l-dopa-induced dyskinesias in unilateral 6-hydroxydopamine-lesioned rats are not modified by excitotoxic lesion of the entopeduncular nucleus and substantia nigra pars reticulata

l -Dopa-induced dyskinesias (LIDs) are a troublesome complication in Parkinson's disease after long-term therapy and a major reason for surgical treatment. LIDs are effectively eliminated by surgery. We aimed to reproduce such effect in the 6-hydroxydopamine (6-OHDA)-lesioned rat model. Single or combined lesions with quinolinic acid were caused in the entopeduncular nucleus (EP) and substantia nigra pars reticulata (SNr) on 6-hydroxydopamine (6-OHDA)-lesioned rats treated for 3 weeks with l -Dopa (6 mg/kg plus 15 mg/kg benserazide, i.p.). l -Dopa administration was continued for a further week following the lesion and abnormal involuntary movements (AIMs) scored at the end of treatment. Neither the individual lesions of the EP and SNr nor the combined lesions had any antidyskinetic effect nor decreased the total number of rotations. These results suggest that excitotoxic lesions of neurons bodies of the output nuclei of the basal ganglia, which destroy cell bodies and spare fibers of passage, do not induce a beneficial reduction of dyskinesias in contrast to thermolytic lesions in humans (which provokes a complete tissue destruction), thus supporting the possibility that other nuclei or systems might be involved in the antidyskinetic effect of pallidotomy. Synapse 67:407–414, 2013. © 2013 Wiley Periodicals, Inc.     

Doxycycline restrains glia and confers neuroprotection in a 6‐OHDA Parkinson model

Neuron–glia interactions play a key role in maintaining and regulating the central nervous system. Glial cells are implicated in the function of dopamine neurons and regulate their survival and resistance to injury. Parkinson's disease is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, decreased striatal dopamine levels and consequent onset of extrapyramidal motor dysfunction. Parkinson's disease is a common chronic, neurodegenerative disorder with no effective protective treatment. In the 6‐OHDA mouse model of Parkinson's disease, doxycycline administered at a dose that both induces/represses conditional transgene expression in the tetracycline system, mitigates the loss of dopaminergic neurons in the substantia nigra compacta and nerve terminals in the striatum. This protective effect was associated with: (1) a reduction of microglia in normal mice as a result of doxycycline administration per se; (2) a decrease in the astrocyte and microglia response to the neurotoxin 6‐OHDA in the globus pallidus and substantia nigra compacta, and (3) the astrocyte reaction in the striatum. Our results suggest that doxycycline blocks 6‐OHDA neurotoxicity in vivo by inhibiting microglial and astrocyte expression. This action of doxycycline in nigrostriatal dopaminergic neuron protection is consistent with a role of glial cells in Parkinson's disease neurodegeneration. The neuroprotective effect of doxycycline may be useful in preventing or slowing the progression of Parkinson's disease and other neurodegenerative diseases linked to glia function.     

Cerebral and somatic NIRS-determined oxygenation in IUGR preterm infants during transition

Background: Fetal growth restriction (intra-uterine growth restriction [IUGR]) has a considerable impact on perinatal morbidity. Preterm IUGR infants are prone to impaired intestine function. Near-infrared spectroscopy (NIRS) has been used to monitor oxygenation status of the brain and of the intestine.

Patients and methods: We conducted a prospective case–control study at our NICU in 20 preterm infants of whom 10 infants complicated by compared with 10 non-IUGR preterm infants. Splanchnic and cerebral regional oximetry values were measured with NIRS. Three hours of consecutive recordings were performed in the first 24?h of life, T0, and during the transitional period, T1. The cerebral/splanchnic oxygenation ratio, CSOR, (cerebral regional saturations [rScO_2]/splanchnic regional saturations [rSsO₂]) was also calculated.

Results: Both in the IUGR and the non-IUGR infants, at T0 and T1 monitoring time-points, the rSO₂ values were higher in the cerebral district when compared to those of the splanchnic area. Comparison of the NIRS parameters between the IUGR and non-IUGR infants at T0 showed no difference in rScO₂, while rSsO₂ was significantly lower in the IUGR group. At T1, rScO₂ was significantly lower and rSsO₂ higher in the IUGR group.

