Biology subjects

Ahmed, S.

Publications and source records attributed to Ahmed, S..

10 recordsLinked to original sources

bioRxiv2018

Guiding dose selection of monoclonal antibodies using a new parameter (AFTIR) for characterizing ligand binding systems

Guiding the dose selection for monoclonal antibody oncology drugs is often done using methods for predicting the receptor occupancy of the drug in the tumor. In this manuscript, previous work on characterizing target inhibition at steady state using the AFIR metric [1] is extended to include a \"target-tissue\" compartment and the shedding of membrane-bound targets. A new potency metric AFTIR (Averarge Free Tissue target to Initial target ratio at steady state) is derived, and it depends on only four key quantities: the equilibrium binding constant, the fold-change in target expression at steady state after binding to drug, the biodistribution of target from circulation to target tissue, and the average drug concentration in circulation. The AFTIR metric is useful for guiding dose selection, for efficiently performing sensitivity analyses, and for building intuition for more complex target mediated drug disposition models. In particular, reducing the complex, physiological model to four key parameters needed to predict target inhibition helps to highlight specific parameters that are the most important to estimate in future experiments to guide drug development.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC=\"FIGDIR/small/432500_fig1.gif\" ALT=\"Figure 1\">\nView larger version (30K):\norg.highwire.dtl.DTLVardef@6895d7org.highwire.dtl.DTLVardef@4566e0org.highwire.dtl.DTLVardef@6515e8org.highwire.dtl.DTLVardef@8168ea_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1C_FLOATNO Graphical Abstract\n\nC_FIG

pharmacology and toxicology↗

bioRxiv2018

Unified single-cell analysis of testis gene regulation and pathology in 5 mouse strains

By removing the confounding factor of cellular heterogeneity, single cell genomics can revolutionize the study of development and disease, but methods are needed to simplify comparison among individuals. To develop such a framework, we assayed the transcriptome in 62,600 single cells from the testes of wildtype mice, and mice with gonadal defects due to disruption of the genes Mlh3, Hormad1, Cul4a or Cnp. The resulting expression atlas of distinct cell clusters revealed novel markers and new insights into testis gene regulation. By jointly analysing mutant and wildtype cells using a model-based factor analysis method, SDA, we decomposed our data into 46 components that identify novel meiotic gene regulatory programmes, mutant-specific pathological processes, and technical effects. Moreover, we identify, de novo, DNA sequence motifs associated with each component, and show that SDA can be used to impute expression values from single cell data. Analysis of SDA components also led us to identify a rare population of macrophages within the seminiferous tubules of Mlh3-/- and Hormad1-/- testes, an area typically associated with immune privilege. We provide a web application to enable interactive exploration of testis gene expression and components at http://www.stats.ox.ac.uk/~wells/testisAtlas.html

genomics↗

bioRxiv2018

DAF-16/Foxo suppresses the transgenerational sterility of prg-1 piRNA mutants via a systemic small RNA pathway

Mutation of the daf-2 insulin/IGF-1 receptor activates the DAF-16/Foxo transcription factor to suppress the transgenerational sterility phenotype of prg-1/piRNA mutants that are deficient for piRNA-mediated genome silencing. As with PRG-1/piRNAs, mutations in the nuclear RNA interference gene nrde-1 compromised germ cell immortality, but deficiency for daf-2 did not suppress the transgenerational sterility of nrde-1 or nrde-4 single mutants or of prg-1; nrde-4 or prg-1; hrde-1 double mutants. NRDE-1 and NRDE-4 promote transcriptional silencing in somatic cells via the nuclear Argonaute protein NRDE-3, which was dispensable for germ cell immortality. However, daf-2 deficiency failed to promote germ cell immortality in prg-1; nrde-3 mutants. Consistently, we found that DAF-16 activity in somatic cells suppressed the transgenerational sterility of prg-1 mutants via the SID-1 dsRNA transmembrane channel that promotes systemic RNAi as well as Dicer, the dsRNA binding protein RDE-4 and the RDRP RRF-3. We conclude that DAF-16 activates a cell-non-autonomous systemic RNAi pathway that promotes small RNA-mediated genome silencing in germ cells to suppress loss of the genomic immune surveillance factor Piwi/PRG-1. Author SummarySmall RNAs can promote genome silencing. The Argonaute protein Piwi interacts with thousands of small RNAs termed piRNAs in germ cells to suppress expression of transposons and foreign genetic elements. However, the Piwi silencing system may be commonly targeted by viral or transposon genomic parasites that seek to suppress the endogenous defences against their expression and replication. Activation of the DAF-16 stress response pathway promotes adult longevity and can also abolish the transgenerational sterility of C. elegans Piwi mutants. We found that DAF-16 accomplishes this by activating a somatic small RNA pathway where small RNAs are initially produced in the soma and are then transported into the germline to suppress expression of a toxic genetic locus in Piwi mutants. Thus, the DAF-16 stress response pathway activates a systemic small RNA cascade to suppress defects in the Piwi/piRNA genome silencing system.

