Inferring pattern-driving intercellular flows from single-cell and spatial transcriptomics.

From single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomics (ST), one can extract high-dimensional gene expression patterns that can be described by intercellular communication networks or decoupled gene modules. These two descriptions of information flow are often assumed to occur independently. However, intercellular communication drives directed flows of information that are mediated by intracellular gene modules, in turn triggering outflows of other signals. Methodologies to describe such intercellular flows are lacking. We present FlowSig, a method that infers communication-driven intercellular flows from scRNA-seq or ST data using graphical causal modeling and conditional independence. We benchmark FlowSig using newly generated experimental cortical organoid data and synthetic data generated from mathematical modeling. We demonstrate FlowSig's utility by applying it to various studies, showing that FlowSig can capture stimulation-induced changes to paracrine signaling in pancreatic islets, demonstrate shifts in intercellular flows due to increasing COVID-19 severity and reconstruct morphogen-driven activator-inhibitor patterns in mouse embryogenesis.

25-Hydroxycholesterol Protects Host against Zika Virus Infection and Its Associated Microcephaly in a Mouse Model.

Zika virus (ZIKV) has become a public health threat due to its global transmission and link to severe congenital disorders. The host immune responses to ZIKV infection have not been fully elucidated, and effective therapeutics are not currently available. Herein, we demonstrated that cholesterol-25-hydroxylase (CH25H) was induced in response to ZIKV infection and that its enzymatic product, 25-hydroxycholesterol (25HC), was a critical mediator of host protection against ZIKV. Synthetic 25HC addition inhibited ZIKV infection in vitro by blocking viral entry, and treatment with 25HC reduced viremia and conferred protection against ZIKV in mice and rhesus macaques. 25HC suppressed ZIKV infection and reduced tissue damage in human cortical organoids and the embryonic brain of the ZIKV-induced mouse microcephaly model. Our findings highlight the protective role of CH25H during ZIKV infection and the potential use of 25HC as a natural antiviral agent to combat ZIKV infection and prevent ZIKV-associated outcomes, such as microcephaly.

Occlusal Evaluation Using Modified Huddart and Bodenham Scoring System Following 2-Stage Palatoplasty With Hotz Plate: A Comparison Among 3 Different Surgical Protocols.

OBJECTIVE: This study aimed to determine if the change in technique of soft palate closure or timing of hard palatal repair induced occlusal changes in patients with complete unilateral cleft lip and palate (CUCLP). DESIGN: Retrospective study. SETTINGS: A medical and dental hospital in Japan. SUBJECTS: A total of 96 patients with CUCLP treated with 2-stage palatoplasty were included in the study and categorized into 3 groups (G1, G2, and G3) according to the protocol used. INTERVENTIONS: G1 underwent soft palate repair using Perko method at 1.5 years of age and hard palate repair using vomer flap procedure at 5.5 years of age. Furlow method was used for soft palate repair in G2 at 1.5 years of age and hard palate repair using vomer flap procedure at 5.5 years of age. The Furlow method was used to repair the soft palate in G3 at 1.5 years of age and vomer flap procedure was used to repair the hard palate at 4 years of age. MAIN OUTCOME MEASURES: Two evaluators assessed the dental arch relationship using the modified Huddart/Bodenham (mHB) index on 2 separate occasions. RESULTS: Intra- (intraclass correlation coefficient [ICC]: 0.962) and inter-examiner (ICC: 0.950) reliability showed very good agreement. The frequency of crossbite present in the major and minor segments gradually decreased with each change in protocol. Mean segmental scores showed no significant difference between 3 protocols (P > .05). Good inter-arch alignment occurred with all 3 surgical protocols (G1:82.6%, G2:89.8%, and G3:91.7%). CONCLUSIONS: There was no significant difference in the dental arch relationship outcomes between the 3 surgical protocols. The dentition status was comparable with all surgical protocols, even after the changes.

MicroRNAs control eyelid development through regulating Wnt signaling.

