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A new xylanase from Paenibacillus barcinonensis BP-23, Xyn11E, has been identified and characterized. Xyn11E has been cloned and heterologously expressed in Escherichia coli. It is a single-domain xylanase belonging to the family 11 of glycosyl hydrolases (GH11) with a predicted molecular weight of 20.652 kDa and an isoelectric point (pI) of 8.7. Substrate specificity, kinetic properties, and mode of action of the purified xylanase were characterized. Xyn11E exhibited high activity toward branched xylans, being beechwood xylan the preferred substrate. The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. Catalytic constants were determined on beechwood xylan, on which Xyn11E showed a Km of 12.98 mg/ml and a Vmax of 3,023 U/mg. The enzyme hydrolyzed long xylooligosaccharides, while oligomers shorter than xylotetraose were not degraded. Products released from glucuronoxylans were shorter than those liberated from cereal arabinoxylans. The xylanase was dependent on P. barcinonensis BP-23 LppX for its expression in an active form. Coexpression of Xyn11E with E. coli chaperones could not replace the need of LppX, which seems to act as a specific chaperone for Xyn11E correct folding. Activity of the enzyme on bleached pulps was evaluated. Xyn11E liberated reducing sugars from ECF and TCF pulps from eucalyptus, sisal, and flax, which makes it a good candidate for the enzymatic-assisted production of high-cellulose-content pulps from paper-grade pulps.

The choice of inotropic agent, particularly in catecholamine-resistant septic shock, remains an area of debate. Here we discuss a recent trial examining the use of vasopressin in a carefully controlled trial setting. Yet more data on the use of drotrecogin alfa (activated) in septic shock are described, as are novel but as yet experimental approaches to the treatment of sepsis. Finally, it is important not to forget to read the latest surviving sepsis guidelines.

Negative ion collision-induced dissociation (CID) of underivatized N-glycans has proved to be a simple, yet powerful method for their structural determination. Recently, we have identified a series of such structures with GalNAc rather than the more common galactose capping the antennae of hybrid and complex glycans. As part of a series of publications describing the negative ion fragmentation of different types of N-glycan, this paper describes their CID spectra and estimated nitrogen cross sections recorded by travelling wave ion mobility mass spectrometry (TWIMS). Most of the glycans were derived from the recombinant glycoproteins gp120 and gp41 from the human immunodeficiency virus (HIV), recombinantly derived from human embryonic kidney (HEK 293T) cells. Twenty-six GalNAc-capped hybrid and complex N-glycans were identified by a combination of TWIMS, negative ion CID, and exoglycosidase digestions. They were present as the neutral glycans and their sulfated and α2→3-linked sialylated analogues. Overall, negative ion fragmentation of glycans generates fingerprints that reveal their structural identity.

Rotavirus is the most common cause of severe diarrhea in infants and children worldwide and is responsible for about 215,000 deaths annually. Over 85% of these deaths originate in low-income/developing countries in Asia and Africa. Therefore, it is necessary to explore the development of vaccines that avoid the use of "living" viruses and furthermore, vaccines that have viral antigens capable of generating powerful heterotypic responses. Our strategy is based on the expression of the fusion of the anti-DEC205 single-chain variable fragment (scFv) coupled by an OLLAS tag to a viral protein (VP6) of Rotavirus in Nicotiana plants. It was possible to express transiently in N. benthamiana and N. sylvestris a recombinant protein consisting of the single chain variable fragment linked by an OLLAS tag to the VP6 protein. The presence of the recombinant protein, which had a molecular weight of approximately 75 kDa, was confirmed by immunodetection, in both plant species and in both cellular compartments (cytoplasm and apoplast) where it was expressed. In addition, the recombinant protein was modeled, and it was observed that some epitopes of interest are exposed on the surface, which could favor their immunogenic response.

Objective A chloroplast transgenic approach was assessed in order to produce a structural protein SPPV117 of sheep pox virus in Nicotiana tabacum for the future development of a plant-based subunit vaccine against sheep pox. Results Two DNA constructs containing SPPV117 coding sequence under the control of chloroplast promoter and terminator of psbA gene or rrn promoter and rbcL terminator were designed and inserted into the chloroplast genome by a biolistic method. The transgenic plants were selected via PCR analysis. Northern and Western blot analysis showed expression of the transgene at transcriptional and translational levels, respectively. The recombinant protein accumulated to about 0.3% and 0.9% of total soluble protein in leaves when expressed from psbA and rrn promoter, respectively. Plant-produced SPPV117 protein was purified using metal affinity chromatography and the protein yield was 19.67 ± 1.25 µg g−1 (FW). The serum of a sheep infected with the virus recognised the chloroplast-produced protein indicating that the protein retains its antigenic properties. Conclusions These results demonstrate that chloroplasts are a suitable system for the production of a candidate subunit vaccine against sheep pox.

Coccidiosis triggered by Eimeria tenella is accompanied by haemorrhagic caecum and high morbidity. Vaccines are preferable choices to replace chemical drugs against coccidiosis. Surface antigens of apicomplexan parasites can adhere to host cells during the infection process. Therefore, truncated fragments coding E. tenella surface antigen 16 (EtSAG16) and 22 (EtSAG22) were cloned into pET-28a prokaryotic vector to express recombinant protein 16 (rEtSAG16) and 22 (rEtSAG22), respectively. Likewise, pEGFP-N1-EtSAG16 and pEGFP-N1-EtSAG22 plasmids were constructed using pEGFP-N1 eukaryotic vector. Further, pEGFP-N1-EtSAG4-16-22 multiple gene plasmid carrying EtSAG4, 16 and 22 were designed as cocktail vaccines to study integral immunoprotective effects. Western blot and RT-PCR (reverse transcription) assay were performed to verify expressions of EtSAG16 and 22 genes. Immunoprotective effects of recombinant protein or DNA vaccine were evaluated using different doses (50 or 100 μg) in vivo. All chickens in the vaccination group showed higher cytokine concentration (IFN-γ and IL-17), raised IgY antibody level, increased weight gain, lower caecum lesion score and reduced oocyst shedding compared with infection control groups (p < 0.05). The highest anticoccidial index (ACI) value 173.11 was from the pEGFP-N1-EtSAG4-16-22 plasmid (50 μg) group. In conclusion, EtSAG16 and 22 might be alternative candidate genes for generating vaccines against E. tenella infection.

