MRI findings are often missed in the diagnosis of Creutzfeldt-Jakob disease

Background

Establishing a confident clinical diagnosis before an advanced stage of illness can be difficult in Creutzfeldt-Jakob disease (CJD) but unlike common causes of dementia, prion diseases can often be diagnosed by identifying characteristic MRI signal changes. However, it is not known how often CJD-associated MRI changes are identified at the initial imaging report, whether the most sensitive sequences are used, and what impact MRI-diagnosis has on prompt referral to clinical trial-like studies.

Methods

We reviewed the MRI scans of 103 patients with CJD referred to the National Prion Clinic since 2007 and reviewed the presence of CJD-associated changes, compared these findings with the formal report from the referring centre and reviewed the types of sequence performed.

Results

In sCJD we found CJD-associated MRI changes in 83 of 91 cases (91% sensitivity). However, the referring centres documented CJD-associated MRI changes in 43 of the sCJD cases (47% sensitivity). The most common region not documented by referring centres was the cortex (23 of 68 sCJD cases), but there was a statistically significant discrepancy in all regions (p<0.0001). Patients in whom MRI abnormalities were missed by the referring hospital were more advanced at the time of recruitment to a clinical trial-like study (p=0.03).

Conclusions

CJD-associated MRI changes are often not documented on the formal investigation report at the referring centre. This is important as delay makes enrolment to clinical trials futile because of highly advanced disease. If a diagnosis of CJD is suspected, even if the initial imaging is reported as normal, a specialist MRI review either by an experienced neuroradiologist or by a prion disease specialist unit could facilitate earlier diagnosis.

     

Reengineering substrate specificity of <Emphasis Type="Italic">E. coli</Emphasis> glutamate dehydrogenase using a position-based prediction method

Objective

To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM).

Results

The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg^?1.

Conclusion

The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.

     

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrP^Sc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion.

Case presentation

We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrP^Sc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrP^Sc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine.

Conclusions

A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

     

Overview and evaluation of 15 years of Creutzfeldt-Jakob disease surveillance in Belgium, 1998-2012

Background

In 1998, following the detection of variant Creutzfeldt-Jakob disease (vCJD) in the UK, Belgium installed a surveillance system for Creutzfeldt-Jakob disease (CJD). The objectives of this system were to identify vCJD cases and detect increases in CJD incidence. Diagnostic confirmation of CJD is based on autopsy after referral by neurologists. Reference centres perform autopsies and report to the surveillance system. The aim of this study was to assess whether the system met its objectives and to assess its acceptability.

Methods

For 1999–2010, we linked surveillance data with hospital discharge data. We calculated the proportion of CJD suspected patients who died in hospitals and were captured by the surveillance system. We surveyed stakeholders on knowledge of the surveillance system, referral practices and acceptability. We compared proportions using the chi-square test and investigated variables associated with capture using a multivariable logistic regression model.

Results

On average 60 % of hospitalised patients who died with suspected CJD were captured by the surveillance system. This proportion did not significantly differ over the years (p?=?0.1). The odds of capture significantly decreased with every 1 year increase in age (OR?=?0.95, 95 % CI 0.92–0.98, p?=?0.001). Eleven percent of surveyed neurologists would not refer suspect vCJD cases for autopsy, nor contact a reference centre for diagnostic support. Sixty-one percent of surveyed neurologists were not familiar with the surveillance system. Awareness of the existence of the system did not impact referral behaviour (p?=?0.18). CJD and vCJD surveillance were considered important by the majority of stakeholders.

Conclusions

Although 40 % of the suspect CJD cases were not referred for autopsy, our data suggest that the Belgian CJD surveillance system meets one of its main objectives: it can detect changes in CJD incidence. However, we do not have sufficient evidence to conclude that the system meets its second objective of detecting vCJD cases arising in Belgium. Although not well known, the system was considered acceptable by its stakeholders.

     

Serum metabolites associated with feed efficiency in black angus steers

Introduction

Improving feed utilization in cattle is required to reduce input costs, increase production, and ultimately improve sustainability of the beef cattle industry. Characterizing metabolic differences between efficient and non-efficient animals will allow stakeholders to identify more efficient cattle during backgrounding.

