Prediction of virus-host interactions and identification of hot spot residues of DENV-2 and SH3 domain interactions.

Dengue virus, particularly serotype 2 (DENV-2), poses a significant global health threat, and understanding the molecular basis of its interactions with host cell proteins is imperative for developing targeted therapeutic strategies. This study elucidated the interactions between proline-enriched motifs and Src homology 3 (SH3) domain. The SH3 domain is pivotal in mediating protein-protein interactions, particularly by recognizing and binding to proline-rich regions in partner proteins. Through a computational pipeline, we analyzed the interactions and binding modes of proline-enriched motifs with SH3 domains, identified new potential DENV-2 interactions with the SH3 domain, and revealed potential hot spot residues, underscoring their significance in the viral life cycle. This comprehensive analysis provides crucial insights into the molecular basis of DENV-2 infection, highlighting conserved and serotype-specific interactions. The identified hot spot residues offer potential targets for therapeutic intervention, laying the foundation for developing antiviral strategies against Dengue virus infection. These findings contribute to the broader understanding of viral-host interactions and provide a roadmap for future research on Dengue virus pathogenesis and treatment.

Faecal microbial transfer and complex carbohydrates mediate protection against COPD.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DESIGN: Using an in vivo mouse model of cigarette smoke (CS)-induced COPD and faecal microbial transfer (FMT), we characterised the faecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in 16 patients with COPD using a randomised, double-blind, placebo-controlled pilot study of inulin supplementation. RESULTS: FMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation, and transfer of CS-associated microbiota after antibiotic-induced microbiome depletion was sufficient to increase lung inflammation while suppressing colonic immunity in the absence of CS exposure. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in CS-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. CONCLUSION: The gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically.

Prokaryotic ncRNAs: Master regulators of gene expression.

ncRNA plays a very pivotal role in various biological activities ranging from gene regulation to controlling important developmental networks. It is imperative to note that this small molecule is not only present in all three domains of cellular life, but is an important modulator of gene regulation too in all these domains. In this review, we discussed various aspects of ncRNA biology, especially their role in bacteria. The last two decades of scientific research have proved that this molecule plays an important role in the modulation of various regulatory pathways in bacteria including the adaptive immune system and gene regulation. It is also very surprising to note that this small molecule is also employed in various processes related to the pathogenicity of virulent microorganisms.

Dynamics of the Queensland Fruit Fly Microbiome through the Transition from Nature to an Established Laboratory Colony.

The transition from nature to laboratory or mass rearing can impose significant physiological and evolutionary impact on insects. The Queensland fruit fly (also known as 'Qfly'), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is a serious economic pest that presents major challenges for horticulture industries in Australia. The sterile insect technique (SIT) is being developed to manage outbreaks in regions that remain free of Qfly and to suppress populations in regions where this species is endemic. The biology of Qfly is intimately connected to its microbiome. Therefore, changes in the microbiome that occur through domestication have implications for SIT. There are numerous studies of the microbiome in Qfly larvae and adults, but there is little information on how the microbiome changes as Qfly laboratory colonies are established. In this study, high-throughput Illumina sequencing was used to assess the Qfly microbiome in colonies reared from wild larvae, collected from fruit, for five generations, on a gel-based larval diet. Beta diversity analysis showed that the bacterial communities from Generation 5 (G5) clustered separately from earlier generations. At the genus level, bacterial communities were significantly different between the generations and mostly altered at G5. However, communities were found similar at phyla to family taxonomic levels. We observed high abundance of Morganella and Burkholderia at the genus level in the larval and pupal stages respectively at G5, but these were not detected in earlier generations. Overall, our findings demonstrate that the domestication process strongly affects the Qfly microbiome and prompts questions about the functional relationship between the Qfly and its microbiome, as well as implications for the performance of insects that have been domesticated and mass-reared for SIT programs.

Oral Administration of Live and Dead Cells of Lactobacillus sakei proBio65 Alleviated Atopic Dermatitis in Children and Adolescents: a Randomized, Double-Blind, and Placebo-Controlled Study.

