Increased deformations are dispensable for cell mechanoresponse in engineered bone analogs mimicking aging bone marrow.

Aged individuals and astronauts experience bone loss despite rigorous physical activity. Bone mechanoresponse is in-part regulated by mesenchymal stem cells (MSCs) that respond to mechanical stimuli. Direct delivery of low intensity vibration (LIV) recovers MSC proliferation in senescence and simulated microgravity models, indicating that age-related reductions in mechanical signal delivery within bone marrow may contribute to declining bone mechanoresponse. To answer this question, we developed a 3D bone marrow analog that controls trabecular geometry, marrow mechanics and external stimuli. Validated finite element (FE) models were developed to quantify strain environment within hydrogels during LIV. Bone marrow analogs with gyroid-based trabeculae of bone volume fractions (BV/TV) corresponding to adult (25%) and aged (13%) mice were printed using polylactic acid (PLA). MSCs encapsulated in migration-permissive hydrogels within printed trabeculae showed robust cell populations on both PLA surface and hydrogel within a week. Following 14 days of LIV treatment (1g, 100 Hz, 1 hour/day), type-I collagen and F-actin were quantified for the cells in the hydrogel fraction. While LIV increased all measured outcomes, FE models predicted higher von Mises strains for the 13% BV/TV groups (0.2%) when compared to the 25% BV/TV group (0.1%). Despite increased strains, collagen-I and F-actin measures remained lower in the 13% BV/TV groups when compared to 25% BV/TV counterparts, indicating that cell response to LIV does not depend on hydrogel strains and that bone volume fraction (i.e. available bone surface) directly affects cell behavior in the hydrogel phase independent of the external stimuli. Overall, bone marrow analogs offer a robust and repeatable platform to study bone mechanobiology.

microRNA-124 directly suppresses Nodal and Notch to regulate mesodermal development.

MicroRNAs regulate gene expression post-transcriptionally by destabilizing and/or inhibiting translation of target mRNAs in animal cells. MicroRNA-124 (miR-124) has been examined mostly in the context of neurogenesis. This study discovers a novel role of miR-124 in regulating mesodermal cell differentiation in the sea urchin embryo. The expression of miR-124 is first detectable at 12hours post fertilization at the early blastula stage, during endomesodermal specification. Mesodermally-derived immune cells come from the same progenitor cells that give rise to blastocoelar cells (BCs) and pigment cells (PCs) that must make a binary fate decision. We determined that miR-124 directly represses Nodal and Notch to regulate BC and PC differentiation. miR-124 inhibition does not impact the dorsal-ventral axis formation, but result in a significant increase in number of cells expressing BC-specific transcription factors (TFs) and a concurrent reduction of differentiated PCs. In general, removing miR-124's suppression of Nodal phenocopies miR124 inhibition. Interestingly, removing miR-124's suppression of Notch leads to an increased number of both BCs and PCs, with a subset of hybrid cells that express both BC- and PC-specific TFs in the larvae. Removal of miR-124's suppression of Notch not only affects differentiation of both BCs and PCs, but also induces cell proliferation of these cells during the first wave of Notch signaling. This study demonstrates that post-transcriptional regulation by miR-124 impacts differentiation of BCs and PCs by regulating the Nodal and Notch signaling pathways.

Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

RNA localization to the mitotic spindle is essential for early development and is regulated by kinesin-1 and dynein.

Mitosis is a fundamental and highly regulated process that acts to faithfully segregate chromosomes into two identical daughter cells. Localization of gene transcripts involved in mitosis to the mitotic spindle might be an evolutionarily conserved mechanism to ensure that mitosis occurs in a timely manner. We identified many RNA transcripts that encode proteins involved in mitosis localized at the mitotic spindles in dividing sea urchin embryos and mammalian cells. Disruption of microtubule polymerization, kinesin-1 or dynein results in lack of spindle localization of these transcripts in the sea urchin embryo. Furthermore, results indicate that the cytoplasmic polyadenylation element (CPE) within the 3'UTR of the Aurora B transcript, a recognition sequence for CPEB, is essential for RNA localization to the mitotic spindle in the sea urchin embryo. Blocking this sequence results in arrested development during early cleavage stages, suggesting that RNA localization to the mitotic spindle might be a regulatory mechanism of cell division that is important for early development.

An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity.

Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multilineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/- HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

microRNA-124 regulates Notch and NeuroD1 to mediate transition states of neuronal development.

MicroRNAs regulate gene expression by destabilizing target mRNA and/or inhibiting translation in animal cells. The ability to mechanistically dissect miR-124's function during specification, differentiation, and maturation of neurons during development within a single system has not been accomplished. Using the sea urchin embryo, we take advantage of the manipulability of the embryo and its well-documented gene regulatory networks (GRNs). We incorporated NeuroD1 as part of the sea urchin neuronal GRN and determined that miR-124 inhibition resulted in aberrant gut contractions, swimming velocity, and neuronal development. Inhibition of miR-124 resulted in an increased number of cells expressing transcription factors (TFs) associated with progenitor neurons and a concurrent decrease of mature and functional neurons. Results revealed that in the early blastula/gastrula stages, miR-124 regulates undefined factors during neuronal specification and differentiation. In the late gastrula/larval stages, miR-124 regulates Notch and NeuroD1 during the transition between neuronal differentiation and maturation. Overall, we have improved the neuronal GRN and identified miR-124 to play a prolific role in regulating various transitions of neuronal development.

