NMR analysis of the CSF and plasma metabolome of rigorously matched amyotrophic lateral sclerosis,Parkinson’s disease and control subjects

Introduction

Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are two severe neurodegenerative disorders for which the disease mechanisms are poorly understood and reliable biomarkers are absent.

Objectives

To identify metabolite biomarkers for ALS and PD, and to gain insights into which metabolic pathways are involved in disease.

Methods

Nuclear magnetic resonance (NMR) metabolomics was utilized to characterize the metabolite profiles of cerebrospinal fluid (CSF) and plasma from individuals in three age, gender, and sampling-date matched groups, comprising 22 ALS, 22 PD and 28 control subjects.

Results

Multivariate analysis of NMR data generated robust discriminatory models for separation of ALS from control subjects. ALS patients showed increased concentrations of several metabolites in both CSF and plasma, these are alanine (CSF fold change = 1.22, p = 0.005), creatine (CSF-fc = 1.17, p = 0.001), glucose (CSF-fc = 1.11, p = 0.036), isoleucine (CSF-fc = 1.24, p = 0.002), and valine (CSF-fc = 1.17, p = 0.014). Additional metabolites in CSF (creatinine, dimethylamine and lactic acid) and plasma (acetic acid, glutamic acid, histidine, leucine, pyruvate and tyrosine) were also important for this discrimination. Similarly, panels of CSF-metabolites that discriminate PD from ALS and control subjects were identified.

Conclusions

The results for the ALS patients suggest an affected creatine/creatinine pathway and an altered branched chain amino acid (BCAA) metabolism, and suggest links to glucose and energy metabolism. Putative metabolic markers specific for ALS (e.g. creatinine and lactic acid) and PD (e.g. 3-hydroxyisovaleric acid and mannose) were identified, while several (e.g. creatine and BCAAs) were shared between ALS and PD, suggesting some overlap in metabolic alterations in these disorders.

     

Association between the blood concentrations of ammonia and carnitine/amino acid of schizophrenic patients treated with valproic acid

Background

Administration of valproic acid (VPA) is complicated with approximately 0.9% of patients developing hyperammonemia, but the pathogenesis of this adverse effect remains to be clarified. The aim of the present study was to search for mechanisms associated with VPA-induced hyperammonemia in the light of changes in serum amino acids concentrations associated with the urea cycle of schizophrenic patients.

Method

Blood samples (10 mL) were obtained from 37 schizophrenic patients receiving VPA for the prevention of violent behaviors in the morning after overnight fast. Blood concentrations of ammonia, VPA, free carnitine, acyl-carnitine, and 40 amino acids including glutamate and citrulline were measured for each patient. Univariate and multivariate regression analyses were performed to identify amino acids or concomitantly administered drugs that were associated with variability in the blood concentrations of ammonia.

Result

The blood ammonia level was positively correlated with the serum glutamate concentration (r = 0.44, p < 0.01) but negatively correlated with glutamine (r = ?0.41, p = 0.01), citrulline (r = ?0.42, p = 0.01), and glycine concentrations (r = ?0.54, p < 0.01). It was also revealed that the concomitant administration of the mood stabilizers (p = 0.04) risperidone (p = 0.03) and blonanserin (p < 0.01) was positively associated with the elevation of the blood ammonia level.

Conclusion

We hypothisized that VPA would elevate the blood ammonia level of schizophrenic patients. The observed changes in serum amino acids are compatible with urea cycle dysfunction, possibly due to reduced carbamoyl-phosphate synthase 1 (CPS1) activity. We conclude that VPA should be prudently prescribed to schizophrenic patients, particularly those receiving mood stabilizers or certain antipsychotics.

     

No diurnal variation of classical and candidate biomarkers of Alzheimer’s disease in CSF

Background

Cerebrospinal fluid (CSF) biomarkers have gained increasing importance in the diagnostic work-up of Alzheimer’s disease (AD). The core CSF biomarkers related to AD pathology (Aβ42, t-tau and p-tau) are currently used in CSF diagnostics, while candidate markers of amyloid metabolism (Aβ38, Aβ40, sAPPα, sAPPβ), synaptic loss (neurogranin), neuroinflammation (YKL-40), neuronal damage (VILIP-1) and genetic risk (apolipoprotein E) are undergoing evaluation. Diurnal fluctuation in the concentration of CSF biomarkers has been reported and may represent a preanalytical confounding factor in the laboratory diagnosis of AD. The aim of the present study was to investigate the diurnal variability of classical and candidate CSF biomarkers in a cohort of neurosurgical patients carrying a CSF drainage.