Several studies suggest that probiotics might be useful in the management of atopic dermatitis (AD). However, the efficacy and comparison between both the administration of viable and non-viable probiotics on alleviation of AD is not well studied. Therefore, the purpose of this study was to evaluate the effect of L. sakei proBio65 live and dead cells when administered (1 × 1010 cells/day) for 12 weeks to children and adolescents (aged 3 to 18) with atopic dermatitis. In this randomized double-blind, placebo-controlled study, ninety patients were recruited and randomly allocated to either the L. sakei proBio65 live cells, L. sakei proBio65 dead cells, or placebo groups. Assessment of efficacy was based on the change in SCORing Atopic Dermatitis (SCORAD) score, Investigators Global Assessment (IGA) score, serum inflammatory markers such as the serum eosinophil (count), IgE, eosinophil cationic protein (ECP), CCL17 (thymus and activation-regulated chemokine [TARC]), and CCL27 (cutaneous T cell-attracting chemokine [CTACK]), and changes in skin condition (moisture and sebum) at baseline, week 6 and week 12. The SCORAD total score decreased in the live cells (p = 0.0015) and dead cell group (p = 0.0017) from the baseline after 12 weeks, whereas there were no significant changes in the placebo group when compared with baseline. The skin sebum content increased in both the live cell (p < 0.0001) and the dead cell group (p < 0.0001), suggesting potential improvements in skin barrier functions. Current data suggested a positive improvement in alleviation of AD symptoms upon oral administration of L. sakei proBio65 in both viable and non-viable forms.

Ribosylation induced structural changes in Bovine Serum Albumin: understanding high dietary sugar induced protein aggregation and amyloid formation.

Non-enzymatic glycation of proteins is believed to be the root cause of high dietary sugar associated pathophysiological maladies. We investigated the structural changes in protein during progression of glycation using ribosylated Bovine Serum Albumin (BSA). Non enzymatic attachment of about 45 ribose molecules to BSA resulted in gradual reduction of hydrophobicity and aggregation as indicated by red-shifted tryptophan fluorescence, reduced ANS binding and lower anisotropy of FITC-conjugated protein. Parallely, there was a significant decrease of alpha helicity as revealed by Circular Dichroism (CD) and Fourier transformed-Infra Red (FT-IR) spectra. The glycated proteins assumed compact globular structures with enhanced Thioflavin-T binding resembling amyloids. The gross structural transition affected by ribosylation led to enhanced thermostability as indicated by melting temperature and Transmission Electron Microscopy. At a later stage of glycation, the glycated proteins developed non-specific aggregates with increase in size and loss of amyloidogenic behaviour. A parallel non-glycated control incubated under similar conditions indicated that amyloid formation and associated changes were specific for ribosylation and not driven by thermal denaturation due to incubation at 37 °C. Functionality of the glycated protein was significantly altered as probed by Isothermal Titration Calorimetry using polyphenols as substrates. The studies demonstrated that glycation driven globular amyloids form and persist as transient intermediates during formation of misfolded glycated adducts. To the best of our knowledge, the present study is the first systematic attempt to understand glycation associated changes in a protein and provides important insights towards designing therapeutics for arresting dietary sugar induced amyloid formation.

Artificial Larval Diet Mediates the Microbiome of Queensland Fruit Fly.

Larval diets used for artificial rearing can have a significant effect on insect biology. The Queensland fruit fly (aka "Qfly"), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is one of the greatest challenges for fruit growers in Australia. The sterile insect technique (SIT) is being developed to manage outbreaks in regions that remain free of Qfly and to reduce populations in regions where this species is endemic. Factory scale rearing is essential for SIT; however, artificial larval diets are known to affect the microbiome of Qfly, which may then affect fly performance. In this study, high-throughput Illumina sequencing was used to assess the Qfly microbiome in colonies reared, for five generations from nature, on two common artificial diets (carrot and gel). At generation five (G5), the microbiome was assessed in larvae, pupae, adult males and adult females and standard fly quality control parameters were assessed together with additional performance measures of mating propensity and survival under nutritional stress. At the genus level, bacterial communities were significantly different between the colonies reared on the two larval diets. However, communities converged at Phyla to family taxonomic levels. Bacterial genera of Morganella, Citrobacter, Providencia, and Burkholderia were highly abundant in all developmental stages of Qfly reared on the gel diet, when compared to the carrot diet. Despite abundance of these genera, a greater percentage of egg hatching, heavier pupal weight and a higher percentage of fliers were found in the Qfly reared on the gel diet. Mating propensity and survival under nutritional stress was similar for adult Qfly that had been reared on the two larval diets. Overall, our findings demonstrate that the artificial larval diet strongly influences the microbiome and quality control measures of Qfly, with likely downstream effects on performance of flies released in SIT programs.

