Miracle-dz/UniFAR-FTUs huggingface dataset:
Abundantly expressed class of noncoding RNAs conserved through the multicellular evolution of dictyostelid social amoebas

Background Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes. One of the most well-studied examples is the development of dictyostelid social amoebae, e.g. Dictyostelium discoideum. However, it is still poorly understood why multicellularity emerged in these amoebae while the great majority of other members of Amoebozoa are unicellular. Previously a novel type of non-coding RNA, Class I RNAs, was identified in D. discoideum and demonstrated to be important for normal multicellular development. In this study we investigated Class I RNA evolution and its connection to multicellular development. Results New Class I RNA genes were identified by constructing a co-variance model combined with a scoring system based on conserved up-stream sequences. Multiple genes were predicted in representatives of each major group of Dictyostelia and expression analysis validated that our search approach can identify expressed Class I RNA genes with high accuracy and sensitivity. Further studies showed that Class I RNAs are ubiquitous in Dictyostelia and share several highly conserved structure and sequence motifs. Class I RNA genes appear to be unique to dictyostelid social amoebae since they could not be identified in searches in outgroup genomes, including the closest known relatives to Dictyostelia. Conclusion Our results show that Class I RNA is an ancient abundant class of ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 million years. Taken together, our current knowledge of Class I RNAs suggests that they may have been involved in evolution of multicellularity in Dictyostelia.
An 85K SNP Array Uncovers Inbreeding and Cryptic Relatedness in an Antarctic Fur Seal Breeding Colony

High density single nucleotide polymorphism (SNP) arrays allow large numbers of individuals to be rapidly and cost-effectively genotyped at large numbers of genetic markers. However, despite being widely used in studies of humans and domesticated plants and animals, SNP arrays are lacking for most wild organisms. We developed a custom 90K Affymetrix Axiom array for an intensively studied pinniped, the Antarctic fur seal (Arctocephalus gazella). SNPs were discovered from a combination of genomic and transcriptomic resources and filtered according to strict criteria. Out of a total of 85,359 SNPs tiled on the array, 75,601 (88.6%) successfully converted and were polymorphic in 274 animals from a breeding colony at Bird Island in South Georgia. Evidence was found for inbreeding, with three genomic inbreeding coefficients being strongly intercorrelated and the proportion of the genome in ROH being non-zero in all individuals. Furthermore, analysis of genomic relatedness coefficients identified multiple second and third order relatives among a sample of ostensibly unrelated individuals. Such “cryptic relatedness” within fur seal breeding colonies may increase the likelihood of consanguinous matings and could therefore have implications for understanding fitness variation and mate choice. Finally, we demonstrate the cross-amplification potential of the array in three related species. Overall, our SNP array will facilitate future studies of Antarctic fur seals and has the potential to serve as a more general resource for the wider pinniped research community.
Programmable RNA Targeting Using CasRx in Flies

CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof-of-principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on Cas ribonuclease function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact in the field. Here we report on our efforts to develop a programmable platform for RNA-targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.
A recombinant gp145 Env glycoprotein from HIV-1 expressed in two different cell lines: Effects on glycosylation and antigenicity

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145 produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.
TMEM70 promotes ATP synthase assembly within cristae via transient interactions with subunit c