Conclusions: Cerebral/splanchnic vascular adaptation of IUGR infants to the extra-uterine environment is characterized by a postnatal persistence of the brain sparing effect with reperfusion in the transitional period.     

Characterization of divergent and atypical canine coronaviruses from Sweden

Summary Field canine coronaviruses (CCVs) identified during a series of outbreaks of gastroenteritis in Swedish dogs were subjected to genetic analysis involving the open reading frame 1b (ORF1b) and the membrane (M) and spike (S) protein genes. Four field viruses originating from the Stockholm region presented identical sequences and segregated separately from other CCVs characterized so far and from GOT/05, the variant recovered in Western Sweden. A recombinant origin of the fifth virus identified in the Stockholm region is suggested. In addition, the five viruses originating from the same geographical area displayed atypical 5′ S gene sequences.     

Microarray-based molecular detection of foot-and-mouth disease, vesicular stomatitis and swine vesicular disease viruses, using padlock probes

The World Organization for Animal Health (Office International des Epizooties, OIE) includes the diseases caused by foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), as "Diseases Notifiable to the OIE". Foot-and-mouth disease (FMD) outbreaks have severe economical as well as social effects and cannot be differentiated from the diseases caused by the other two viruses on the basis of clinical symptoms. Efficient laboratory techniques are therefore required for detection and identification of the viruses causing similar vesicular symptoms in swine. A rapid method is described using padlock probes and microarrays to detect simultaneously and differentiate the three viruses in a single reaction, as well as providing serotype information in cases of VSV infection. The padlock probe/microarray assay detected successfully and identified 39 cDNA samples of different origin representing the three viruses. The results were in complete agreement with identities and serotypes determined previously. This novel virus detection method is discussed in terms of usefulness and further development.     

Universal detection of hepatitis E virus by two real-time PCR assays: TaqMan and Primer-Probe Energy Transfer

Hepatitis E virus (HEV) is a major cause of food- and waterborne diseases in countries with poor sanitation. Furthermore, travellers to such countries are also at risk of contracting the virus. Noteworthily, during the last decade an increasing number of non-travel-related cases were recorded even in countries with high sanitary standards. An alternative, direct route of infection, from animals to humans (zoonotic transmission) is suspected to be the cause of recent cases of hepatitis E. In order to provide rapid and sensitive methods for detecting the virus in various hosts, two real-time PCR methods were developed and compared: a TaqMan and Primer-Probe Energy Transfer (PriProET) assay. These highly sensitive novel methods provide valuable diagnostic tools to investigate zoonotic transmission, to detect the virus in the food chain and in research related to the potential of hepatitis E virus to cross the species barrier. The results show that the two novel PCR assays are robust, highly sensitive and specific for broad range detection of the four genotypes of HEV. Compared to PriProET, the TaqMan assay appears to perform slightly better, with higher fluorescence values for positive samples. However, the PriProET has the benefit of better tolerating the point mutations in the target nucleic acids. Thus, it provides a more powerful tool to detect new virus variants. These new molecular diagnostic assays are practical tools that can be employed in the area of public health, for disease diagnosis and for tracking outbreaks. In basic research the methods provide new tools to study HEV biology, including virus-host interactions and transmission between various host species.     

Development of a real-time RT-PCR assay for improved detection of Borna disease virus

Borna disease virus (BDV) is a non-segmented, negative-stranded RNA virus, which infects cells of the central nervous system (CNS) in many different species. BDV is the causative agent of the neurological disorders in horses and sheep termed classical Borna disease (BD), as well as staggering disease in cats. At present, the diagnosis staggering disease or feline BD is made by histopathology or immunohistochemistry of the CNS. In order to obtain a better clinical diagnostic tool, a duplex real-time RT-PCR assay (rRT-PCR) was developed. TaqMan probes and primers specific for the BDV P and BDV L genes were designed by aligning the sequences of known BDV strains. After optimisation, the sensitivity and specificity of the rRT-PCR were established. The detection limit was set to 10-100 viral genomic copies per reaction and the assay detects the BDV strains V and He/80, as well as the most divergent BDV strain known so far, No/98. Furthermore, the system detected feline BDV variants in five naturally infected cats and a feline isolate used in experimental infection of cats. This rRT-PCR assay will be a powerful tool in further studies of BDV, including epidemiological screening and diagnosis.     

