Single-cell transcriptomic analysis identifies an immune-prone population in erythroid precursors during human ontogenesis.

Nonimmune cells can have immunomodulatory roles that contribute to healthy development. However, the molecular and cellular mechanisms underlying the immunomodulatory functions of erythroid cells during human ontogenesis remain elusive. Here, integrated, single-cell transcriptomic studies of erythroid cells from the human yolk sac, fetal liver, preterm umbilical cord blood (UCB), term UCB and adult bone marrow (BM) identified classical and immune subsets of erythroid precursors with divergent differentiation trajectories. Immune-erythroid cells were present from the yolk sac to the adult BM throughout human ontogenesis but failed to be generated in vitro from human embryonic stem cells. Compared with classical-erythroid precursors, these immune-erythroid cells possessed dual erythroid and immune regulatory networks, showed immunomodulatory functions and interacted more frequently with various innate and adaptive immune cells. Our findings provide important insights into the nature of immune-erythroid cells and their roles during development and diseases.

A direct fiber approach to model sclera collagen architecture and biomechanics.

Sclera collagen fiber microstructure and mechanical behavior are central to eye physiology and pathology. They are also complex, and are therefore often studied using modeling. Most models of sclera, however, have been built within a conventional continuum framework. In this framework, collagen fibers are incorporated as statistical distributions of fiber characteristics such as the orientation of a family of fibers. The conventional continuum approach, while proven successful for describing the macroscale behavior of the sclera, does not account for the sclera fibers are long, interwoven and interact with one another. Hence, by not considering these potentially crucial characteristics, the conventional approach has only a limited ability to capture and describe sclera structure and mechanics at smaller, fiber-level, scales. Recent advances in the tools for characterizing sclera microarchitecture and mechanics bring to the forefront the need to develop more advanced modeling techniques that can incorporate and take advantage of the newly available highly detailed information. Our goal was to create a new computational modeling approach that can represent the sclera fibrous microstructure more accurately than with the conventional continuum approach, while still capturing its macroscale behavior. In this manuscript we introduce the new modeling approach, that we call direct fiber modeling, in which the collagen architecture is built explicitly by long, continuous, interwoven fibers. The fibers are embedded in a continuum matrix representing the non-fibrous tissue components. We demonstrate the approach by doing direct fiber modeling of a rectangular patch of posterior sclera. The model integrated fiber orientations obtained by polarized light microscopy from coronal and sagittal cryosections of pig and sheep. The fibers were modeled using a Mooney-Rivlin model, and the matrix using a Neo-Hookean model. The fiber parameters were determined by inversely matching experimental equi-biaxial tensile data from the literature. After reconstruction, the direct fiber model orientations agreed well with the microscopy data both in the coronal plane (adjusted R2 = 0.8234) and in the sagittal plane (adjusted R2 = 0.8495) of the sclera. With the estimated fiber properties (C10 = 5746.9 MPa; C01 = -5002.6 MPa, matrix shear modulus 200 kPa), the model's stress-strain curves simultaneously fit the experimental data in radial and circumferential directions (adjusted R2's 0.9971 and 0.9508, respectively). The estimated fiber elastic modulus at 2.16% strain was 5.45 GPa, in reasonable agreement with the literature. During stretch, the model exhibited stresses and strains at sub-fiber level, with interactions among individual fibers which are not accounted for by the conventional continuum methods. Our results demonstrate that direct fiber models can simultaneously describe the macroscale mechanics and microarchitecture of the sclera, and therefore that the approach can provide unique insight into tissue behavior questions inaccessible with continuum approaches.

A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells.

