Aging-Related Protein Alterations in the Brain.

Aging is an intrinsic aspect of an organism's life cycle and is characterized by progressive physiological decline and increased susceptibility to mortality. Many age-associated disorders, including neurological disorders, are most commonly linked with the aging process, such as Alzheimer's disease (AD). This review aims to provide a comprehensive overview of the effects of aging and AD on the molecular pathways and levels of different proteins in the brain, including metalloproteins, neurotrophic factors, amyloid proteins, and tau proteins. AD is caused by the aggregation of amyloid proteins in the brain. Factors such as metal ions, protein ligands, and the oligomerization state of amyloid precursor protein significantly influence the proteolytic processing of amyloid-ß protein precursor (AßPP). Tau, a disordered cytosolic protein, serves as the principal microtubule-associated protein in mature neurons. AD patients exhibit decreased levels of nerve growth factor within their nervous systems and cerebrospinal fluid. Furthermore, a significant increase in brain-derived neurotrophic factor resulting from the neuroprotective effect of glial cell line-derived neurotrophic factor suggests that the synergistic action of these proteins plays a role in inhibiting neuronal degeneration and atrophy. The mechanism through which Aß and AßPP govern Cu2+ transport and their influence on Cu2+ and other metal ion pools requires elucidation in future studies. A comprehensive understanding of the influence of aging and AD on molecular pathways and varying protein levels may hold the potential for the development of novel diagnostic and therapeutic methods for the treatment of AD.

Computational design of experimentally validated multi-epitopes vaccine against hepatitis E virus: An immunological approach.

Hepatitis E virus (HEV) is one of the leading acute liver infections triggered by viral hepatitis. Patients infected with HEV usually recover and the annual death rate is negligible. Currently, there is no HEV licensed vaccine available globally. This study was carried out to design a multi-epitope HEV peptide-based vaccine by retrieving already experimentally validated epitopes from ViPR database leading to epitope prioritization. Epitopes selected as potential vaccine candidates were non-allergen, immunogenic, soluble, non-toxic and IFN gamma positive. The epitopes were linked together by AAY linkers and the linker EAAAK was used to join adjuvant with epitopes. Toll-like receptor (TLR)-4 agonist was used as an adjuvant to boost efficacy of the vaccine. Furthermore, codon optimization followed by disulfide engineering was performed to analyse the designed vaccine's structural stability. Computational modeling of the immune simulation was done to examine the immune response against the vaccine. The designed vaccine construct was docked with TLR-3 receptor for their interactions and then subjected to molecular dynamic simulations. The vaccine model was examined computationally towards the capability of inducing immune responses which showed the induction of both humoral and cell mediated immunity. Taken together, our study suggests an In-silico designed HEV based multi-epitope peptide-based vaccine (MEPV) that needs to be examined in the wet lab-based data that can help to develop a potential vaccine against HEV.

Role of Thioredoxin System in Regulating Cellular Redox Status in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia and a public health problem. It exhibits significant oxidative stress and redox alterations. The antioxidant enzyme systems defend the cellular environment from oxidative stress. One of the redox systems is the thioredoxin system (TS), which exerts decisive control over the cellular redox environment. We aimed to review the protective effects of TS, which include thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. In the following, we discussed the physiological functioning and the role of the TS in maintaining the cellular redox-homeostasis in the AD-damaged brain. Trx protects the cellular environment from oxidative stress, while TrxR is crucial for the cellular detoxification of reactive oxygen species in the brain. However, TS dysregulation increases the susceptibility to cellular death. The changes in Trx and TrxR levels are significantly associated with AD progression. Though the data from human, animal, and cellular models support the neuroprotective role of TS in the brain of AD patients, the translational potential of these findings to clinical settings is not yet applied. This review summarizes the current knowledge on the emerging role of the TrxR-Trx system in AD.

Potential of nanotechnology to replace cancer stem cells.

