M. Shioya, S. Obayashi, H. Tabunoki, K. Arima, Y. Saito, T. Ishida and J. Satoh (2010) Neuropathology and Applied Neurobiology36, 320–330 Aberrant microRNA expression in the brains of ...neurodegenerative diseases: miR‐29a decreased in Alzheimer disease brains targets neurone navigator 3
Aims: MicroRNAs (miRNAs) are small non‐coding RNAs that regulate translational repression of target mRNAs. Accumulating evidence indicates that various miRNAs, expressed in a spatially and temporally controlled that manner in the brain plays a key role in neuronal development. However, at present, the pathological implication of aberrant miRNA expression in neurodegenerative events remains largely unknown. To identify miRNAs closely associated with neurodegeneration, we performed miRNA expression profiling of brain tissues of various neurodegenerative diseases. Methods: We initially studied the frontal cortex derived from three amyotrophic lateral sclerosis patients by using a microarray of 723 human miRNAs. This was followed by enlargement of study population with quantitative RT‐PCR analysis (n = 21). Results: By microarray analysis, we identified up‐regulation of miR‐29a, miR‐29b and miR‐338‐3p in amyotrophic lateral sclerosis brains, but due to a great interindividual variation, we could not validate these results by quantitative RT‐PCR. However, we found significant down‐regulation of miR‐29a in Alzheimer disease (AD) brains. The database search on TargetScan, PicTar and miRBase Target identified neurone navigator 3 (NAV3), a regulator of axon guidance, as a principal target of miR‐29a, and actually NAV3 mRNA levels were elevated in AD brains. MiR‐29a‐mediated down‐regulation of NAV3 was verified by the luciferase reporter assay. By immunohistochemistry, NAV3 expression was most evidently enhanced in degenerating pyramidal neurones in the cerebral cortex of AD. Conclusions: These observations suggest the hypothesis that underexpression of miR‐29a affects neurodegenerative processes by enhancing neuronal NAV3 expression in AD brains.
Influenza viruses can mutate genetically and cause a range of respiratory ailments. The H275Y mutation in the neuraminidase (NA) gene reduces the effectiveness of oseltamivir, a widely used drug for ...the treatment of Influenza A and B virus infection. The World Health Organization (WHO) recommends single‐nucleotide polymorphism assays to detect this mutation. The present study aims to estimate the prevalence of H275Y mutation conferring oseltamivir resistance in Influenza A(H1N1)pdm09 virus among hospitalized patients from June 2014 to December 2021. Following the WHO protocol, allelic discrimination real‐time RT‐PCR was performed for 752 samples. Out of the 752 samples, 1 tested positive for Y275 gene mutation by allelic discrimination real‐time RT‐PCR. In samples of years 2020 and 2021, neither the H275 nor Y275 genotype was detected. Sequencing of the NA gene of all negative samples showed a mismatch between the NA sequence and the probes used in the allelic discrimination assay. Also, Y275 mutation was detected in only 1 sample from 2020. The prevalence of oseltamivir resistance was estimated as 0.27% among the Influenza A(H1N1)pdm09 patients during 2014−2021. The study highlights that the WHO‐recommended probes for detecting H275Y mutation may not be useful to detect 2020 and 2021 circulating strains of Influenza A(H1N1)pdm09, emphasizing the need for continuous monitoring of mutations in the influenza virus.
•High sensitivity of Simplexa™ COVID-19 Direct assay.•100 % specificity of this new assay.•“Almost perfect” agreement with Corman’s method (κ = 0.938).•High-speed detection, all-in-one reagent mix ...and no separate extraction required.•Promising for laboratory diagnosis of COVID-19 and field application.
So far, one of the major drawbacks of the available molecular assays for the diagnosis of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) is the need for viral nucleic acid extraction from clinical specimens.
The aim of this study was to evaluate the performances of a newly designed real-time RT-PCR (Simplexa™ COVID-19 Direct assay), that is established with an all-in-one reagent mix and no separate extraction required.