Alternative splicing (AS) leads to transcriptome diversity in eukaryotic cells and is one of the key regulators driving cellular differentiation. Although AS is of crucial importance for normal hematopoiesis and hematopoietic malignancies, its role in early hematopoietic development is still largely unknown. Here, by using high-throughput transcriptomic analyses, we show that pervasive and dynamic AS takes place during hematopoietic development of human pluripotent stem cells (hPSCs). We identify a splicing factor switch that occurs during the differentiation of mesodermal cells to endothelial progenitor cells (EPCs). Perturbation of this switch selectively impairs the emergence of EPCs and hemogenic endothelial progenitor cells (HEPs). Mechanistically, an EPC-induced alternative spliced isoform of NUMB dictates EPC specification by controlling NOTCH signaling. Furthermore, we demonstrate that the splicing factor SRSF2 regulates splicing of the EPC-induced NUMB isoform, and the SRSF2-NUMB-NOTCH splicing axis regulates EPC generation. The identification of this splicing factor switch provides a new molecular mechanism to control cell fate and lineage specification.

Stretch-Induced Uncrimping of Equatorial Sclera Collagen Bundles.

Stretch-induced collagen uncrimping underlies the nonlinear mechanical behavior of the sclera according to what is often called the process of recruitment. We recently reported experimental measurements of sclera collagen crimp and pressure-induced uncrimping. Our studies, however, were cross-sectional, providing statistical descriptions of crimp with no information on the effects of stretch on specific collagen bundles. Data on bundle-specific uncrimping is necessary to better understand the effects of macroscale input on the collagen microscale and tissue failure. Our goal in this project was to measure bundle-specific stretch-induced collagen uncrimping of sclera. Three goat eyes were cryosectioned sagittally (30 µm). Samples of equatorial sclera were isolated, mounted to a custom uni-axial stretcher and imaged with polarized light microscopy at various levels of clamp-to-clamp stretch until failure. At each stretch level, local strain was measured using image tracking techniques. The level of collagen crimping was determined from the bundle waviness, defined as the circular standard deviation of fiber orientation along a bundle. Eye-specific recruitment curves were then computed using eye-specific waviness at maximum stretch before sample failure to define fibers as recruited. Nonlinear mixed effect models were used to determine the associations of waviness to local strain and recruitment to clamp-to-clamp stretch. Waviness decreased exponentially with local strain (p < 0.001), whereas bundle recruitment followed a sigmoidal curve with clamp-to-clamp stretch (p < 0.001). Individual bundle responses to stretch varied substantially, but recruitment curves were similar across sections and eyes. In conclusion, uni-axial stretch caused measurable bundle-specific uncrimping, with the sigmoidal recruitment pattern characteristic of fiber-reinforced soft tissues.

R gene triplication confers European fodder turnip with improved clubroot resistance.

Clubroot is one of the most important diseases for many important cruciferous vegetables and oilseed crops worldwide. Different clubroot resistance (CR) loci have been identified from only limited species in Brassica, making it difficult to compare and utilize these loci. European fodder turnip ECD04 is considered one of the most valuable resources for CR breeding. To explore the genetic and evolutionary basis of CR in ECD04, we sequenced the genome of ECD04 using de novo assembly and identified 978 candidate R genes. Subsequently, the 28 published CR loci were physically mapped to 15 loci in the ECD04 genome, including 62 candidate CR genes. Among them, two CR genes, CRA3.7.1 and CRA8.2.4, were functionally validated. Phylogenetic analysis revealed that CRA3.7.1 and CRA8.2.4 originated from a common ancestor before the whole-genome triplication (WGT) event. In clubroot susceptible Brassica species, CR-gene homologues were affected by transposable element (TE) insertion, resulting in the loss of CR function. It can be concluded that the current functional CR genes in Brassica rapa and non-functional CR genes in other Brassica species were derived from a common ancestral gene before WGT. Finally, a hypothesis for CR gene evolution is proposed for further discussion.

[Biosynthetic pathways of triterpenes in medicinal plants and key enzyme genes: a review].