Stem cells, which were initially identified in the 1900s, are distinct cells with the potential to replenish themselves as well as differentiate into specialised cells with certain forms and functions. Cancer stem cells play a significant role in the growth and recurrence of the tumours and, similar to normal stem cells, are capable of proliferating and differentiating. Traditional cancer treatments are ineffective against cancer stem cells, which leads to tumour regrowth. Cancer stem cells are thought to emerge as a result of epithelial-to-mesenchymal transition pathways. Brain, prostate, pancreatic, blood, ovarian, lung, liver, melanomas, AML, and breast cancer stem cells are among the most prevalent cancer forms. This review aims to comprehend the possibility of using specific forms of nanotechnology to replace cancer stem cells. In terms of nanotechnology, magnetic nanoparticles can deliver medications, especially to the target region without harming healthy cells, and they are biocompatible. In order to kill glioma cancer stem cells, the gold nanoparticles bond with DNA and function as radio sensitizers. In contrast, liposomes can circulate and traverse biological membranes and exhibit high therapeutic efficacy, precise targeting, and better drug release. Similar to carbon nanotubes, grapheme, and grapheme oxide, these substances can be delivered specifically when utilized in photothermal therapy. Recent treatments including signaling pathways and indicators targeted by nanoparticles are being researched. Future research in nanotechnology aims to develop more effective and targeted medicinal approaches. The results of the current investigation also showed that this technology's utilization will improve medical therapy and treatment.

Oxidative Stress and Dopaminergic Metabolism: A Major PD Pathogenic Mechanism and Basis of Potential Antioxidant Therapies

Reactive oxygen species (ROS)-induced oxidative stress triggers the vicious cycle leading to the degeneration of dopaminergic neurons in the nigra pars compacta. ROS produced during the metabolism of dopamine is immediately neutralized by the endogenous antioxidant defense system (EADS) under physiological conditions. Aging decreases the vigilance of EADS and makes the dopaminergic neurons more vulnerable to oxidative stress. As a result, ROS left over by EADS oxidize the dopamine-derived catechols and produces a number of reactive dopamine quinones, which are precursors to endogenous neurotoxins. In addition, ROS causes lipid peroxidation, uncoupling of the electron transport chain, and DNA damage, which lead to mitochondrial dysfunction, lysosomal dysfunction, and synaptic dysfunction. The mutations in genes such as DNAJC6, SYNJ1, SH3GL2, LRRK2, PRKN, and VPS35 caused by ROS have been associated with synaptic dysfunction and the pathogenesis of Parkinson's disease (PD). The available drugs that are used against PD can only delay the progression of the disease, but they produce various side effects. Through their antioxidant activity, flavonoids can substantiate the EADS of dopaminergic neurons and disrupt the vicious cycle incepted by oxidative stress. In this review, we show how the oxidative metabolism of dopamine generates ROS and dopamine-quinones, which then exert unrestrained OS, causing mutations in several genes involved in the proper functioning of mitochondrion, synapse, and lysosome. Besides, we also present some examples of approved drugs used for the treatment of PD, therapies in the clinical trial phase, and an update on the flavonoids that have been tested to boost the EADS of dopaminergic neurons.

Comparative impact of streptozotocin on altering normal glucose homeostasis in diabetic rats compared to normoglycemic rats.

Diabetes mellitus is a syndrome and an endocrine disorder, primarily considered as a loss of glucose homeostasis because of the insulin action and/or secretion or both. Currently there are more than 150 million people in the world affected by diabetes mellitus with a higher share of Asian and European countries. The current study aimed to investigate the comparative altering properties of streptozotocin (STZ), based on up-turn and down-turn configuration of biochemical, toxicological and hematological parameters in comparison with normoglycemic male albino rats. This comparative study was conducted among normoglycemic and STZ based induced-type 2 diabetic male albino rats groups. The male albino rats were intra-peritoneally injected with STZ with the dose rate of 65 mg/kg body weight for one time to developed type 2 diabetic model. Biochemical (blood glucose, uric acid, urea and creatinine), toxicological (AST, ALT and ALP) and hematological parameters (red and white blood cells) and their functional indices were evaluated in type 2 diabetic induced group along with normoglycemic rats. The STZ based induced- type 2 diabetic rats showed statistically significance (p < 0.001) higher level in the blood glucose, alongwith the change in the levels of biochemical parameters including urea, uric acid, and creatinine. Toxicological parameters comprising AST, ALT and ALP were also shown significance (p < 0.001) as sufficient after experimental evaluation of biologically important parameter in STZ based induced-type 2 diabetic rats. Likewise, the red blood cells, white blood cells and their efficient components were exposed significantly insufficient after the injecting of STZ to induce the rats as type 2 diabetic. The results of the current study indicates the comparatively higher levels of variation among biochemical, toxicological and hematological parameters in STZ based Induced-type 2 diabetic model as compared to normoglycemic group.