Method

Samples were collected from a cohort of 13 neurosurgical patients from either ventricular (n?=?6) or lumbar (n?=?7) CSF drainage at six time points during the day, 1–7 days following the neurosurgical intervention. Concentrations of the core biomarkers were determined by immunoassays.

Results

Although absolute values largely varied among subjects, none of the biomarkers showed significant diurnal variation. Site of drainage (lumbar vs. ventricular) did not influence this result. The different immunoassays used for tau and Aβ markers provided similar results.

Conclusion

Time of day at CSF collection does not ultimately affect the concentration levels of classical and candidate AD biomarkers. Similar trends were found when using different immunoassays, thus corroborating the consistency of the data.

     

Genotyping and Persistence of <Emphasis Type="Italic">Candida albicans</Emphasis> from Pregnant Women with Vulvovaginal Candidiasis

Objective

To study Candida albicans genotypes using RAPD and their susceptibility to fluconazole in healthy pregnant women and in vulvovaginal candidiasis (VVC) patients after topical treatment with clotrimazole.

Methods

Vaginal swabs were collected at t = 0 and t = 1 (1 month later) in pregnant women (control group, n = 33), and before (t = 0), at 1 month (t = 1) and at 2 months (t = 2) after clotrimazole treatment in pregnant women with VVC.

Results

Candida albicans was isolated in 30% of healthy pregnant women and 80% of patients with VVC. A high genetic heterogeneity was observed in C. albicans genotypes between individuals. In patients with VVC, topical antifungal treatment with clotrimazole was clinically effective, but only in a 62% C. albicans was eradicated. In patients in which C. albicans was not eradicated, this microorganism persisted for 1 or 2 months after the antifungal treatment. The persistent colonies were not associated with a specific genotype, but they were associated with higher MICs in comparison with colonies isolated from the control group.

Conclusions

Therapy with topical clotrimazole, despite a good clinical outcome, could not eradicate completely C. albicans allowing the persistence of genotypes, with higher MICs to fluconazole. More studies with higher number of patients are needed to validate this preliminary finding.

     

Improved production of carotenoid-free welan gum in a genetic-engineered <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ATCC31555

Objective

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.

Results

The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l^?1 compared to 21 g ± 0.4 g l^?1 in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.

Conclusions

Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Serum protein pattern associated with organ damage and lupus nephritis in systemic lupus erythematosus revealed by PEA immunoassay

Background

Systemic lupus erythematosus (SLE) is a remarkably heterogeneous autoimmune disease. Despite tremendous efforts, our knowledge of serum protein patterns in severe SLE phenotypes is still limited. We investigated the serum protein pattern of SLE, with special emphasis on irreversible organ damage and active lupus nephritis (LN) as assessed by renal Systemic Lupus Erythematosus Disease Activity Index.

Methods

We used proximity extension immunoassay (PEA, Proseek Multiplex, Olink) to assess the serum levels of ninety-two inflammation-related proteins in Czech patients with SLE (n = 75) and age-matched healthy control subjects (n = 23). Subgroup analysis was carried out on the basis of organ damage (with/without, 42/33) and biopsy-proven LN (with/without, 27/48; active LN, n = 13; inactive LN, n = 14).

Results

Of thirty deregulated proteins between SLE and the healthy controls (P _corr  < 0.05), the top upregulated proteins in SLE were sirtuin 2, interleukin 18 (IL18), and caspase 8 (P _corr  < 0.0006). Of these, sirtuin 2 and caspase 8 had not yet been reported with SLE. Elevated levels of IL8, CCL2/MCP1, CCL11, and MMP10 (P _corr  < 0.05) were detected in patients with organ damage for which the serum levels of CCL11 and MMP10 were particularly informative in organ damage prediction. Comparing patients based on LN, elevated levels of CSF1, sIL15RA, sCD40, sCX3CL1, caspase 8, sIL18R1, bNGF, and GDNF (P _corr  < 0.05) were detected in active LN. Except GDNF, all LN-associated markers showed usefulness in prediction of active renal disease.

Conclusions

This highly sensitive PEA analysis identified the serum pattern of SLE, organ damage, and active LN, with many novel candidate proteins detected. Their exact role and suitability as biomarkers in SLE deserve further investigation.

     

Characterization of an ene-reductase from <Emphasis Type="Italic">Meyerozyma guilliermondii</Emphasis> for asymmetric bioreduction of α,β-unsaturated compounds

Objectives

To characterize a novel ene-reductase from Meyerozyma guilliermondii and achieve the ene-reductase-mediated reduction of activated C=C bonds.