This article comparatively reports the workability of Escherichia coli BL21(DE3) and Pseudomonas putida KT2440 cell factories for the expression of three model autodisplayed cellulases (i.e., endoglucanase, BsCel5A; exoglucanase, CelK; β-glucosidase, BglA). The differentiation of the recombinant cells was restricted to their cell growth and enzyme expression/activity attributes. Comparatively, the recombinant E. coli showed higher cell growth rates but lower enzyme activities than the recombinant P. putida. However, the endo-, exoglucanase, and β-glucosidase on the surfaces of both cell factories showed activity over a broad range of pH (4-10) and temperature (30-100 °C). The pH and temperature optima were pH 6, 60 °C (BsCel5A); pH 6, 60-70 °C (CelK); and pH 6, 50 °C (BglA). Overall, the P. putida cell factory with autodisplayed enzymes demonstrated higher bioactivity and remarkable biochemical characteristics and thus was chosen for the saccharification of filter paper. A volumetric blend of the three cellulases with P. putida as the host yielded a ratio of 1:1:1.5 of endoglucanase, exoglucanase, and β-glucosidase, respectively, as the optimum blend composition for filter paper degradation. At an optical density (578 nm) of 50, the blend generated a maximum sugar yield of about 0.7 mg/ml (~ 0.08 U/g) from Whatman filter paper (Ø 6 mm, ~ 2.5 mg) within 24 h.

Recombinant granulocyte colony–stimulating factor (G-CSF) protein produced in Escherichia coli has been widely used for the treatment of neutropenia induced by chemotherapy for decades. In E. coli cells, G-CSF is usually expressed as inactive inclusion bodies, which requires costly and inefficient denaturation and refolding steps to obtain the protein in its active form. However, following the findings of previous studies, we here successfully produced G-CSF in E. coli as non-classical inclusion bodies (ncIBs), which contained likely correctly folded protein. The ncIBs were easily dissolved in 0.2% N-lauroylsarcosine solution and then directly applied to a Ni-NTA affinity chromatography column to get G-CSF with high purity (> 90%). The obtained G-CSF was demonstrated to have a similar bioactivity with the well-known G-CSF containing product Neupogen (Amgen, Switzerland). Our finding clearly verified that the G-CSF production from ncIBs is a feasible approach to improve the yield and lower the cost of G-CSF manufacturing process.

Trueperella pyogenes (T. pyogenes) is a common opportunistic pathogen of many livestock and play an important regulation role during multibacterial infection and interaction with the host by its primary virulence factor pyolysin (PLO). The purpose of this study was to investigate the regulation role of PLO which serve as a combinational pathogen with lipopolysaccharide (LPS) during endometritis. In this study, the expression of bioactive recombinant PLO (rPLO) in a prokaryotic expression system and its purification are described. Moreover, we observed that rPLO inhibited the innate immune response triggered by LPS and that methyl-β-cyclodextrin (MBCD) abrogated this inhibitory effect in goat endometrium stromal cells (gESCs). Additionally, we show from pharmacological and genetic studies that rPLO-induced autophagy represses gene expression by inhibiting NLRP3 inflammasome activation. Importantly, this study reported that ATF6 serves as a primary regulator of the cellular inflammatory reaction to rPLO. Overall, these observations suggest that T. pyogenes PLO could create an immunosuppressive environment for other pathogens invasion by regulating cellular signaling pathways. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00422-5.

Paragonimiasis is a foodborne trematode infection that affects 23 million people, mainly in Asia. Lung fluke infections lead frequently to chronic cough with fever and hemoptysis, and are often confused with lung cancer or tuberculosis. Paragonimiasis can be efficiently treated with praziquantel, but diagnosis is often delayed, and patients are frequently treated for other conditions. To improve diagnosis, we selected five Paragonimus kellicotti proteins based on transcriptional abundance, recognition by patient sera, and conservation among trematodes and expressed them as His-fusion proteins in Escherichia coli. Sequences for these proteins have 76–99% identity with amino acid sequences for orthologs in the genomes of Paragonimus westermani, Paragonimus heterotremus, and Paragonimus miyazakii. Immunohistology studies showed that antibodies raised to four recombinant proteins bound to the tegument of adult P. kellicotti worms, at the parasite host interface. Only a known egg antigen was absent from the tegument but present in developing and mature eggs. We evaluated the diagnostic potential of these antigens by Western blot with sera from patients with paragonimiasis (from MO and the Philippines), fascioliasis, and schistosomiasis, and with sera from healthy North American controls. Two recombinant proteins (a cysteine protease and a myoglobin) showed the highest sensitivity and specificity as diagnostic antigens, and they detected antibodies in sera from paragonimiasis patients with early or mature infections. In contrast, antibodies to egg yolk ferritin appeared to be specific marker for patients with adult fluke infections that produce eggs. Our study has identified and localized antigens that are promising for serodiagnosis of human paragonimiasis.

Porcine deltacoronavirus (PDCoV) has been recently identified as an emerging enteropathogenic coronavirus that mainly infects newborn piglets and causes enteritis, diarrhea and high mortality. Although coronavirus N proteins have multifarious activities, the subcellular localization of the PDCoV N protein is still unknown. Here, we produced mouse monoclonal antibodies against the PDCoV N protein. Experiments using anti-haemagglutinin antibodies and these monoclonal antibodies revealed that the PDCoV N protein is shuttled into the nucleolus in both ectopic PDCoV N-expressing cells and PDCoV-infected cells. The results of deletion mutagenesis experiments demonstrated that the predicted nucleolar localization signal at amino acids 295–318 is critical for nucleolar localization. Cumulatively, our study yielded a monoclonal antibody against the PDCoV N protein and revealed a mechanism by which the PDCoV N protein translocated into the nucleolus. The tolls and findings from this work will facilitate further investigations on the functions of the PDCoV N protein.

Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role. Electronic supplementary material The online version of this article (10.1007/s12192-020-01134-9) contains supplementary material, which is available to authorized users.

Objectives Noroviruses (NoVs) are major cause of acute viral gastroenteritis in worldwide, and the lack of a cell culture system that must be considered the virus like particles (VLPs) are used as an effective vaccine development. Materials and methods In the present study, we investigated the expression of the major capsid protein (VP1) of the Genogroup II, genotype 17 (GII.17) NoV, using recombinant baculovirus system in insect cells, as well as a saliva binding blockade assay to detect their protective potency. Results Our results showed that GII.17 VLPs could be successfully generated in sf9 insect cells, and electron microscopic revealed that GII.17 VLPs appeared as spherical particles with a − 35 nm diameter. Immunized mice with purified VLPs produced GII.17 specific sera and could efficiently block GII.17 VLPs binding to the saliva histo-blood group antigens (HBGAs). Conclusions Together, these results suggested that GII.17 VLPs represent a promising vaccine candidate against NoV GII.17 infection and strongly support further preclinical and clinical studies.