Biogenic Synthesis of Silver Nanoparticles Using Catharanthus roseus and Its Cytotoxicity Effect on Vero Cell Lines.

Background: Type 2 diabetes mellitus (DM2) is a chronic and sometimes fatal condition which affects people all over the world. Nanotherapeutics have shown tremendous potential to combat chronic diseases­including DM2­as they enhance the overall impact of drugs on biological systems. Greenly synthesized silver nanoparticles (AgNPs) from Catharanthus roseus methanolic extract (C. AgNPs) were examined primarily for their cytotoxic and antidiabetic effects. Methods: Characterization of C. AgNPs was performed by UV−vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and atomic force microscopy (AFM). The C. AgNPs were trialed on Vero cell line and afterwards on an animal model (rats). Results: The C. AgNPs showed standard structural and functional characterization as revealed by FTIR and XRD analyses. The zetapotential analysis indicated stability while EDX analysis confirmed the formation of composite capping with Ag metal. The cytotoxic effect (IC50) of C. AgNPs on Vero cell lines was found to be 568 g/mL. The animal model analyses further revealed a significant difference in water intake, food intake, body weight, urine volume, and urine sugar of tested rats after treatment with aqueous extract of C. AgNPs. Moreover, five groups of rats including control and diabetic groups (NC1, PC2, DG1, DG2, and DG3) were investigated for their blood glucose and glycemic control analysis. Conclusions: The C. AgNPs exhibited positive potential on the Vero cell line as well as on experimental rats. The lipid profile in all the diabetic groups (DG1-3) were significantly increased compared with both of the control groups (p < 0.05). The present study revealed the significance of C. AgNPs in nanotherapeutics.

Naegleria fowleri from Pakistan Has Type-2 Genotype.

Background: Primary amoebic meningoencephalitis (PAM) is an acute and fulminant CNS infection caused by Naegleria fowleri. Recreational activities and ritual ablution with contaminated warm fresh water are the main reason of PAM. Pakistan ranked the second most affected country, where most of the PAM incidences were reported from Karachi, Pakistan. Methods: In May, 2019, a 28-yr-old suspected PAM patient came to the Imam Zain-Ul-Abdin Hospital, Karachi. Biochemical and cytological investigations of patient's CSF were carried out at Karachi Diagnostic Center and Molecular Biology Lab. Sequencing of Naegleria sp. specific (ITS) primer-based amplicons was performed from both patient's CSF and water samples followed by multiple sequence alignment and phylogenetic studies. Results: Biochemical and cytological investigations of patient's CSF showed 5 mg/dl glucose, 240 mg/dl total protein and 2260/mm3 TLC suggesting acute meningoencephalitis. PCR-based analyses of patient's CSF and his residential tap water samples using Naegleria sp. specific (ITS) and N. fowleri specific primers revealed the presence of N. fowleri DNA. Nucleotide sequences of ITS primer-based amplicons from both patient's CSF and water samples were submitted in GenBank under the accession numbers MT726981.1 and MT726226.1, respectively. According to phylogenetic analysis, N. fowleri isolate from Pakistan has shown the least node age of seven. Conclusion: Here, for the very first time in Pakistan, N. fowleri genotype has been identified as type-2. Phylogenetic analysis showed that N. fowleri isolate from Pakistan is among the latest descendants, i.e., evolved later in life.

Biosynthesis of Polyhydroxyalkanoates (PHAs) by the Valorization of Biomass and Synthetic Waste.

Synthetic pollutants are a looming threat to the entire ecosystem, including wildlife, the environment, and human health. Polyhydroxyalkanoates (PHAs) are natural biodegradable microbial polymers with a promising potential to replace synthetic plastics. This research is focused on devising a sustainable approach to produce PHAs by a new microbial strain using untreated synthetic plastics and lignocellulosic biomass. For experiments, 47 soil samples and 18 effluent samples were collected from various areas of Punjab, Pakistan. The samples were primarily screened for PHA detection on agar medium containing Nile blue A stain. The PHA positive bacterial isolates showed prominent orange-yellow fluorescence on irradiation with UV light. They were further screened for PHA estimation by submerged fermentation in the culture broth. Bacterial isolate 16a produced maximum PHA and was identified by 16S rRNA sequencing. It was identified as Stenotrophomonas maltophilia HA-16 (MN240936), reported first time for PHA production. Basic fermentation parameters, such as incubation time, temperature, and pH were optimized for PHA production. Wood chips, cardboard cutouts, plastic bottle cutouts, shredded polystyrene cups, and plastic bags were optimized as alternative sustainable carbon sources for the production of PHAs. A vital finding of this study was the yield obtained by using plastic bags, i.e., 68.24 ± 0.27%. The effective use of plastic and lignocellulosic waste in the cultivation medium for the microbial production of PHA by a novel bacterial strain is discussed in the current study.