genetics↗

bioRxiv2018

Deficiency for Piwi results in transmission of a heritable stress that promotes longevity via DAF-16/Foxo

The C. elegans Argonaute protein PRG-1/Piwi and associated piRNAs protect metazoan genomes by silencing transposons and other types of foreign DNA. As prg-1 mutants are propagated, their fertility deteriorates prior to the onset of a reproductive arrest phenotype that resembles a starvation-induced stress response. We found that late-generation prg-1 mutants with substantially reduced fertility were long-lived, whereas early- or mid-generation prg-1 mutants had normal lifespans. Loss of the stress response transcription factor DAF-16 caused mid- or late-generation prg-1 mutants to live very short lives, whereas overexpression of DAF-16 enabled both mid- and late-generation prg-1 mutants to live long. Cytoplasmic P-bodies that respond to stress increased in long-lived late-generation prg-1 mutants and were transmitted to F1 but not F2 cross-progeny. Moreover, moderate levels of heritable stress shorten late-generation prg-1 mutant longevity when DAF-16 or P bodies are deficient. Together, these results suggest that the longevity of late-generation prg-1 mutants is a hormetic stress response. However, dauer larvae that occur in response to stress were not observed in late-generation prg-1 mutants. Small germ cell nucleoli that depended on germline DAF-16 were present in late-generation prg-1 mutants but were not necessary for their longevity. We propose that prg-1 mutant germ cells transmit a form of heritable stress, high levels of which promote longevity and strongly reduce fertility. The heritable stress transmitted by PRG-1/Piwi mutant germ cells may be generally relevant to epigenetic inheritance of longevity. Core message of paperprg-1/Piwi mutants with strongly reduced fertility live long and longevity is transmitted for one generation to F1 cross progeny. Stress granules are increased and germ cell nucleoli are small for long-lived Piwi mutants and their F1 progeny. Loss of daf-16 stress response transcription factor or dcap-1 P body protein causes very short life for worms when prg-1 mutant fertility is moderately reduced, whereas moderate fertility is sufficient to extend lifespan when somatic DAF-16 is overexpressed. We propose that prg-1 mutant germ cells transmit a heritable epigenetic factor that is stressful and elicits two hormetic stress responses: reproductive arrest and longevity.

genetics↗

bioRxiv2018

Transgenerational sterility of small RNA genome silencing mutants in response to germ granule dysfunction

In several species, Piwi/piRNA genome silencing defects lead to immediate sterility accompanied by heterochromatin dysfunction and transposon-induced genomic instability, which may cause Piwi mutant sterility. In C. elegans, Piwi pathway mutants transmit a heritable stress through germ cells that induces sterility after growth for several generations. We found that sterile Piwi pathway mutant germ cells displayed inconsistent increases in DNA damage but consistently altered perinuclear germ granules that are known to promote fertility. Germ granule dysfunction did not elicit transposon expression but was sufficient to induce multiple phenotypes found in sterile Piwi silencing mutants, including germline atrophy and regrowth. Furthermore, loss of the germ granule component PGL-1 accelerated sterility in response to deficiency for prg-1/Piwi. Restoration of germ granule function to sterile pgl-1 mutants restored their fertility. Together, our results suggest that germ granule defects may promote an adult reproductive arrest phenotype that is responsible for Piwi/piRNA mutant sterility.

developmental biology↗

bioRxiv2018

The conserved phosphatase GSP-2/PP1 promotes germline immortality via small RNA-mediated genome silencing during meiosis