Triterpenes, with high diversity and a wide range of sources, can be found in many medicinal plants. They have been found free or as glycosides/esters by combining with sugars. Most of them act as signaling molecules and function in stress response. They are also the material basis for the therapeutic effect of various medicinal plants. Modern pharmacological research has shown that they have the anti-inflammatory, antibacterial, antiviral, anti-tumor, fertility-regulating, and immunomodulatory effects. They top plant natural products in both quantity and diversity, and among them, tetrachyclic triterpenes and pentachyclic triterpenes are most abundant. The first step of the structural diversification is the cyclization 2,3-oxidosqualene, which is catalyzed by oxidosqualene cyclases(OSCs). Numerous OSCs exist, each with a specific cyclization mechanism, and thus over 100 different cyclic triterpene skeletons have been found in nature. This study reviewed the research on the biosynthetic pathways of triterpenes in medicinal plants, regulatory mechanisms of the pathways, and the key enzymes, and analyzed the expression regulation and structural characteristics of key enzyme genes involved in the synthetic pathways. This study is expected to serve as a reference for further research on triterpenes, such as the directional regulation of metabolic flow and heterologous biosynthesis and lay a basis for the regulation of triterpene synthesis and the selection of high-quality germplasm. This study also provides basic materials for further research and development of triterpenes from medicinal plants.

Fine mapping and candidate gene analysis of a major locus controlling ovule abortion and seed number per silique in Brassica napus L.

KEY MESSAGE: A major QTL controlling ovule abortion and SN was fine-mapped to a 80.1-kb region on A8 in rapeseed, and BnaA08g07940D and BnaA08g07950D are the most likely candidate genes. The seed number per silique (SN), an important yield determining trait of rapeseed, is the final consequence of a complex developmental process including ovule initiation and the subsequent ovule/seed development. To explore the genetic mechanism regulating the natural variation of SN and its related components, quantitative trait locus (QTL) mapping was conducted using a doubled haploid (DH) population derived from the cross between C4-146 and C4-58B, which showed significant differences in SN and aborted ovule number (AON), but no obvious differences in ovule number (ON). QTL analysis identified 19 consensus QTLs for six SN-related traits across three environments. A novel QTL on chromosome A8, un.A8, which associates with multiple traits, except for ON, was stably detected across the three environments. This QTL explained more than 50% of the SN, AON and percentage of aborted ovules (PAO) variations as well as a moderate contribution on silique length (SL) and thousand seed weight (TSW). The C4-146 allele at the locus increases SN and SL but decreases AON, PAO and TSW. Further fine mapping narrowed down this locus into an 80.1-kb interval flanked by markers BM1668 and BM1672, and six predicted genes were annotated in the delimited region. Expression analyses and DNA sequencing showed that two homologs of Arabidopsis photosystem I subunit F (BnaA08g07940D) and zinc transporter 10 precursor (BnaA08g07950D) were the most promising candidate genes underlying this locus. These results provide a solid basis for cloning un.A8 to reduce the ovule abortion and increase SN in the yield improvement of rapeseed.

Systematic elucidation of the mechanism of Jingyin granule in the treatment of Novel Coronavirus (COVID-19) Pneumonia via Network Pharmacology.

Background: Jingyin granule is one of the widely used traditional Chinese medicine mixture composed of multiple herbs in the treatment of respiratory system diseases. The mechanism of its therapeutic effects has still been obscure. The aim of this study is to use the network pharmacology approach for identification of the main active ingredients of Jingyin granule against COVID-19 targets and to explore their therapeutic mechanism. Material and Method: In this study, the ingredients of Jingyin granule were evaluated by the usage of Traditional Chinese Medicine Systems Pharmacology Database and Traditional Chinese Medicine Integrated Database, and the interactions between potential gene targets and ingredients were identified using the SwissTargetPrediction database. Meanwhile the possible efficient targets COVID-19 acts on were identified via Online Mendelian Inheritance in Man database, DisGeNET database and GeneCards database. In addition, functions, components and pathways were identified by Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Protein interaction, ingredients-targets network was established. Results: Our findings showed that numerous ingredients of Jingyin granule could act on COVID-19 with 88 target genes. GO enrichment analysis, KEGG pathway analysis, and protein-protein interaction network revealed that these targets were interrelated with regulation of immune function, directly targeting disease genes. Conclusions: Jingyin granule could be utilized to exert systematic pharmacological effects. Jingyin granule could directly target the major genes, and also regulate the immune system, acting as oblique disease treatment.