Glioblastoma is the most common primary tumor of the central nervous system that develops chemotherapy resistance. Previous studies showed that Allicin could inhibit multiple cancer cells including glioblastoma, but the function of Allicin in glioblastoma is still unclear. Our work aimed to investigate the underlying molecular mechanism. The results showed that miR-486-3p levels were greatly increased in glioblastoma during Allicin treatment. Overexpression of miR-486-3p increased chemosensitivity to temozolomide (TMZ) in vitro and in vivo. O6-methylguanine-DNA methyltransferase (MGMT) was identified as a direct target of miR-486-3p, and miR-486-3p overexpression prevented the protein translation of MGMT. Moreover, overexpression of MGMT restored miR-486-3p-induced chemosensitivity to TMZ. Taken together, our studies revealed that Allicin could upregulate miR-486-3p and enhance TMZ sensitivity in glioblastoma. The results suggested that in the future, Allicin can be used as an adjuvant therapy with TMZ to improve the prognosis of patients, and miR-486-3p may be a potential target for glioblastoma treatment to improve the curative effects. Electronic supplementary material The online version of this article (10.1007/s12017-020-08592-5) contains supplementary material, which is available to authorized users.

In biotechnological processes, technical failures in the upstream process often lead to batch loss. It is of great interest to investigate the empirical impact of technical failures to understand and mitigate their impact accurately and reduce economic damage. We investigated the impact in the upstream and downstream of a recombinant antibody fragment inclusion body production process chain to provide integrated empirical data and knowledge. First, we provided a reproducible process chain that yielded high inclusion body content, high specific product titer, and a refolding yield of 30%. The inclusion body downstream proved to be of high reproducibility. Through the intended introduction of technical failures, we were not only able to shed more light on the empirical responses in the upstream and downstream, but also on process-boosting parameters that would have been neglected. Herein, a short increase in temperature during the cultivation clearly increased the refolding yield. Electronic supplementary material The online version of this article (10.1007/s00449-019-02158-x) contains supplementary material, which is available to authorized users.

The heat-inducible expression system has been widely used to produce recombinant proteins in Escherichia coli. However, the rise in temperature affects cell growth, activates the bacterial Heat-Shock Response (HSR), and promotes the formation of insoluble protein aggregates known as inclusion bodies (IBs). In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis), compared with cultures grown at 30 °C (without induction). Also, the expression of the major E. coli chaperones (DnaK and GroEL) was analyzed. We found that almost twice maximum biomass and rESAT-6 production were obtained in bioreactors (~ 3.29 g/L of biomass and ~ 0.27 g/L of rESAT-6) than in shake flasks (~ 1.41 g/L of biomass and ~ 0.14 g/L of rESAT-6) when induction was carried out at 42 °C, but similar amounts of rESAT-6 were obtained from cultures induced at 39 °C (~ 0.14 g/L). In all thermo-induced conditions, rESAT-6 was trapped in IBs. Furthermore, DnaK was preferably expressed in the soluble fraction, while GroEL was present in IBs. Importantly, IBs formed at 39 °C, in both shake flasks and bioreactors, were more susceptible to degradation by proteinase-K, indicating a lower amyloid content compared to IBs formed at 42 °C. Our work presents evidence that the culture scale and the induction temperature modify the E. coli metabolic response, expression of chaperones, and structure of the IBs during rESAT-6 protein production in a thermoinducible system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-019-01006-x) contains supplementary material, which is available to authorized users.

The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 °C + 1%M provided the most stable viability. By contrast, the temperature of 18 °C + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.

YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960–962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG oxygenases are of interest from a biocatalytic perspective. Electronic supplementary material The online version of this article (10.1007/s00792-018-1016-9) contains supplementary material, which is available to authorized users.

Encysted embryos of Artemia are among the most stress-resistant eukaryotes partly due to the massive amount of a cysteine-rich protein termed artemin. High number of cysteine residues in artemin and their intramolecular spatial positions motivated us to investigate the role of the cysteine residues in the chaperone-like activity of artemin. According to the result of Ellman’s assay, there are nine free thiols (seven buried and two exposed) and one disulfide bond per monomer of artemin. Subsequent theoretical analysis of the predicted 3D structure of artemin confirmed the data obtained by the spectroscopic study. Native and reduced/modified forms of artemin were also compared with respect to their efficiency in chaperoning activity, tertiary structure, and stability. Since the alkylation and reduction of artemin diminished its chaperone activity, it appears that its chaperoning potential depends on the formation of intermolecular disulfide bond and the presence of cysteine residues. Comparative fluorescence studies on the structure and stability of the native and reduced protein revealed some differences between them. Due to the redox-dependent functional switching of artemin from the less to more active form, it can be finally suggested as a redox-dependent chaperone.

In paper industry, xylanases are used to increase the pulp properties in bleaching process as its eco-friendly nature. The xylanases activity is hindered by high temperature and alkaline conditions with high enzyme production cost in the paper industry. Here, XynHB, an alkaline stable xylanase from Bacillus pumilus HBP8 was mutated at N188A to XynHBN188A. Expressed mutant in E. coli showed 1.5-fold higher xylanase activity than XynHB at 60 °C. The mutant expressed in Pichia pastoris was glycosylated, remained stable for 30 min at 60 °C. XynHBN188A optimized based on codon usage bias for P. pastoris (xynHBN188As) showed an increase of 39.5% enzyme activity. The strain Y16 forming the largest hydrolysis halo in the xylan plate was used in shake flask experiments produced an enzyme activity of 6,403 U/ml. The Y16 strain had 9 copies of the recombinant xynHBN188As gene in the genome revealed by qPCR. The enzymatic activity increased to 48,241 U/ml in a 5 L fermentor. Supplement of 15 U/g xylanase enhanced the brightness of paper products by 2% in bleaching experiment, and thereby improved the tensile strength and burst factor by 13% and 6.5%, respectively. XynHBN188As has a great potential in paper industries.

