Heterozygous mutations in the glucocerebrosidase gene (GBA) represent the strongest common genetic risk factor for Parkinson's disease (PD), the second most common neurodegenerative disorder. However, the molecular mechanisms underlying this association are still poorly understood. Here, we have analyzed ten independent induced pluripotent stem cell (iPSC) lines from three controls and three unrelated PD patients heterozygous for the GBA-N370S mutation, and identified relevant disease mechanisms. After differentiation into dopaminergic neurons, we observed misprocessing of mutant glucocerebrosidase protein in the ER, associated with activation of ER stress and abnormal cellular lipid profiles. Furthermore, we observed autophagic perturbations and an enlargement of the lysosomal compartment specifically in dopamine neurons. Finally, we found increased extracellular α-synuclein in patient-derived neuronal culture medium, which was not associated with exosomes. Overall, ER stress, autophagic/lysosomal perturbations, and elevated extracellular α-synuclein likely represent critical early cellular phenotypes of PD, which might offer multiple therapeutic targets.
<p>Figure S1: BCRA2 p.Y3035S Pedigrees. Figure S2: Western blot of ectopically expressed full-length wildtype and mutant hBRCA2 protein in VC8 DR-GFP cells subjected to HDR assay. Figure S3: Purification of wildtype and mutant BRCA2 from human cells. Figure S4: Position of BRCA2 DNA binding domain (DBD) variants on a ribbon diagram of the murine DBD crystal structure.</p>
Bamboo is one of the fastest-growing plants commonly used in food, fibre, paper, biofuel, ornamental and medicinal industries. Natural hybridization in bamboo is rare due to its long vegetative period followed by gregarious flowering and death of the entire population. In the current study, a new bamboo species, Bambusa changningensis, shows intermediate characteristics of Dendrocalamus farinosus and B. rigida morphologically, but it is unknown whether B. changningensis is a natural hybrid. Moreover, B. changningensis has been identified as a superior variety of Sichuan Province with high pulping yield, fibre length and width. Therefore, we analyzed the morphological characteristics, DNA markers, DNA barcoding and chloroplast genomes to identify the hybrid origin of B. changningensis and possible maternal parent. We have developed the transcriptomic data for B. changningensis and mined the SSR loci. The putative parental lines and hybrid were screened for 64 SSR makers and identified that SSR14, SSR28, SSR31 and SSR34 markers showed both alleles of the parental species in B. changningensis, proving heterozygosity. Sequencing nuclear gene GBSSI partial regions and phylogenetic analysis also confirm the hybrid nature of B. changningensis. Further, we have generated the complete chloroplast genome sequence (139505 bp) of B. changningensis. By analyzing the cp genomes of both parents and B. changningensis, we identified that B. rigida might be the female parent. In conclusion, our study identified that B. changningensis is a natural hybrid, providing evidence for bamboo's natural hybridization. This is the first report on confirming a natural bamboo hybrid and its parents through SSR and chloroplast genome sequence.
Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract.IMPORTANCE EAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5.
Tissue-specific novel transcripts expressed during sexual development were examined by RT-PCR, quantitative RT-PCR (qRT-PCR), and in situ hybridization to provide data for chicken genomics. Public databases for transcript data have been constructed with known and unknown sequences of various tissues from different animals. However, the expression patterns and functions of the transcripts are less known. From the The Institute for Genomics Research Gallus gallus library, we examined 291 tentative consensus (TC) sequences that assembled 100% with transcripts by RT-PCR during male and female sexual development from Embryonic Day 6 to 25 wk of age. We found 85 TC sequences that were specific to testicular development; of these, 43 TC sequences were exclusively upregulated in 25-wk-old testis. Another 52 TC sequences were not specific to one tissue, but occurred in the testis and ovary at different developmental ages. Twelve testis-specific TC sequences upregulated in 25-wk-old testis were randomly selected and further examined with qRT-PCR. For precise localization, these 12 testis-specific TC sequences were examined by in situ hybridization with 25-wk-old adult testis. Six TC sequences were strongly expressed in secondary spermatocytes and haploid spermatids until spermatozoa release. Another six TC sequences were differentially expressed in the adluminal compartment of seminiferous tubules. Among the testis-specific TC sequences, TC120901 is a known gene, phospholipase C, zeta (PLCZ1). Our data provide potential insight into gene expression and genomic information on novel transcripts that are important to avian reproduction.
Bacterial strain M19T was isolated from the gut of a wood-feeding termite, Nasutitermes hainanensis. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain M19T was related to members of the genus Lactococcus, with sequence similarities ranging from 84.8 to 95.5 %. Comparison of housekeeping gene ropB sequences revealed that strain M19T was well separated from Lactococcus fujiensis JCM 16395T and Lactococcus hircilactis 117T. The isolate was Gram-stain-positive, catalase-negative and non-motile. Cells were coccoid or ovoid-shaped, and occurred singly, in pairs or as short chains. Growth of strain M19T occurred at 10-40 °C and at pH 5.0-7.5. The DNA G+C content of strain M19T was 39.6 mol% and the major fatty acids were C16 : 0, cyclo-C19 : 0ω8c, C18 : 1ω9c, summed feature 7 and summed feature 8. Based on the phylogenetic, chemotaxonomic and phenotypic data presented, strain M19T represents a novel species of the genus Lactococcus, for which the name Lactococcus nasutitermitis sp. nov. is proposed. The type strain is M19T ( = CGMCC 1.15204T = NBRC 111537T).