Results

The gene encoding an ene-reductase was cloned from M. guilliermondii. Sequence homology analysis showed that MgER shared the maximal amino acid sequence identity of 57 % with OYE2.6 from Scheffersomyces stipitis. MgER showed the highest specific activity at 30 °C and pH 7 (100 mM sodium phosphate buffer), and excellent stereoselectivities were achieved for the reduction of (R)-carvone and ketoisophorone. Under the reaction conditions (30 °C and pH 7.0), 150 mM (R)-carvone could be completely converted to (2R,5R)-dihydrocarvone within 22 h employing purified MgER as catalyst, resulting in a yield of 98.9 % and an optical purity of >99 % d.e.

Conclusion

MgER was characterized as a novel ene-reductase from yeast and showed great potential for the asymmetric reduction of activated C=C bonds of α,β-unsaturated compounds.

     

Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress.

Results

Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g^-1 dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain.

Conclusion

The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.

     

Functional alterations of myeloid cells during the course of Alzheimer’s disease

Background

Neuroinflammation is a hallmark of neurodegenerative disease and a significant component of the pathology of Alzheimer’s disease (AD). Patients present with extensive microgliosis along with elevated pro-inflammatory signaling in the central nervous system and periphery. However, the role of peripheral myeloid cells in mediating and influencing AD pathogenesis remains unresolved.

Methods

Peripheral myeloid cells were isolated from peripheral blood of patients with prodromal AD (n =?44), mild AD dementia (n =?25), moderate/severe AD dementia (n =?28), and age-matched controls (n =?54). Patients were evaluated in the clinic for AD severity and categorized using Clinical Dementia Rating (CDR) scale resulting in separation of patients into prodromal AD (CDR0.5) and advancing forms of AD dementia (mild-CDR1 and moderate/severe-CDR2/3). Separation of peripheral myeloid cells into mature monocytes or immature MDSCs permitted the delineation of population changes from flow cytometric analysis, RNA phenotype analysis, and functional studies using T cell suppression assays and monocyte suppression assays.

Results

During stages of AD dementia (CDR1 and 2/3) peripheral myeloid cells increase their pro-inflammatory gene expression while at early stages of disease (prodromal AD—CDR0.5) pro-inflammatory gene expression is decreased. MDSCs are increased in prodromal AD compared with controls (16.81% vs 9.53%) and have markedly increased suppressive functions: 42.4% suppression of activated monocyte-produced IL-6 and 78.16% suppression of T cell proliferation. In AD dementia, MDSC populations are reduced with decreased suppression of monocyte IL-6 (5.22%) and T cell proliferation (37.61%); the reduced suppression coincides with increased pro-inflammatory signaling in AD dementia monocytes.

Conclusions

Peripheral monocyte gene expression is pro-inflammatory throughout the course of AD, except at the earliest, prodromal stages when pro-inflammatory gene expression is suppressed. This monocyte biphasic response is associated with increased numbers and suppressive functions of MDSCs during the early stages and decreased numbers and suppressive functions in later stages of disease. Prolonging the early protective suppression and reversing the later loss of suppressive activity may offer a novel therapeutic strategy.

     

Cerebrospinal fluid Presenilin-1 increases at asymptomatic stage in genetically determined Alzheimer’s disease

Background

Presenilin-1 (PS1), the active component of the intramembrane γ-secretase complex, can be detected as soluble heteromeric aggregates in cerebrospinal fluid (CSF). The aim of this study was to examine the different soluble PS1 complexes in the lumbar CSF (CSF-PS1) of individuals with Alzheimer’s disease (AD), particularly in both symptomatic and asymptomatic genetically determined AD, in order to evaluate their potential as early biomarkers.

Methods

Western blotting, differential centrifugation and co-immunoprecipitation served to determine and characterize CSF-PS1 complexes. We also monitored the assembly of soluble PS1 into complexes in a cell model, and the participation of Aβ in the dynamics and robustness of the stable PS1 complexes.

Results

There was an age-dependent increase in CSF-PS1 levels in cognitively normal controls, the different complexes represented in similar proportions. The total levels of CSF-PS1, and in particular the proportion of the stable 100–150 kDa complexes, increased in subjects with autosomal dominant AD that carried PSEN1 mutations (eight symptomatic and six asymptomatic ADAD) and in Down syndrome individuals (ten demented and ten non-demented DS), compared with age-matched controls (n?=?23), even prior to the appearance of symptoms of dementia. The proportion of stable CSF-PS1 complexes also increased in sporadic AD (n?=?13) and mild-cognitive impaired subjects (n?=?12), relative to age-matched controls (n?=?17). Co-immunoprecipitation demonstrated the association of Aβ oligomers with soluble PS1 complexes, particularly the stable complexes.