BACKGROUND: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. RESULTS: It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer fl/fl ;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer fl/fl ;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial-mesenchymal interactions in eyelid development. CONCLUSIONS: miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.

Use of malleoli as an indicator for flatfoot in patients with Down syndrome: development of a simple and non-invasive evaluation method through medial longitudinal arch.

[Purpose] Flatfoot often presents in patients with Down syndrome, and it can be diagnosed using a simple radiograph. Consequently, due to radiograph limitations, alternative non-invasive testing must be determined. Conventionally, arch height ratio can be used for evaluation of the medial longitudinal arch, where the foot is evaluated by detecting the navicular bone on the foot surface. However, detection of the navicular tuberosity is difficult and even though the detection is relatively straightforward for patients without intellectual disability, measuring navicular bone is more difficult in patients with intellectual disability, such as those who have Down syndrome and are uncooperative with a tester. Therefore, we evaluated arch height ratio using the malleoli instead of the navicular bone to determine whether malleoli testing was appropriate for patients with Down syndrome that have an intellectual disability. [Participants and Methods] We conducted a retrospective study of 16 pairs of feet in 16 patients with Down syndrome, diagnosed with flatfoot. The height to the centre of the talo-navicular joint and that of the malleoli from the sole were measured on radiographs using weight-bearing conditions. [Results] The age range was 5.2 to 25.3 years. There was a correlation between the height of the navicular bone and that of the medial and lateral malleoli. [Conclusion] We conclude that the medial and lateral malleoli can substitute navicular bone as a landmark diagnosis test for flatfoot. Considering the close physical distance between the medial malleolus and navicular bone, and the association between the tibia and medial longitudinal arch, the medial malleolus may provide a better landmark in patients with Down syndrome with it being potentially less invasive for uncooperative patients.

Regional regulation of Filiform tongue papillae development by Ikkα/Irf6.

BACKGROUND: Non-gustatory filiform papillae play critical roles in helping to grip food, drawing food to the esophagus, cleaning the mouth, and spreading saliva. The molecular mechanisms of filiform tongue papillae development however are not fully understood. RESULTS: We found Ikkα and Irf6 expression in developing tongue epithelium, and describe here specific tongue abnormalities in mice with mutation of these genes, indicating a role for Ikkα and Irf6 in filiform papillae development. Ikkα and Irf6 mutant tongues showed ectopic vertical epithelium at the midline, while lateral sides of mutant tongues adhered to the oral mucosa. Both the ectopic median vertical epithelium and adhered epithelium exhibited the presence of filiform tongue papillae, whereas epithelium between the median vertical epithelium and adhered tongue showed a loss of filiform tongue papillae. Timing of filiform papillae development was found to be slightly different between the midline and lateral regions of the wild-type tongue. CONCLUSIONS: Filiform papillae thus develop through distinct molecular mechanisms between the regions of tongue dorsum in the medio-lateral axis, with some filiform papillae developing under the control of Ikkα and Irf6. Developmental Dynamics 245:937-946, 2016. © 2016 Wiley Periodicals, Inc.

Spatially heterogeneous choroid plexus transcriptomes encode positional identity and contribute to regional CSF production.

A sheet of choroid plexus epithelial cells extends into each cerebral ventricle and secretes signaling factors into the CSF. To evaluate whether differences in the CSF proteome across ventricles arise, in part, from regional differences in choroid plexus gene expression, we defined the transcriptome of lateral ventricle (telencephalic) versus fourth ventricle (hindbrain) choroid plexus. We find that positional identities of mouse, macaque, and human choroid plexi derive from gene expression domains that parallel their axial tissues of origin. We then show that molecular heterogeneity between telencephalic and hindbrain choroid plexi contributes to region-specific, age-dependent protein secretion in vitro. Transcriptome analysis of FACS-purified choroid plexus epithelial cells also predicts their cell-type-specific secretome. Spatial domains with distinct protein expression profiles were observed within each choroid plexus. We propose that regional differences between choroid plexi contribute to dynamic signaling gradients across the mammalian cerebroventricular system.