Mitochondrial ATP-synthesis is catalyzed by F1Fo-ATP synthase, an enzyme of dual genetic origin. TMEM70, a transmembrane protein mutated in neonatal encephalocardiomyopathies, is required for assembly/stability of ATP-synthase, but its precise role remains largely unknown. Herein, we demonstrate a role of TMEM70 in the formation of the c-ring (an oligomer of 8 c-subunits/Su.c) essential for Fo-mediated proton transport. Immunoprecipitation and two-dimensional blue-native/SDS-PAGE reveal interactions between TMEM70 and Su.c not yet assembled into the ATP synthase, and provide evidence for oligomerization of TMEM70 into homo-oligomers of 8 TMEM70 dimers. Analysis of ATPsynthase assembly in cells devoid of TMEM70, mitochondrial DNA or F1 subunits indicate that TMEM70 oligomers provide a scaffold for c-ring assembly, regardless of the presence of other ATP synthase subunits or of mitochondrial bioenergetics status. Expansion superresolution microscopy depicting mitochondrial ultrastructure reveals the specific localization of TMEM70 within inner membrane cristae. This indicates that ATP synthase, localizing to the edge of cristae, is assembled within inner cristae membranes rather than at the inner boundary membrane that is apposed to the outer membrane.
A molecular approach to studying Hymenoptera diets using polistine wasps

The study of animal diets has benefited from the rise of high-throughput DNA sequencing applied to stomach content or faecal samples. The latter can be fresh samples used to describe recent meals, or older samples, which can inform about past feeding activities. For most invertebrates, however, it is difficult to access ‘historical’ samples, due to the small size of the animals and the absence of permanent defecation sites. Therefore, sampling must be repeated to account for seasonal variation and to capture the overall diet of a species. This study develops a method to describe the overall diet of nest-building Hymenoptera based on a single sampling event, by analysing prey DNA from faeces accumulated in brood cells. We collected 48 nests from two species of introduced paper wasps (Polistes chinensis, and P. humilis) in the urban and peri-urban areas of Auckland, New Zealand, and selected two samples per nest. One from brood cells in the outer layer of the nest to represent the most recent diet, and one from brood cells in an inner layer to represent older diet. Diet differed between species, although both fed mainly on Thysanoptera, Lepidoptera and Acariformes. Prey taxa identified to species level included both agricultural pests and native species. Prey communities consumed were significantly different between inner and outer nest samples suggesting seasonal variation in prey availability and/or a diversification of the wasps’ diet as the colony grows. We also show for the first time potential predation of marine organisms by Polistes wasps. Our study provides field evidence that polistine wasps feed on agricultural pests, supporting the hypothesis that some social wasp species could have a suppressing effect on agricultural pests. The proposed methodology is readily applicable to other nest-building Hymenoptera and has the potential to provide comprehensive knowledge about their diet with minimum sampling effort. Such knowledge is Essential to measure the ecological impact of invasive Vespidae and support the conservation of native invertebrate biodiversity.
Altered skeletal muscle glucose–fatty acid flux in amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis is characterized by the degeneration of upper and lower motor neurons, yet an increasing number of studies in both mouse models and patients with amyotrophic lateral sclerosis suggest that altered metabolic homeostasis is also a feature of disease. Pre-clinical and clinical studies have shown that modulation of energy balance can be beneficial in amyotrophic lateral sclerosis. However, the capacity to target specific metabolic pathways or mechanisms requires detailed understanding of metabolic dysregulation in amyotrophic lateral sclerosis. Here, using the superoxide dismutase 1, glycine to alanine substitution at amino acid 93 (SOD1G93A) mouse model of amyotrophic lateral sclerosis, we demonstrate that an increase in whole-body metabolism occurs at a time when glycolytic muscle exhibits an increased dependence on fatty acid oxidation. Using myotubes derived from muscle of amyotrophic lateral sclerosis patients, we also show that increased dependence on fatty acid oxidation is associated with increased whole-body energy expenditure. In the present study, increased fatty acid oxidation was associated with slower disease progression. However, within the patient cohort, there was considerable heterogeneity in whole-body metabolism and fuel oxidation profiles. Thus, future studies that decipher specific metabolic changes at an individual patient level are essential for the development of treatments that aim to target metabolic pathways in amyotrophic lateral sclerosis.
Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes

Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the absence of CSF1R. Here, we analyzed mpeg1:GFP–expressing macrophages in csf1r-deficient zebrafish and report that embryonic macrophages emerge followed by their developmental arrest. In larvae, mpeg1+ cell numbers then increased showing two distinct types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, although numbers also increased in csf1r-mutants, exclusively amoeboid mpeg1+ cells were present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. They expressed macrophage-associated genes, but also showed decreased phagocytic gene expression and increased epithelial-associated gene expression, characteristic of metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that juvenile csf1r-deficient zebrafish exhibit systemic macrophage depletion. Thus, Csf1r deficiency disrupts embryonic to adult macrophage development. Csf1r-deficient zebrafish are viable and permit analyzing the consequences of macrophage loss throughout life.
The Ncm-1 gene for resistance to Cucumber mosaic virus in yellow lupin (Lupinus luteus): molecular studies and marker development

Cucumber mosaic virus (CMV) is an important virus pathogen of lupins in Australia which causes serious yield losses of up to 60% in epidemic years. In commercially grown lupin (Lupinus angustifolius and L. luteus) crops CMV is spread non-persistently by aphid vectors, but it can also be seed borne and this extends virus infection into successive generations. Resistance to CMV has been identified in L. luteus cv. Wodjil and is the conferred by the Ncm-1 gene. The aims of this research were to study the Ncm-1 gene in order to gain a better understanding of resistance in yellow lupin, and to develop a molecular marker linked to Ncm-1 for use in marker assisted selection. Previously published data by Jones et al (1996) identified Ncm-1 as being a single dominant resistance gene, however, phenotypic analysis of CMV infection in a segregating L. luteus mapping population in this thesis was consistent with the Ncm-1 gene being a dominant gene modified by at least one other minor gene. The polygenic nature of CMV resistance in this genetic background was further supported by AFLP analysis which identified one major and one minor QTL associated with resistance. A PCR based approach, using degenerate primers designed on conserved disease resistance protein motifs, was used to identify resistance gene analogues (RGA) in L. luteus. Comparative analysis revealed that RGAs isolated from L. luteus were members of the TIR-NBS-LRR class of R proteins and were similar to the TMV resistance gene N identified in tobacco and the RT4-4 CMV resistance gene from pepper. Extensive comparative analysis using the genomes of model species (including Medicago truncatula, Glycine max, Arabidopsis thaliana and Lotus japonicus) was explored and validated the assignment from L. luteus RGAs to the category of candidate gene for CMV resistance. The RGAs identified in L. luteus were found to be highly conserved in both the CMV resistant and susceptible varieties tested. SNPs which resulted in non-synonymous mutations were identified using cDNA based 5’ RACE and used to develop a single nucleotide primer extension (SNuPE) assays for MALDI-ToF mass spectrophotometric analysis. As SNuPE is based on the allele specific extension of a single nucleotide, genotyping is highly accurate and provides co-dominant information. Two SNuPE assays were developed based on the RGAs isolated and validated on bulked samples from two L.luteus populations segregating for CMV resistance. One assay, SNuPE A267→C was found to associate with CMV resistance. This co-dominant assay is the first of its kind reported for yellow lupin.
Antifungal Efficacy of Routine and Newer Irrigants on Candida Albicans Biofilm Colonization in Young and Old Human Root Canal Dentin: An Ex Vivo Study

