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In this study, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was cloned from Tetragenococcus halophilus CY54. The translated PepA from T. halophilus CY54 showed very low similarities with PepAs from Lactobacillus and Lactococcus genera. The pepA from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) using pET26b(+). The recombinant PepA was purified by using an Ni- NTA column. The size of the recombinant PepA was 39.13 kDa as determined by SDS-PAGE, while its optimum pH and temperature were pH 5.0 and 60°C, respectively. In addition, the PepA was completely inactivated by 1 mM EDTA, indicating its metallopeptidase nature. The Km and Vmax of the PepA were 0.98 ± 0.006 mM and 0.1 ± 0.002 mM/min, respectively, when Glu-pNA was used as the substrate. This is the first report on PepA from Tetragenococcus species.

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Toxin-antitoxin (TA) systems are small genetic elements which encode toxin proteins that interfere with vital cellular functions. PepA1 and PepG1 toxin proteins, known also as SprA1 and SprG1, are type I TA. In Staphylococcus aureus (S. aureus), their expression without the antitoxin counterparts (SprA1AS and SprF1), is lethal to the pathogen. Molecular Dynamics (MD) simulation was performed for PepA1 and PepG1 to understand their dynamic state, conformational changes, and their toxicity. The protein structures were constructed and used for MD simulation and the conformational changes, stability, flexibility, fluctuations, hydrophobicity, and role of their dynamic state on function prediction were studied extensively by GROMACS MD simulation analysis tools. In silico study indicated that the PepA1 and PepG1 proteins change their structural conformation from an open to closed state where PepA1 conformational changes were faster (10 ns) than PepG1 (20 ns) while PepG1 exerted more stability and flexibility than PepA1. According to SASA values, PepG1 is more hydrophobic than the PepA1 and forms fewer hydrogen bonds than PepA1. The in vivo study with PepA1 and PepG1 proteins provided evidence that both the conformation changes between the open and closed states and the amino acid sequence are crucial for peptide toxicity.

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INTRODUCTION: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) receptors play a central role in neurotransmission and neuronal function. A positron emission tomography (PET) tracer for AMPA receptors, [11C]K-2, was recently developed by us to visualize AMPA receptors in the living human brain. [11C]K-2 is a derivative of 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difuluoro-phenoxyacetamide (PEPA), and is labeled with the radioactive isotope 11C, which has a short half-life. PET drugs are usually labeled with 18F because of its long half-life. Therefore, we screened and identified potential 18F-labeled PET drugs for AMPA receptors (AMPA-PET drugs), which could provide an image equivalent to that of [11C]K-2. METHODS: Derivatives of K-2 labeled with 18F were synthesized and administered to rats and PET imaging was performed. The transferability of each compound to the brain and its correlation with the PET image of [11C]K-2 were evaluated from the obtained PET images. Furthermore, the specific binding ability of promising compounds to the AMPA receptor was evaluated by the PET imaging of rats, which we specifically knocked down the expression of AMPA by the lentivirus-mediated introduction of short hairpin RNA (shRNA) targeted to subunits of the AMPA receptor (GluA1-A3). The specific binding ability was also evaluated through electrophysiological experiments with acute brain slices. RESULTS: Some of the synthesized 18F-labeled candidate compounds showed a distribution similar to that of K-2, with reasonable transferability to the brain. In addition, from the evaluation of the specific binding ability to the AMPA receptor, a promising structure of an 18F-labeled AMPA PET drug was identified. This study also revealed that the alkylation of the sulfonamide group of PEPA enhances brain transferability.

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Renal impairment in Multiple Myeloma (MM) represents one of the most important factors that influences patient survival. In fact, before the introduction of modern chemotherapy, less than 25% of patients with acute kidney injury (AKI) and MM who required dialysis recovered sufficient renal function to become independent from dialysis, with a median overall survival of less than 1 year. There are many other factors involved in determining patient survival. In this study we aimed to investigate the role of double filter-based extracorporeal treatment for removal of serum free light chains (sFLC) in acute myeloma kidney (AKI for MM) and to evaluate patient overall survival. All patients received Bortezomib-based chemotherapy and extracorporeal treatment for sFLC removal. For each session 2 dialyzers of the same kind were used. The dialytic dose was not related to the degree of renal function but to the removal of sFLC. The factors that have been found to be significantly associated with lower mortality were reduction of sFLC at day 12 and day 30, >50% reduction of sFLC at day 30, number of sessions and independence from dialysis. Among baseline characteristics, albumin level was statistically associated with the patients' outcome. Our analysis highlights the importance of the early treatment for removal of sFLC in AKI for MM. These results indicate that the early removal of sFLC can improve patient's outcome.