Fruit host-dependent fungal communities in the microbiome of wild Queensland fruit fly larvae.

Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect 'host'.

Microbiome of the Queensland Fruit Fly through Metamorphosis.

Bactroceratryoni (Froggatt) (Queensland fruit fly, or "Qfly") is a highly polyphagous tephritid fruit fly and a serious economic pest in Australia. Qfly biology is intimately linked to the bacteria and fungi of its microbiome. While there are numerous studies of the microbiome in larvae and adults, the transition of the microbiome through the pupal stage remains unknown. To address this knowledge gap, we used high-throughput Next-Generation Sequencing (NGS) to examine microbial communities at each developmental stage in the Qfly life cycle, targeting the bacterial 16S rRNA and fungal ITS regions. We found that microbial communities were similar at the larval and pupal stage and were also similar between adult males and females, yet there were marked differences between the larval and adult stages. Specific bacterial and fungal taxa are present in the larvae and adults (fed hydrolyzed yeast with sugar) which is likely related to differences in nutritional biology of these life stages. We observed a significant abundance of the Acetobacteraceae at the family level, both in the larval and pupal stages. Conversely, Enterobacteriaceae was highly abundant (> 80%) only in the adults. The majority of fungal taxa present in Qfly were yeasts or yeast-like fungi. In addition to elucidating changes in the microbiome through developmental stages, this study characterizes the Qfly microbiome present at the establishment of laboratory colonies as they enter the domestication process.

Effect of a bioactive product SEL001 from Lactobacillus sakei probio65 on gut microbiota and its anti-colitis effects in a TNBS-induced colitis mouse model.

This study underpins the therapeutic potential of SEL001, a bioactive product isolated from Lactobacillus sakei probio65, in terms of its anti-inflammatory properties and its effect on gut-microbiota in a TNBS-induced ulcerative colitis mouse model. Ulcerative colitis was developed in mice by intra rectal administration of trinitrobenzene sulfonic acid. Bioactive product SEL001 (50 mg/kg b.w.) was administered orally. Myeloperoxidase activity was measured using 3,3', 5,5'-tetramethylbenzidine. The entire colon was sampled for post-mortem clinical assessment. Colonic injury was assessed through histological and histomorphometric examinations. The 454 pyrosequencing and QIIME pipeline were used for gut microbiota analysis and statistical analysis were conducted using R. mRNA extraction from colon tissue and RT-PCR approaches were employed to determine the changes in the level of specific biomarker genes associated with UC. The results depict that SEL001 significantly lowered pro-inflammatory cytokines, including CD4, TNF-α, and interleukin-6. Examination of clinical and histopathological traits revealed that SEL001 was effective and potent in reducing the inflammatory signatures of UC to a similar extent as did by the standard drug mesalamine (5-ASA). Pyro-sequencing 16S data revealed that the reduction in the major member of phylum Firmicutes, which has been previously associated with a higher risk of UC. The SEL001, an anti-inflammatory bioactive product originated from a probiotic strain L. sakei probio65 could be an alternative therapeutic agent for treatment of UC.

Next-Generation Sequencing reveals relationship between the larval microbiome and food substrate in the polyphagous Queensland fruit fly.

Insects typically host substantial microbial communities (the 'microbiome') that can serve as a vital source of nutrients and also acts as a modulator of immune function. While recent studies have shown that diet is an important influence on the gut microbiome, very little is known about the dynamics underpinning microbial acquisition from natural food sources. Here, we addressed this gap by comparing the microbiome of larvae of the polyphagous fruit fly Bactrocera tryoni ('Queensland fruit fly') that were collected from five different fruit types (sapodilla [from two different localities], hog plum, pomegranate, green apple, and quince) from North-east to South-east Australia. Using Next-Generation Sequencing on the Illumina MiSeq platform, we addressed two questions: (1) what bacterial communities are available to B. tryoni larvae from different host fruit; and (2) how does the microbiome vary between B. tryoni larvae and its host fruit? The abundant bacterial taxa were similar for B. tryoni larvae from different fruit despite significant differences in the overall microbial community compositions. Our study suggests that the bacterial community structure of B. tryoni larvae is related less to the host fruit (diet) microbiome and more to vertical transfer of the microbiome during egg laying. Our findings also suggest that geographic location may play a quite limited role in structuring of larval microbiomes. This is the first study to use Next-Generation Sequencing to analyze the microbiome of B. tryoni larvae together with the host fruit, an approach that has enabled greatly increased resolution of relationships between the insect's microbiome and that of the surrounding host tissues.