HIV-associated CD8+ T-cell Skin Infiltrative Disease and EBV-associated Polymorphic B-cell Lymphoproliferative Disorder in an AIDS Patient Who Improved Dramatically with Antiretroviral Therapy Alone.

Human immunodeficiency virus (HIV)-associated CD8+ T-cell skin infiltrative disease with severe erythroderma has rarely been reported. While HIV-positive patients are prone to develop lymphoma, which is often associated with Epstein-Barr virus, polymorphic lymphoproliferative disorder is rare, accounting for <5% of cases. We herein report a 41-year-old HIV-positive man who presented with a fever, erythroderma, and lymphadenopathy and was diagnosed with the coexistence of both diseases. His condition improved significantly with continued antiretroviral therapy. This case suggests that HIV-induced immunodeficiency is central to the pathogenesis of both entities and that improvement of the immunodeficient state is an effective treatment.

BMP4 sufficiency to induce choroid plexus epithelial fate from embryonic stem cell-derived neuroepithelial progenitors.

Choroid plexus epithelial cells (CPECs) have essential developmental and homeostatic roles related to the CSF and blood-CSF barrier they produce. Accordingly, CPEC dysfunction has been implicated in many neurological disorders, such as Alzheimer's disease, and transplant studies have provided proof-of-concept for CPEC-based therapies. However, such therapies have been hindered by the inability to expand or generate CPECs in culture. During development, CPECs differentiate from preneurogenic neuroepithelial cells and require bone morphogenetic protein (BMP) signaling, but whether BMPs suffice for CPEC induction is unknown. Here we provide evidence for BMP4 sufficiency to induce CPEC fate from neural progenitors derived from mouse embryonic stem cells (ESCs). CPEC specification by BMP4 was restricted to an early time period after neural induction in culture, with peak CPEC competency correlating to neuroepithelial cells rather than radial glia. In addition to molecular, cellular, and ultrastructural criteria, derived CPECs (dCPECs) had functions that were indistinguishable from primary CPECs, including self-assembly into secretory vesicles and integration into endogenous choroid plexus epithelium following intraventricular injection. We then used BMP4 to generate dCPECs from human ESC-derived neuroepithelial cells. These findings demonstrate BMP4 sufficiency to instruct CPEC fate, expand the repertoire of stem cell-derived neural derivatives in culture, and herald dCPEC-based therapeutic applications aimed at the unique interface between blood, CSF, and brain governed by CPECs.

Vascular ring anomaly with a right patent ductus arteriosus and a left aortic arch in a juvenile cat.

This paper reports the clinical findings and surgical treatment of feline right patent ductus arteriosus (RPDA) with a left aortic arch. A two-month-old female Maine Coon was referred for an investigation of regurgitation after weaning. RPDA with a left aortic arch was diagnosed based on the echocardiographic and computed tomography (CT) findings. A right-fourth intercostal thoracotomy was found to be an appropriate approach to the duct. Preoperative diagnosis is crucial and diagnostic imaging, including radiography, echocardiography, and cardiac CT examination, is essential for determining if the aortic arch is right or left.

Brain organoid-on-a-chip to create multiple domains in forebrain organoids.

Brain organoids are three-dimensionally reconstructed brain tissue derived from pluripotent stem cells in vitro. 3D tissue cultures have opened new avenues for exploring development and disease modeling. However, some physiological conditions, including signaling gradients in 3D cultures, have not yet been easily achieved. Here, we introduce Brain Organoid-on-a-Chip platforms that generate signaling gradients that in turn enable the induction of topographic forebrain organoids. This creates a more continuous spectrum of brain regions and provides a more complete mimic of the human brain for evaluating neurodevelopment and disease in unprecedented detail.

The Fbw7 tumor suppressor regulates nuclear factor E2-related factor 1 transcription factor turnover through proteasome-mediated proteolysis.