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For designing and evaluating the dialyzer and investigating the optimal therapeutic conditions, in vitro studies bring us many useful findings. In hemodialysis, however, the membrane fouling due to protein molecules reduces solute removal performance. Therefore, we investigated a method for replicating the fouling in dialyzers in aqueous experiments. After the albumin solution was circulated in the test circuit with a dialyzer, a glutaraldehyde solution was pumped into the dialyzer to immobilize albumin on the hollow fiber membrane. Under various immobilization conditions, the permeability of creatinine and vitamin B12 was evaluated by dialysis experiments. The creatinine clearance after immobilization of albumin was decreased, suggesting pore plugging by our fouling replication method. The glutaraldehyde crosslinked albumin molecules that adhered them to the membrane firmly. Moreover, the degree of fouling may be controlled by changing the concentration of albumin solution and the volume of glutaraldehyde solution used for immobilization. Our fouling replication method was applied to three types of polyester polymer alloy (PEPA) dialyzers and one polysulfone (PSf) dialyzer. This method enables to evaluate the permeability of various dialyzers with fouling in vitro that will be of great help in collecting data for designing dialyzers.

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The significant number of people with latent and active tuberculosis infection requires further efforts to develop new vaccines or improve the Bacillus Calmette-Guérin (BCG), which is the only approved vaccine against this disease. In this study, we developed a recombinant fusion protein (PEPf) containing high-density immunodominant epitope sequences from Rv0125, Rv2467, and Rv2672 Mycobacterium tuberculosis (Mtb) proteases that proved immunogenic and used it to develop a recombinant BCG vaccine expressing the fusion protein. After challenging using Mtb, a specific immune response was recalled, resulting in a reduced lung bacterial load with similar protective capabilities to BCG. Thus BCG PEPf failed to increase the protection conferred by BCG. The PEPf was combined with Advax4 adjuvant and tested as a subunit vaccine using a prime-boost strategy. PEPf + Advax4 significantly improved protection after Mtb challenge, with a reduction in bacterial load in the lungs. Our results confirm that Mtb proteases can be used to develop vaccines against tuberculosis and that the use of the recombinant PEPf subunit protein following a prime-boost regimen is a promising strategy to improve BCG immunity.

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OBJECTIVES: Neonates who develop moderate to severe encephalopathy following perinatal asphyxia will benefit from therapeutic hypothermia. Current National Institute of Child Health and Human Development (NICHD) criteria for identifying encephalopathic neonates needing therapeutic hypothermia has high specificity. This results in correctly identifying neonates who have already developed moderate to severe encephalopathy but miss out many potential beneficiaries who progress to develop moderate to severe encephalopathy later. The need is therefore not just to diagnose encephalopathy, but to predict development of encephalopathy and extend the therapeutic benefit for all eligible neonates. The primary objective of the study was to develop and validate the statistical model for prediction of moderate to severe encephalopathy following perinatal asphyxia and compare with current NICHD criteria. METHODS: The study was designed as prospective observational study. It was carried out in a single center Level 3 perinatal unit in India. Neonates>35 weeks of gestation and requiring resuscitation at birth were included. Levels of resuscitation and blood gas lactate were used to determine the pre-test probability, Thompson score between 3 and 5 h of life was used to determine post-test probability of developing encephalopathy. Primary outcome measure: Validation of Prediction of Encephalopathy in Perinatal Asphyxia (PEPA) score by Holdout method. RESULTS: A total of 55 babies were included in the study. The PEPA score was validated by Holdout method where the fitted receiver-operating characteristic (ROC) area for the training and test sample were comparable (p=0.758). The sensitivity and specificity of various PEPA scores for prediction of encephalopathy ranged between 74 and 100% in contrast to NICHD criteria which was 42%. PEPA score of 30 had a best combination of sensitivity and specificity of 95 and 89% respectively. CONCLUSIONS: PEPA score has a higher sensitivity than NICHD criteria for prediction of Encephalopathy in asphyxiated neonates.