Trehalose mediated stabilisation of cellobiase aggregates from the filamentous fungus Penicillium chrysogenum.

Extracellular fungal cellobiases develop large stable aggregates by reversible concentration driven interaction. In-vitro addition of trehalose resulted in bigger cellobiase assemblies with increased stability against heat and dilution induced dissociation. In presence of 0.1 M trehalose, the size of aggregates increased from 344 nm to 494 nm. The increase in size was also observed in zymography of cellobiase. Activation energy of the trehalose stabilised enzyme (Ea = 220.9 kJ/mol) as compared to control (Ea = 257.734 kJ/mol), suggested enhanced thermostability and also showed increased resistance to chaotropes. Purified cellobiase was found to contain 196.27 µg of sugar/µg of protein. It was proposed that presence of glycan on protein's surface impedes and delays trehalose docking. Consequently, self-association of cellobiase preceded coating by trehalose leading to stabilisation of bigger cellobiase aggregates. In unison with the hypothesis, ribosylated BSA failed to get compacted by trehalose and developed into bigger aggregates with average size increasing from 210 nm to 328 nm. Wheat Germ Lectin, in presence of trehalose, showed higher molecular weight assemblies in DLS, native-PAGE and fluorescence anisotropy. This is the first report of cross-linking independent stabilisation of purified fungal glycosidases providing important insights towards understanding the aggregation and stability of glycated proteins.

Acute and sublethal effects of organophosphate insecticide chlorpyrifos on freshwater fish Oreochromis niloticus.

Chlorpyrifos is a widely used organosphosphate insecticide in India. Residue of the insecticide is frequently detected in trace to moderate concentration in food grains and in surface water of different freshwater ecosystems of the country. In this study, 96 h LC50 of the technical grade (94% a.i.) and commercial formulation (20% EC) of chlorpyrifos to freshwater fish Oreochromis niloticus were determined as 90.0 and 42.0 µg/L based on 2 h actual concentration of chlorpyrifos in water. About 96 h exposure to sublethal concentrations (0, 12.0 and 25.0 µg/L) of the commercial formulation (20% EC) of chlorpyrifos reduced the level of hepatic glycogen, activities of alkaline phosphatase, acetylcholinesterase, and catalase in liver and elevated the level of plasma glucose and activities of hepatic acid phosphatase, aspartate aminotransferase, and alanine aminotransferase in O. niloticus. About 28-day exposure to these sub-lethal concentrations caused anemia in fish, while 90 days exposure reduced growth of the fish and carcass concentration of crude protein and crude lipid as compared to control. It was concluded from this study that commercial formulation of chlorpyrifos (20% EC) was highly toxic to O. niloticus. Exposure to sub-lethal concentrations of the insecticide could induce oxidative stress and anemia resulting in reduced growth of the fish.

Attenuation of inflammatory responses by (+)-syringaresinol via MAP-Kinase-mediated suppression of NF-κB signaling in vitro and in vivo.

We examined the anti-inflammatory effects of (+)-syringaresinol (SGRS), a lignan isolated from Rubia philippinensis, in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells using enzyme-based immuno assay, Western blotting, and RT-PCR analyses. Additionally, in vivo effects of SGRS in the acute inflammatory state were examined by using the carrageenan-induced hind paw edema assay in experimental mice. As a result, treatment with SGRS (25, 50, and 100 µM) inhibited protein expression of lipopolysaccharide-stimulated inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF-κB) as well as production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and interleukin-6 (IL-6) induced by LPS. Moreover, SGRS also reduced LPS-induced mRNA expression levels of iNOS and COX-2, including NO, PGE2, TNF-α, IL-1ß, and IL-6 cytokines in a dose-dependent fashion. Furthermore, carrageenan-induced paw edema assay validated the in vivo anti-edema effect of SGRS. Interestingly, SGRS (30 mg/kg) suppressed carrageenan-induced elevation of iNOS, COX-2, TNF-α, IL-1ß, and IL-6 mRNA levels as well as COX-2 and NF-κB protein levels, suggesting SGRS may possess anti-inflammatory activities.

Depletion of Cr(VI) from aqueous solution by heat dried biomass of a newly isolated fungus Arthrinium malaysianum: A mechanistic approach.