Small heat shock proteins (sHSPs) are heat shock proteins sized 12–43 kDa that can protect proteins from denaturation, particularly under high temperature; sHSPs thus increase the heat tolerance capability of an organisms enabling survival in adverse climates. sHSP20 is overexpressed in Oenococcus oeni in response to low temperatures. However, we found that overexpression of sHSP20 in Escherichia coli BL21 increased the microbial survival ratio at 50 °C by almost 2 h. Adding sHSP20 to the glutamate dehydrogenase solution significantly increased the stability of the enzyme at high temperature (especially at 60–70 °C), low pH values (especially below 6.0), and high concentration of metal ions of Ga2+, Zn2+, Mn2+, and Fe3+. Notably, the coexpression of sHSP20 significantly enhanced soluble expression of laccase from Phomopsis sp. XP-8 (CCTCCM209291) in E. coli without codon optimization, as well as the activity and heat stability of the expressed enzyme. In addition to the chaperone activity of sHSP20 in the gene containing host in vivo and the enzyme heat stability in vitro, our study indicated the capability of coexpression of sHSP20 to increase the efficiency of prokaryotic expression of fungal genes and the activity of expressed enzymes.

Biodiesels produced from vegetable oils have a major quality problem due to the presence of steryl glucosides (SGs), which form precipitates that clog filters and cause engine failures. Recently, we described an enzymatic process for removing SGs from biodiesel. However, industrial adoption of this technology was hindered by the cost of the steryl glucosidase (SGase) enzyme used. Here we report the development and validation at the pilot scale of a cost-efficient process for manufacturing the SGase. First, we tested various low-cost carbon sources for the Escherichia coli producing strain, ultimately developing a fed-batch fermentation process that utilizes crude glycerol as a feedstock. Next, we designed an efficient process for isolating the SGase. That process uses a novel thermolysis approach in the presence of a non-ionic detergent, centrifugation to separate the solids, and ultrafiltration to concentrate and formulate the final product. Our cost analysis indicates that on a large scale, the dose of enzyme required to eliminate SGs from each ton of biodiesel will have a manufacturing cost below $1. The new process for manufacturing the SGase, which will lead to biodiesels of a higher quality, should contribute to facilitate the global adoption of this renewable fuel. Our technology could also be used to manufacture other thermostable proteins in E. coli. Fil: Eberhardt, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina Fil: Aguirre, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina Fil: Paoletti, Luciana Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina Fil: Hails, Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina Fil: Braia, Mauricio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina Fil: Ravasi, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina Fil: Peirú, Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina. Keclon S.A.; Argentina Fil: Menzella, Hugo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Procesos Biotecnológicos y Químicos Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos Rosario; Argentina. Keclon S.A.; Argentina

Parkinson’s disease is characterized by degeneration of substantia nigra dopamine neurons and by intraneuronal aggregates, primarily composed of misfolded α-synuclein. The α-synuclein aggregates in Parkinson’s patients are suggested to first appear in the olfactory bulb and enteric nerves and then propagate, following a stereotypic pattern, via neural pathways to numerous regions across the brain. We recently demonstrated that after injection of either mouse or human α-synuclein fibrils into the olfactory bulb of wild-type mice, α-synuclein fibrils recruited endogenous α-synuclein into pathological aggregates that spread transneuronally to over 40 other brain regions and subregions, over 12 months. We previously reported the progressive spreading of α-synuclein aggregates, between 1 and 12 months following α-synuclein fibril injections, and now report how far the pathology has spread 18- and 23-month post-injection in this model. Our data show that between 12 and 18 months, there is a further increase in the number of brain regions exhibiting pathology after human, and to a lesser extent mouse, α-synuclein fibril injections. At both 18 and 23 months after injection of mouse and human α-synuclein fibrils, we observed a reduction in the density of α-synuclein aggregates in some brain regions compared to others at 12 months. At 23 months, no additional brain regions exhibited α-synuclein aggregates compared to earlier time points. In addition, we also demonstrate that the induced α-synucleinopathy triggered a significant early neuron loss in the anterior olfactory nucleus. By contrast, there was no loss of mitral neurons in the olfactory bulb, even at 18 month post-injection. We speculate that the lack of continued progression of α-synuclein pathology is due to compromise of the neural circuitry, consequential to neuron loss and possibly to the activation of proteolytic mechanisms in resilient neurons of wild-type mice that counterbalances the spread and seeding by degrading pathogenic α-synuclein. Electronic supplementary material The online version of this article (10.1007/s00401-017-1792-9) contains supplementary material, which is available to authorized users.

Depilation/unhairing is the crucial but heavy pollution process in leather industry. Traditional inorganic sulfide treatment was the most widely used depilation technique in the past decades, which was usually detrimental to leather quality and resulted in serious environmental pollution. Using biocatalysts to substitute inorganic sulfide showed great advantages in environment protection and unhairing efficiency. Keratinolytic protease is one of the excellent biocatalysts to hydrolyze disulfide bond-rich proteins of hair and has little damage to leather. Biological treatment with keratinolytic proteases could largely reduce the quantity and toxicity of wastewater effluent from the leather industry. But low thermostability and substrate specificity or specific activity of these enzymes limited their practical application. Therefore, recent progresses on protein engineering strategies (site-directed mutagenesis, protein fusion, N/C-terminus truncation, and domain swapping) used to enhance the keratinolytic enzyme performance were presented.

Exposure to divalent metals such as iron and manganese is thought to increase the risk for Parkinson’s disease (PD). Under normal circumstances, cellular iron and manganese uptake is regulated by the divalent metal transporter 1 (DMT1). Accordingly, alterations in DMT1 levels may underlie the abnormal accumulation of metal ions and thereby disease pathogenesis. Here, we have generated transgenic mice overexpressing DMT1 under the direction of a mouse prion promoter and demonstrated its robust expression in several regions of the brain. When fed with iron-supplemented diet, DMT1-expressing mice exhibit rather selective accumulation of iron in the substantia nigra, which is the principal region affected in human PD cases, but otherwise appear normal. Alongside this, the expression of Parkin is also enhanced, likely as a neuroprotective response, which may explain the lack of phenotype in these mice. When DMT1 is overexpressed against a Parkin null background, the double-mutant mice similarly resisted a disease phenotype even when fed with iron- or manganese-supplemented diet. However, these mice exhibit greater vulnerability toward 6-hydroxydopamine-induced neurotoxicity. Taken together, our results suggest that iron accumulation alone is not sufficient to cause neurodegeneration and that multiple hits are required to promote PD. Electronic supplementary material The online version of this article (doi:10.1007/s12017-017-8451-0) contains supplementary material, which is available to authorized users.