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The barrier performance and structural lightening of organic materials are increasingly desired and constitute a major challenge for manufacturers, particularly for transport and packaging. A promising technique which tends to emerge in recent years is that of multinanolayer coextrusion. The advantage is that it can produce multilayers made of thousands of very thin layers, leading to new properties due to crystalline morphology changes induced by confinement. This paper is focusing on the study of multinanolayered films with alternated polyethylene (PE), compatibilizer (PEgMA) and polyamide 6 (PA6) layers and made by a forced assembly coextrusion process equipped with layer multiplying elements (LME). PE/PA6 multilayer films consisting of 5 to 2049 layers (respectively 0 to 9 LME) were successfully obtained with well-organized multilayered structure. The evolution of the morphology and the microstructure of these two semi-crystalline polymers, when the thickness of each polymer layer decreases from micro-scale to nano-scale, was correlated to the water and gas transport properties of the PE/PA multilayers. The expected improvement of barrier properties was limited due to the on-edge orientation of crystals in very thin PE and PA6 layers. Despite this change of crystalline morphology, a slight improvement of the gas barrier properties was shown by comparing experimental results with permeabilities predicted on the basis of a serial model developed by considering a PE/PA6 interphase. This interphase observed by TEM images and the on-edge crystal orientation in multilayers were evidenced from mechanical properties showing an increase of the stiffness and the strength.

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BACKGROUND: In primary care, patients play a crucial role in managing care processes and handling drug treatment. A decisive factor for success is their health literacy, and several interventions have been introduced to support patients in fulfilling their responsibility. OBJECTIVE: The aim of this study is to assess the influence of such an intervention (ie, a medication module) within a patient-led electronic health record on patients' health literacy. METHODS: We conducted a randomized controlled study among community-dwelling patients with type 2 diabetes mellitus. Patients were recruited from primary care practices. After randomization, patients either had access to an internet-based medication module allowing them to store their medication information, look up drug information, and print a medication schedule (intervention group), or they received an information brochure on the importance of medication schedules (control group). After 4-8 weeks, all patients were invited to attend a structured medication review (ie, follow-up visit). Data were collected via questionnaires before the start of the intervention and during the follow-up visit. The main outcome measure was the mean difference in health literacy between baseline and follow-up assessments of patients in the control and intervention groups. RESULTS: Of 116 recruited patients, 107 (92.2%) completed the follow-up assessment and were eligible for intention-to-treat analyses. Only 73 patients, of which 29 were in the intervention group, followed the study protocol and were eligible for per-protocol analysis. No differences in overall health literacy were observed in either the intention-to-treat or in the per-protocol cohorts. Reasons for a null effect might be that the cohort was not particularly enriched with participants with low health literacy, thus precluding measurable improvement (ie, ceiling effect). Moreover, the success of implementation was considered poor because both the correct application of the study procedure (ie, randomization according to the protocol and dropout of 29 patients) and the actual interaction with the medication module was modest (ie, dropout of 9 patients). CONCLUSIONS: The conduct of this randomized controlled study was challenging, leaving it open whether inadequate implementation, too short of a duration, or insufficient efficacy of the intervention, as such, contributed to the null effect of this study. This clearly outlines the value of piloting complex interventions and the accompanying process evaluations.

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The mash of sweet potato shochu (Japanese distilled spirit) has a low pH value because the shochu koji mold produces a large amount of citric acid, which prevents germ contamination. In this study, we examined acid protease PepA's role in shochu production. For this purpose, we constructed pepA deletion and overexpression strains, using a black koji mold Aspergillus luchuensis RIB 2604 (NBRC 4314), with the Agrobacterium-mediated transformation method. The rice koji, prepared using a pepA disruptant (ΔpepA) and pepA-overexpressing strain (OEpepA), demonstrated 1/2- and 24-fold acid protease activities compared to that prepared using the parental strain, respectively. A small-scale test of sweet potato shochu brewing indicated the mash of ΔpepA had a lower amino acid concentration, while the mash of OEpepA had a higher concentration than that produced by the parental strain. Therefore, the mash amino acid concentrations were proportional to these strains' acid proteases activities. After distilling these mashes, we examined each shochu's aroma components. Shochu prepared using ΔpepA had relatively higher aroma components, such as alcohol and ester, compared to that prepared using parental strains. Meanwhile, shochu prepared using OEpepA had lower aroma components than that prepared using the parental strains. Based on these results, the amount of shochu aroma components showed an inverse correlation to the acid protease activity in the mash. Thus, the koji mold's acid protease content had a greater influence on the aroma qualities of sweet potato shochu. Accordingly, we have discussed the possibility of the breeding of shochu koji mold with acid protease as an indicator.

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Chemotherapy and extracorporeal treatment reduce serum free light chains (FLCs) allowing the recovery of acute kidney injury (AKI) caused by myeloma cast nephropathy (MCN). We report the first case of recovery from AKI in a patient with MCN who underwent the removal of FLCs using the PEPA filter, with an undisclosed cut-off, combined with chemotherapy for multiple myeloma (MM).