Antibiotics, Acid and Heat Tolerance of Honey adapted Escherichia coli, Salmonella Typhi and Klebsiella pneumoniae.

The medicinal importance of honey has been known for many decades due to its antimicrobial properties against life-threatening bacteria. However, previous studies revealed that microorganisms are able to develop adaptations after continuous exposure to antimicrobial compounds. The present study was conducted to explore the impact of subinhibitory concentrations of branded honey (Marhaba) and unbranded honey (extracted from Ziziphus mauritiana plant) locally available in Pakistan on Escherichia coli ATCC 10536, Salmonella Typhi and Klebsiella pneumoniae by investigating the development of self- or cross-resistance to antibiotics (gentamicin, kanamycin and imipenem). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of autoclaved honeys were determined. The bacterial cells of E. coli ATCC 10536, S. Typhi and K. pneumoniae were subjected to honey adaptation by exposing to » × MIC (4 passages) and ½ × MIC (4 passages) of both honeys. Moreover, tolerance to low pH and high temperature was also studied in adapted and unadapted cells. The decreasing trend in growth pattern (OD600nm) of E. coli ATCC 10536, S. Typhi and K. pneumoniae was observed with increases in the concentration of honeys (6.25-50% v/v) respectively. Our results showed that continuous exposure of both honeys did not lead to the development of any self- or cross-resistance in tested bacteria. However, percent survival to low pH was found to be significantly higher in adapted cells as compared to unadapted cells. The results indicate that both branded honey (Marhaba) and unbranded honey (extracted from Ziziphus mauritiana plant) were effective in controlling the growth of tested pathogenic bacteria. However, the emergence of tolerance to adverse conditions (pH 2.5, temperature 60 °C) deserves further investigation before proposing honey as a better antibacterial agent in food fabrication/processing, where low pH and high temperatures are usually implemented.

Self-Redirection of Metabolic Flux Toward Squalene and Ethanol Pathways by Engineered Yeast.

We have previously reported that squalene overproducing yeast self-downregulate the expression of the ethanol pathway (non-essential pathway) to divert the metabolic flux to the squalene pathway. In this study, the effect of co-production of squalene and ethanol on other non-essential pathways (fusel alcohol pathway, FA) of Saccharomyces cerevisiae was evaluated. However, before that, 13 constitutive promoters, like IRA1p, PET9p, RHO1p, CMD1p, ATP16p, USA3p, RER2p, COQ1p, RIM1p, GRS1p, MAK5p, and BRN1p, were engineered using transcription factor bindings sites from strong promoters HHF2p (-300 to -669 bp) and TEF1p (-300 to -579 bp), and employed to co-overexpress squalene and ethanol pathways in S. cerevisiae. The FSE strain overexpressing the key genes of the squalene pathway accumulated 56.20 mg/L squalene, a 16.43-fold higher than wild type strain (WS). The biogenesis of lipid droplets was stimulated by overexpressing DGA1 and produced 106 mg/L squalene in the FSE strain. AFT1p and CTR1p repressible promoters were also characterized and employed to downregulate the expression of ERG1, which also enhanced the production of squalene in FSE strain up to 42.85- (148.67 mg/L) and 73.49-fold (255.11 mg/L) respectively. The FSE strain was further engineered by overexpressing the key genes of the ethanol pathway and produced 40.2 mg/mL ethanol in the FSE1 strain, 3.23-fold higher than the WS strain. The FSE1 strain also self-downregulated the expression of the FA pathway up to 73.9%, perhaps by downregulating the expression of GCN4 by 2.24-fold. We demonstrate the successful tuning of the strength of yeast promoters and highest coproduction of squalene and ethanol in yeast, and present GCN4 as a novel metabolic regulator that can be manipulated to divert the metabolic flux from the non-essential pathway to engineered pathways.

Studying the Influence of Apple Peel Polyphenol Extract Fortification on the Characteristics of Probiotic Yoghurt.

