Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

Clusterin confers gmcitabine resistance in pancreatic cancer

Objective

To measure clusterin expression in pancreatic cancer tissues and cell lines and to evaluate whether clusterin confers resistance to gmcitabine in pancreatic cancer cells.

Methods

Immunohistochemistry for clusterin was performed on 50 primary pancreatic cancer tissues and 25 matched backgrounds, and clusterin expression in 5 pancreatic cancer cell lines was quantified by Western blot and PT-PCR. The correlation between clusterin expression level and gmcitabine IC50 in pancreatic cancer cell lines was evaluated. The effect of an antisense oligonucleotide (ASO) against clusterin(OGX-011) on gmcitabine resistance was evaluated by MTT assays. Xenograft model was used to demonstrate tumor growth.

Results

Pancreatic cancer tissues expressed significantly higher levels of clusterin than did normal pancreatic tissues (P < 0.01). Clusterin expression levels were correlated with gmcitabine resistance in pancreatic cancer cell lines, and OGX-011 significantly decreased BxPc-3 cells resistance to gmcitabine (P < 0.01). In vivo systemic administration of AS clusterin and gmcitabine significantly decreased the s.c. BxPC-3 tumor volume compared with mismatch control ODN plus gmcitabine.

Conclusion

Our finding that clusterin expression was significantly higher in pancreatic cancer than in normal pancreatic tissues suggests that clusterin may confer gmcitabine resistance in pancreatic cancer cells.

     

The effect of unhealthy <Emphasis Type="Italic">β</Emphasis>-cells on insulin secretion in pancreatic islets

Background

Insulin secreted by pancreatic islet β-cells is the principal regulating hormone of glucose metabolism and plays a key role in controlling glucose level in blood. Impairment of the pancreatic islet function may cause glucose to accumulate in blood, and result in diabetes mellitus. Recent studies have shown that mitochondrial dysfunction has a strong negative effect on insulin secretion.

Methods

In order to study the cause of dysfunction of pancreatic islets, a multiple cell model containing healthy and unhealthy cells is proposed based on an existing single cell model. A parameter that represents the function of mitochondria is modified for unhealthy cells. A 3-D hexagonal lattice structure is used to model the spatial differences among β-cells in a pancreatic islet. The β-cells in the model are connected through direct electrical connections between neighboring β-cells.

Results

The simulation results show that the low ratio of total mitochondrial volume over cytoplasm volume per β-cell is a main reason that causes some mitochondria to lose their function. The results also show that the overall insulin secretion will be seriously disrupted when more than 15% of the β-cells in pancreatic islets become unhealthy.

Conclusion

Analysis of the model shows that the insulin secretion can be reinstated by increasing the glucokinase level. This new discovery sheds light on antidiabetic medication.

     

<Emphasis Type="Italic">massPix</Emphasis>: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics

Introduction

Mass spectrometry imaging (MSI) experiments result in complex multi-dimensional datasets, which require specialist data analysis tools.

Objectives

We have developed massPix—an R package for analysing and interpreting data from MSI of lipids in tissue.

Methods

massPix produces single ion images, performs multivariate statistics and provides putative lipid annotations based on accurate mass matching against generated lipid libraries.

Results

Classification of tissue regions with high spectral similarly can be carried out by principal components analysis (PCA) or k-means clustering.

Conclusion

massPix is an open-source tool for the analysis and statistical interpretation of MSI data, and is particularly useful for lipidomics applications.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Genotypic and metabolic approaches towards the segregation of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strains producing different antibiotic resistant enzymes

Introduction

Genotype and metabolomic variation are important for bacterial survival and adaptation to environmental changes.

Objectives

In this study, we compared the relationship among Klebsiella pneumoniae strains based on their genotypic and metabolic profiles. In addition, we also evaluated the association of the relationship with beta-lactamase production.

Methods

A total of 53 K. pneumoniae strains isolated in 2013–2014 from a tertiary teaching hospital in Malaysia were subjected to antimicrobial susceptibility testing (AST) via disk diffusion method and beta-lactamase production confirmation. The bacterial strains were also typed genotypically and metabolically via REP-PCR and ¹H-NMR spectroscopy respectively. The concordance of the matrices derived based on genotypic and metabolic characterization was measured based on Spearman’s rank correlation.