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A challenging problem in systems biology is the reconstruction of gene regulatory networks from postgenomic data. A variety of reverse engineering methods from machine learning and computational statistics have been proposed in the literature. However, deciding on the best method to adopt for a particular application or data set might be a confusing task. The present chapter provides a broad overview of state-of-the-art methods with an emphasis on conceptual understanding rather than a deluge of mathematical details, and the pros and cons of the various approaches are discussed. Guidance on practical applications with pointers to publicly available software implementations are included. The chapter concludes with a comprehensive comparative benchmark study on simulated data and a real-work application taken from the current plant systems biology.

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The aminopeptidase A (PepA; EC 3.4.11.7) belongs to the group of metallopeptidases with two bound metal ions per subunit (M1M2(PepA)) and is specific for the cleavage of N-terminal glutamic (Glu) and aspartic acid (Asp) and, in low amounts, serine (Ser) residues. Our group recently characterized the first PepA from a Lactobacillus strain. However, the characterization was performed using synthetic para-nitroaniline substrates and not original peptide substrates, as was done in the current study. Prior to the characterization using original peptide substrates, the PepA purified was converted to its inactive apo-form and eight different metal ions were tested to restore its activity. It was found that five of the metal ions were able to reactivate apo-PepA: Co2+, Cu2+, Mn2+, Ni2+ and Zn2+. Interestingly, depending on the metal ion used for reactivation, the activity and the pH and temperature profile differed. Exemplarily, MnMn(PepA), NiNi(PepA) and ZnZn(PepA) had an activity optimum using MES buffer (50mM, pH 6.0) and 60°C, whereas the activity optimum changed to Na/K-phosphate-buffer (50mM, pH 7.0) and 55°C for CuCu(PepA). However, more important than the changes in optimum pH and temperature, the kinetic properties of PepA were affected by the metal ion used. While all PepA variants could release N-terminal Glu or Asp, only CoCo(PepA), NiNi(PepA) and CuCu(PepA) could release Ser from the particular peptide substrate. In addition, it was found that the enzyme efficiency (Vmax/KM) and catalytic mechanism (positive cooperative binding (Hill coefficent; n), substrate inhibition (KIS)) were influenced by the metal ion. Exemplarily, a high cooperativity (n>2),KIS value >20mM and preference for N-terminal Glu were detected for CuCu(PepA). In summary, the results suggested that an exchange of the metal ion can be used for tailoring the properties of PepA for specific hydrolysis requirements.

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Recently, intensive care physicians have focused on continuous hemodiafiltration with a cytokine-adsorbing hemofilter in the treatment of sepsis. We aimed to establish extracorporeal circulation in a rat sepsis model to evaluate the cytokine removal properties of mini-modules using two types of membrane materials. Rats were divided into polyester polymer alloy (PEPA) and cellulose triacetate (CTA) groups as membrane materials of mini-modules. One hour after 0.1 mg/kg of lipopolysaccharide administration, continuous hemofiltration (CHF) was started in each group. Plasma interleukin-6 (IL-6), an important mediator of sepsis, was measured over time during hemofiltration. The peak IL-6 concentration in PEPA group was approximately 13,000 pg/mL, in comparison to approximately 31,000 pg/mL in CTA group. IL-6 clearance in PEPA group was much more than CTA group. Since IL-6 was not detected in the filtrate in PEPA group, it was considered that IL-6 was adsorbed to the membrane. In conclusion, our results suggest that CHF with PEPA hemofilter can be suitable for removing IL-6 from the blood stream efficiently.

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Vaccination programs for childhood diseases, such as measles, mumps and rubella have greatly contributed to decreasing the incidence and impact of those diseases. Nonetheless, despite long vaccination programmes across the world, mumps has not yet been eradicated in those countries: indeed, large outbreaks continue. For example, in Scotland large outbreaks occurred in 2004, 2005, and 2015, despite introducing the MMR (Measles-Mumps-Rubella) vaccine more than 20 years ago. There are indications that this vaccine-preventable disease is re-emerging in highly vaccinated populations. Here we investigate whether the resurgence of mumps is due to waning immunity, and further, could a booster dose be the solution to eradicate mumps or would it just extend the period of waning immunity? Using mathematical modeling we enhance a seasonally-structured disease model with four scenarios: no vaccination, vaccinated individuals protected for life, vaccinated individuals at risk of waning immunity, and introduction of measures to increase immunity (a third dose, or a better vaccine). The model is parameterised from observed clinical data in Scotland 2004-2015 and the literature. The results of the four scenarios are compared with observed clinical data 2004-2016. While the force of infection is relatively sensitive to the duration of immunity and the number of boosters undertaken, we conclude that periodic large outbreaks of mumps will be sustained for all except the second scenario. This suggests that the current protocol of two vaccinations is optimal in the sense that while there are periodic large outbreaks, the severity of cases in vaccinated individuals is less than in unvaccinated individuals, and the size of the outbreaks does not decrease sufficiently with a third booster to make economic sense. This recommendation relies on continuous efforts to maintain high levels of vaccination uptake.