The aim of the current study was to evaluate the effect of apple peel polyphenol extract (APPE) on the physicochemical and microbiological properties of probiotic yoghurt. Five concentrations of APPE were added in probiotic yoghurt as: (1) CTL, control without APPE; (2) AE1, addition of 1% APPE; (3) AE2, addition of 2% APPE; (4) AE3, addition of 3% APPE; (5) AE4, addition of 4% APPE; and (6) AE5, addition of 5% APPE. The prepared probiotic yoghurt was stored at 4 °C for 21 days and analyzed for physicochemical and microbiological properties. The initial viable count of L. bulgaricus, S. thermophilus, B. lactis and L. acidophilus were similar in all yoghurt samples at day 1. The maximum viability loss of probiotics was observed in CTL (p < 0.05). The lowest viability loss of probiotics was observed in AE5 samples (p < 0.05). The acidity, water holding capacity and viscosity were increased with the addition of APPE. No significant effects were observed on milk fat and total solid contents of probiotic yoghurt with the addition of APPE. The total phenolic contents of probiotic yoghurt increased significantly as 0.59, 0.71, 0.97, 1.18, 1.35 in AE1, AE2, AE3, AE4 and AE5, samples respectively. It was observed that AE3 and AE4 samples had better taste, flavour and colour with good texture. The survival of probiotics and antioxidant activity of the yoghurts were enhanced with the addition of APPE. In conclusion, apple peels could be successfully used as prebiotic in yoghurt with increased viable counts of probiotics.

Assessment of Antimicrobial Features of Selenium Nanoparticles (SeNPs) Using Cyclic Voltammetric Strategy.

The emerging biomedical applications of selenium nanoparticles (SeNPs) require facile and efficient strategy to assess its interactions with cell membrane. In this study, an efficient and reproducible microwave assisted method was used to synthesize SeNPs with controllable size distributions. The physical properties of the emergent structures, such as morphology, structure, and size were studied. The antimicrobial applications of SeNPs were assessed by electrochemical analyses that entailed the systematic acquisition of cyclic voltammetry data. Our results demonstrate a straightforward method to predict the integrity of bacterial cell membranes following the administration of SeNP treatments.

Biosorption of lead by cotton shells powder: Characterization and equilibrium modeling study.

Lead (Pb) is a toxic heavy metal causing serious health risks to humans and animals. In the present study, cotton (Gossypium hirsutum L.) shells powder was used as adsorbent for the treatment of synthetic Pb-contaminated water. The batch scale biosorption capacity of cotton shells powder was evaluated to study the effects of Pb concentrations, adsorbent doses and contact time at constant pH (6) and temperature (25 °C). Results revealed that sorption of Pb increased (q = 0.09-9.60 mg/g) with increasing Pb concentration (1-15 mg/L) and contact time (15-90 min) while decreasing adsorbent dose (1-0.1 g/100 mL). The maximum Pb removal (90%) was achieved at Pb concentration (1 mg/L), contact time (90 min) and adsorbent dose (1 g/100 mL). Freundlich isotherm model proved best fit for Pb sorption (R2 = 0.99). The cotton shells powder has microporous structure confirmed by SEM, and has BET surface area (45 m2/g) and pore size (2.3 µm). These surface moieties along with various functional groups (C-H, C-O, C=O, O-H, S=O) confirmed by FTIR analysis might involve in Pb removal by complexation and ion exchange mechanisms. The cotton shells powder biomass could be considered as promising adsorbent for the removal of Pb from contaminated water.

Seroprevalence and Risk Factors of Toxoplasma gondii in Wild Birds of Punjab Province, Pakistan.

Toxoplasma gondii is a protozoan parasite of veterinary and human public health importance for which birds act as an intermediate host. No information is available about the epidemiology of T. gondii in wild birds of Pakistan. The present study was designed to determine the seroprevalence and risk factors associated with T. gondii antibodies in wild birds of District Kasur, Punjab Province, Pakistan. A total of 200 wild birds of 28 species were captured from four tehsils (administrative subdistricts of districts) of the district Kasur and their serum samples screened for the presence of T. gondii antibodies using a latex agglutination test (cut-off value: 1:64). Twenty-five (13%) individual birds and 13 (46%) of the bird species were seropositive for T. gondii antibodies. There were statistical differences in T. gondii prevalence between adults and young (15% and 7%, respectively, P=0.001) and healthy and sick (11% and 50%, respectively, P=0.000) while there were not differences between genders, sites, urbanicity, and tehsils. The present study provides evidence of T. gondii antibodies in wild birds of Pakistan.