Results

Spearman’s correlation rank showed that there is a weak but significant negative correlation between the genetic fingerprints and metabolic profiles of K. pneumoniae. Specifically, K. pneumoniae strains were clustered into five major clusters based on REP-PCR where most of the carbapenem resistant K. pneumoniae (CRKP) strains made up the major cluster. In contrast, metabolic patterns of the three groups (i.e. CRKP, extended spectrum beta-lactamase producing K. pneumoniae (ESBL), susceptible) of K. pneumoniae were clearly differentiated on PLS-DA score plots derived from ¹H-NMR spectroscopy.

Conclusion

Overall, this study showed that metabolomic profiling using ¹H-NMR spectroscopy is able to discriminate K. pneumoniae strains based on their beta-lactamase production status.

     

The metabolic switch can be activated in a recombinant strain of <Emphasis Type="Italic">Streptomyces lividans</Emphasis> by a low oxygen transfer rate in shake flasks

Background

In Streptomyces, understanding the switch from primary to secondary metabolism is important for maximizing the production of secondary metabolites such as antibiotics, as well as for optimizing recombinant glycoprotein production. Differences in Streptomyces lividans bacterial aggregation as well as recombinant glycoprotein production and O-mannosylation have been reported due to modifications in the shake flask design. We hypothetized that such differences are related to the metabolic switch that occurs under oxygen-limiting conditions in the cultures.

Results

Shake flask design was found to affect undecylprodigiosin (RED, a marker of secondary metabolism) production; the RED yield was 12 and 385 times greater in conventional normal Erlenmeyer flasks (NF) than in baffled flasks (BF) and coiled flasks (CF), respectively. In addition, oxygen transfer rates (OTR) and carbon dioxide transfer rates were almost 15 times greater in cultures in CF and BF as compared with those in NF. Based on these data, we obtained respiration quotients (RQ) consistent with aerobic metabolism for CF and BF, but an RQ suggestive of anaerobic metabolism for NF.

Conclusion

Although the metabolic switch is usually related to limitations in phosphate and nitrogen in Streptomyces sp., our results reveal that it can also be activated by low OTR, dramatically affecting recombinant glycoprotein production and O-mannosylation and increasing RED synthesis in the process.

     

Evaluation of <Emphasis Type="Italic">in silico</Emphasis> algorithms for use with ACMG/AMP clinical variant interpretation guidelines

Background

The American College of Medical Genetics and American College of Pathologists (ACMG/AMP) variant classification guidelines for clinical reporting are widely used in diagnostic laboratories for variant interpretation. The ACMG/AMP guidelines recommend complete concordance of predictions among all in silico algorithms used without specifying the number or types of algorithms. The subjective nature of this recommendation contributes to discordance of variant classification among clinical laboratories and prevents definitive classification of variants.

Results

Using 14,819 benign or pathogenic missense variants from the ClinVar database, we compared performance of 25 algorithms across datasets differing in distinct biological and technical variables. There was wide variability in concordance among different combinations of algorithms with particularly low concordance for benign variants. We also identify a previously unreported source of error in variant interpretation (false concordance) where concordant in silico predictions are opposite to the evidence provided by other sources. We identified recently developed algorithms with high predictive power and robust to variables such as disease mechanism, gene constraint, and mode of inheritance, although poorer performing algorithms are more frequently used based on review of the clinical genetics literature (2011–2017).

Conclusions

Our analyses identify algorithms with high performance characteristics independent of underlying disease mechanisms. We describe combinations of algorithms with increased concordance that should improve in silico algorithm usage during assessment of clinically relevant variants using the ACMG/AMP guidelines.

     

Resveratrol induces insulin gene expression in mouse pancreatic α-cells

Background

Type 1 and type 2 diabetes are characterized by loss of β-cells; therefore, β-cell regeneration has become one of the primary approaches to diabetes therapy. Resveratrol, a naturally occurring polyphenolic compound, has been shown to improve glycaemic control in diabetic patients, but its action on pancreatic α-cells is not well understood.

Findings

Using mouse α-cells (αTC9), we showed that resveratrol induces expression of pancreatic β-cell genes such as Pdx1 and Ins2 in a SirT1-dependent manner. The mRNA and protein levels of insulin were further increased by histone deacetylase (HDAC) inhibition.

Conclusion

In summary, we provide new mechanistic insight into the anti-diabetic action of resveratrol through its ability to express β-cell genes in α-cells.