Late embryogenesis abundant (LEA) proteins constitute a large protein family that is closely associated with resistance to abiotic stresses in multiple organisms and protect cells against drought and other stresses. Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts. This bacterium possesses two genes, here named lea1 and lea2, coding for avLEA1 and avLEA2 proteins, both containing 20-mer motifs characteristic of eukaryotic plant LEA proteins. In this study, we found that disruption of the lea1 gene caused a loss of the cysts’ viability after 3 months of desiccation, whereas at 6 months, wild-type or lea2 mutant strain cysts remained viable. Vegetative cells of the lea1 mutant were more sensitive to osmotic stress; cysts developed by this mutant were also more sensitive to high temperatures than cysts or vegetative cells of the wild type or of the lea2 mutant. Expression of lea1 was induced several fold during encystment. In addition, the protective effects of these proteins were assessed in Escherichia coli cells. We found that E. coli cells overexpressing avLEA1 were more tolerant to salt stress than control cells; finally, in vitro analysis showed that avLEA1 protein was able to prevent the freeze thaw-induced inactivation of lactate dehydrogenase. In conclusion, avLEA1 is essential for the survival of A. vinelandii in dry conditions and for protection against hyper-osmolarity, two major stress factors that bacteria must cope with for survival in the environment. This is the first report on the role of bacterial LEA proteins on the resistance of cysts to desiccation.

Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term ‘intrinsic repair', and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.

Purpose Identification of human insulin analogs’ impurity with a mass shift +14 Da in comparison to a parent protein. Methods The protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing. Results The misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs. Conclusions Although there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing. Electronic supplementary material The online version of this article (10.1007/s11095-019-2601-z) contains supplementary material, which is available to authorized users.

Abnormal features of the systemic lupus erythematosus (SLE)-derived neutrophils, promoted aberrant immune response, have inspired new studies of the induction of autoimmunity and the development of organ damage in SLE. In this study, we explore the effect of milk fat globule-EGF factor 8 (MFG-E8) on the aberrant nitrification features in pristane-induced lupus. SLE patients and mice with pristane-induced lupus develop autoantibodies associated with MFG-E8 overproduction. However, the deletion of MFG-E8 leads to uncontrolled early pulmonary and peritoneal inflammation and tissue damage in mice with pristane-induced lupus. Consistent with these findings, MFG-E8-deficient mice that are exposed to pristane show enhanced neutrophil accumulation and increased neutrophil death, including apoptosis, necrosis and NETosis, as well as impaired phagocytosis of macrophages. The consequences are the expansion of diffuse pulmonary hemorrhage, increased anti-nuclear antibody, anti-dsDNA antibody and anti-neutrophil cytoplasmic antibody levels, and enhanced immune complexes deposition and neutrophil extracellular traps (NETs) formation in the lung and kidney tissues of MFG-E8-deficient mice exposed to pristane. In patients with SLE and mice with pristane-induced lupus, neutrophil accumulation is elevated, which depends on higher expression of the surface receptor CXCR2. After pretreatment with recombinant MFG-E8, the surface expression of CXCR2 on neutrophil is downregulated, and the MFG-E8 deletion increase CXCR2 expression by ~40%. These studies indicate that MFG-E8 reduces neutrophil migration and NETosis via downregulating surface CXCR2 expression in parallel with its role in the phagocytosis of apoptotic neutrophils, suggesting that MFG-E8 may serve as a therapeutic agent for attenuating the early inflammatory responses of SLE and protect patients from lupus-related damage.

RNA-binding motif proteins (RBMs) belong to RNA-binding proteins that display extraordinary posttranscriptional gene regulation roles in various cellular processes, including development, growth, and stress responses. Nevertheless, only a few examples of the roles of RBMs are known in insects, particularly in Apis cerana cerana. In the present study, we characterized the novel RNA-binding motif protein 11 from Apis cerana cerana, which was named AccRBM11 and whose promoter sequence included abundant potential transcription factor binding sites that are connected to responses to adverse stress and early development. Quantitative PCR results suggested that AccRBM11 was expressed at highest levels in 1-day postemergence worker bees. AccRBM11 mRNA and protein levels were higher in the poison gland and the epidermis than in other tissues. Moreover, levels of AccRBM11 transcription were upregulated upon all the simulation of abiotic stresses. Furthermore, Western blot analysis indicated that AccRBM11 protein expression levels could be induced under some abiotic stressors, a result that did not completely in agree with the qRT-PCR results. It is also noteworthy that the expression of some genes that connected with development or stress responses were remarkably suppressed when AccRBM11 was silenced, which suggested that AccRBM11 might play a similar role in development or stress reactions with the above genes. Taken together, the data presented here provide evidence that AccRBM11 is potentially involved in the regulation of development and some abiotic stress responses. We expect that this study will promote future research on the function of RNA-binding proteins.

Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications. Electronic supplementary material The online version of this article (doi:10.1007/s12033-016-9944-z) contains supplementary material, which is available to authorized users.

The pseudokinase, MLKL (mixed-lineage kinase domain-like), is the most terminal obligatory component of the necroptosis cell death pathway known. Phosphorylation of the MLKL pseudokinase domain by the protein kinase, receptor interacting protein kinase-3 (RIPK3), is known to be the key step in MLKL activation. This phosphorylation event is believed to trigger a molecular switch, leading to exposure of the N-terminal four-helix bundle (4HB) domain of MLKL, its oligomerization, membrane translocation and ultimately cell death. To examine how well this process is evolutionarily conserved, we analysed the function of MLKL orthologues. Surprisingly, and unlike their mouse, horse and frog counterparts, human, chicken and stickleback 4HB domains were unable to induce cell death when expressed in murine fibroblasts. Forced dimerization of the human MLKL 4HB domain overcame this defect and triggered cell death in human and mouse cell lines. Furthermore, recombinant proteins from mouse, frog, human and chicken MLKL, all of which contained a 4HB domain, permeabilized liposomes, and were most effective on those designed to mimic plasma membrane composition. These studies demonstrate that the membrane-permeabilization function of the 4HB domain is evolutionarily conserved, but reveal that execution of necroptotic death by it relies on additional factors that are poorly conserved even among closely related species.