N-Glycosylation enhances functional and structural stability of recombinant ß-glucuronidase expressed in Pichia pastoris.

Recombinant ß-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein, encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked carbohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylated GUS were compared under various conditions such as temperature, pH, organic solvents, detergents and chaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of maximum enzymatic activity against various types of denaturants compared with the deglycosylated. The conformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by using the denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase in its conformational stability than the deglycosylated counterpart. These results affirmed the key role of N-glycosylation on the structural and functional stability of ß-glucuronidase and could have potential applications in the functional enhancement of industrial enzymes.

Effects of a non-conservative sequence on the properties of ß-glucuronidase from Aspergillus terreus Li-20.

We cloned the ß-glucuronidase gene (AtGUS) from Aspergillus terreus Li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (GAMG) and glycyrrhetinic acid (GA). Based on sequence alignment, the C-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence AtGUS(-3t) (1-592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS(-3t) were expressed in E. coli BL21, producing AtGUS-E and AtGUS(-3t)-E, respectively. At the similar optimum temperature (55°C) and pH (AtGUS-E, 6.6; AtGUS(-3t)-E, 7.0) conditions, the thermal stability of AtGUS(-3t)-E was enhanced at 65°C, and the metal ions Co(2+), Ca(2+) and Ni(2+) showed opposite effects on AtGUS-E and AtGUS(-3t)-E, respectively. Furthermore, Km of AtGUS(-3t)-E (1.95 mM) was just nearly one-seventh that of AtGUS-E (12.9 mM), whereas the catalytic efficiency of AtGUS(-3t)-E was 3.2 fold higher than that of AtGUS-E (7.16 vs. 2.24 mM s(-1)), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of AtGUS. Conformational analysis illustrated significant difference in the secondary structure between AtGUS-E and AtGUS(-3t)-E by circular dichroism (CD). The results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme.

N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum ß-d-glucuronidase.

To study the influence of N-linked carbohydrate moiety on the catalytic and biochemical properties of glycosylated enzyme, a recombinant ß-d-glucuronidase (PGUS-P) from Penicillium purpurogenum as a model glycoprotein, was deglycosylated with peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic deglycosylation procedure resulted in the complete removal of carbohydrate moiety. Compared with the glycosylated PGUS-P, the deglycosylated PGUS-P exhibited 20-70% higher activity (p<0.05) within pH 6-9, but 15-45% lower activity (p<0.05) at 45-70°C. The apparent decrease in the thermal stability of the deglycosylated enzyme was reflected by a decrease in the denaturation temperature (T(d)) values determined by differential scanning calorimetry (DSC). The removal of N-linked glycans also reduced enzyme's sensitivity to certain metal ions. The deglycosylated PGUS-P displayed lower K(m) vaules, but higher k(cat)/K(m) ratios than the glycosylated isoform towards glycyrrhizin. The consequent conformational changes were also determined by circular dichroism (CD) and fluorescence spectroscopy which revealed no significant difference in the secondary but a slight dissimilarity between the tertiary structures of both isoforms of PGUS-P.

Transcriptional profiling of protein expression related genes of Pichia pastoris under simulated microgravity.

The physiological responses and transcription profiling of Pichia pastoris GS115 to simulated microgravity (SMG) were substantially changed compared with normal gravity (NG) control. We previously reported that the recombinant P. pastoris grew faster under SMG than NG during methanol induction phase and the efficiencies of recombinant enzyme production and secretion were enhanced under SMG, which was considered as the consequence of changed transcriptional levels of some key genes. In this work, transcriptiome profiling of P. pastoris cultured under SMG and NG conditions at exponential and stationary phases were determined using next-generation sequencing (NGS) technologies. Four categories of 141 genes function as methanol utilization, protein chaperone, RNA polymerase and protein transportation or secretion classified according to Gene Ontology (GO) were chosen to be analyzed on the basis of NGS results. And 80 significantly changed genes were weighted and estimated by Cluster 3.0. It was found that most genes of methanol metabolism (85% of 20 genes) and protein transportation or secretion (82.2% of 45 genes) were significantly up-regulated under SMG. Furthermore the quantity and fold change of up-regulated genes in exponential phase of each category were higher than those of stationary phase. The results indicate that the up-regulated genes of methanol metabolism and protein transportation or secretion mainly contribute to enhanced production and secretion of the recombinant protein under SMG.