Objectives

This study used an untargeted metabolomics approach to determine differences in serum metabolites between animals of low and high residual feed intake.

Methods

Residual feed intake was determined for 50 purebred Angus steers and 29 steers were selected for the study steers based on low versus high feed efficiency. Blood samples were collected from steers and analyzed using untargeted metabolomics via mass spectrometry. Metabolite data was analyzed using Metaboanalyst, visualized using orthogonal partial least squares discriminant analysis, and p-values derived from permutation testing. Non-esterified fatty acids, urea nitrogen, and glucose were measured using commercially available calorimetric assay kits. Differences in metabolites measured were grouped by residual feed intake was measured using one-way analysis of variance in SAS 9.4.

Results

Four metabolites were found to be associated with differences in feed efficiency. No differences were found in other serum metabolites, including serum urea nitrogen, non-esterified fatty acids, and glucose.

Conclusions

Four metabolites that differed between low and high residual feed intake have important functions related to nutrient utilization, among other functions, in cattle. This information will allow identification of more efficient steers during backgrounding.

     

Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model

Objectives

To investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish.

Results

Here we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout.

Conclusions

The transgenic zebrafish with expressed Cas9, is more efficient in genome editing. And the results of ZFERV knockout indicated that ERV may affect the vertebral development by Notch1/Delta D signal pathway.

     

Smad4 SUMOylation is essential for memory formation through upregulation of the skeletal myopathy gene <Emphasis Type="Italic">TPM2</Emphasis>

Background

Smad4 is a critical effector of TGF-β signaling that regulates a variety of cellular functions. However, its role in the brain has rarely been studied. Here, we examined the molecular mechanisms underlying the post-translational regulation of Smad4 function by SUMOylation, and its role in spatial memory formation.

Results

In the hippocampus, Smad4 is SUMOylated by the E3 ligase PIAS1 at Lys-113 and Lys-159. Both spatial training and NMDA injection enhanced Smad4 SUMOylation. Inhibition of Smad4 SUMOylation impaired spatial learning and memory in rats by downregulating TPM2, a gene associated with skeletal myopathies. Similarly, knockdown of TPM2 expression impaired spatial learning and memory, while TPM2 mRNA and protein expression increased after spatial training. Among the TPM2 mutations associated with skeletal myopathies, the TPM2E122K mutation was found to reduce TPM2 expression and impair spatial learning and memory in rats.

Conclusions

We have identified a novel role of Smad4 SUMOylation and TPM2 in learning and memory formation. These results suggest that patients with skeletal myopathies who carry the TPM2E122K mutation may also have deficits in learning and memory functions.

     

Type 2 diabetes increases oocyte mtDNA mutations which are eliminated in the offspring by bottleneck effect

Background

Diabetes induces many complications including reduced fertility and low oocyte quality, but whether it causes increased mtDNA mutations is unknown.

Methods

We generated a T2D mouse model by using high-fat-diet (HFD) and Streptozotocin (STZ) injection. We examined mtDNA mutations in oocytes of diabetic mice by high-throughput sequencing techniques.

Results

T2D mice showed glucose intolerance, insulin resistance, low fecundity compared to the control group. T2D oocytes showed increased mtDNA mutation sites and mutation numbers compared to the control counterparts. mtDNA mutation examination in F1 mice showed that the mitochondrial bottleneck could eliminate mtDNA mutations.

Conclusions

T2D mice have increased mtDNA mutation sites and mtDNA mutation numbers in oocytes compared to the counterparts, while these adverse effects can be eliminated by the bottleneck effect in their offspring. This is the first study using a small number of oocytes to examine mtDNA mutations in diabetic mothers and offspring.

     

Isolated and repeated stroke-like episodes in a middle-aged man with a mitochondrial ND3 T10158C mutation: a case report

Background

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, is the most common phenotype of mitochondrial disease. It often develops in childhood or adolescence, usually before the age of 40, in a maternally-inherited manner. Mutations in mitochondrial DNA (mtDNA) are frequently responsible for MELAS.