Genomic silencing can promote germ cell immortality, or transgenerational maintenance of the germ line, via mechanisms that may occur during mitosis or meiosis. Here we report that the gsp-2 PP1/Glc7 phosphatase promotes germ cell immortality. We identified a separation-of-function allele of C. elegans GSP-2 that caused a meiosis-specific chromosome segregation defect and defects in transgenerational small RNA-induced genome silencing. GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. Sterile gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents and histone phosphorylation defects in oocytes, similar to small RNA genome silencing mutants. Epistasis and RNA analysis suggested that GSP-2 functions downstream of small RNAs. We conclude that a meiosis-specific function of GSP-2/LAB-1 ties small RNA-mediated silencing of the epigenome to germ cell immortality. Given that hemizygous genetic elements can drive transgenerational epigenomic silencing, and given that LAB-1 promotes pairing of homologous chromosomes and localizes to the interface between homologous chromosomes during pachytene, we suggest that discontinuities at this interface could promote nuclear silencing in a manner that depends on GSP-2.\n\nAuthor SummaryThe germ line of an organism is considered immortal in its capacity to give rise to an unlimited number of future generations. To protect the integrity of the germ line, mechanisms act to suppress the accumulation of transgenerational damage to the genome or epigenome. Loss of germ cell immortality can result from mutations that disrupt the small RNA-mediated silencing pathway that helps to protect the integrity of the epigenome. Here we report for the first time that the C. elegans protein phosphatase GSP-2 that promotes core chromosome biology functions during meiosis is also required for germ cell immortality. Specifically, we identified a partial loss of function allele of gsp-2 that exhibits defects in meiotic chromosome segregation and is also dysfunctional for transgenerational small RNA-mediated genome silencing. Our results are consistent with a known role of Drosophila Protein Phosphatase 1 in heterochromatin silencing, and point to a meiotic phosphatase function that is relevant to germ cell immortality, conceivably related to its roles in chromosome pairing or sister chromatid cohesion.

genetics↗

bioRxiv2018

Atlastins mediate selective autophagy of the endoplasmic reticulum

The selective lysosomal degradation (autophagy) of entire organelles is required for cellular homeostasis, and its dysregulation is involved in degenerative disorders such as Parkinsons Disease. While autophagy of mitochondria (mitophagy) is becoming better understood, other forms of organelle autophagy are relatively unexplored. Here we develope multiple quantitative assays to measure organelle autophagy using flow cytometry, microscopy, and Western blotting. Focusing on autophagy of the endoplasmic reticulum (ER-phagy), we show that these assays allow facile measurement of ER-phagy, and that ER-phagy is inhibited by knockdown of either core autophagy components or the recently reported FAM134B ER-phagy receptor. Using these assays, we further identify that Atlastins, the ER-resident GTPases involved in ER membrane morphology, are key positive effectors of ER-phagy. Atlastin-depleted cells have decreased ER-phagy under starvation conditions, and Atlastins role in ER-phagy requires both a functional GTPase domain and proper ER localization. The three Atlastin family members functionally compensate for one another during ER-phagy and may form heteromeric complexes with one another. We also find that Atlastins act downstream of the FAM134B ER-phagy receptor. We propose that during ER-phagy, Atlastins remodel ER membrane to separate pieces of FAM134B-marked ER for efficient autophagosomal engulfment. Human mutations in Atlastins led to hereditary spastic paraplegia, and our results suggest that this disease may be linked to deficiencies in ER-phagy rather than ER morphology.

