Proteomics investigation of molecular mechanisms affected by EnBase culture system in anti-VEGF fab fragment producing E. coli BL21 (DE3)

Aggregation of recombinant proteins, a major problem in E. coli expression system, is improved by using EnBase culture system based on slow release of glucose. In the present study, to understand the intracellular mechanisms involved in increased solubility of the target recombinant protein through EnBase system, the effect of this system was investigated on E. coli cells proteome profile. The proteome profile of E. coli cells cultured in EnBase and conventional batch mode was analyzed by two-dimensional gel electrophoresis. The proteins with significant expressional changes were identified through MALDI-TOF/TOF mass spectrometry. In EnBase system, the expressions of carbon metabolism-related proteins, sugar transport system-related proteins, and amino acids metabolism-related proteins were significantly altered. Furthermore, the expression of Thioredoxin 1 as the facilitator of protein folding was up-regulated in EnBase system that could be related to the increased solubility of recombinant protein.

The proteomics analysis of E. coli cells cultured in EnBase system revealed that Thioredoxin 1 can be a potential candidate for future studies aiming at increased anti-VEGF fab fragment solubility. Studying proteomics is a valuable tool for revealing the target proteins that play the central role in EnBase culture system for increasing the solubility.     

Hepatitis C virus core or NS3/4A protein expression preconditions hepatocytes against oxidative stress and endoplasmic reticulum stress

Objectives: The occurrence of oxidative stress and endoplasmic reticulum (ER) stress in hepatitis C virus (HCV) infection has been demonstrated and play an important role in liver injury. During viral infection, hepatocytes must handle not only the replication of the virus, but also inflammatory signals generating oxidative stress and damage. Although several mechanisms exist to overcome cellular stress, little attention has been given to the adaptive response of hepatocytes during exposure to multiple noxious triggers.

Methods: In the present study, Huh-7 cells and hepatocytes expressing HCV Core or NS3/4A proteins, both inducers of oxidative and ER stress, were additionally challenged with the superoxide anion generator menadione to mimic external oxidative stress. The production of reactive oxygen species (ROS) as well as the response to oxidative stress and ER stress were investigated.

Results: We demonstrate that hepatocytes diminish oxidative stress through a reduction in ROS production, ER-stress markers (HSPA5 [GRP78], sXBP1) and apoptosis (caspase-3 activity) despite external oxidative stress. Interestingly, the level of the autophagy substrate protein p62 was downregulated together with HCV Core degradation, suggesting that hepatocytes can overcome excess oxidative stress through autophagic degradation of one of the stressors, thereby increasing cell survival.

Duscussion: In conclusion, hepatocytes exposed to direct and indirect oxidative stress inducers are able to cope with cellular stress associated with viral hepatitis and thus promote cell survival.     

A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization

The gene encoding copper-dependent laccase from Bacillus subtilis strain R5 was cloned and expressed in Escherichia coli. Initially the recombinant protein was produced in insoluble form as inclusion bodies. Successful attempts were made to produce the recombinant protein in soluble and active form. The laccase activity of the recombinant protein was highly dependent on the presence of copper ions in the growth medium and microaerobic conditions during protein production. The purified enzyme exhibited highest activity at 55 °C and pH 7.0. The recombinant protein was highly thermostable, albeit from a mesophilic source, with a half-life of 150 min at 80 °C. Similar to temperature, the recombinant protein was stable in the presence of organic solvents and protein denaturants such as urea. Furthermore, the recombinant protein was successfully utilized for the degradation of various synthetic dyes reflecting its potential use in treatment of wastewater in textile industry.

Abbreviations: ABTS,2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid; CBB, Coomassie brilliant blue; SGZ, syringaldazine; DMP, 2,2-dimethoxy phenol.     

The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins

Introduction: Glycosylation is recognized as a Critical Quality Attribute for therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and therapeutic replacement enzymes. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for their discovery, development and quality control. The aim of this review is to highlight relevant and recent advances in analytical technologies for characterization of biotherapeutic glycoproteins.

Areas covered: The review gives an overview of the glycosylation trends of biotherapeutics approved in 2016 and 2017 by FDA. It describes current and novel analytical technologies for characterization of therapeutic glycoproteins and is explored in the context of released glycan, glycopeptide or intact glycoprotein analysis. Ultra performance liquid chromatography, mass spectrometry and capillary electrophoresis technologies are explored in this context.