Regulation of cell division requires the concerted function of proteins and protein complexes that properly mediate cytoskeletal dynamics. NudC is an evolutionarily conserved protein of undetermined function that associates with microtubules and interacts with several key regulators of mitosis, such as polo-kinase 1 (Plk1) and dynein. NudC is essential for proper mitotic progression, and homologs have been identified in species ranging from fungi to humans. In this paper, we report the characterization of the Caenorhabditis elegans NudC homolog, NUD-1, as a protein exhibiting molecular chaperone activity. All NudC/NUD-1 proteins share a conserved p23/HSP20 domain predicted by three-dimensional modeling [Garcia-Ranea, Mirey, Camonis, Valencia, FEBS Lett 529(2-3):162-167, 2002]. We demonstrate that nematode NUD-1 is able to prevent the aggregation of two substrate proteins, citrate synthase (CS) and luciferase, at stoichiometric concentrations. Further, NUD-1 also protects the native state of CS from thermal inactivation by significantly reducing the inactivation rate of this enzyme. To further determine if NUD-1/substrate complexes were productive or simply "dead-end" unfolding intermediates, a luciferase refolding assay was utilized. Following thermal denaturation, rabbit reticulocyte lysate and ATP were added and luciferase activity measured. In the presence of NUD-1, nearly all of the luciferase activity was regained, indicating that unfolded intermediates complexed with NUD-1 could be refolded. These studies represent the first functional evidence for a member of this mitotically essential protein family as having chaperone activity and facilitates elucidation of the role such proteins play in chaperone complexes utilized in cell division. C. elegans NUD-1 is a member of an evolutionary conserved protein family of unknown function involved in the regulation of cytoskeletal dynamics. NUD-1 and its mammalian homolog, NudC, function with the dynein motor complex to ensure proper cell division, and knockdown or overexpression of these proteins leads to disruption of mitosis. In this paper, we show that NUD-1 possesses ATP-independent chaperone activity comparable to that of small heat shock proteins and cochaperones and that changes in phosphorylation state functionally alter chaperone activity in a phosphomimetic NUD-1 mutant.

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death. The role of extracellular α-synuclein (α-syn) in the initiation and the spreading of neurodegeneration in Parkinson's disease (PD) has been studied extensively over the past 10 years. However, the nature of the α-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric α-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric α-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, α-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric α-syn. The causal relationship between α-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on α-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of α-syn fibrillization (Tolcapone) or replacing monomeric α-syn by monomeric β-synuclein in α-syn mixture composition prevent α-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added α-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of α-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular α-syn play central roles in α-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against α-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies.

Objectives To use the glucose–fructose oxidoreductase (GFOR) from Zymomonas mobilis and expressed in Escherichia coli for lactobionic acid production by conversion of lactose from whey. Results The highest concentrations of lactobionic acid (3.2 mg ml−1) during oxidation of whey-derived lactose by E. coli was at 24 h. Introduction of GFOR gene from Z. mobilis, into E. coli improved enzyme yields compared to what is obtainable by fermentation of the donor strain. The production of lactobionic acid by E. coli was 2.6-times higher than by Z. mobilis. Conclusions Recombinants of E. coli overexpressing the GFOR gene from Z. mobilis produced higher amount of lactobionoic acid from whey-derived lactose.

Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H(2)O(2), and HgCl(2). The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H(2)O(2) in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.

In Leishmania donovani, the HSP90 chaperone complex plays an essential role in the control of the parasite's life cycle, general viability and infectivity. Several of the associated co-chaperones were also shown to be essential for viability and/or infectivity to mammalian cells. Here, we identify and describe the co-chaperone P23 and distinguish its function from that of the structurally related small heat shock protein HSP23. P23 is expressed constitutively and associates itself with members of the HSP90 complex, i.e. HSP90 and Sti1. Viable P23 gene replacement mutants could be raised and confirmed as null mutants without deleterious effects on viability under a variety of physiological growth conditions. The null mutant also displays near-wild-type infectivity, arguing against a decisive role played by P23 in laboratory settings. However, the P23 null mutant displays a marked hypersensitivity against HSP90 inhibitors geldanamycin and radicicol. P23 also appears to affect the radicicol resistance of a HSP90 Leu33-Ile mutant described previously. Therefore, the annotation of L. donovani P23 as HSP90-interacting co-chaperone is confirmed.

Purpose Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis. Methods For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed. Results Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro—one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine. Conclusions The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro.

Recombinant human follicle stimulating hormone (r-hFSH) is effective and safe for controlled ovarian stimulation. Bemfola(®) (Finox AG, Burgdorf, Switzerland), a new biosimilar r-hFSH, has proven comparable non-clinical pharmacological profiles to those of the widely used Gonal-f(®) (Serono Pharma S.p.A., Bari, Italy). The objective of this study was to show that Bemfola(®) yields comparable clinical pharmacokinetic (PK) and safety profiles to Gonal-f(®) in healthy female subjects. In this randomized, Phase I trial conducted in healthy female volunteers (N = 32), a 2-period, balanced 2-treatment crossover design was used. A single subcutaneous dose of 225 IU Bemfola(®) or Gonal-f(®) was administered in each treatment period per sequence. Blood was collected for pharmacokinetic analysis until 10 days after each r-hFSH treatment. For down-regulation of endogenous FSH subjects were given a depot injection with leuprolide acetate prior to the study drug in either sequence. Pharmacokinetic data was available for 23 subjects. No appreciable differences in key PK parameters were detected between the r-hFSH products as per non-compartmental PK analysis [i.e. for Bemfola(®) and Gonal-f(®) respectively AUC0-192 424.90 and 432.75 IU h/L, C max 0.98 and 0.95 IU/L, T max 24.0 h (range 6.0-24.0) and 24.0 h (range 9.0-24.0), t 1/2 43.58 h [standard deviation (SD 14.17)] and 42.58 h (SD 16.47), and K e 0.0075 1/h (SD 0.003) and 0.0077 1/h (SD 0.002)]. Subgroup analysis for estradiol (E2) response was similar for Bemfola(®) and Gonal f(®) (AUC(0--120) p = 0.21 and C max p = 0.82). No major safety issues were identified and no immunogenic reaction to r-hFSH was observed. The results of this study indicate that a single dose of Bemfola(®) exhibits pharmacokinetic and safety profiles comparable to Gonal-f(®) in healthy young women.

Rice husk is one of the most abundant types of lignocellulosic biomass. Because of its significant amount of sugars, such as cellulose and hemicellulose, it can be used for the production of biofuels such as bioethanol. However, the complex structure of lignocellulosic biomass, consisting of cellulose, hemicellulose and lignin, is resistant to degradation, which limits biomass utilization for ethanol production. The protection of cellulose by lignin contributes to the recalcitrance of lignocelluloses to hydrolysis. Therefore, we conducted steam-explosion treatment as pretreatment of rice husk. However, recombinant Escherichia coli KO11 did not ferment the reducing sugar solution obtained by enzymatic saccharification of steam-exploded rice husk. When the steam-exploded rice husk was washed with hot water to remove inhibitory substances and M9 medium (without glucose) was used as a fermentation medium, E. coli KO11 completely fermented the reducing sugar solution obtained by enzymatic saccharification of hot water washing-treated steam-exploded rice husk to ethanol. We report here the efficient production of bioethanol using steam-exploded rice husk.