cell biology↗

bioRxiv2018

Household Transmission Study of Cryptosporidiosis in Bangladesh

BackgroundCryptosporidium, an apicomplexan protozoa, is a leading contributor to diarrheal morbidity and mortality in children under five years old worldwide. As there is no vaccine and no approved drug for Cryptosporidium spp. in young children, preventing parasite transmission is crucial. We undertook a pilot case-control study to define the extent of person-to-person transmission of cryptosporidiosis within families in an urban and rural community in Bangladesh.\n\nMethodsWe enrolled 48 case families with a Cryptosporidium-infected child aged 6-18 months. Controls were age-sex matched Cryptosporidium-negative children (n=12). Once children were identified, we enrolled all household members. We then followed these individuals for 8 weeks, with weekly surveillance stools and testing with qPCR for Cryptosporidium spp.\n\nFindingsIn the 48 case families, the rate of secondary infections with Cryptosporidium was 18.6% (22/118) compared to 0 new infections (0/35) in the 12 control families. In the 22 urban Mirpur households, the secondary attack rate was 30% (18/60) in cases compared to 0% (0/14) in controls (chi-square p = 0.018). In contrast, in the 21 rural Mirzapur households, the secondary attack rate was 6.9% (4/58) in case households compared to 0% (0/21) in controls (chi-square p = 0.22). Genotyping by gp60 demonstrated infection with the same subspecies in five of six families. Serologic response to Cryptosporidium infection was associated with younger age, longer duration of infection, and C hominis gp60_IbA9G3R2 infection.\n\nInterpretationThe high rate of secondary infection in Mirpur suggests that person-to-person transmission is likely a major source of Cryptosporidium infection for young children living in this region. GP 60 genotyping demonstrated direction of infection in 2 households, and concurrent infection in five households. Further work is needed to understand the differences in parasite transmissibility and immunity to different genotypes.

microbiology↗

bioRxiv2017

GrandPrix: Scaling up the Bayesian GPLVM for single-cell data

MotivationThe Gaussian Process Latent Variable Model (GPLVM) is a popular approach for dimensionality reduction of single-cell data and has been used for pseudotime estimation with capture time information. However current implementations are computationally intensive and will not scale up to modern droplet-based single-cell datasets which routinely profile many tens of thousands of cells.\n\nResultsWe provide an efficient implementation which allows scaling up this approach to modern single-cell datasets. We also generalize the application of pseudotime inference to cases where there are other sources of variation, such as branching dynamics. We apply our method on microarray, nCounter, RNA-seq, qPCR and droplet-based datasets from different organisms. The model converges an order of magnitude faster compared to existing methods whilst achieving similar levels of estimation accuracy. Further, we demonstrate the flexibility of our approach by extending the model to higher-dimensional latent spaces that can be used to simultaneously infer pseudotime and other structure such as branching. Thus, the model has the capability of producing meaningful biological insights about cell ordering as well as cell fate regulation.\n\nAvailabilitySoftware available at github.com/ManchesterBioinference/GrandPrix.

bioinformatics↗

bioRxiv2017

Intrinsic and Modifiable Contributors to Distal Radius Microstructure, Macrostructure and Strength in Premenopausal Women

While weight-bearing and resistive exercise modestly increases aBMD, the precise relationship between physical activity and bone microstructure, and strain in humans is not known. Previously, we established a voluntary upper-extremity loading model that assigns a persons target force based on their subject-specific, continuum FE-estimated radius bone strain. Here, our purpose was to quantify the inter-individual variability in radius microstructure and FE-estimated strain explained by site-specific mechanical loading history, and to determine whether variability in strain is captured by aBMD, a clinically relevant measure of bone density and fracture risk. Seventy-two women aged 21-40 were included in this cross-sectional analysis. High resolution peripheral quantitative computed tomography (HRpQCT) was used to measure macro- and micro-structure in the distal radius. Mean energy equivalent strain in the distal radius was calculated from continuum finite element models generated from clinical resolution CT images of the forearm. Areal BMD was used in a nonlinear regression model to predict FE strain. Hierarchical linear regression models were used to assess the predictive capability of intrinsic (age, height) and modifiable (body mass, grip strength, physical activity) predictors. Fifty-one percent of the variability in FE bone strain was explained by its relationship with aBMD, with higher density predicting lower strains. Age and height explained up to 31.6% of the variance in microstructural parameters. Body mass explained 9.1% and 10.0% of the variance in aBMD and bone strain, respectively, with higher body mass indicative of greater density. Overall, results suggest that meaningful differences in bone structure and strain can be predicted by subject characteristics.\n\nHighlightsO_LIAreal bone mineral density (aBMD) explains 51% of the variability in bone strain.\nC_LIO_LIAdult bone loading predicts greater cortical porosity and trabecular density.\nC_LIO_LIGreater body mass predicts greater aBMD and lower bone strain.\nC_LI

physiology↗