Conclusions

Our data suggest that CSF-PS1 complexes may be useful as an early biomarker for AD, reflecting the pathology at asymptomatic state.

     

Expression,purification and characterization of a thermostable leucine dehydrogenase from the halophilic thermophile <Emphasis Type="Italic">Laceyella sacchari</Emphasis>

Objective

A potential thermotolerant l-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized.

Results

Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C.

Conclusion

It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.

     

One pot simultaneous preparation of both enantiomer of β-amino alcohol and vicinal diol via cascade biocatalysis

Objectives

To investigate the efficiency of a new cascade biocatalysis system for the conversion of R, S-β-amino alcohols to enantiopure vicinal diol and β-amino alcohol.

Results

An efficient cascade biocatalysis was achieved by combination of a transaminase, a carbonyl reductase and a cofactor regeneration system. An ee value of > 99% for 2-amino-2-phenylethanol and 1-phenyl-1, 2-ethanediol were simultaneously obtained with 50% conversion from R, S-2-amino-2-phenylethanol. The generality of the cascade biocatalysis was further demonstrated with the whole-cell approaches to convert 10–60 mM R, S-β-amino alcohol to (R)- and (S)-diol and (R)- and (S)-β-amino alcohol in 90–99% ee with 50–52% conversion. Preparative biotransformation was demonstrated at a 50 ml scale with mixed recombinant cells to give both (R)- and (S)-2-amino-2-phenylethanol and (R)- and (S)-1-phenyl-1, 2-ethanediol in > 99% ee and 40–42% isolated yield from racemic 2-amino-2-phenylethanol.

Conclusions

This cascade biocatalysis system provides a new practical method for the simultaneous synthesis of optically pure vicinal diol and an β-amino alcohol.

     

Left ventricular outflow tract velocity time integral outperforms ejection fraction and Doppler-derived cardiac output for predicting outcomes in a select advanced heart failure cohort

Background

Left ventricular outflow tract velocity time integral (LVOT VTI) is a measure of cardiac systolic function and cardiac output. Heart failure patients with low cardiac output are known to have poor cardiovascular outcomes. Thus, extremely low LVOT VTI may predict heart failure patients at highest risk for mortality.

Methods

Patients with heart failure and extremely low LVOT VTI were identified from a single-center database. Baseline characteristics and heart failure related clinical outcomes (death, LVAD) were obtained at 12 months. Correlation between clinical endpoints and the following variables were analyzed: ejection fraction (EF), pulmonary artery systolic pressure (PASP), NYHA class, renal function, Doppler cardiac output (CO), and LVOT VTI.

Results

Study cohort consisted of 100 patients. At the 12-month follow up period, 30 events (28 deaths, 2 LVADs) were identified. Occurrence of death and LVAD implantation was statistically associated with a lower LVOT VTI (p = 0.039) but not EF (p = 0.169) or CO (p = 0.217). In multivariate analysis, LVOT VTI (p = 0.003) remained statistically significant, other significant variables were age (p = 0.033) and PASP (p = 0.022). Survival analysis by LVOT VTI tertile demonstrated an unadjusted hazard ratio of 4.755 (CI 1.576-14.348, p = 0.006) for combined LVAD and mortality at one year.

Conclusions

Extremely low LVOT VTI strongly predicts adverse outcomes and identifies patients who may benefit most from advanced heart failure therapies.

     

Root strength and density decay after felling in a Silver Fir-Norway Spruce stand in the Italian Alps

Aims

Forests induce a mechanical reinforcement of soil, generally quantified in terms of additional root cohesion (c _r ), which decreases due to root decay after felling. The aim of this work is providing new field data on soil reinforcement by roots after trees cutting.

Methods

The present work investigated c _r decay in a mixed Silver Fir-Norway Spruce (Abies alba Mill. Picea abies (L.) Karst.) stand in the Italian Alps over a period of 3 years after felling by monitoring the two c _r driving variables: root tensile resistance and root density.

Results

Results showed that a significant difference in root resistance occurred only 3 years after felling, whereas the decrease in the number of roots was significant in the second year. The degradation process was more rapid in shallower layers and for thinner roots, as a consequence of the pattern of biological activity rate. The reduction of c _r after felling was, for a reference profile depth of 70 cm, 55 % in the first 2 years and another 16 % in the third year.