Role of radially aligned scleral collagen fibers in optic nerve head biomechanics.

Collagen fibers organized circumferentially around the canal in the peripapillary sclera are thought to provide biomechanical support to the sensitive tissues within the optic nerve head (ONH). Recent studies have demonstrated the existence of a family of fibers in the innermost sclera organized radially from the scleral canal. Our goal was to determine the role of these radial fibers in the sensitivity of scleral canal biomechanics to acute increases in intraocular pressure (IOP). Following the same general approach of previous parametric sensitivity studies, we created nonlinear generic finite element models of a posterior pole with various combinations of radial and circumferential fibers at an IOP of 0 mmHg. We then simulated the effects of normal and elevated IOP levels (15 and 30 mmHg). We monitored four IOP-induced geometric changes: peripapillary sclera stretch, scleral canal displacement, lamina cribrosa displacement, and scleral canal expansion. In addition, we examined the radial (maximum tension) and through-thickness (maximum compression) strains within the ONH tissues. Our models predicted that: 1) radial fibers reduced the posterior displacement of the lamina, especially at elevated IOP; 2) radial fibers reduced IOP-induced radial strain within the peripapillary sclera and retinal tissue; and 3) a combination of radial and circumferential fibers maintained strains within the ONH at a level similar to those conferred by circumferential fibers alone. In conclusion, radial fibers provide support for the posterior globe, additional to that provided by circumferential fibers. Most importantly, a combination of both fiber families can better protect ONH tissues from excessive IOP-induced deformation than either alone.

The bZIP Transcription Factor GmbZIP15 Negatively Regulates Salt- and Drought-Stress Responses in Soybean.

Soybean (Glycine max), as an important oilseed crop, is constantly threatened by abiotic stress, including that caused by salinity and drought. bZIP transcription factors (TFs) are one of the largest TF families and have been shown to be associated with various environmental-stress tolerances among species; however, their function in abiotic-stress response in soybean remains poorly understood. Here, we characterized the roles of soybean transcription factor GmbZIP15 in response to abiotic stresses. The transcript level of GmbZIP15 was suppressed under salt- and drought-stress conditions. Overexpression of GmbZIP15 in soybean resulted in hypersensitivity to abiotic stress compared with wild-type (WT) plants, which was associated with lower transcript levels of stress-responsive genes involved in both abscisic acid (ABA)-dependent and ABA-independent pathways, defective stomatal aperture regulation, and reduced antioxidant enzyme activities. Furthermore, plants expressing a functional repressor form of GmbZIP15 exhibited drought-stress resistance similar to WT. RNA-seq and qRT-PCR analyses revealed that GmbZIP15 positively regulates GmSAHH1 expression and negatively regulates GmWRKY12 and GmABF1 expression in response to abiotic stress. Overall, these data indicate that GmbZIP15 functions as a negative regulator in response to salt and drought stresses.

Enhanced architecture and implementation of spectrum shaping codes.

Spectral shaping codes are modulation codes widely used in communication and data storage systems. This research enhances the algorithms employed in constructing spectral shaping codes for hardware implementation. We present a parallel scrambling calculation with a time complexity of O(1). Second, in the minimum accumulated signal power (MASP) module, the sine-cosine accumulation needs to be determined by remainder with time complexity O(n2). We offer reduced MASP computations for short bit-width data, ROM storage, and addition pipelines. It can remove the remainder operation, reducing accumulated complexity to O(1). In addition, we present a search algorithm to generate segmented lines to replace the square operations in the MASP module. By employing the search algorithm and shift operations, we can reduce the complexity of the square from O(n2) to O(1). The implementation results reveal that the original and proposed MASPs yield nearly identical spectrum nulls. The encoder-decoder of the spectral shaping codes with proposed approaches consumes just 6% of the hardware resources when carried out with a Spartan6 XC6SLX25.