For the first time, the heat dried biomass of a newly isolated fungus Arthrinium malaysianum was studied for the toxic Cr(VI) adsorption, involving more than one mechanism like physisorption, chemisorption, oxidation-reduction and chelation. The process was best explained by the pseudo-second order kinetic model and Redlich-Peterson isotherm with maximum predicted biosorption capacity (Q m ) of 100.69 mg g-1. Film-diffusion was the rate-controlling step and the adsorption was spontaneous, endothermic and entropy-driven. The mode of interactions between Cr(VI) ions and fungal biomass were investigated by several methods [Fourier Transform-Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD) and Energy-Dispersive X-ray spectroscopy (EDX)]. X-ray Photoelectron Spectroscopy (XPS) studies confirmed significant reduction of Cr(VI) into non-toxic Cr(III) species. Further, a modified methodology of Atomic Force Microscopy was successfully attempted to visualize the mycelial ultra-structure change after chromium adsorption. The influence of pH, biomass dose and contact time on Cr(VI) depletion were evaluated by Response Surface Model (RSM). FESEM-EDX analysis also exhibited arsenic (As) and lead (Pb) peaks on fungus surface upon treating with synthetic solutions of NaAsO2 and Pb(NO3)2 respectively. Additionally, the biomass could also remove chromium from industrial effluents, suggesting the fungal biomass as a promising adsorbent for toxic metals removal.

Trehalose induced structural modulation of Bovine Serum Albumin at ambient temperature.

Trehalose is a well-known protein stabilizing osmolyte. The present study has been designed to understand the interaction of trehalose with BSA at ambient temperature. Steady state fluorescence and life-time analysis along with CD, DLS and ITC have been employed to show that trehalose causes surface-associated structural perturbation of BSA to promote its compaction. Trehalose at 0.1M concentration resulted in increased solvent exposure of one of the two tryptophans of BSA with a 5nm redshift in emission and enhanced susceptibility to acrylamide quenching with an increase in KSV from 2.61M-1to 5.16M-1. 0.5M trehalose resulted in reduced accessibility of tryptophan and destabilization of ANS binding (Forster radius increased from 24Å to 27.36Å for tryptophan-ANS FRET) indicating shielding of BSA in trehalose matrix. Simultaneously, there was compaction of BSA as shown by increased alpha-helicity from 45.85% to 48.81%, decreased thioflavin-T binding and reduction in hydrodynamic radius from 9.69nm to 6.59nm. Trehalose induced solution viscosity resulted in significant decrease in binding affinity of BSA towards curcumin and resveratrol. The results are in unison with the preferential exclusion and vitrification models to explain protein stabilization by trehalose and also points at the structure-function trade-off of proteins in presence of trehalose.

Antioxidant efficacy and the upregulation of Nrf2-mediated HO-1 expression by (+)-lariciresinol, a lignan isolated from Rubia philippinensis, through the activation of p38.

The aim of the present study was to examine the antioxidative activity of (+)-lariciresinol (LRSL), an optically active lignan isolated from Rubia philippinensis in several in vitro assays. LRSL was also subjected to evaluate its inhibitory effect against the generation of reactive oxygen species (ROS) in murine macrophage (RAW 264.7) cells. The results showed that LRSL possessed very strong radical scavenging activity and reducing power, as well as inhibited ROS generation in a dose-dependent manner without showing any cytotoxicity. The transcriptional and translational levels of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were markedly higher in the sample treated group. LRSL treatment also increased the transcriptional and translational activities of NF-E2-related factor-2 (Nrf-2) with a corresponding increase in the transcriptional and translational activities of the heme oxygenase-1 (HO-1). LRSL activated p38 and treatments with SB239063 (a p38 inhibitor) suppressed the LRSL-induced activation of Nrf2, resulting in a decrease in HO-1 expression. Collectively, the data demonstrated that LRSL has potent antioxidative activity, decreasing ROS generation in RAW 264.7 cells and increasing the transcriptional and translational levels of antioxidant enzymes by activating Nrf2-mediated HO-1 induction via p38 signaling.

Inhibition of melanogenesis by jineol from Scolopendra subspinipes mutilans via MAP-Kinase mediated MITF downregulation and the proteasomal degradation of tyrosinase.