Conclusions

The findings of this study, providing new data on the decrease of c _r after felling, can be introduced into geotechnical models allowing a better estimation of the stability of forest hillslopes.

     

Characterization of a novel <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> xanthine dehydrogenase expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To characterize a novel xanthine dehydrogenase (XDH) from Acinetobacter baumannii by recombinant expression in Escherichia coli and to assess its potential for industrial applications.

Results

The XDH gene cluster was cloned from A. baumannii CICC 10254, expressed heterologously in E. coli and purified to homogeneity. The purified recombinant XDH consisted of two subunits with the respective molecular weights of 87 kDa and 56 kDa according to SDS-PAGE. XDH catalysis was optimum at pH 8.5 and 40–45 °C, was stable under alkaline conditions (pH 7–11) and the half-inactivation temperature was 60 °C. The K _m, turnover number and catalytic efficiency for xanthine were 25 μM, 69 s^?1 and 2.7 μM^?1 s^?1, respectively, which is an improvement over XDHs characterized previously. A. baumannii XDH is less than 50 % identical to previously identified XDH orthologs from other species, and is the first from the Acinetobacter genus to be characterized.

Conclusion

The novel A. baumannii enzyme was found to be among the most active, thermostable and alkaline-tolerant XDH enzymes reported to date and has potential for use in industrial applications.

     

Delivery of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> HpaA to gastrointestinal mucosal immune sites using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its immune efficacy in mice

Objective

To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis.

Results

The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P < 0.05).

Conclusions

A novel engineered L. lactis strain was developed that efficiently produces whole HpaA protein with desired antigenicity and potent immunogenicity. It provides a basis for approaches to L. lactis-delivered anti-H. pylori vaccination.

     

Tumour infiltrating lymphocytes correlate with improved survival in patients with oesophageal adenocarcinoma

Background

Oesophageal adenocarcinoma (OAC) is increasingly common in the west, and survival remains poor at 10–15 % at 5 years. Immune responses are increasingly implicated as a determining factor of tumour progression. The ability of lymphocytes to recognise tumour antigens provides a mechanism for a host immune attack against cancer providing a potential treatment strategy.

Materials and Methods

Tumour infiltrating lymphocytes (TILs: CD3+, CD4+, CD8+ and FOXp3+) were assessed by immunohistochemistry using tissue microarrays in a contemporary and homogeneous cohort of OAC patients (n = 128) undergoing curative treatment.

Results

Multivariate analysis identified three independent prognostic factors for improved cancer-specific survival (CSS): increased CD8+ TILs (p = 0.003), completeness of resection (p < 0.0001) and lower pathological N stage (p < 0.0001). Independent prognostic factors for favourable disease-free survival included surgery-only treatment (p = 0.015), completeness of resection (p = 0.001), increased CD8+ TILs (p < 0.0001) and reduced pathological N stage (p < 0.0001). Higher levels of TILs in the pathological specimen were associated with significant pathological response to neoadjuvant chemotherapy (NAC). On multivariate analysis increased levels of CD4+ (p = 0.017) and CD8+ TILs (p = 0.005) were associated with significant local tumour regression and lymph node downstaging, respectively.

Discussion

Our results establish an association of TILs and survival in OAC, as seen in other solid tumours, and identify particular TIL subsets that are present at higher levels in patients who responded to NAC compared to non-responders. These findings highlight potential therapeutic strategies in EAC based on utilising the host immunological response and highlight the immune responses biomarker potential.

     

Expression and biochemical characterization of a novel type I pullulanase from <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Objectives

To identify novel pullulanases from microorganisms and to investigate their biochemical characterizations.

Results

A novel pullulanase gene (BmPul) from Bacillus megaterium WW1210 was cloned and heterologously expressed in Escherichia coli. The gene has an ORF of 2814 bp encoding 937 amino acids. The recombinant pullulanase (BmPul) was purified to homogeneity and biochemically characterized. BmPul has an MW of approx. 112 kDa as indicated by SDS-PAGE. Optimum conditions were at 55 °C and pH 6.5. The enzyme was stable below 40 °C and from pH 6.5?8.5. The Km values of BmPul towards pullulan and amylopectin were 3.3 and 3.6 mg/ml, respectively. BmPul hydrolyzed pullulan to yield mainly maltotriose, indicating that it should be a type I pullulanase.

Conclusions

A novel type I pullulanase from Bacillus megaterium was identified, heterologously expressed and biochemically characterized. Its properties makes this enzyme as a good candidate for the food industry.