Comparing continuum and direct fiber models of soft tissues. An ocular biomechanics example reveals that continuum models may artificially disrupt the strains at both the tissue and fiber levels.

Collagen fibers are the main load-bearing component of soft tissues but difficult to incorporate into models. Whilst simplified homogenization models suffice for some applications, a thorough mechanistic understanding requires accurate prediction of fiber behavior, including both detailed fiber-level strains and long-distance transmission. Our goal was to compare the performance of a continuum model of the optic nerve head (ONH) built using conventional techniques with a fiber model we recently introduced which explicitly incorporates the complex 3D organization and interaction of collagen fiber bundles [1]. To ensure a fair comparison, we constructed the continuum model with identical geometrical, structural, and boundary specifications as for the fiber model. We found that: 1) although both models accurately matched the intraocular pressure (IOP)-induced globally averaged displacement responses observed in experiments, they diverged significantly in their ability to replicate specific 3D tissue-level strain patterns. Notably, the fiber model faithfully replicated the experimentally observed depth-dependent variability of radial strain, the ring-like pattern of meridional strain, and the radial pattern of circumferential strain, whereas the continuum model failed to do so; 2) the continuum model disrupted the strain transmission along each fiber, a feature captured well by the fiber model. These results demonstrate limitations of the conventional continuum models that rely on homogenization and affine deformation assumptions, which render them incapable of capturing some complex tissue-level and fiber-level deformations. Our results show that the strengths of explicit fiber modeling help capture intricate ONH biomechanics. They potentially also help modeling other fibrous tissues.

Comparing continuum and direct fiber models of soft tissues: An ocular biomechanics example reveals that continuum models may artificially disrupt the strains at both the tissue and fiber levels.

Collagen fibers are the main load-bearing component of soft tissues but difficult to incorporate into models. Whilst simplified homogenization models suffice for some applications, a thorough mechanistic understanding requires accurate prediction of fiber behavior, including both detailed fiber-level strains and long-distance transmission. Our goal was to compare the performance of a continuum model of the optic nerve head (ONH) built using conventional techniques with a fiber model we recently introduced which explicitly incorporates the complex 3D organization and interaction of collagen fiber bundles [1]. To ensure a fair comparison, we constructed the continuum model with identical geometrical, structural, and boundary specifications as for the fiber model. We found that: 1) although both models accurately matched the intraocular pressure (IOP)-induced globally averaged displacement responses observed in experiments, they diverged significantly in their ability to replicate specific 3D tissue-level strain patterns. Notably, the fiber model faithfully replicated the experimentally observed depth-dependent variability of radial strain, the ring-like pattern of meridional strain, and the radial pattern of circumferential strain, whereas the continuum model failed to do so; 2) the continuum model disrupted the strain transmission along each fiber, a feature captured well by the fiber model. These results demonstrate limitations of the conventional continuum models that rely on homogenization and affine deformation assumptions, which render them incapable of capturing some complex tissue-level and fiber-level deformations. Our results show that the strengths of explicit fiber modeling help capture intricate ONH biomechanics. They potentially also help modeling other fibrous tissues. STATEMENT OF SIGNIFICANCE: Understanding the mechanics of fibrous tissues is crucial for advancing knowledge of various diseases. This study uses the ONH as a test case to compare conventional continuum models with fiber models that explicitly account for the complex fiber structure. We found that the fiber model captured better the biomechanical behaviors at both the tissue level and the fiber level. The insights gained from this study demonstrate the significant potential of fiber models to advance our understanding of not only glaucoma pathophysiology but also other conditions involving fibrous soft tissues. This can contribute to the development of therapeutic strategies across a wide range of applications.

