Selective Disruption of Drp1-Independent Mitophagy and Mitolysosome Trafficking by an Alzheimer’s Disease Relevant Tau Modification in a Novel C. elegans Model

Background: Accumulation of inappropriately phosphorylated tau into neurofibrillary tangles (NFT) is a defining feature of Alzheimer’s Disease (AD), with specific epitopes such as Tau pT231 emerging early in the development of tau pathology. Previously, we demonstrated that a phosphomimetic mutant (T231E) of human tau drove the loss of neuronal function and structural integrity with age in a novel C. elegans single-copy gene insertion AD model. A critical finding was that T231E, unlike wild type tau, suppressed oxidative stress-induced mitochondrial autophagy, or mitophagy. Regulation of mitochondrial morphology by fission is important for mitophagy, which has been reported to be dysregulated by AD-relevant tau species. Dynamin Related Protein 1 (Drp1) is a GTPase that plays a central role in mediating mitochondrial fission, and its altered function may contribute to AD pathogenesis. Methods: Genetically-encoded fluorescent biosensors and dynamic imaging approaches were combined with a genomic drp-1(-) loss-of-function and transgenic tau mutants to derive a comprehensive in vivo analysis of age-associated changes in mitochondria and mitolysosome (ML) morphology, abundance, neurite trafficking, and stress-induced mitophagy. Results: Strain expressing disease-associated PTM mimetic Tau T231E demonstrated a surprisingly selective effect on ML development and trafficking, with no effect on lysosomes or autolysosomes, and a subtle effect on mitochondria that was apparent mainly in older animals. Unexpectedly, we found that drp-1(-) mutants mount a robust mitophagy response to oxidative stress, consistent with recent observations that adaptive mitophagy may occur independent of the canonical DRP1 pathway. Moreover, T231E continued to suppress oxidative stress-induced mitophagy in the drp-1(-) background. Conclusions: Our C. elegans single-copy gene insertion model unveils multiple levels of selectivity – phenotypic selectivity for mutations that mimic pathologic tauopathy-associated PTM and physiologic selectivity for organelles that contain damaged mitochondria. In addition, our novel findings provide compelling support for DRP1-independent mechanisms playing a pivotal role in regulating mitochondrial dynamics and function in the context of AD-relevant tau species and age-associated stress.

Impact of Amplification Efficiency Approaches on Telomere Length Measurement via Quantitative-Polymerase Chain Reaction

Background: Precise determination of amplification efficiency is critical for reliable conversion of within-sample changes in fluorescence occurring on a logarithmic scale to between-sample differences in DNA content occurring on a linear scale. This endeavor is especially challenging for the telomere length (TL) quantitative-PCR (qPCR) assay, where amplification efficiency can vary between reactions targeting telomeric repeats (T) and those targeting a single-copy gene (S) to calculate TL as the T/S ratio.Methods: We compared seven different approaches toward estimating amplification efficiency, including the standard-curve method utilized by the qPCR instrument software, and alternative approaches which estimate efficiency on a reaction-by-reaction basis using the stand-alone program LinRegPCR. After calculating T/S ratios using efficiency estimates from each approach (N = 363), we tested their relative performance on metrics of assay precision and correlates of external validity including chronological age (age range = 1–72 years), across tissues within-person (leukocyte-buccal), and between parents and offspring.Results: Estimated amplification efficiency for telomere reactions was significantly lower than estimates for single-copy gene reactions. Efficiency estimates for both reaction sets were significantly higher when estimated with the standard-curve method utilized by the qPCR instrument relative to estimates reconstructed during the log-linear phase with LinRegPCR. While estimates of single-copy gene efficiency reconstructed using LinRegPCR measured within 90% of perfect exponential doubling (E = 1.92), estimates generated using the standard-curve method were inflated beyond 100% (E = 2.10–2.12), indicating poor fidelity. Despite differences in raw value, TL measurements calculated with LinRegPCR efficiency estimates exhibited similar relationships with external validity correlates to measurements generated using the qPCR instrument software.Conclusion: Since methods to estimate amplification efficiency can vary across qPCR instruments, we suggest that future analyses empirically consider external methods of efficiency calculations such as LinRegPCR, and that already generated data be re-analyzed to glean possible improvements.

Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

MetaBinner: a high-performance and stand-alone ensemble binning method to recover individual genomes from complex microbial communities

Binning is an essential procedure during metagenomic data analysis. However, the available individual binning methods usually do not simultaneously fully use different features or biological information. Furthermore, it is challenging to integrate multiple binning results efficiently and effectively. Therefore, we developed an ensemble binner, MetaBinner, which generates component results with multiple types of features and utilizes single-copy gene (SCG) information for k-means initialization. It then utilizes a two-step ensemble strategy based on SCGs to integrate the component results. Extensive experimental results over three large-scale simulated datasets and one real-world dataset demonstrate that MetaBinner outperforms other state-of-the-art individual binners and ensemble binners. MetaBinner is freely available at https://github.com/ziyewang/MetaBinner.

Polyploidization within the Funariaceae—a key principle behind speciation, sporophyte reduction and the high variance of spore diameters?

Although being recognized as a major force behind speciation in flowering plants, the evolutionary relevance of genome duplication (polyploidization) remains largely unexplored in mosses. Phylogenetic and-genomic insights from the model organism Physcomitrella patens and closely related species revealed that polyploidization, likely via hybridization (allopolyploidization), gives rise to new species within the Funariaceae. Based on the phylogenetic analysis of the nuclear single copy gene BRK1 combined with the measurement of DNA content by flow cytometry, we identified Entosthodon hungaricus as such an allopolyploid species. Together with Physcomitrium pyriforme, Physcomitrium eurystomum and Physcomitrium collenchymatum, which were identified previously as species that likely arose by hybridization, E. hungaricus represents an additional allopolyploid lineage of a species complex that is characterized by convergent sporophyte reduction and a considerable variance in spore sizes. Based on morphological and cytological data from 18 species, we highlight the potential impact of polyploidization on the size of the spores and on sporophyte architecture.

