kindling in mice. In addition, the increased levels of MDA and glutathione were also reduced by curcumin in kindled animals. These results suggest that curcumin appears to possess protective activity against kindling in mice.

The present study was performed to examine the effect of Eugenia caryophyllata (Myrtaceae) on learning and memory, and also evaluate whether it can modulate oxidative stress in mice. Passive avoidance step-down task and elevated plus-maze were used to assess learning and memory in scopolamine-treated mice. Oxidative stress parameters were also assessed in brain samples by estimating the malondialdehyde (MDA) and reduced glutathione (GSH) levels at the end of the study. Scopolamine (0.3 mg/kg, i. p.) produced impairment of acquisition memory as evidenced by a decrease in step-down latency and an increase in transfer latency on day 1, and also impairment of retention of memory on day 2. Pretreatment with clove oil (0.05 mL/kg and 0.1 mL/kg) for 3 weeks significantly reversed the increase in acquisition latency and all the doses (0.025, 0.05, 0.1 mL/kg, i. p.) reversed the increase in retention latency induced by scopolamine (0.3 mg/kg, i. p.) in elevated plus-maze. However, 0.05 mL/kg clove oil attenuated memory deficits in the passive avoidance step-down task. Brain samples showed a significant decrease in MDA levels in the group treated with clove oil (0.05 and 0.025 mL/kg). GSH levels were also increased in clove oil-treated mice though the results were not significant. Thus, it can be concluded that clove oil can reverse the short-term and long-term memory deficits induced by scopolamine (0.3 mg/kg, i. p.) and this effect can, to some extent, be attributed to decreased oxidative stress.

We report a method for continuously producing the nitroaldol, the nitrostyrene, or the Michael product by performing the Henry reaction over a fixed bed reactor that is packed with primary or secondary amine-functionalized nanoporous materials. The % conversion of the reactants as well as the % selectivity to the particular product were found to be strongly dependent on the residence time of the reactants in the reactor (weight hourly spatial velocity or WHSV) as well as the type of reactant, catalyst and reaction temperature used. When a 0.08 M p-hydroxybenzaldehyde solution in nitromethane was passed over the fixed bed reactor containing primary amine-functionalized mesoporous silica catalyst by postgrafting in toluene (AP-T) at 90 °C with WHSV of 0.20, the reactor continuously and selectively generated for hours the p-hydroxy-β-nitrostyrene product with 100% selectivity at 31% reactant conversion (or with 90% selectivity at 88% reactant conversion for WHSV of 0.10). The remaining 12% product in the latter case was the Michael product. The corresponding primary amine-functionalized sample prepared by postgrafting of 3-aminopropyltrimethoxysilane (APTS) in isopropanol (AP-I) also gave similar results with slightly higher efficiency and selectivity to p-hydroxy-β-nitrostyrene. When the same reactant solution was passed over the bed-reactor packed with secondary amine grafted mesoporous silica catalyst by postgrafting in toluene (MAP-T) with WHSV of 0.25 at 90 °C, the reactor also continuously produced selectively the p-hydroxy-β-nitrostyrene product but less efficiently; i.e. with 91% selectivity at 21 reactant conversion for WHSV of 0.20 (or with 85% selectivity at 34% reactant conversion for WHSV of 0.10). Here also, the remaining product was the Michael addition product. Increasing the reaction temperature of the reactor containing the primary amine catalyst to 150 °C at WHSV of 0.10 for p-hydroxybenzaldehyde reactant led to the reversal of the product type from being 90% p-hydroxy-β-nitrostyrene to >85% Michael product with ∼100% reactant conversion. Raising the reaction temperature of the reactor containing a secondary amine catalyst for p-hydroxybenzaldehyde reactant also increasingly favored the formation of the Michael product. When changing the reactant to 0.08 M p-nitrobenzaldehyde, the reactor packed with secondary amine catalyst resulted in the nitroalcohol product with 90% selectivity at 40% reactant conversion for WHSV of 0.15. These results indicate that higher WHSV lead to greater selectivity to a particular product; however, lower WHSV and higher temperatures favor greater reactant conversion reaching as high as ∼100% in all the cases although they can be accompanied by less % selectivity. By simply adjusting the WSHV's or the temperatures to optimum values, one of the products can be exclusively generated in a continuous manner. The continuous reactor and the catalysts were proven to catalyze the reactions and give the respective product(s) continuously for several days. This method can be used as a route for the mass production of industrially and pharmaceutically important p-substituted nitroalcohol, nitrostyrene, or Michael addition product with high selectivity, by simply packing mesoporous catalysts within a fixed bed reactor.

Combination of opioid and potassium channel openers holds immense potential for the treatment for most acute and chronic pain. Therefore, the study was performed to assess the interaction between morphine and K(+) -channel openers. Swiss albino mice of either sex weighing between 25 and 30 g were used for the study. The study assesses the interaction between morphine and K(+) -channel openers (cromakalim, diazoxide and minoxidil), when administered intraperitoneally, using formalin and tail-flick tests in mice. Both morphine and K(+) -channel openers produced significant antinociception at higher doses in both the behavioral tests. Lower doses of morphine and K(+) -channel openers had no significant effect on tail-flick latency, while the same drugs had significant antinociceptive effect on formalin test. The combination of lower doses of morphine and K(+) -openers was observed to have significant antinociceptive effect in both the behavioral tests. Administration of naloxone prior to morphine or K(+) -channel openers antagonized the analgesic effect of morphine but not of K(+) -channel openers, whereas prior administration of glibenclamide antagonized the effect of both morphine and K(+) -channel openers. The study, therefore, suggests that the common site of action of morphine and K(+) -channel openers is at the levels of K(+) -channels rather than at the level of receptors. However, such interaction depends on the differential sensitivity to different pain stimulus.

The newspaper is comprised of (w w −1 ) holocellulose (70.0%) with substantial amount of lignin (16.0%). Bioconversion of the carbohydrate component of newspaper to sugars by enzymatic saccharification, and its fermentation to ethanol was investigated. Of various enzymatic treatments using cellulase, xylanase and laccase, cellulase enzyme system was found to deink the newspaper most efficiently. The saccharification of deinked paper pulp using enzyme cocktail containing exoglucanase (20 U g −1 ), β-glucosidase (60 U g −1 ) and xylanase (80 U g −1 ) resulted in 59.8% saccharification. Among additives, 1% (v v −1 ) Tween 80 and 10 mol m −3 CoCl 2 improved the enzymatic hydrolysis of newspaper maximally, releasing 14.64 g L −1 sugars. The fed batch enzymatic saccharification of the newspaper increased the sugar concentration in hydrolysate from 14.64 g L −1 to 38.21 g L −1 . Moreover, the batch and fed batch enzymatic hydrolysates when fermented with Saccharomyces cerevisiae produced 5.64 g L −1 and 14.77 g L −1 ethanol, respectively.