The lower limit of detection (LOD) for both target genes resulted the same: 3.2 (CI: 2.9–3.8) log10 cp/mL and 0.40 (CI: 0.2–1.5) TCID50/mL for S gene while 3.2 log10 (CI: 2.9–3.7) log10 cp/mL and 0.4 (CI: 0.2–1.3) TCID50/mL for ORF1ab. The LOD obtained with extracted viral RNA for both S gene or ORF1ab was 2.7 log10 cp/mL. Crossreactive analysis performed in 20 nasopharyngeal swabs confirmed a 100% of clinical specificity of the assay. Clinical performances of Simplexa™ COVID-19 Direct assay were assessed in 278 nasopharyngeal swabs tested in parallel with Corman's method. Concordance analysis showed an "almost perfect" agreement in SARS-CoV-2 RNA detection between the two assays, being κ = 0.938; SE = 0.021; 95% CI = 0.896-0.980.
The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, enabling highspeed detection in just over one hour, which is significantly faster than the up to five hours currently required by traditional extraction followed by amplification technologies, thus allowing prompt decision making regarding isolation of infected patients.
The COVID-19 outbreak caused by SARS-CoV-2 in Wuhan (China) in December 2019 is currently spreading rapidly and globally. We recently reported a laboratory diagnostic protocol for SARS-CoV-2 based on ...real-time RT-PCR assays using two primer sets, N and N2. On 30–31 January 2020, the protocol and reagents for these assays were distributed to local public health institutes and quarantine depots in Japan, and nationwide, SARS-CoV-2 diagnostic testing was started. For further validation, the assays were compared with the commercially available kits using SARS CoV-2 viral RNA and the clinical specimens obtained from COVID19-suspected individuals. The LightMix Modular SARS and Wuhan CoV E-gene (LN S&W-E) assay was highly sensitive for SARS-CoV-2, as was the N2 set, and both assays had perfectly consistent results with the clinical specimens. While the LM S&W-E set targets the highly conserved region of the E gene in SARS-CoV and SARS-CoV-2, the N2 set was designed to target the unique region in the SARS-CoV-2 N gene. Thus, the N2 set has high specificity and sensitivity for SARS-CoV-2 detection. These indicate that the protocol using the N and N2 sets is comparable to commercially available kits and is reliable for the laboratory diagnosis of COVID-19.
Estrogen hormones interact with cellular
ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct
ER subtypes,
ERα (NR3A1) and
ERβ (NR3A2). The
...ERβ subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of
ERα have not been demonstrated in any fish species. In the rainbow trout (
Oncorhynchus mykiss), the only
ER described is an isoform of the
ERα subtype (i.e.
ERα1, NR3A1a). The purpose of this study was to determine whether the gene for the other
ERα isoform,
ERα2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to
ERα1. Both the rainbow trout
ERβ subtypes,
ERβ1 NR3A2a and
ERβ2, NR3A2b which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the
ERαs (∼
40% similarity).
ERβ1 and
ERβ2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear
ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish
ER gene sequences was undertaken to understand the evolution of fish
ERs. The results show a single
ERα subtype clade, with the closest relative to rainbow trout
ERα2 being rainbow trout
ERα1, suggesting a recent, unique duplication event to create these two isoforms. For the
ERβ subtype there are two distinct subclades, one represented by the
ERβ1 isoform and the other by the
ERβ2 isoform. The rainbow trout
ERβ1 and
ERβ2 are not closely associated with each other, but instead fall into their respective
ERβ subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four
ER isoforms (
ERα1,
ERα2,
ERβ1, and
ERβ2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four
ERs were detected in every tissue examined. The liver tended to have the highest
ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear
ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.