Lamina Cribrosa Insertions Into the Sclera Are Sparser, Narrower, and More Slanted in the Anterior Lamina.

Purpose: The lamina cribrosa (LC) depends on the sclera for support. The support must be provided through the LC insertions. Although a continuous insertion over the whole LC periphery is often assumed, LC insertions are actually discrete locations where LC collagenous beams meet the sclera. We hypothesized that LC insertions vary in number, size, and shape by quadrant and depth. Methods: Coronal cryosections through the full LCs from six healthy monkey eyes were imaged using instant polarized light microscopy. The images were registered into a stack, on which we manually marked LC insertion outlines, nothing their position in-depth and quadrant (inferior, superior, nasal, or temporal). From the marks, we determined the insertion number, width, angle to the canal wall (90 degrees = perpendicular), and insertion ratio (fraction of LC periphery represented by insertions). Using linear mixed effect models, we determined if the insertion characteristics were associated with depth or quadrant. Results: Insertions in the anterior LC were sparser, narrower, and more slanted than those in deeper LC (P values < 0.001). There were more insertions spanning a larger ratio of the canal wall in the middle LC than in the anterior and posterior (P values < 0.001). In the nasal quadrant, the insertion angles were significantly smaller (P < 0.001). Conclusions: LC insertions vary substantially and significantly over the canal. The sparser, narrower, and more slanted insertions of the anterior-most LC may not provide the robust support afforded by insertions of the middle and posterior LC. These variations may contribute to the progressive deepening of the LC and regional susceptibility to glaucoma.

Direct measurements of collagen fiber recruitment in the posterior pole of the eye.

Collagen is the main load-bearing component of the peripapillary sclera (PPS) and lamina cribrosa (LC) in the eye. Whilst it has been shown that uncrimping and recruitment of the PPS and LC collagen fibers underlies the macro-scale nonlinear stiffening of both tissues with increased intraocular pressure (IOP), the uncrimping and recruitment as a function of local stretch have not been directly measured. This knowledge is crucial to understanding their functions in bearing loads and maintaining tissue integrity. In this project we measured local stretch-induced collagen fiber bundle uncrimping and recruitment curves of the PPS and LC. Thin coronal samples of PPS and LC of sheep eyes were mounted and stretched biaxially quasi-statically using a custom system. At each step, we imaged the PPS and LC with instant polarized light microscopy and quantified pixel-level (1.5 µm/pixel) collagen fiber orientations. We used digital image correlation to measure the local stretch and quantified collagen crimp by the circular standard deviation of fiber orientations, or waviness. Local stretch-recruitment curves of PPS and LC approximated sigmoid functions. PPS recruited more fibers than the LC at the low levels of stretch. At 10% stretch the curves crossed with 75% bundles recruited. The PPS had higher uncrimping rate and waviness remaining after recruitment than the LC: 0.9º vs. 0.6º and 3.1º vs. 2.7º. Altogether our findings support describing fiber recruitment of both PPS and LC with sigmoid curves, with the PPS recruiting faster and at lower stretch than the LC, consistent with a stiffer tissue. STATEMENT OF SIGNIFICANCE: Peripapillary sclera (PPS) and lamina cribrosa (LC) collagen recruitment behaviors are central to the nonlinear mechanical behavior of the posterior pole of the eye. How PPS and LC collagen fibers recruit under stretch is crucial to develop constitutive models of the tissues but remains unclear. We used image-based stretch testing to characterize PPS and LC collagen fiber bundle recruitment under local stretch. We found that fiber-level stretch-recruitment curves of PPS and LC approximated sigmoid functions. PPS recruited more fibers at a low stretch, but at 10% bundle stretch the two curves crossed with 75% bundles recruited. We also found that PPS and LC fibers had different uncrimping rates and non-zero waviness's when recruited.