Expert commentary: There is a need for the biopharmaceutical industry to incorporate novel state of the art analytical technologies into existing and new therapeutic glycoprotein workflows for safer and more efficient biotherapeutics and for the improvement of future biotherapeutic design. Additionally, at present, there is no ‘gold-standard’ approach to address all the regulatory requirements and as such this will involve the use of orthogonal glycoanalytical technologies with a view to gain diagnostic information about the therapeutic glycoprotein.     

Rapid evaluation of 1-kestose producing β-fructofuranosidases from Aspergillus species and enhancement of 1-kestose production using a PgsA surface-display system

1-Kestose is a key prebiotic fructooligosaccharide (FOS) sugar. Some β-fructofuranosidases (FFases) have high transfructosylation activity, which is useful for manufacturing FOS. Therefore, obtaining FFases that produce 1-kestose efficiently is important. Here, we established a rapid FFase evaluation method using Escherichia coli that display different FFases fused to a PgsA anchor protein from Bacillus subtilis. E. coli cell suspensions expressing the PgsA-FFase fusion efficiently produce FOS from sucrose. Using this screening technique, we found that the E. coli transformant expressing Aspergillus kawachii FFase (AkFFase) produced a larger amount of 1-kestose than those expressing FFases from A. oryzae and A. terreus. Saturation mutagenesis of AkFFase was performed, and the mutant G85W was obtained. The E. coli transformant expressing AkFFase G85W markedly increased production of 1-kestose. Our results indicate that the surface display technique using PgsA is useful for screening of FFases, and AkFFase G85W is likely to be suitable for 1-kestose production.

Abbreviations: AkFFase: Aspergillus kawachii FFase; AoFFase: Aspergillus oryzae FFase; AtFFase: Aspergillus terreus FFase; FFase: β-fructofuranosidase; FOS: fructooligosaccharide; fructosylnystose: 1^F-β-fructofuranosylnystose     

Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1

Introduction: Protein glycosylation is recognized as an important post-translational modification, with specific substructures having significant effects on protein folding, conformation, distribution, stability and activity. However, due to the structural complexity of glycans, elucidating glycan structure-function relationships is demanding. The fine detail of glycan structures attached to proteins (including sequence, branching, linkage and anomericity) is still best analysed after the glycans are released from the purified or mixture of glycoproteins (glycomics). The technologies currently available for glycomics are becoming streamlined and standardized and many features of protein glycosylation can now be determined using instruments available in most protein analytical laboratories.

Areas covered: This review focuses on the current glycomics technologies being commonly used for the analysis of the microheterogeneity of monosaccharide composition, sequence, branching and linkage of released N- and O-linked glycans that enable the determination of precise glycan structural determinants presented on secreted proteins and on the surface of all cells.

Expert commentary: Several emerging advances in these technologies enabling glycomics analysis are discussed. The technological and bioinformatics requirements to be able to accurately assign these precise glycan features at biological levels in a disease context are assessed.     

Global views of proteasome-mediated degradation by mass spectrometry

Introduction: Degradation of proteins by cellular proteasomes is critical for the fidelity of protein homeostasis and proper cell function. Indeed, perturbations in proteasome function, as well as the degradation of specific substrates, are associated with a variety of human diseases. Yet, monitoring and analyzing protein degradation in a high throughput manner in physiology and pathology remains limited.

Areas covered: Here we discuss several of the recently developed mass spectrometry-based methods for studying proteasome-mediated cellular degradation and discuss their advantages and limitations. We highlight Mass Spectrometry Analysis of Proteolytic Peptides (MAPP), a method designed to purify and identify proteasome-cleaved cellular proteins as a novel approach in molecular and clinical profiling of human disease.

Expert opinion: The recent improvement of proteomics technologies now offers an unprecedented ability to study disease in clinical settings. Expanding clinical studies to include the degradation landscape will provide a new resolution to complement the cellular proteome. In turn, this holds promise to provide both new disease targets and novel peptide biomarkers which will further enhance personalized proteomics.     