     

Study on the Clinical Features and Prognosis of <Emphasis Type="Italic">Penicilliosis marneffei</Emphasis> Without Human Immunodeficiency Virus Infection

Objective

To improve the diagnosis and treatment of Penicilliosis marneffei without human immunodeficiency virus infection.

Methods

Analyze and review the clinical features, diagnosis and treatment of six cases of P. marneffei without human immunodeficiency virus infection at The First Affiliated Hospital of Fujian Medical University.

Results

Two cases were diagnosed in the ENT Department, three cases in the respiratory department and one case in the dermatological department. Penicillium marneffei infection was confirmed by sputum culture, blood culture and tissue biopsy. After definite diagnosis, one refused further treatment, and others showed significant improvement.

Conclusion

Penicilliosis marneffei is insidious onset and easy to be escaped and misdiagnosed. To achieve early diagnosis and appropriate treatment, doubtful cases should be alerted for the diagnoses as P. marneffei.

     

Plant growth-promoting bacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> FR1 secrets a novel type of extracellular polyhydroxybutyrate polymerase involved in abiotic stress response in plants

Objectives

Identification of novel microbial factors contributing to plant protection against abiotic stress.

Results

The genome of plant growth-promoting bacterium Pseudomonas fluorescens FR1 contains a short mobile element encoding a novel type of extracellular polyhydroxybutyrate (PHB) polymerase (PhbC) associated with a type I secretion system. Genetic analysis using a phbC mutant strain and plants showed that this novel extracellular enzyme is related to the PHB production in planta and suggests that PHB could be a beneficial microbial compound synthesized during plant adaptation to cold stress.

Conclusion

Extracellular PhbC can be used as a new tool for improve crop production under abiotic stress.

     

Performance in daily activities,cognitive impairment and perception in multiple sclerosis patients and their caregivers

Background

The relationship between cognitive assessment results in multiple sclerosis (MS) and performance in daily activities (DAs) remains unclear. Our study aimed to evaluate the relationship between cognitive functions (CF) measured by tests, performance in DAs, and the perception of CF in patients and their caregivers (CG) in MS.

Methods

The Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) battery was used to evaluate cognitive status. We created an ad hoc questionnaire (DaQ) to assess performance in DAs not requiring specific motor skills. We used the Multiple Sclerosis Neuropsychological Questionnaire (MSNQ) to measure each patient self-judgment and caregiver’s perception of CF.

Results

Forty-nine patients and their caregivers were included in the study. Significant correlations were found between the BICAMS and the DaQ (Symbol Digit Modalities Test (SDMT): r?=???0.48, p?<?0.001; California Verbal Learning Test (CVLT): r?=???0.33, p?=?0.01; Brief Visual Memory Test (BVMT-R): r?=???0.42; p?=?0.002); patients self-judgment (SDMT: r?=???0.38, p?=?0.004; CVLT: r?=???0.26, p?=?0.03); caregiver perception of patient’s CF (SDMT: r?=???0.52, p?<?0.001; CVLT: r?=???0.3, p?=?0.01; BVMT-R: r?=???0.42, p?=?0.002). The difference in perception between the patients and their caregivers was related to patient age (p?=?0.001) and severity of cognitive impairment (p?=?0.03).

Conclusions

Cognitive assessment results show a significant correlation with performance in daily activities and with patients and, especially, caregiver perception of cognitive impairment. These data support the importance of a routine evaluation of cognitive function in MS that includes an anamnestic evaluation of patients, and, when possible, consideration of the caregiver’s point of view.

     

A versatile <Emphasis Type="Italic">Trichoderma reesei</Emphasis> expression system for the production of heterologous proteins

Objectives

To develop a versatile Trichoderma reesei (teleomorph Hypocrea jecorina) expression system for the high-purity production of heterologous proteins.

Results

The versatile T. reesei expression system is based on xyn1 and xyn2 promoters, A824V transition in XYRI, and a bicomponent carbon source strategy. Red fluorescent protein gene rfp and alkaline endoglucanase EGV gene egv3 from Humicola insolens were used as reporter genes to test our versatile expression system

Conclusions

The versatile T. reesei expression system can be applied to produce heterologous proteins with high purity and high yield.

     

Genetic improvement of <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> for enhanced biological removal of phosphate

Objectives

To improve its phosphate accumulating abilities for phosphate recycling from wastewater, a magnetotactic bacterium, Magnetospirillum gryphiswaldense, was genetically modified to over-express polyphosphate kinase.