Characterization of genes encoding novel peptidases in the biocontrol fungus <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> CECT 2413 using the TrichoEST functional genomics approach

Proteolytic enzymes (EC 3.4) secreted by Trichoderma strains are receiving increasing attention because of their potential implication in the Trichoderma biocontrol abilities. We have used an expressed sequence tag (EST) approach to identify genes encoding extracellular peptidases in T. harzianum CECT 2413 grown under several biocontrol-related conditions. Based on BlastX results and Gene Ontology annotation, a total of 61 (among 3478) unique sequences (unisequences) were predicted to encode enzymes with peptidase activity, three corresponding to secreted peptidases already known from this Trichoderma strain (PAPA, PRA1 and P6281). Further manual screening based on the functional identity and cellular location of the best matches revealed ten unisequences encoding novel extracellular peptidases. We report the characterization of the corresponding genes as well as a potential orthologous gene of the intracellular peptidase PAPB from T. asperellum. In each case, full-length coding sequences were obtained, and deduced proteins were compared at phylogenetic level with peptidases from other organisms. T. harzianum CECT 2413 novel peptidases included six serine endopeptidases (EC 3.4.21) belonging to the families S1, S8 and S53, three aspartic endopeptidases (EC 3.4.23) of the family A1, one metallo-endopeptidase (EC 3.4.24) of the family M35, and one aminopeptidase (EC 3.4.11) of the family M28. Results obtained by Northern blot analyses demonstrated that the genes within a family are differentially regulated in response to different culture conditions, suggesting that they have diverse functional roles.     

An electronic nose in the discrimination of patients with asthma and controls

BACKGROUND: Exhaled breath contains thousands of volatile organic compounds (VOCs) that could serve as biomarkers of lung disease. Electronic noses can distinguish VOC mixtures by pattern recognition. OBJECTIVE: We hypothesized that an electronic nose can discriminate exhaled air of patients with asthma from healthy controls, and between patients with different disease severities. METHODS: Ten young patients with mild asthma (25.1 +/- 5.9 years; FEV(1), 99.9 +/- 7.7% predicted), 10 young controls (26.8 +/- 6.4 years; FEV(1), 101.9 +/- 10.3), 10 older patients with severe asthma (49.5 +/- 12.0 years; FEV(1), 62.3 +/- 23.6), and 10 older controls (57.3 +/- 7.1 years; FEV(1), 108.3 +/- 14.7) joined a cross-sectional study with duplicate sampling of exhaled breath with an interval of 2 to 5 minutes. Subjects inspired VOC-filtered air by tidal breathing for 5 minutes, and a single expiratory vital capacity was collected into a Tedlar bag that was sampled by electronic nose (Cyranose 320) within 10 minutes. Smellprints were analyzed by linear discriminant analysis on principal component reduction. Cross-validation values (CVVs) were calculated. RESULTS: Smellprints of patients with mild asthma were fully separated from young controls (CVV, 100%; Mahalanobis distance [M-distance], 5.32), and patients with severe asthma could be distinguished from old controls (CVV, 90%; M-distance, 2.77). Patients with mild and severe asthma could be less well discriminated (CVV, 65%; M-distance, 1.23), whereas the 2 control groups were indistinguishable (CVV, 50%; M-distance, 1.56). The duplicate samples replicated these results. CONCLUSION: An electronic nose can discriminate exhaled breath of patients with asthma from controls but is less accurate in distinguishing asthma severities. CLINICAL IMPLICATION: These findings warrant validation of electronic noses in diagnosing newly presented patients with asthma.