Lignocellulose contains a large amount of cellulose but is recalcitrant to enzymatic hydrolysis, which yields sugars for fuels or chemicals. Various pretreatment methods are used to improve the enzymatic digestibility of cellulose in lignocellulose. Depending on the lignocellulose types and pretreatment methods, biomass compositions and physical properties significantly vary. Therefore, customized enzyme mixtures have to be employed for the efficient hydrolysis of pretreated lignocellulose. Here, using three recombinant model enzymes consisting of endoglucanase, cellobiohydrolase, and xylanase with a fixed amount of β-glucosidase, the optimal formulation of enzyme mixtures was designed for two differently pretreated rice straws (acid-pretreated or alkali-pretreated rice straw) by the mixture design methodology. As a result, different optimal compositions for the enzyme mixtures were employed depending on the type of pretreatment of rice straw. These results suggest that customized enzyme mixtures for pretreated lignocellulosic biomass are necessary to obtain increased sugar yields and should be considered in the industrial utilization of lignocellulose.

Chaperone Hsp70 can cross the plasma membrane of living cells using mechanisms that so far have not received much research attention. Searching the part of the molecule that is responsible for transport ability of Hsp70, we found a cationic sequence composed of 20 amino acid residues on its surface, KST peptide, which was used in further experiments. We showed that KST peptide enters living cells of various origins with the same efficiency as the full-length chaperone. KST peptide is capable of carrying cargo with a molecular weight 30 times greater than its own into cells. When we compared the membrane-crossing activity of KST peptide in complex with Avidin (KST–Av complex) with that of similarly linked canonical TAT peptide, we found that TAT peptide penetrated SK-N-SH human neuroblastoma cells at a similar rate and efficiency as the KST peptide. Furthermore, KST peptide can carry protein complexes consisting of a specific antibody coupled to the peptide through the Avidin bridge. An antibody to Hsp70 delivered to SK-N-SH cells with high expression level of Hsp70 reduced the protective power of the chaperone and sensitized the cells to the pro-apoptotic effect of staurosporine. We studied the mechanisms of penetration of KST–Av and full-length Hsp70 inside human neuroblastoma SK-N-SH and human erythroleukemia K-562 cells and found that both used an active intracellular transport mechanism that included vesicular structures and negatively charged lipid membrane domains. Competition analysis of intracellular transport showed that the chaperone reduced intracellular penetration of KST peptide and conversely KST peptide prevented Hsp70 transport in a dose-dependent manner. Electronic supplementary material The online version of this article (doi:10.1007/s12192-014-0554-z) contains supplementary material, which is available to authorized users.

Mutations in the human DJ-1/PARK7 gene are associated with familial Parkinson’s disease. DJ-1 belongs to a large, functionally diverse family with homologues in all biological kingdoms. Several activities have been demonstrated for DJ-1: an antioxidant protein, a redox-regulated molecular chaperone and a modulator of multiple cellular signalling pathways. The majority of functional studies have focussed on human DJ-1 (hDJ-1), but studies on DJ-1 homologues in Drosophila melanogaster, Caenorhabditis elegans, Dugesia japonica and Escherichia coli also provide evidence of a role for DJ-1 as an antioxidant. Here, we show that dehydration is a potent inducer of a dj-1 gene in the anhydrobiotic nematode Panagrolaimus superbus. Our secondary structure and homology modelling analyses shows that recombinant DJ-1 protein from P. superbus (PsuDJ-1.1) is a well-folded protein, which is similar in structure to the hDJ-1. PsuDJ-1.1 is a heat stable protein; with T1/2 unfolding transition values of 76 and 70 °C obtained from both circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) measurements respectively. We found that PsuDJ-1.1 is an efficient antioxidant that also functions as a ‘holdase’ molecular chaperone that can maintain its chaperone function in a reducing environment. In addition to its chaperone activity, PsuDJ-1.1 may also be an important non-enzymatic antioxidant, capable of providing protection to P. superbus from oxidative damage when the nematodes are in a desiccated, anhydrobiotic state.

Apoptosis is programmed cell death triggered by activation of death receptors or cellular stress. Activation of caspases is the hallmark of apoptosis. Arrestins are best known for their role in homologous desensitization of G protein-coupled receptors (GPCRs). Arrestins quench G protein activation by binding to activated phosphorylated GPCRs. Recently, arrestins have been shown to regulate multiple signalling pathways in G protein-independent manner via scaffolding signalling proteins. Here we demonstrate that arrestin-2 isoform is cleaved by caspases during apoptosis induced via death receptor activation or by DNA damage at evolutionarily conserved sites in the C-terminus. Caspase-generated arrestin-2-(1-380) fragment translocates to mitochondria increasing cytochrome C release, which is the key checkpoint in cell death. Cells lacking arrestin-2 are significantly more resistant to apoptosis. The expression of wild-type arrestin-2 or its cleavage product arrestin-2-(1-380), but not of its caspase-resistant mutant, restores cell sensitivity to apoptotic stimuli. Arrestin-2-(1-380) action depends on tBID: at physiological concentrations, arrestin-2-(1-380) directly binds tBID and doubles tBID-induced cytochrome C release from isolated mitochondria. Arrestin-2-(1-380) does not facilitate apoptosis in BID knockout cells, whereas its ability to increase caspase-3 activity and facilitate cytochrome C release is rescued when BID expression is restored. Thus, arrestin-2-(1-380) cooperates with another product of caspase activity, tBID, and their concerted action significantly contributes to cell death.

Protein therapeutics have emerged as a major new class of pharmaceuticals. One important shelf-life-limiting factor of biopharmaceuticals is methionine oxidation, and therefore, it is important that analytical methods are able to thoroughly characterize all possible oxidized variants. Here, we present a fast and sensitive method to perform online methionine oxidation site assignment using granulocyte colony-stimulating factor (filgrastim) as a model. The method is based on top-down MS using the all-ion fragmentation mode of the Exactive benchtop mass spectrometer. Conditions that provide information on the intact mass of the protein as well as on fragment ions that allow unambiguous site assignment of methionine oxidation in filgrastim variants as low as 0.12 % of total peak area in a chromatographic time scale were identified. Using this method, we performed methionine oxidation site assignment in H2O2-stressed filgrastim and in filgrastim which was stored at intended conditions, respectively. We show that the relative abundance of oxidation species observed in filgrastim stored under intended conditions differs strikingly from the oxidized species observed after H2O2 stress. Additionally, we report an oxidized filgrastim variant that has not been previously described in the literature. Figure A top-down approach on an Exactive benchtop mass spectrometer in all-ion fragmentation mode is a highly attractive alternative to the traditional approach of isolation/bottom-up analysis for methionine oxidation site assignement in biopharmaceuticals. With a sensitivity as low as 0.12 % of total peak area and a throughput of about one sample per hour, the method is highly suitable for a thorough characterization of oxidized methionine residues