Impact of elevated IOP on lamina cribrosa oxygenation; A combined experimental-computational study on monkeys.

Purpose: Our goal is to evaluate how lamina cribrosa (LC) oxygenation is affected by the tissue distortions resulting from elevated IOP. Design: Experimental study on monkeys. Subjects: Four healthy monkey eyes with OCT scans with IOP of 10 to 50 mmHg, and then with histological sections of LC. Methods: Since in-vivo LC oxygenation measurement is not yet possible, we used 3D eye-specific numerical models of the LC vasculature which we subjected to experimentally-derived tissue deformations. We reconstructed 3D models of the LC vessel networks of 4 healthy monkey eyes from histological sections. We also obtained in-vivo IOP-induced tissue deformations from a healthy monkey using OCT images and digital volume correlation analysis techniques. The extent that LC vessels distort under a given OCT-derived tissue strain remains unknown. We biomechanics-based mapping techniques: cross-sectional and isotropic. The hemodynamics and oxygenations of the four vessel networks were simulated for deformations at several IOPs up to 60mmHg. The results were used to determine the effects of IOP on LC oxygen supply, assorting the extent of tissue mild and severe hypoxia. Main Outcome Measures: IOP-induced deformation, vasculature structure, blood supply, and oxygen supply for LC region. Result: IOP-induced deformations reduced LC oxygenation significantly. More than 20% of LC tissue suffered from mild hypoxia when IOP reached 30 mmHg. Extreme IOP(>50mmHg) led to large severe hypoxia regions (>30%) in the isotropic mapping cases. Conclusion: Our models predicted that moderately elevated IOP can lead to mild hypoxia in a substantial part of the LC, which, if sustained chronically, may contribute to neural tissue damage. For extreme IOP elevations, severe hypoxia was predicted, which would potentially cause more immediate damage. Our findings suggest that despite the remarkable LC vascular robustness, IOP-induced distortions can potentially contribute to glaucomatous neuropathy.

The Robust Lamina Cribrosa Vasculature: Perfusion and Oxygenation Under Elevated Intraocular Pressure.

Purpose: Elevated intraocular pressure (IOP) is thought to cause lamina cribrosa (LC) blood vessel distortions and potentially collapse, adversely affecting LC hemodynamics, reducing oxygenation, and triggering, or contributing to, glaucomatous neuropathy. We assessed the robustness of LC perfusion and oxygenation to vessel collapses. Methods: From histology, we reconstructed three-dimensional eye-specific LC vessel networks of two healthy monkey eyes. We used numerical simulations to estimate LC perfusion and from this the oxygenation. We then evaluated the effects of collapsing a fraction of LC vessels (0%-36%). The collapsed vessels were selected through three scenarios: stochastic (collapse randomly), systematic (collapse strictly by the magnitude of local experimentally determined IOP-induced compression), and mixed (a combination of stochastic and systematic). Results: LC blood flow decreased linearly as vessels collapsed-faster for stochastic and mixed scenarios and slower for the systematic one. LC regions suffering severe hypoxia (oxygen <8 mm Hg) increased proportionally to the collapsed vessels in the systematic scenario. For the stochastic and mixed scenarios, severe hypoxia did not occur until 15% of vessels collapsed. Some LC regions had higher perfusion and oxygenation as vessels collapsed elsewhere. Some severely hypoxic regions maintained normal blood flow. Results were equivalent for both networks and patterns of experimental IOP-induced compression. Conclusions: LC blood flow was sensitive to distributed vessel collapses (stochastic and mixed) and moderately vulnerable to clustered collapses (systematic). Conversely, LC oxygenation was robust to distributed vessel collapses and sensitive to clustered collapses. Locally normal flow does not imply adequate oxygenation. The actual nature of IOP-induced vessel collapse remains unknown.