Taxonomical notes on Chinese camaenids with description of three new species (Gastropoda: Pulmonata)

The present study deals with four Chinese camaenid species based on museum collections and newly obtained materials. Pseudiberus liuae Wu, n. sp., diagnosed by two long mucous glands and the smallest shell size in the genus and inhabiting bare rock like other congeneric members, is described from southern Gansu. Aegista (Plectotropis) wardi (Preston, 1912) is conchologically re-described and moved out of Aegista Alber, 1860 to Pseudiberus Ancey, 1887 based on the keeled periphery and the absence of hairs, scales or their scars on the teleoconch, which are present in Aegista but partially absent in Pseudiberus. The first Chinese fluorescent snail Bradybaena qixiaensis Wu & Asami n. sp. is reported from Nanjing, Jiangsu. The species shares many characters with the Japanese fluorescent snail Bradybaena pellucida Kuroda & Habe, 1953 but is distinct in the pattern of microsculpture on the internal surface of the penis. Nesiohelix yeni Wu & Asami n. sp., sympatric with N. moreletiana (Heude, 1882), is distinguished from its congener by possession of a bubble-shaped penial caecum.

Pseudiberus liuae Wu in Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:3C9299AA-5089-4E43-9B26-85A0D6C23B66

Bradybaena qixiaensis Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:7991C4D5-5E0B-46DF-8B19-BE26511806CD

Nesiohelix yeni Wu & Asami, In Press

LSID urn:lsid:zoobank.org:act:68CFF173-AACC-4DA8-B347-9ABB5CA569A3     

Proteomics and metabolomics in biomarker discovery for cardiovascular diseases: progress and potential

Introduction: The process of discovering novel biomarkers and potential therapeutic targets may be shortened using proteomic and metabolomic approaches.

Areas covered: Several complementary strategies, each one presenting different advantages and limitations, may be used with these novel approaches. In vitro studies show how cells involved in cardiovascular disease react, although the phenotype of cultured cells differs to that occurring in vivo. Tissue analysis either in human specimens or animal models may show the proteins that are expressed in the pathological process, although the presence of structural proteins may be confounding. To identify circulating biomarkers, analyzing the secretome of cultured atherosclerotic tissue, analysis of blood cells and/or plasma may be more straightforward. However, in the latter approach, high-abundant proteins may mask small molecules that could be potential biomarkers. The study of sub-proteomes such as high-density lipoproteins may be useful to circumvent this limitation. Regarding metabolomics, most studies have been performed in small populations, and we need to perform studies in large populations in order to discover robust biomarkers.

Expert commentary: It is necessary to involve the clinicians in these areas to improve the design of clinical studies, including larger populations, in order to obtain consistent novel biomarkers.     

Phytochemicals,antioxidant activity and hepatoprotective effect of ginger (Zingiber officinale) on diethylnitrosamine toxicity in rats

Context: Chronic liver damage has serious medical consequences.

Objective: To investigate the hepatoprotective effect of dry Zingiber officinale (ginger) and its essential (volatile) oil against diethylnitrosamine (DEN) toxicity in rats.

Materials and methods: Phenols and flavonoids components were characterized in dry ginger using HPLC-UV instrument while ginger essential oil (E.O.) was investigated via GC-MS technique. Antioxidant activity was determined in vitro. In rat model, ginger was administrated for 2 months. Lipid profile, antioxidant biomarkers, liver functions and histopathology were assessed.

Results: Chlorogenic acid (63.85?ppm) and hesperidin (156.91?ppm) are among the major phenolic and flavonoid constituents in dry ginger. Curcumene (15.21%) and linalool (13.47%) represent the main E.O. constituents. In rats treated with ginger E.O., a significant elevation in serum HDL (31.14%) was accompanied by a decrease in LDL (55.14%). A significant decrease in serum ALT and ALP was reported (56.85% and 53.84%, respectively). Serum GSH-Px activity has significantly increased 75.06%. Meanwhile, E.O. showed anticancer potential against HepG2 cell line (IC₅₀?=?40?µg/mL). Liver histopathological examinations confirmed the protective effect against abnormalities.

Conclusion: Ginger was able to reduce the severity of DEN-cytotoxicity in rats, which suggests a novel antioxidant role originating from this medicinal plant.     

Inhibitory effects of Eucalyptus saligna leaf litter on grassland species: physical versus chemical factors

Background: In Southern Brazil, large areas of grassland have been replaced by Eucalyptus plantations. Vegetation is scarce under plantations which may be associated with Eucalyptus releasing of allelochemicals.

Aims: To investigate effects of Eucalyptus saligna leaf litter on the colonisation and development of the ground layer by grassland species and if these effects were related to allelopathy.

Methods: We assessed the effects of Eucalyptus litter on the establishment of species of the native grassland community and on seeded test species (Paspalum notatum and Lotus corniculatus – introduced). We tested the impact of the addition of E. saligna leaf litter, artificial leaves (shading and mechanical impediment) and shading. We also tested the phytotoxicity of the soil from E. saligna plantations.