Human peripheral blood monocytes were exposed to single or pairs of cell stress proteins (CSPs), specifically Hsp10, Hsp27, Hsp60 and Hsp70—the former two having anti-inflammatory actions while the latter pair being assumed to be pro-inflammatory in activity. This study was to test if these proteins exhibited any network behaviour. To control for possible lipopolysaccharide contamination, polymyxin B was used. Surprisingly, at concentrations higher than 1 μg/ml, polymyxin B itself could induce cytokine synthesis. A number of commercial sources of the molecular chaperones were tested, and marked variations in monocyte cytokine synthesis were found. All four CSPs stimulated the same profile of IL-1/IL-6 synthesis and IL-10/TNF-α synthesis although the kinetics of production of these two pairs of cytokines were very different. A key question was whether extracellular molecular chaperones exhibited network behaviour. To test this, monocytes were cultured with suboptimal concentrations of single CSP and pairs of CSP to look for additive, synergistic or antagonistic cell responses. The major finding was that pairs of molecular chaperones, including chaperones thought to stimulate monocyte cytokine synthesis, could produce significant antagonistic cellular responses. This demonstrates that extracellular CSPs constitute an additional potent layer within the complex cytokine network and furthermore suggests that monocytes have evolved to dampen their immune responses upon exposure to extracellular networks of CSPs—perhaps as a mechanism for protecting cells against detrimental cellular stress responses.

Interleukin-24 (IL-24), a member of the IL-10 cytokine family whose physiological function remains largely unknown, has been shown to induce apoptosis when expressed in an adenoviral background. It is yet little understood, why IL-24 alone induced apoptosis only in a limited number of tumor cell lines. Analyzing an influenza A virus vector expressing IL-24 for its oncolytic potential revealed enhanced pro-apoptotic activity of the chimeric virus compared with virus or IL-24 alone. Interestingly, IL-24-mediated enhancement of influenza-A-induced apoptosis did not require viral replication but critically depended on toll-like receptor 3 (TLR3) and caspase-8. Immunoprecipitation of TLR3 showed that infection by influenza A virus induced formation of a TLR3-associated signaling complex containing TRIF, RIP1, FADD, cFLIP and pro-caspase-8. Co-administration of IL-24 decreased the presence of cFLIP in the TLR3-associated complex, converting it into an atypical, TLR3-associated death-inducing signaling complex (TLR3 DISC) that induced apoptosis by enabling caspase-8 activation at this complex. The sensitizing effect of IL-24 on TLR3-induced apoptosis, mediated by influenza A virus or the TLR3-specific agonist poly(I:C), was also evident on tumor spheroids. In conclusion, rather than acting as an apoptosis inducer itself, IL-24 sensitizes cancer cells to TLR-mediated apoptosis by enabling the formation of an atypical DISC which, in the case of influenza A virus or poly(I:C), is associated with TLR3.

Oxidative stress may be the most significant threat to the survival of living organisms. Glutathione S-transferases (GSTs) serve as the primary defences against xenobiotic and peroxidative-induced oxidative damage. In contrast to other well-defined GST classes, the Omega-class members are poorly understood, particularly in insects. Here, we isolated and characterised the GSTO2 gene from Apis cerana cerana (AccGSTO2). The predicted transcription factor binding sites in the AccGSTO2 promoter suggested possible functions in early development and antioxidant defence. Real-time quantitative PCR (qPCR) and western blot analyses indicated that AccGSTO2 was highly expressed in larvae and was predominantly localised to the brain tissue in adults. Moreover, AccGSTO2 transcription was induced by various abiotic stresses. The purified recombinant AccGSTO2 exhibited glutathione-dependent dehydroascorbate reductase and peroxidase activities. Furthermore, it could prevent DNA damage. In addition, Escherichia coli overexpressing AccGSTO2 displayed resistance to long-term oxidative stress exposure in disc diffusion assays. Taken together, these results suggest that AccGSTO2 plays a protective role in counteracting oxidative stress.

Hsp70 is involved in immune responses against infectious pathogens, thermal, and osmotic stress. To understand the immune defense mechanisms of the mud crab Scylla paramamosain, genomic DNA, transcript level and antimicrobial activities of Hsp70 were analyzed. Genomic DNA sequence analysis revealed one intron in this gene. Furthermore, six SNPs were detected by direct sequencing from 30 samples in this study. Hsp70 mRNA was expressed in almost all tissues examined. By using the quantitative real-time PCR, the expression level of Hsp70 in hemocytes showed a clear time-dependent expression pattern during the 96 h after stimulated by Vibrio alginolyticus. Then, recombinant Hsp70 was obtained by using the bacterial expression system, but no obvious antimicrobial activity has been found for the protein in the antimicrobial tests. After osmotic stress, the expression of Hsp70 in hemocytes showed this gene was induced by the high salinity (30 ‰) for at least 96 h. Hsp70 mRNA expression in hemocytes was analyzed after thermal stress at 6 h, the highest and the lowest expression level of Hsp70 was observed at 36 and 15 °C, respectively. These results indicated that Hsp70 was inducible by bacterial, osmotic, and thermal stress, and therefore plays an important role, different from antibacterial peptide, in innate immune responses of S. paramamosain.

The level of vitamin D-binding protein (DBP) is increased in the cerebrospinal fluid of patients with Alzheimer's disease (AD), suggesting a relationship with its pathogenesis. In this study, we investigated whether and how DBP is related to AD using several different approaches. A pull-down assay and a surface plasmon resonance binding assay indicated direct interactions between purified DBP and amyloid beta (Aβ), which was confirmed in the brain of AD patients and transgenic AD model mice by immunoprecipitation assay and immunohistochemical double-staining method. Moreover, atomic force microscopic examination revealed that DBP reduced Aβ aggregation in vitro. DBP also prevented Aβ-mediated death in cultured mouse hippocampal HT22 cell line. Finally, DBP decreased Aβ-induced synaptic loss in the hippocampus and rescued memory deficits in mice after injection of Aβ into the lateral ventricle. These results provide converging evidence that DBP attenuates the harmful effects of Aβ by a direct interaction, and suggest that DBP is a promising therapeutic agent for the treatment of AD.