In this study, the authors investigated the anti-melanogenic effects of 3,8-dihydroxyquinoline (jineol) isolated from Scolopendra subspinipes mutilans, the mechanisms responsible for its inhibition of melanogenesis in melan-a cells, and its antioxidant efficacy. Mushroom tyrosinase activities and melanin contents were determined in melan-a cells, and the protein and mRNA levels of MITF, tyrosinase, TYRP-1, and TYRP-2 were assessed. Jineol exhibited significant, concentration-dependent antioxidant effects as determined by DPPH, ABTS, CUPRAC, and FRAP assays. Jineol significantly inhibited mushroom tyrosinase activity by functioning as an uncompetitive inhibitor, and markedly inhibited melanin production and intracellular tyrosinase activity in melan-a cells. In addition, jineol abolished the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF, thereby blocking melanin production and interfering with the phosphorylations of ERK1/2 and p38. Furthermore, specific inhibitors of ERK1/2 and p38 prevented melanogenesis inhibition by jineol, and the proteasome inhibitor (MG-132) prevented jineol-induced reductions in cellular tyrosinase levels. Taken together, jineol was found to stimulate MAP-kinase (ERK1/2 and p38) phosphorylation and the proteolytic degradation pathway, which led to the degradations of MITF and tyrosinase, and to suppress the productions of melanin.

Evaluation of in vitro antioxidant, anticancer and in vivo antitumour activity of Termitomyces clypeatus MTCC 5091.

CONTEXT: Termitomyces clypeatus (Lyophyllaceae) is a filamentous edible mushroom, having ethnomedicinal uses. However, information about the antioxidant, anticancer and antitumour properties of this mushroom remains to be elucidated. OBJECTIVE: The study examines the in vitro antioxidant, anticancer and in vivo antitumour activity of T. clypeatus. MATERIALS AND METHODS: Antioxidant activity was evaluated with seven in vitro assays. Cytotoxicity of T. clypeatus was tested against a panel of cancer cells lines including U373MG, MDA-MB-468, HepG2, HL-60, A549, U937, OAW-42 and Y-79 using MTT assay. The antitumour activity of aqueous extract was evaluated against Ehrlich ascites carcinoma (EAC) tumour model in Swiss albino mice. RESULTS: HPLC analysis of aqueous extract revealed the presence of sugar entities. Termitomyces clypeatus showed excellent in vitro antioxidant activity. Termitomyces clypeatus was found cytotoxic against all cancer cells, among which it showed higher activity against U937 (IC50 25 ± 1.02 µg/mL). Treatment of EAC-bearing mice with varied doses of aqueous extract significantly (p < 0.01) reduced tumour volume, viable tumour cell count and improved haemoglobin content, RBC count, mean survival time, tumour inhibition and % increase life span. The enhanced antioxidant status in treated animals was evident from the decline in the levels of lipid peroxidation, increased levels of glutathione, catalase and superoxide dismutase. DISCUSSION: The analyzed data indicate that the aqueous extract of T. clypeatus exhibits significant antitumour activity, which might be due to the antioxidant effects on EAC bearing hosts. CONCLUSION: Termitomyces clypeatus possesses anticancer activity, valuable for application in food and drug products.

AkP from mushroom Termitomyces clypeatus is a proteoglycan specific protease with apoptotic effect on HepG2.

Termitomyces clypeatus is an edible mushroom, prized for its therapeutic values and as producer of industrially important enzymes. However, the biomedical efficacies of anticancer proteases have not been reported yet. The present study aimed to purify and characterize a serine protease (AkP) from T. clypeatus for investigating cytotoxic potency on HepG2, Hep3B, and compared the effect on normal hepatic L-02 cells. Purification and biochemical characterization of AkP were evaluated by three stage chromatography, 1D/2D-SDS-PAGE, 1D zymography, far-UV CD spectral analysis, N-terminal sequencing, MALDI-TOF/MS-MS analysis and enzyme kinetics studies. AkP could cleave the growth promoting cell surface proteoglycans of HepG2, corroborated by RP-HPLC analysis. AkP (IC50: 75±1.18nM) mediated anti-proliferative activity solely on HepG2 cells through the induction of apoptosis. Augmentation of apoptosis was attributed to up-regulation of p53 and Bax protein expression succeeded by caspase-3 activation. Serine protease inhibitor phenyl methane sulfonyl fluoride (PMSF) inhibited both its proteolytic activity and cytotoxicity on HepG2. These findings demonstrate that AkP could be an effective biomolecule for killing of cancer cells by p53 restoration and surface proteoglycans cleavage.