Case presentation

A 55-year-old man, who had no family or past history of mitochondrial disorders, suddenly developed bilateral visual field constriction and repeated stroke-like episodes. He ultimately presented with cortical blindness, recurrent epilepsy and severe cognitive impairment approximately 6 months after the first episode. Genetic analysis of biopsied biceps brachii muscle, but not of peripheral white blood cells, revealed a T10158C mutation in the mtDNA-encoded gene of NADH dehydrogenase subunit 3 (ND3), which has previously been thought to be associated with severe or fatal mitochondrial disorders that develop during the neonatal period or in infancy.

Conclusion

A T10158C mutation in the ND3 gene can cause atypical adult-onset stroke-like episodes in a sporadic manner.

     

Italian cohort of patients affected by inflammatory bowel disease is characterised by variation in glycerophospholipid,free fatty acids and amino acid levels

Background

Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn’s disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression.

Objectives

The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples.

Methods

A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis.

Results

Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A₂ and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease.

Conclusions

Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease.

     

Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector

Objective

To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein.

Results

The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE.

Conclusions

The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.

     

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.

     

NMR-based metabolomics of transgenic and non-transgenic sweet orange reveals different responses in primary metabolism during citrus canker development

Introduction

Citrus canker, a disease caused by Xanthomonas axonopodis pv. citri (Xac) bacteria, has been responsible for extensive economic losses in citriculture. In this work, we report the metabolic responses of citrus plants during disease development. This information can be useful for understanding the natural mechanism of plant defense beyond helping design new varieties and/or genetically modified genotypes for tolerance/resistance against citrus canker.

Objectives

To understand how primary metabolism is affected in two sweet orange genotypes during citrus canker development.

Methods

¹H NMR spectroscopy together with chemometrics was used to evaluate the metabolic changes caused by Xac infection at various time points (days 4, 12 and 20) in Citrus sinensis L. Osbeck leaves from non-transgenic and transgenic plants expressing the antibacterial peptide sarcotoxin.

Results

The results revealed a high level of metabolic similarity between the studied genotypes without Xac infection. However, after Xac infection, the plants responded differently to disease development. The non-transgenic genotype showed altered early precursors of some secondary metabolites (tryptophan, tyrosine and putrescine) in addition to signaling metabolites of biotic stress (putrescine and dimethylamine), and the drastic reduction of gluconeogenesis was the overall metabolic cost for defense. The transgenic genotype suffered late metabolic changes due to the protective stoichiometric role of sarcotoxin. In addition, the oxidative stress response was more balanced in transgenic than in non-transgenic plants.

Conclusion

An NMR-based metabolomic approach was useful for understanding plant–pathogen interactions in citrus canker. Our findings provide valuable preliminary insights into different stages of citrus canker development.

     

YKL-40 in the brain and cerebrospinal fluid of neurodegenerative dementias

Background

YKL-40 (also known as Chitinase 3-like 1) is a glycoprotein produced by inflammatory, cancer and stem cells. Its physiological role is not completely understood but YKL-40 is elevated in the brain and cerebrospinal fluid (CSF) in several neurological and neurodegenerative diseases associated with inflammatory processes. Yet the precise characterization of YKL-40 in dementia cases is missing.

Methods

In the present study, we comparatively analysed YKL-40 levels in the brain and CSF samples from neurodegenerative dementias of different aetiologies characterized by the presence of cortical pathology and disease-specific neuroinflammatory signatures.

Results

YKL-40 was normally expressed in fibrillar astrocytes in the white matter. Additionally YKL-40 was highly and widely expressed in reactive protoplasmic cortical and perivascular astrocytes, and fibrillar astrocytes in sporadic Creutzfeldt-Jakob disease (sCJD). Elevated YKL-40 levels were also detected in Alzheimer’s disease (AD) but not in dementia with Lewy bodies (DLB). In AD, YKL-40-positive astrocytes were commonly found in clusters, often around β-amyloid plaques, and surrounding vessels with β-amyloid angiopathy; they were also distributed randomly in the cerebral cortex and white matter. YKL-40 overexpression appeared as a pre-clinical event as demonstrated in experimental models of prion diseases and AD pathology.CSF YKL-40 levels were measured in a cohort of 288 individuals, including neurological controls (NC) and patients diagnosed with different types of dementia. Compared to NC, increased YKL-40 levels were detected in sCJD (p < 0.001, AUC = 0.92) and AD (p < 0.001, AUC = 0.77) but not in vascular dementia (VaD) (p > 0.05, AUC = 0.71) or in DLB/Parkinson’s disease dementia (PDD) (p > 0.05, AUC = 0.70). Further, two independent patient cohorts were used to validate the increased CSF YKL-40 levels in sCJD. Additionally, increased YKL-40 levels were found in genetic prion diseases associated with the PRNP-D178N (Fatal Familial Insomnia) and PRNP-E200K mutations.