NeuroD1 localizes to the presumptive ganglia and gut of the sea urchin larvae.

NeuroD is a transcription factor (TF) that plays a dual role in vertebrate neurogenesis and glucose homeostasis in the pancreas. We identified a NeuroD antibody developed against human that cross-reacts with the sea urchin NeuroD1. NeuroD1 protein localizes to the presumptive ganglia and neurofilament structures in the ciliary band of the sea urchin larvae. In addition, we also observed NeuroD1 in the perinuclear region in the sea urchin gut which is analogous to the mammalian pancreas. These results suggest that NeuroD1 may play an evolutionarily conserved role in the invertebrate sea urchin.

Robust automatic hexahedral cartilage meshing framework enables population-based computational studies of the knee.

Osteoarthritis of the knee is increasingly prevalent as our population ages, representing an increasing financial burden, and severely impacting quality of life. The invasiveness of in vivo procedures and the high cost of cadaveric studies has left computational tools uniquely suited to study knee biomechanics. Developments in deep learning have great potential for efficiently generating large-scale datasets to enable researchers to perform population-sized investigations, but the time and effort associated with producing robust hexahedral meshes has been a limiting factor in expanding finite element studies to encompass a population. Here we developed a fully automated pipeline capable of taking magnetic resonance knee images and producing a working finite element simulation. We trained an encoder-decoder convolutional neural network to perform semantic image segmentation on the Imorphics dataset provided through the Osteoarthritis Initiative. The Imorphics dataset contained 176 image sequences with varying levels of cartilage degradation. Starting from an open-source swept-extrusion meshing algorithm, we further developed this algorithm until it could produce high quality meshes for every sequence and we applied a template-mapping procedure to automatically place soft-tissue attachment points. The meshing algorithm produced simulation-ready meshes for all 176 sequences, regardless of the use of provided (manually reconstructed) or predicted (automatically generated) segmentation labels. The average time to mesh all bones and cartilage tissues was less than 2 min per knee on an AMD Ryzen 5600X processor, using a parallel pool of three workers for bone meshing, followed by a pool of four workers meshing the four cartilage tissues. Of the 176 sequences with provided segmentation labels, 86% of the resulting meshes completed a simulated flexion-extension activity. We used a reserved testing dataset of 28 sequences unseen during network training to produce simulations derived from predicted labels. We compared tibiofemoral contact mechanics between manual and automated reconstructions for the 24 pairs of successful finite element simulations from this set, resulting in mean root-mean-squared differences under 20% of their respective min-max norms. In combination with further advancements in deep learning, this framework represents a feasible pipeline to produce population sized finite element studies of the natural knee from subject-specific models.

Limitations of mouse models for sickle cell disease conferred by their human globin transgene configurations.

We characterized the human ß-like globin transgenes in two mouse models of sickle cell disease (SCD) and tested a genome-editing strategy to induce red blood cell fetal hemoglobin (HbF; α2γ2). Berkeley SCD mice contain four to 22 randomly arranged, fragmented copies of three human transgenes (HBA1, HBG2-HBG1-HBD-HBBS and a mini-locus control region) integrated into a single site of mouse chromosome 1. Cas9 disruption of the BCL11A repressor binding motif in the γ-globin gene (HBG1 and HBG2; HBG) promoters of Berkeley mouse hematopoietic stem cells (HSCs) caused extensive death from multiple double-strand DNA breaks. Long-range sequencing of Townes SCD mice verified that the endogenous Hbb genes were replaced by single-copy segments of human HBG1 and HBBS including proximal but not some distal gene-regulatory elements. Townes mouse HSCs were viable after Cas9 disruption of the HBG1 BCL11A binding motif but failed to induce HbF to therapeutic levels, contrasting with human HSCs. Our findings provide practical information on the genomic structures of two common mouse SCD models, illustrate their limitations for analyzing therapies to induce HbF and confirm the importance of distal DNA elements in human globin regulation. This article has an associated First Person interview with the first author of the paper.

Persistent Parental RNAi in the Beetle Tribolium castaneum Involves Maternal Transmission of Long Double-Stranded RNA.

Parental RNA interference (pRNAi) is a powerful and widely used method for gene-specific knockdown. Yet in insects its efficacy varies between species, and how the systemic response is transmitted from mother to offspring remains elusive. Using the beetle Tribolium castaneum, an RT-qPCR strategy to distinguish the presence of double-stranded RNA (dsRNA) from endogenous mRNA is reported. It is found that injected dsRNA is directly transmitted into the egg and persists throughout embryogenesis. Despite this depletion of dsRNA from the mother, it is shown that strong pRNAi can persist for months before waning at strain-specific rates. In seeking the receptor proteins for cellular uptake of long dsRNA into the egg, a phylogenomics profiling approach of candidate proteins is also presented. A visualization strategy based on taxonomically hierarchical assessment of orthology clustering data to rapidly assess gene age and copy number changes, refined by sequence-based evidence, is demonstrated. Repeated losses of SID-1-like channel proteins in the arthropods, including wholesale loss in the Heteroptera (true bugs), which are nonetheless highly sensitive to pRNAi, are thereby documented. Overall, practical considerations for insect pRNAi against a backdrop of outstanding questions on the molecular mechanism of dsRNA transmission for long-term, systemic knockdown are elucidated.

