BCL11A enhancer edited hematopoietic stem cells persist in rhesus monkeys without toxicity

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from sickle cell disease (SCD) patients induces fetal hemoglobin (HbF) without detectable toxicity as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited HSPCs in four non-human primates. We utilized a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer or control locus (AAVS1) targeted cells. Biallelic BCL11A enhancer editing resulted in robust gamma-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Non-homologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we find that edited HSCs can persist for at least 101 weeks post-transplant, and biallelic edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach

An extreme case of plant-insect co-diversification: figs and fig-pollinating wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale co-phylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on average, wasps had sequences from 77% of six genes (5.6kb), figs had sequences from 60% of five genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based co-phylogenetic analyses further support the co-diversification hypothesis. Biogeographic analyses indicate that the presentday distribution of fig and pollinator lineages is consistent with an Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term co-diversification

Accelerating functional gene discovery in osteoarthritis

Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease

Phylogenetic Analysis of Sec7-Domain–containing Arf Nucleotide Exchangers

The eukaryotic family of ADP-ribosylation factor (Arf) GTPases plays a key role in the regulation of protein trafficking, and guanine-nucleotide exchange is crucial for Arf function. Exchange is stimulated by members of another family of proteins characterized by a 200-amino acid Sec7 domain, which alone is sufficient to catalyze exchange on Arf. Here, we analyzed the phylogeny of Sec7-domain–containing proteins in seven model organisms, representing fungi, plants, and animals. The phylogenetic tree has seven main groups, of which two include members from all seven model systems. Three groups are specific for animals, whereas two are specific for fungi. Based on this grouping, we propose a phylogenetically consistent set of names for members of the Sec7-domain family. Each group, except for one, contains proteins with known Arf exchange activity, implying that all members of this family have this activity. Contrary to the current convention, the sensitivity of Arf exchange activity to the inhibitor brefeldin A probably cannot be predicted by group membership. Multiple alignment reveals group-specific domains outside the Sec7 domain and a set of highly conserved amino acids within it. Determination of the importance of these conserved elements in Arf exchange activity and other cellular functions is now possible

Stress-Induced Transcriptomic Changes in Females with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Reveal Disrupted Immune Signatures

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, complex multi-organ illness characterized by unexplained debilitating fatigue and post-exertional malaise (PEM), which is defined as a worsening of symptoms following even minor physical or mental exertion. Our study aimed to evaluate transcriptomic changes in ME/CFS female patients undergoing an exercise challenge intended to precipitate PEM. Our time points (baseline before exercise challenge, the point of maximal exertion, and after an exercise challenge) allowed for the exploration of the transcriptomic response to exercise and recovery in female patients with ME/CFS, as compared to healthy controls (HCs). Under maximal exertion, ME/CFS patients did not show significant changes in gene expression, while HCs demonstrated altered functional gene networks related to signaling and integral functions of their immune cells. During the recovery period (commonly during onset of PEM), female ME/CFS patients showed dysregulated immune signaling pathways and dysfunctional cellular responses to stress. The unique functional pathways identified provide a foundation for future research efforts into the disease, as well as for potential targeted treatment options

Stress-Induced Transcriptomic Changes in Females with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Reveal Disrupted Immune Signatures

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, complex multi-organ illness characterized by unexplained debilitating fatigue and post-exertional malaise (PEM), which is defined as a worsening of symptoms following even minor physical or mental exertion. Our study aimed to evaluate transcriptomic changes in ME/CFS female patients undergoing an exercise challenge intended to precipitate PEM. Our time points (baseline before exercise challenge, the point of maximal exertion, and after an exercise challenge) allowed for the exploration of the transcriptomic response to exercise and recovery in female patients with ME/CFS, as compared to healthy controls (HCs). Under maximal exertion, ME/CFS patients did not show significant changes in gene expression, while HCs demonstrated altered functional gene networks related to signaling and integral functions of their immune cells. During the recovery period (commonly during onset of PEM), female ME/CFS patients showed dysregulated immune signaling pathways and dysfunctional cellular responses to stress. The unique functional pathways identified provide a foundation for future research efforts into the disease, as well as for potential targeted treatment options

Sex-Dependent Transcriptional Changes in Response to Stress in Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Pilot Project

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multi-symptom illness characterized by debilitating fatigue and post-exertional malaise (PEM). Numerous studies have reported sex differences at the epidemiological, cellular, and molecular levels between male and female ME/CFS patients. To gain further insight into these sex-dependent changes, we evaluated differential gene expression by RNA-sequencing (RNA-Seq) in 33 ME/CFS patients (20 female, 13 male) and 34 matched healthy controls (20 female and 14 male) before, during, and after an exercise challenge intended to provoke PEM. Our findings revealed that pathways related to immune-cell signaling (including IL-12) and natural killer cell cytotoxicity were activated as a result of exertion in the male ME/CFS cohort, while female ME/CFS patients did not show significant enough changes in gene expression to meet the criteria for the differential expression. Functional analysis during recovery from an exercise challenge showed that male ME/CFS patients had distinct changes in the regulation of specific cytokine signals (including IL-1β). Meanwhile, female ME/CFS patients had significant alterations in gene networks related to cell stress, response to herpes viruses, and NF-κβ signaling. The functional pathways and differentially expressed genes highlighted in this pilot project provide insight into the sex-specific pathophysiology of ME/CFS

Beta-amylase gene variability in introgressive wheat lines: [preprint]

Variability of the beta-amylase gene in bread wheat, artificial amphidiploids, and derived introgression wheat lines was analyzed. Variation in homeologous beta-amylase sequences caused by the presence of MITE (Miniature Inverted-Repeat Transposable Element) and its footprint has been identified in bread wheat. The previously unknown location of MITE in Triticum urartu and T. aestivum L. beta-amylase gene has been found. These species have a MITE sequence in the third intron of beta-amylase, as opposed to Aegilops comosa and a number of other Triticeae species, which have it in the fourth intron. These two MITEs from Ae. comosa and T. aestivum were shown to have low identity scores. Miosa, an artificial amphidiploid, which has the M genome from Ae. comosa was shown to lose the MITE sequences. This loss might be caused by genomic shock due to allopolyploidization