Capturing sclera anisotropy using direct collagen fiber models. Linking microstructure to macroscopic mechanical properties.

Because of the crucial role of collagen fibers on soft tissue mechanics, there is great interest in techniques to incorporate them in computational models. Recently we introduced a direct fiber modeling approach for sclera based on representing the long-interwoven fibers. Our method differs from the conventional continuum approach to modeling sclera that homogenizes the fibers and describes them as statistical distributions for each element. At large scale our method captured gross collagen fiber bundle architecture from histology and experimental intraocular pressure-induced deformations. At small scale, a direct fiber model of a sclera sample reproduced equi-biaxial experimental behavior from the literature. In this study our goal was a much more challenging task for the direct fiber modeling: to capture specimen-specific 3D fiber architecture and anisotropic mechanics of four sclera samples tested under equibiaxial and four non-equibiaxial loadings. Samples of sclera from three eyes were isolated and tested in five biaxial loadings following an approach previously reported. Using microstructural architecture from polarized light microscopy we then created specimen-specific direct fiber models. Model fiber orientations agreed well with the histological information (adjusted R2's>0.89). Through an inverse-fitting process we determined model characteristics, including specimen-specific fiber mechanical properties to match equibiaxial loading. Interestingly, the equibiaxial properties also reproduced all the non-equibiaxial behaviors. These results indicate that the direct fiber modeling method naturally accounted for tissue anisotropy within its fiber structure. Direct fiber modeling is therefore a promising approach to understand how macroscopic behavior arises from microstructure.

Discovery of Novel Phenoxyaryl Pyridones as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with High Selectivity for the Second Bromodomain (BD2) to Potentially Treat Acute Myeloid Leukemia.

Bromodomain-selective BET inhibition has emerged as a promising strategy to improve the safety profiles of pan-BET inhibitors. Herein, we report the discovery of potent phenoxyaryl pyridones as highly BD2-selective BET inhibitors. Compound 23 (IC50 = 2.9 nM) exhibited a comparable BRD4 BD2 inhibitory activity relative to 10 (IC50 = 1.0 nM) and remarkably improved selectivity over BRD4 BD1 (23: 2583-fold; 10: 344-fold). This lead compound significantly inhibited the proliferation of acute myeloid leukemia (AML) cell lines through induction of G0/G1 arrest and apoptosis in vitro. Excellent in vivo antitumor efficacy with 23 was achieved in an MV;411 mouse xenograft model. Pleasingly, compound 23 (hERG IC50 > 30 µM) mitigated the inhibition of the human ether-à-go-go-related gene (hERG) ion channel compared with 10 (hERG IC50 = 2.8 µM). This work provides a promising BD2-selective lead for the development of more effective and safe BET inhibitors as anticancer agents.

Erythroid-intrinsic activation of TLR8 impairs erythropoiesis in inherited anemia.

Inherited non-hemolytic anemia is a group of rare bone marrow disorders characterized by erythroid defects. Although concerted efforts have been made to explore the underlying pathogenetic mechanisms of these diseases, the understanding of the causative mutations are still incomplete. Here we identify in a diseased pedigree that a gain-of-function mutation in toll-like receptor 8 (TLR8) is implicated in inherited non-hemolytic anemia. TLR8 is expressed in erythroid lineage and erythropoiesis is impaired by TLR8 activation whereas enhanced by TLR8 inhibition from erythroid progenitor stage. Mechanistically, TLR8 activation blocks annexin A2 (ANXA2)-mediated plasma membrane localization of STAT5 and disrupts EPO signaling in HuDEP2 cells. TLR8 inhibition improves erythropoiesis in RPS19+/- HuDEP2 cells and CD34+ cells from healthy donors and inherited non-hemolytic anemic patients. Collectively, we identify a gene implicated in inherited anemia and a previously undescribed role for TLR8 in erythropoiesis, which could potentially be explored for therapeutic benefit in inherited anemia.