Macropores are an important part of soil structure. However, in alpine regions, the effects of soil macropores on soil properties and vegetation growth are not clear. We used the X-ray computed tomography (CT) method to obtain 3D images and visualize the distribution and morphology of soil macropores. By combining principal component analysis (PCA) and stepwise regression methods, we studied the relationships between soil macropores and both soil properties and vegetation growth in three types of grassland [alpine degraded steppe (ADS), alpine typical steppe (ATS), and alpine meadow steppe (AMS)] on the Tibetan Plateau. More tubular and continuous macropores occurred in the soil profiles of the AMS and ATS than in that of the ADS. In addition, the AMS soil had the highest macropore number (925 ± 189), while the ADS soil had the lowest macropore number (537 ± 137). PCA and correlation analysis suggested that macroporosity (MP) has significant positive correlations with the contents of soil organic matter, total nitrogen (TN), available phosphorus (AP) and total phosphorus (TP) ( p < 0.05). The two parameters with the greatest influence on aboveground and belowground biomass were the shape factor ( p < 0.05) and MP ( p < 0.05), respectively. However, there was no significant correlation between plant diversity and soil macropores. We conclude that the irregularity of soil macropores restricts the growth space of roots and causes plants to sacrifice the accumulation of aboveground biomass for that of roots to find suitable sites for nutrient and water absorption.
A flock of budgerigars (Melopsittacus undulates) was purchased from a licensed breeder and quarantined at a zoologic facility within the United States in 2016. Following 82 deaths within the flock, the remaining 66 birds were depopulated because of ongoing clinical salmonellosis despite treatment. Gross necropsy was performed on all 66 birds. Histopathologic examination was performed on 10 birds identified with gross lesions and 10 birds without. Pathologic findings were most often observed in the liver, kidney, and spleen. Lesions noted in the livers and spleens were consistent with published reports of salmonellosis in psittacine species. Multisystemic changes associated with septicemia were not noted, most likely because of antibiotic intervention before euthanasia. Of the 20 budgerigars evaluated by histopathology, six had large basophilic intranuclear inclusion bodies within tubular epithelia in a portion of the kidneys. Electronic microscopy, next-generation sequencing, Sanger sequencing, and phylogenetic analyses were used to identify and categorize the identified virus as a novel siadenovirus strain BuAdV-1 USA-IA43444-2016. The strain was 99% similar to budgerigar adenovirus 1 (BuAdV-1), previously reported in Japan, and to a psittacine adenovirus 5 recently identified in a U.S. cockatiel. Salmonella typhimurium carriers were identified via polymerase chain reaction (PCR) and bacterial culture and compared with viral carriers identified via PCR. Inclusion bodies and Salmonella detection were significant in birds with gross lesions versus those without; however, there was no correlation between budgerigars positive with siadenovirus by PCR and concurrent Salmonella infection. Identifying subclinical siadenovirus strain BuAdV-1 USA-IA43444-2016 infection in this flock significantly differs from a previous report of clinical illness in five budgerigars resulting in death caused by BuAdV-1 in Japan. S. typhimurium remains a significant pathogen in budgerigars, and zoonotic concerns prompted depopulation to mitigate the public health risks of this flock.
Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors play critical roles in mediating host immunity to pathogen attack. We use tomato Sw-5b::tospovirus as a model system to study the specific role of the compartmentalized plant NLR in dictating host defenses against the virus at different infection steps. We demonstrated here that tomato NLR Sw-5b distributes to the cytoplasm and nucleus, respectively, to play different roles in inducing host resistances against tomato spotted wilt orthotospovirus (TSWV) infection. The cytoplasmic-enriched Sw-5b induces a strong cell death response to inhibit TSWV replication. This host response is, however, insufficient to block viral intercellular and long-distance movement. The nuclear-enriched Sw-5b triggers a host defense that weakly inhibits viral replication but strongly impedes virus intercellular and systemic movement. Furthermore, the cytoplasmic and nuclear Sw-5b act synergistically to dictate a full host defense of TSWV infection. We further demonstrated that the extended N-terminal Solanaceae domain (SD) of Sw-5b plays critical roles in cytoplasm/nucleus partitioning. Sw-5b NLR controls its cytoplasm localization. Strikingly, the SD but not coil-coil domain is crucial for Sw-5b receptor to import into the nucleus to trigger the immunity. The SD was found to interact with importins. Silencing both importin α and β expression disrupted Sw-5b nucleus import and host immunity against TSWV systemic infection. Collectively, our findings suggest that Sw-5b bifurcates disease resistances by cytoplasm/nucleus partitioning to block different infection steps of TSWV. The findings also identified a new regulatory role of extra domain of a plant NLR in mediating host innate immunity.
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