Results: Species richness, diversity, plant height, cover and biomass were lower in E. saligna leaves and artificial leaves treatments than in the control (absence of litter). The test species showed lower biomass and higher mortality in treatment plots with E. saligna leaves, artificial leaves and shading treatments than in the control. Allelochemicals did not accumulate in soil at phytotoxic levels.

Conclusions: Eucalyptus saligna leaf litter suppresses the establishment of grassland vegetation, but the effects are mainly physical. Field evaluations with appropriate controls should be more extensively used in allelopathy investigations.     

Fruit production along roads and footpaths in an Atlantic rain forest area

Background: Pathways (footpaths and roads) in forests are associated with edge effects, affecting forest structure and composition and associated wildlife. However, little is known about how edge effects along pathways may impact the dynamics of fruit production and their availability for frugivores.

Aim: We related pathway width as a proxy for edge effects to fruit production. Our underlying hypothesis was that pathway width would be positively related to fruit production.

Methods: We observed fruit production along three pathways of different widths – 2, 10 and 20 m wide – and in a control area of undisturbed forest in an Atlantic rain forest stand monthly over a 2-year period.

Results: The number of species and individuals-bearing fruit was higher along the wider pathways than along the narrowest pathway and in the control area. The amount of zoochorous fruits was higher in the control area than along pathways, and the widest pathway had higher non-zoochorous fruits production. Fruiting peaks occurred along pathways, while fruiting in the control area was aseasonal.

Conclusions: Pathway width is related to fruit type and its quantity and temporal availability. These effects extend towards the forest interior beyond 35 m. The presence of paths affects food resources for frugivores and thus can contribute to reconfiguring the spatio-temporal distribution of the fauna.     

A new species of the genus Megasis Guenée, 1845 from Turkey (Lepidoptera: Pyralidae)

A new species in the family Pyralidae is described: Megasis kocaki sp. n., of which the adult, head and the male genitalia are illustrated. The new species is compared with the closely allied Megasis hyrcanella Ragonot, 1893.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:0136DD7F-0E91-4114-9B3B-A42E6114A985     

Polymorphisms and expression of IL-32: impact on genetic susceptibility and clinical outcome of lung cancer

Context: Polymorphisms of IL-32 related closely to tumoregenesis.

Materials and methods: Two IL-32 polymorphisms (rs12934561 and rs28372698) and mRNA expression were conducted by SNP genotype assay and real-time PCR in 423 lung cancer patients and 437 controls.

Results: T allele of rs28372698 associated significantly with poor prognosis in moderate and well-differentiated lung cancer patients. TT genotype of rs12934561 related closely to poor survival status in squamous carcinoma. IL-32 mRNA expression decreased in lung cancer.

Discussion and conclusion: Our study indicates the importance of IL-32 polymorphism and mRNA expression in susceptibility and influence of survival status in lung cancer.     

Proteomics approach to identify biomarkers for upper gastrointestinal cancer

Introduction: The prognosis for patients with upper gastrointestinal cancers remains dismal despite the development of multimodality therapies that incorporate surgery, chemotherapy, and radiotherapy. Early diagnosis and personalized treatment should lead to better prognosis. Given the advances in proteomic technologies over the past decades, proteomics promises to be the most effective technique to identify novel diagnostics and therapeutic targets.

Areas covered: For this review, keywords were searched in combination with ‘proteomics’ and ‘gastric cancer’ or ‘esophageal cancer’ in PubMed. Studies that evaluated proteomics associated with upper gastrointestinal cancer were identified through reading, with several studies quoted at second hand. We summarize the proteomics involved in upper gastrointestinal cancer and discuss potential biomarkers and therapeutic targets.

Expert commentary: In particular, the development of mass spectrometry has enabled detection of multiple proteins and peptides in more biological samples over a shorter time period and at lower cost than was previously possible. In addition, more sophisticated protein databases have allowed a wider variety of proteins in samples to be quantified. Novel biomarkers that have been identified by new proteomic technologies should be applied in a clinical setting.     

Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.     

A new species of Hercostomus Loew, 1857 (Diptera: Dolichopodidae) from Turkey

A new species of Hercostomus is described from Turkey: Hercostomus anatoliensis sp. n. The new species is close to Hercostomus parvilamellatus (Macquart, 1827), but is distinguished by its genitalia.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:38B210E3-610D-4BAB-91AC-A212A8054474