The World Health Organization (WHO) designated Omicron (B.1.1.529 lineage) of SARS-CoV-2 as a new variant of concern (VOC) on 26th of November 2021. The risk to public health conferred by the Omicron ...variant is still not completely clear, although its numerous gene mutations raise concern about its potential for increased transmissibility and immune escape. In this study, we describe our development of two single nucleotide polymorphism (SNP) genotyping assays targeting the G339D or T547K mutation of the spike protein for screening of the Omicron variant. A specificity test revealed that the two assays successfully discriminated the Omicron variant from the Delta variant and Alpha variant each with one nucleotide mismatch. In addition, a sensitivity test showed that the G339D and T547K assays detected at least 2.60 and 3.36 RNA copies of the Omicron variant, respectively, and 1.59 RNA copies of the Delta variant. These results demonstrated that both assays could be useful for detecting and discriminating the Omicron variant from other strains. In addition, because of the rapid and unpredictable evolution of SARS-CoV-2, combining our assays with previously developed assays for detecting other mutations may lead to a more accurate diagnosis system.
20‐Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during ...the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR‐B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR‐B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR‐B1 isoform in Spodoptera litura. Quantitative real‐time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue‐specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone‐inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.
Silencing of GmEcR could significantly downregulate the transcriptional level of ecdysone‐inducible genes, result in increased mortality during metamorphosis and prolonged prepupal duration. These data show that GmEcR plays key roles in the expression of genes involved in 20E signal transduction and directly or indirectly affect the development of G. molesta.
Highlights
Ecdysone receptor isoform (GmEcR) was identified in Grapholita molesta.
GmEcRs is crucial to ensure normal metamorphosis in G. molesta.
GmEcRs gene might be directly or indirectly controlled the expression of ecdysone‐inducible genes.
Genetic variability in the promoter and 3' region of the SNCA gene coding α-synuclein modulates the risk to develop sporadic Parkinson's disease (PD). Whether this is mediated by regulating ...α-synuclein expression levels remains unknown. Therefore, we analyzed levels of α-synuclein in blood and human post mortem brain tissue including the substantia nigra using quantitative real-time reverse transcriptase-polymerase chain reaction and enzyme linked immunosorbent assay in vivo. Single nucleotide polymorphism (SNP) rs356219, a tagging SNP for a disease-associated haplotype in the 3' region of the SNCA gene, has a significant effect on SNCA mRNA levels in the substantia nigra and the cerebellum. Further, the "protective" genotype 259/259 of the PD-associated promoter repeat NACP-Rep1 is associated with lower protein levels in blood than genotypes 261/261, 259/261, and 259/263. In conclusion, we provide evidence that α-synuclein levels are influenced by genetic variability in the promoter and 3' region of the SNCA gene in vivo.--Fuchs, J., Tichopad, A., Golub, Y., Munz, M., Schweitzer, K. J., Wolf, B., Berg, D., Mueller, J. C., Gasser, T. Genetic variability in the SNCA gene influences α-synuclein levels in the blood and brain.
Novel negative‐stranded RNA (nsRNA) viruses have been recently identified in multiple agronomic crops, including pome fruit trees. Citrus concave gum‐associated virus (CCGaV), citrus virus A (CiVA) ...and apple rubbery wood viruses 1 and 2 (ARWV1 and 2) are examples of such viruses. Given the novelty and lack of information about these pathogens in Californian orchards, in this study, real‐time RT‐PCR assays for CCGaV, CiVA, ARWV1 and 2 were developed and employed in a field survey. Initially, the new assays were challenged against a comprehensive set of positive and negative samples, previously analysed by high‐throughput sequencing (HTS), to determine specificity. Aiming to investigate the presence of nsRNA viruses in California apple and pear orchards, 186 samples were collected from 21 different locations. As a result, 79 (42%) samples were found to be infected by these viruses in single or mixed infections. The incidence of each virus in relation to the total number of samples was 36%, 15%, 11% and 0% for ARWV2, CCGaV, ARWV1 and CiVA respectively. Overall, not considering the no detected CiVA, the other three nsRNA viruses were widely distributed among sampled orchards. To further validate the reliability of the new real‐time RT‐PCR assays, six samples tested positive during the survey were screened by previously described detection assays and HTS. This is the first detection of these nsRNA viruses in California, which may represent an issue in apple and pear production.
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