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The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His6-tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0-6.5) and temperature (60-65 °C) were comparable to the His6-tag harboring PepA; the KM value was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time.

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Peptide assimilation in Helicobacter pylori necessitates a coordinated working of the peptide transport systems (PepTs) and aminopeptidase (PepA). We found that H. pylori hydrolyzes two detector peptides, L-phenylalanyl-L-3-thiaphenylalanine (PSP) and L-phenylalanyl-L-2- sulfanilylglycine (PSG), primarily before intake and excludes their antibacterial effects, whereas Escherichia coli readily transports them with resultant growth inhibition. PSP assimilation by H. pylori was inhibited by aminopeptidase inhibitor bestatin, but not by dialanine or cyanide-m-chlorophenylhydrazone, contrary to that of E. coli. RT- and qRT-PCR analyses showed that H. pylori may express first the PepTs (e.g., DppA and DppB) and then PepA. In addition, western blot analysis of PepA suggested that the bacterium secretes PepA in response to specific inducers.

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The EWSR1 (EWS RNA-binding protein 1/Ewing Sarcoma Break Point Region 1) gene encodes a RNA/DNA binding protein that is ubiquitously expressed and involved in various cellular processes. EWSR1 deficiency leads to impairment of development and accelerated senescence but the mechanism is not known. Herein, we found that EWSR1 modulates the Uvrag (UV radiation resistance associated) gene at the post-transcription level. Interestingly, EWSR1 deficiency led to the activation of the DROSHA-mediated microprocessor complex and increased the level of Mir125a and Mir351, which directly target Uvrag. Moreover, the Mir125a- and Mir351-mediated reduction of Uvrag was associated with the inhibition of autophagy that was confirmed in ewsr1 knockout (KO) MEFs and ewsr1 KO mice. Taken together, our data indicate that EWSR1 is involved in the post-transcriptional regulation of Uvrag via a miRNA-dependent pathway, resulting in the deregulation of autophagy inhibition. The mechanism of Uvrag and autophagy regulation by EWSR1 provides new insights into the role of EWSR1 deficiency-related cellular dysfunction.

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Polypropylene (PP) is a general-purpose plastic, but some applications are constrained by its high flammability. Thus, flame retardant PP is urgently demanded. In this article, intumescent flame retardant PP (IFRPP) composites with enhanced fire safety were prepared using 1-oxo-4-hydroxymethyl-2,6,7-trioxa-1-phosphabicyclo [2.2.2] octane (PEPA) functionalized graphene oxide (PGO) as synergist. The PGO was prepared through a mild chemical reaction by the covalent attachment of a caged-structure organic compound, PEPA, onto GO nanosheets using toluene diisocynate (TDI) as the intermediary agent. The novel PEPA-functionalized graphene oxide not only improves the heat resistance of GO but also converts GO and PEPA from hydrophobic to hydrophilic materials, which leads to even distribution in PP. In our case, 7 wt% addition of PGO as one of the fillers for IFRPP composites significantly reduces its inflammability and fire hazards when compared with PEPA, by the improvement of first release rate peak (PHRR), total heat release, first smoke release rate peak (PSRR) and total smoke release, suggesting its great potential as the IFR synergist in industry. The reason is mainly attributed to the barrier effect of the unburned graphene sheets, which protects by the decomposition products of PEPA and TDI, promotes the formation of graphitized carbon and inhibits the heat and gas release.

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The electronic properties of biomolecules and their hybrids with inorganic materials can be utilized for the fabrication of nanoelectronic devices. Here, we report the charge transport behavior of protein-shelled inorganic nanoparticles combined with graphene and demonstrate their possible application as a bionanohybrid capacitor. The conductivity of PepA, a bacterial aminopeptidase used as a protein shell (PS), and the platinum nanoparticles (PtNPs) encapsulated by PepA was measured using a field effect transistor (FET) and a graphene-based FET (GFET). Furthermore, we confirmed that the electronic properties of PepA-PtNPs were controlled by varying the size of the PtNPs. The use of two poly(methyl methacrylate) (PMMA)-coated graphene layers separated by PepA-PtNPs enabled us to build a bionanohybrid capacitor with tunable properties. The combination of bioinorganic nanohybrids with graphene is regarded as the cornerstone for developing flexible and biocompatible bionanoelectronic devices that can be integrated into bioelectric circuits for biomedical purposes.

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