Conclusions

Our results unequivocally demonstrate that in neurodegenerative dementias, YKL-40 is a disease-specific marker of neuroinflammation showing its highest levels in prion diseases. Therefore, YKL-40 quantification might have a potential for application in the evaluation of therapeutic intervention in dementias with a neuroinflammatory component.

     

Metabolomic profiling of maternal hair suggests rapid development of intrahepatic cholestasis of pregnancy

Introduction

Intrahepatic cholestasis of pregnancy (ICP) is a common maternal liver disease; development can result in devastating consequences, including sudden fetal death and stillbirth. Currently, recognition of ICP only occurs following onset of clinical symptoms.

Objective

Investigate the maternal hair metabolome for predictive biomarkers of ICP.

Methods

The maternal hair metabolome (gestational age of sampling between 17 and 41 weeks) of 38 Chinese women with ICP and 46 pregnant controls was analysed using gas chromatography–mass spectrometry.

Results

Of 105 metabolites detected in hair, none were significantly associated with ICP.

Conclusion

Hair samples represent accumulative environmental exposure over time. Samples collected at the onset of ICP did not reveal any metabolic shifts, suggesting rapid development of the disease.

     

Complicated Gitelman syndrome and autoimmune thyroid disease: a case report with a new homozygous mutation in the SLC12A3 gene and literature review

Background

Gitelman syndrome (GS) is an inherited autosomal recessive renal tubular disorder characterized by low levels of potassium and magnesium in the blood, decreased excretion of calcium in the urine, and elevated blood pH. GS is caused by an inactivating mutation in the SLC12A3 gene, which is located on the long arm of chromosome 16 (16q13) and encodes a thiazide-sensitive sodium chloride cotransporter (NCCT).

Case presentation

A 45-year-old man with Graves’ disease complicated by paroxysmal limb paralysis had a diagnosis of thyrotoxic periodic paralysis for 12 years. However, his serum potassium level remained low despite sufficiently large doses of potassium supplementation. Finally, gene analysis revealed a homozygous mutation in the SLC12A3 gene. After his thyroid function gradually returned to normal, his serum potassium level remained low, but his paroxysmal limb paralysis resolved.

Conclusions

GS combined with hyperthyroidism can manifest as frequent episodes of periodic paralysis; to date, this comorbidity has been reported only in eastern Asian populations. This case prompted us to more seriously consider the possibility of GS associated with thyroid dysfunction.

     

<Emphasis Type="Italic">Enterococcus faecalis</Emphasis> promotes osteoclast differentiation within an osteoblast/osteoclast co-culture system

Objective

To investigate the effects of heat-killed Enterococcus faecalis ATCC 29212 and P25RC clinical strain (derived from an obturated root canal with apical periodontitis) on osteoclast differentiation within an osteoblast/osteoclast co-culture system.

Results

Heat-killed E. faecalis significantly increased the proportion of multinucleated osteoclastic cells (MNCs) within the co-culture system. The IL-6 level was significantly increased upon exposure to heat-killed E. faecalis. Gene expression levels of NFATc1 and cathepsin K were significantly up-regulated compared to the untreated control. EphrinB2 and EphB4 expressions at both the mRNA and protein levels were also significantly upregulated compared to the untreated control.

Conclusions

Heat-killed E. faecalis can induce osteoclast differentiation within the osteoblast/osteoclast co-culture system in vitro, possibly through ephrinB2-EphB4 bidirectional signaling.