Results

Polyphosphate kinase was over-expressed in the bacterium. The recombinant strain accumulated ninefold more polyphosphate from synthetic wastewater compared to original wild type. The magnetic property of the recombinant M. gryphiswaldense strain was retained.

Conclusions

The recombinant M. gryphiswaldense can be used for phosphate removal and recovery in bioremediation.

     

Determination of Azole Resistance and TR<Subscript>34</Subscript>/L98H Mutations in Isolates of <Emphasis Type="Italic">Aspergillus</Emphasis> Section <Emphasis Type="Italic">Fumigati</Emphasis> from Turkish Cystic Fibrosis Patients

Background

Aspergillus fumigatus is the species section Fumigati most frequently isolated from the respiratory tract of cystic fibrosis (CF) patients. Recent studies suggest that mutations in the Cyp51 gene, particularly TR₃₄/L98H, are responsible for azole resistance.

Objectives and Methods

The focus of this study was on section Fumigati isolates isolated from the respiratory tract samples of CF patients. More specifically, the goal was to detect A. fumigatus isolates, test their antifungal susceptibility to itraconazole, voriconazole and posaconazole, and finally determine the presence of TR₃₄/L98H and other mutations in the isolates Cyp51A gene.

Results and Conclusions

A set of 31 isolates of Aspergillus section Fumigati were obtained from the sputum samples of 6 CF patients and subsequently identified to species level by microsatellite genotyping. All isolates were determined as A. fumigatus and involved 14 different genotypes. The minimal inhibitory concentrations to the three azoles were determined by the E-test method, and the Cyp51A gene was sequenced. One of the genotypes was found to be resistant to all azoles but no mutations were detected in the Cyp51A gene, especially the TR₃₄/L98H mutation. Therefore, mutations in genes other than Cyp51A or other distinct mechanisms may be responsible for this reported multiazole resistance found in a Turkish CF patient.

     

Synthesis of rosmarinic acid analogues in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS).

Results

Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues.

Conclusion

This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.

     

Role of <Emphasis Type="Italic">PUF60</Emphasis> gene in Verheij syndrome: a case report of the first Chinese Han patient with a de novo pathogenic variant and review of the literature

Background

Verheij syndrome is a rare microdeletion syndrome of chromosome 8q24.3 that harbors PUF60, SCRIB, and NRBP2 genes. Subsequently, loss of function mutations in PUF60 have been found in children with clinical features significantly overlapping with Verheij.

Case presentation

Here we present the first Chinese Han patient with a de novo nonsense variant (c.1357C?>?T, p.Gln453*) in PUF60 by clinical whole exome sequencing. The 5-year-old boy presents with dysmorphic facial features, intellectual disability, and growth retardation but without apparent cardiac, renal, ocular, and spinal anomalies.

Conclusions

Our finding contributes to the understanding of the genotype and phenotype in PUF60 related disorder.

     

Development of a novel engineered antibody targeting <Emphasis Type="Italic">Neisseria</Emphasis> species

Objectives

A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).

Results

Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.

Conclusions

The recombinant scFv could detect Neisseria strains at 10⁶ CFU/ml.

     

The bacterial community associated with rose-scented geranium (<Emphasis Type="Italic">Pelargonium graveolens</Emphasis>) leaves responds to anthracnose symptoms

Background

The fungus Colletotrichum is a plant pathogen that causes the anthracnose disease, resulting in huge losses in various crops including the rose-scented geranium (Pelargonium graveolens). Although the bacterial community associated with plants has an important role in the establishment of plant diseases, little is known about what happens in P. graveolens.

Aims

To increase the knowledge about the bacterial community associated with P. graveolens and its relationship with anthracnose disease symptoms.

Methods

Quantitative PCR and high-throughput sequencing were combined to determine the presence of the fungus Colletotrichum and to reveal the bacterial communities associated with different plant parts – root, stem and leaf – and in the rhizosphere and bulk soil, and also to determine the respective bacterial communities associated with P. graveolens leaves symptomatic and asymptomatic for anthracnose disease.

Results

The fungus Colletotrichum was detected in all plant parts and in the surrounding soil. Bacterial communities varied spatially in plants, and the disease symptoms also influenced the composition of the bacterial community. Abundances of operational taxonomic units (OTUs) assigned to the phylum Actinobacteria and to the genus Streptococcus were greatly increased in asymptomatic leaves.

Conclusions

The bacterial community associated to geranium leaves responds to anthracnose symptoms.