The purpose of this study was to evaluate the effect of routine and newer endodontic irrigants on Candida albicans biofilm colonization in Young and Old human root canal dentin. Eighty intact mandibular premolars were used in this study and divided based on Age into two groups. Forty teeth were from young subjects (Group I) and the remaining 40 from old subjects (Group II). Dentin disc samples of 4mm were prepared from each tooth, standardized using gates glidden drill #2 and autoclaved for sterility. The forty samples each in the young and old group were divided into 4 subgroups with various irrigation protocols. The experimental irrigants were: A) 17% EDTA +5.25% NaOCl, B) 100% Octenisept, C) 17% EDTA +5.25% NaOCl + 1% Clotrimazole and D) Phosphate buffer saline. After the irrigation the experimental samples were inoculated with C.albicans and incubated for 72hrs. Out of the 10 samples in each subgroup, 8 samples were analyzed by the Colony forming unit method and 2 samples were analyzed by the Confocal laser scanning microscope. In the CFU method, aliquots from the experimental samples were plated on Sabouraud dextrose agar plates and the colony forming units were counted as a measure of antifungal activity. In the CLSM method, fungal viability was demonstrated using special dyes SYTO 9 and Propidium iodide. The results showed that Octenisept was the most effective irrigant against C.albicans followed by addition of 1% Clotrimazole to 17% EDTA+5.25% NaOCl in both age groups. The other irrigant subgroups were less effective in both age groups. The results of this study also indicates that higher amount of fungi are found in old root dentin as demonstrated by the CFU method and confirmed by the CLSM method. CONCLUSION: Under the limitations of this ex vivo study it can be concluded that: 1. Octenisept used as a single irrigant and the addition of an antifungal agent (1% Clotrimazole) to 17% EDTA + 5.25% NaOCl, were found to perform as the two best irrigation regimes among the irrigants tested in both age groups to reduce C.albicans adherence in root canal dentin. 2. Higher amount of fungi were found in old root dentin for all the irrigants tested as compared to the young group. 3. Confocal laser scanning microscopic evaluation to demonstrate fungal viability has been explored and confirmed in this investigation. Further studies can be undertaken to investigate the use of Octenisept and Clotrimazole as a final rinse following routine endodontic irrigants in clinical trials against C.albicans.
Minimizing biosignal recording sites for hybrid noninvasive brain/neural robot control

Noninvasive brain/neural controlled robots are promising tools to improve autonomy and quality of life in severe paralysis, but require biosignal recordings, such as electroencephalography (EEG) and electrooculography (EOG), from various sites distributed over the user’s head. This limits the applicability and practicality of noninvasive brain/neural robot control on an everyday basis. It would thus be very desirable to minimize the number of necessary recording sites paving the way for miniaturized, headset-like EEG/EOG systems that users with hemiplegia can mount by themselves. Here, we introduce a novel EEG/EOG brain/neural robot control strategy using only scalp electrodes placed near cortical sensorimotor areas. The strategy was tested across 16 healthy volunteers engaging in an EEG/EOG brain/neural control task. Classification accuracies were compared using scalp electrodes only vs. the conventional electrode placements across the scalp and face. To evaluate whether cranial muscle artifacts impede classification accuracy, participants were asked to chew during the task. We found that brain/neural classification accuracy was comparable and that chewing did not impact classification accuracies when using scalp electrodes only. Our results suggest that the proposed new strategy allows for reliable EEG/EOG-based brain/neural robot control, a critical prerequisite to broaden the use of noninvasive brain/neural assistive and rehabilitative technologies.
Subcellular localization of the J-protein Sis1 regulates the heat shock response

Cells exposed to heat shock induce a conserved gene expression program – the heat shock response (HSR) – encoding chaperones like Hsp70 and other protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1 – a co-chaperone for Hsp70 – controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the cortical ER. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network. One sentence summary Localization of the J-protein Sis1 to a subcellular network of proteostasis factors activates the heat shock response.
Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers to modulate transcription and here we use Hi-C experiments to show that it blurs TAD boundaries and increases the proximity of intermediate-range (~1 Mb) genomic sequences and enhancer-promoter interactions. To understand whether NuRD alters the dynamics of 3D genome structure, we developed an approach to segment and extract key biophysical parameters from 3D single-molecule trajectories of the NuRD complex determined using live-cell imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound enhancers move faster, increasing the overall volume of the nucleus that these key regulatory regions explore. Interestingly, we also uncovered a rare fast-diffusing state of NuRD bound enhancers that exhibits directed motion. The NuRD complex reduces the amount of time that enhancers remain in this fast-diffusing state, which we propose might otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local regions of the genome to which it binds.