Persistent Parental RNAi in the Beetle Tribolium castaneum Involves Maternal Transmission of Long Double-Stranded RNA (Advanced Genetics 3/03).

dsRNA Uptake In article 2100064 by Kristen A. Panfilio and co-workers, the cuticle exoskeleton of flour beetle larvae reveals normal anatomy (above: head-to-tail in blue-to-red) and long-term parental RNAi knockdown (below), here showing a mirror-image duplication of the abdomen (red termini to yellow center). Strong knockdown can persist for months despite transmission of full-length double-stranded RNA (dsRNA) from the mother into the egg, depleting maternal dsRNA levels.

Emerging Gene-Editing Modalities for Osteoarthritis.

Osteoarthritis (OA) is a pathological degenerative condition of the joints that is widely prevalent worldwide, resulting in significant pain, disability, and impaired quality of life. The diverse etiology and pathogenesis of OA can explain the paucity of viable preventive and disease-modifying strategies to counter it. Advances in genome-editing techniques may improve disease-modifying solutions by addressing inherited predisposing risk factors and the activity of inflammatory modulators. Recent progress on technologies such as CRISPR/Cas9 and cell-based genome-editing therapies targeting the genetic and epigenetic alternations in OA offer promising avenues for early diagnosis and the development of personalized therapies. The purpose of this literature review was to concisely summarize the genome-editing options against chronic degenerative joint conditions such as OA with a focus on the more recently emerging modalities, especially CRISPR/Cas9. Future advancements in novel genome-editing therapies may improve the efficacy of such targeted treatments.

Development of a statistical shape-function model of the implanted knee for real-time prediction of joint mechanics.

Outcomes of total knee arthroplasty (TKA) are dependent on surgical technique, patient variability, and implant design. Non-optimal design or alignment choices may result in undesirable contact mechanics and joint kinematics, including poor joint alignment, instability, and reduced range of motion. Implant design and surgical alignment are modifiable factors with potential to improve patient outcomes, and there is a need for robust implant designs that can accommodate patient variability. Our objective was to develop a statistical shape-function model (SFM) of a posterior stabilized implanted knee to instantaneously predict joint mechanics in an efficient manner. Finite element methods were combined with Latin hypercube sampling and regression analyses to produce modeling equations relating nine implant design and six surgical alignment parameters to tibiofemoral (TF) joint mechanics outcomes during a deep knee bend. A SFM was developed and TF contact mechanics, kinematics, and soft tissue loads were instantaneously predicted from the model. Average normalized root-mean-square error predictions were between 2.79% and 9.42%, depending on the number of parameters included in the model. The statistical shape-function model generated instantaneous joint mechanics predictions using a maximum of 130 training simulations, making it ideally suited for integration into a patient-specific design and alignment optimization pipeline. Such a tool may be used to optimize kinematic function to achieve more natural motion or minimize implant wear, and may aid the engineering and clinical communities in improving patient satisfaction and surgical outcomes.

The folic acid metabolism gene mel-32/Shmt is required for normal cell cycle lengths in Caenorhabditis elegans.

Neural tube defects are common and serious birth defects in which the brain and/or spinal cord are exposed outside the body. Supplementation of foods with folic acid, an essential vitamin, is linked to a lower risk of neural tube defects; however, the mechanisms by which folic acid influence neural tube defect risk are unclear. Our research seeks to identify the basic cellular roles of known folic acid metabolism genes during morphogenesis using the roundworm Caenorhabditis elegans (C. elegans) as a simple model system. Here, we used live imaging to characterize defects in embryonic development when mel-32 is depleted. mel-32 is an essential folic acid metabolism gene in C. elegans and a homolog to the mammalian enzyme serine hydroxymethyltransferase (Shmt). Disruption of mel-32 resulted in a doubling or tripling of cell cycle lengths and a lack of directed cell movement during embryogenesis. However, the order of cell divisions, as determined by lineage analysis, is unchanged compared to wild type embryos. These results suggest that mel-32/Shmt is required for normal cell cycle lengths in C. elegans.

Assortative mating for major affective disorder.

The families of 26 married bipolar manic depressives were examined in detail. The rate of affective disorder in their spouses, and the parents and siblings of their spouses, was ascertained. The prevalence of affective disorder among the parents and siblings and of the spouses of age- and social class-matched schizophrenics and well controls was also ascertained. Finally, the prevalence of affective and other psychiatric disorders in the adult offspring of the bipolar probands was ascertained and related to the presence or absence of affective disorder in the spouse. We did not find clear evidence of assortative mating for major affective disorder, although dual mating for affective disorder had the expected result of increasing the risk for affective disorder in the adult offspring.