Unequal contribution of two paralogous CENH3 variants in cowpea centromere function

In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.

Mosaic fungal individuals have the potential to evolve within a single generation

Although cells of mushroom-producing fungi typically contain paired haploid nuclei (n + n), most Armillaria gallica vegetative cells are uninucleate. As vegetative nuclei are produced by fusions of paired haploid nuclei, they are thought to be diploid (2n). Here we report finding haploid vegetative nuclei in A. gallica at multiple sites in southeastern Massachusetts, USA. Sequencing multiple clones of a single-copy gene isolated from single hyphal filaments revealed nuclear heterogeneity both among and within hyphae. Cytoplasmic bridges connected hyphae in field-collected and cultured samples, and we propose nuclear migration through bridges maintains this nuclear heterogeneity. Growth studies demonstrate among- and within-hypha phenotypic variation for growth in response to gallic acid, a plant-produced antifungal compound. The existence of both genetic and phenotypic variation within vegetative hyphae suggests that fungal individuals have the potential to evolve within a single generation in response to environmental variation over time and space.

Reclassification of Brucella ciceri as later heterotypic synonyms of Brucella intermedia

Recently Hördt et al. 2020 proposed to merge Ochrobactrum and Brucella genera based on up to date phylogenomic evidence and overall genomic divergence among Brucella-Ochrobactrum clade. This led to the description of the new combinations Brucella ciceri comb. nov., basonym: Ochrobactrum ciceri Imran et al. 2010 and Brucella intermedia comb. nov., basonym: Ochrobactrum intermedium Velasco et al. 1998. However, the type species for Brucella ciceri DSM 22292T and Brucella intermedia LMG 3301T show whole-genome coherence at the species level (ANI = 98.21 %, Mash D = 0.0154006, dDDH relatedness >70%), suggesting that may belong to the same genomospecies. Also, both taxa formed a single clade in the phylogenomic tree based on single-copy gene sequences. Previously reported phenotypic data offer a context where both taxa are highly related supporting this synonymy. Therefore, Brucella ciceri should be reclassified as later heterotypic synonyms of Brucella intermedia, which has priority. The species description is consequently amended.

A Synthetic Single-Stranded Dual-Template Oligonucleotide as a Reference Standard for Monochrome Multiplex qPCR Measurements of Average Telomere Length

ABSTRACTQuantitative PCR is frequently used to measure average telomere length (TL) relative to the TL of a reference DNA sample of the investigator’s choosing. This makes comparisons of TLs across studies and laboratories difficult. Here we demonstrate that a single synthetic single-stranded dual-template oligonucleotide (DTO) containing both a telomere repeat sequence (T) and a segment of the human beta-globin (HBB) single copy gene (S) can be used as a universal reference standard for monochrome multiplex quantitative PCR (MMqPCR) measurements of average TL using SYBR Green I as the only fluorescent reporter dye. A set of twelve concentrations of the DTO is prepared by serial 3-fold dilutions, to a lowest concentration of ~20 copies per μl. The 5 highest concentrations are used for the T standard curve, and the 5 lowest concentrations are used for the S standard curve. For each reaction 5 μl containing approximately 3 ng of genomic DNA (or one of the DTO dilutions) is mixed with 5 μl of a 2x MasterMix containing the primers for T and S amplification, and MMqPCR is performed. The design of the primers and thermal cycling profile allows all T amplification signals to be collected before exponential amplification of the S signal begins. Exponential amplification from S is then carried out in a temperature range that keeps the telomere product fully melted and therefore unable to influence the S amplification signal. The T value for each DNA sample is the Standard Curve DTO dilution that contains the same number of copies of the telomere sequence as the experimental sample, and the S value is the DTO dilution that contains the same number of copies of the single copy gene sequence as the experimental sample. Dividing the first dilution by the second dilution yields an absolute T/S ratio, since it is expressed relative to the fixed 1:1 T/S ratio that is built into the DTO by design. Absolute T/S ratios for average TL in 48 human DNA samples determined by this method correlated strongly with mean Terminal Restriction Fragment (mTRF) lengths for the same DNA samples determined by the Southern Blot method (R-squared = 0.801). This DTO and the accompanying protocol may facilitate the standardization of average telomere length measurements and analyses across laboratories.

Exceptionally Long-Lived Individuals (ELLI) Demonstrate Slower Aging Rate Calculated by DNA Methylation Clocks as Possible Modulators for Healthy Longevity

Exceptionally long-lived individuals (ELLI) who are the focus of many healthy longevity studies around the globe are now being studied in Israel. The Israeli Multi-Ethnic Centenarian Study (IMECS) cohort is utilized here for assessment of various DNA methylation clocks. Thorough phenotypic characterization and whole blood samples were obtained from ELLI, offspring of ELLI, and controls aged 53–87 with no familial exceptional longevity. DNA methylation was assessed using Illumina MethylationEPIC Beadchip and applied to DNAm age online tool for age and telomere length predictions. Relative telomere length was assessed using qPCR T/S (Telomere/Single copy gene) ratios. ELLI demonstrated juvenile performance in DNAm age clocks and overall methylation measurement, with preserved cognition and relative telomere length. Our findings suggest a favorable DNA methylation profile in ELLI enabling a slower rate of aging in those individuals in comparison to controls. It is possible that DNA methylation is a key modulator of the rate of aging and thus the ELLI DNAm profile promotes healthy longevity.