Nuclear factor E2-related factor 1 (Nrf1) is a basic leucine zipper transcription factor that plays important roles in cellular stress response and development. Currently, the mechanism regulating Nrf1 expression is poorly understood. We report here that Nrf1 is a short-lived protein that is targeted by F-box protein Fbw7, which is the substrate-specifying component of SCF (Skp1-Cul1-Fbox protein-Rbx1)-type ubiquitin ligase for degradation via the ubiquitin-proteasome pathway. We show that Fbw7 directly binds Nrf1 through a Cdc4 phosphodegron and that enforced expression of Fbw7 promotes the ubiquitination and degradation of Nrf1. Conversely, depletion of endogenous Fbw7 leads to decreased Nrf1 ubiquitination and accumulation of Nrf1 protein. Accordingly, expression of Fbw7 leads to down-regulation of antioxidant response element-driven gene activation, whereas disruption of Fbw7-mediated destabilization of Nrf1 leads to increased antioxidant response element-driven gene expression. Together, these data identify Fbw7 as a regulator of Nrf1 expression and reveal a novel function of Fbw7 in cellular stress response.

Mapping the Ethical Issues of Brain Organoid Research and Application.

In 2008, researchers created human three-dimensional neural tissue - known as the pioneering work of "brain organoids." In recent years, some researchers have transplanted human brain organoids into animal brains for applicational purposes. With these experiments have come many ethical concerns. It is thus an urgent task to clarify what is ethically permissible and impermissible in brain organoid research. This paper seeks (1) to sort out the ethical issues related to brain organoid research and application and (2) to propose future directions for additional ethical consideration and policy debates in the field. Toward (1), this paper first outlines the current state of brain organoid research, and then briefly responds to previously raised related ethical concerns. Looking next at anticipated scientific developments in brain organoid research, we will discuss (i) ethical issues related to in vitro brain organoids, (ii) ethical issues raised when brain organoids form complexes or have relationships with other entities, and (iii) ethical issues of research ethics and governance. Finally, in pursuit of (2), we propose research policies that are mindful of the ethics of brain organoid research and application and also suggest the need for an international framework for research and application of brain organoids.

Color M-Mode Echocardiography for Non-Invasive Assessment of the Intraventricular Pressure in Dogs Before and After Ductus Arteriosus Occlusion: A Retrospective Study.

Background: Novel non-invasive evaluation of the intraventricular pressure differences and gradients (IVPD and IVPG) by color M-mode echocardiography (CMME) is a promising method in diastolic function evaluation. Patent ductus arteriosus (PDA) is a congenital heart defect which is associated with increased preload. The present work provides a clinical trial for the assessment of IVPD and IVPG changes in dogs before and after surgical occlusion of PDA. Materials and Methods: A total of 12 client-owned dogs were enrolled in this study. PDA was confirmed using echocardiography, and all dogs underwent PDA occlusion. Conventional echocardiography and CMME were conducted on each patient on the operation day (Pre-PDA) and 48 h after its occlusion (Post-PDA). The total IVPD and total IVPG, as well as segmental intraventricular pressure (basal, mid-to-apical, mid, and apical) were measured from Euler's equation using specific software (MATLAB). Data were analyzed for variability and for the difference between pre- and post-PDA. The effect of PDA occlusion on the measured variables was calculated using biserial ranked correlation (rc). Results: There was a significant reduction in end-diastolic volume, fraction shortening, stroke volume, and mitral inflow velocities (early and late) after PDA closure. CMME was feasible in all dogs, and the CMME indices showed moderate variability, except for the apical segment of IVPD and IVPG. After PDA closure, in comparison with the pre-PDA occlusion, there was a significant reduction in total IVPD (2.285 ± 0.374 vs. 1.748 ± 0.436 mmHg; P = 0.014), basal IVPD (1.177 ± 0.538 vs. 0.696 ± 0.144 mmHg; P = 0.012), total IVPG (1.141 ± 0.246 vs. 0.933 ± 0.208 mmHg; P = 0.032), and basal IVPG (0.578 ± 0.199 vs. 0.377 ± 0.113 mmHg; P = 0.001); meanwhile, mid, mid-to-apical, and apical segments of both IVPD and IVPG showed non-significant difference. The magnitude of PDA occlusion on the measured variables was clinically relevant and associated with a large effect size on total and basal IVPD and IVPG (rc > 0.6). Conclusion: The current clinical study revealed matched response of IVPD and IVPG to the reduced preload rather than left ventricular relaxation. This result is an initial step in the clinical utility of CMME-derived IVPD and IVPG measurements in the diastolic function evaluation in dogs with PDA that warrants further clinical studies.

Clip ligation for treatment of patent ductus arteriosus occlusion in three cats.

Patent ductus arteriosus (PDA) is a rare congenital cardiovascular anomaly in cats. Due to their small body, intercostal thoracotomy is the most common option to close the PDA. However, few reports detail the surgical technique for ligating PDA in kittens. In this case report, three cats weighing 1.4 kg, 1.2 kg, and 2.9 kg were diagnosed PDA. Clip ligation via left fourth intercostal thoracotomy was performed and the cats were successfully treated. Postoperative echocardiography showed no residual flow in any of the cases. This case report highlights clip occlusion for small cats with PDA could be safe and effective.

Plasma Chymase Activity Reflects the Change in Hemodynamics Observed after the Surgical Treatment of Patent Ductus Arteriosus in Dogs.

Chymase is a protease stored in mast cell granules that produces angiotensin II (ANG II) from angiotensin I (ANG I) and is associated with tissue injury, inflammation, and remodeling, especially involving the cardiovascular system. As cardiovascular events occur, chymase is activated by degranulation to the extracellular matrix. Although chymase has been suggested to be associated with cardiovascular disease progression, there are not enough reports in veterinary medicine. Patent ductus arteriosus (PDA) is a common congenital cardiac disease in veterinary medicine. Almost all cases of PDA can be treated surgically to prevent the development of congestive heart disease and/or pulmonary hypertension. The aims of the present study were to measure chymase activity before and after PDA occlusions, and to investigate the relationships between the congestive and hemodynamic states of PDA and chymase activity. In the present study, 17 puppies diagnosed with PDA were included and all puppies completely recovered to the level of healthy dogs. Chymase activity significantly decreased at 2 months after the operation, along with the echocardiography parameters of congestion. Therefore, plasma chymase activity may be useful as a novel predictor for understanding the hemodynamics of PDA in veterinary medicine.

Hemodynamic Effects of Protamine Infusion in Dogs with Myxomatous Mitral Valve Disease Undergoing Mitral Valvuloplasty.

Protamine, an antagonizing agent to heparin, is indispensable for dogs undergoing cardiopulmonary bypass. Protamine-induced hypotension (PIH) during cardiac anesthesia has been reported in humans. The purpose of this study was to describe the hemodynamic effect of protamine administration in dogs during cardiac surgery in clinical cases. Study design: Retrospective, clinical, cohort study. A total of 14 client-owned dogs who suffered heart failure due to medically uncontrolled myxomatous mitral valve disease (MMVD) were included in this study. The severity of MMVD was classified according to American College of Veterinary Internal Medicine staging (ACVIM: stage B2, C, D) and dogs undergoing mitral valve surgery. Records with clinical data for dogs treated between July 2019 to August 2020 were examined for age, sex, breed, body weight, concurrent diseases, hospitalization, anesthetic record, and mortality within 3 months after the operation. PIH was defined as mean arterial pressure (MAP) lowered by 20% of that before protamine infusion. To evaluate the effect of protamine on hemodynamic variables, each of the other values was compared with values at the beginning of protamine infusion. MAP decreased by 41.0 and 45.7% in two dogs (14.3%) compared with pressure before protamine infusion. Others did not show obvious alteration in hemodynamic variables. Epinephrine treatment alleviated hypotension in one dog. Another dog with systemic hypotension concomitant with elevated central venous pressure did not respond to epinephrine treatment and a reboot of extracorporeal circulation was required. Reheparinization and reinstitution of cardiopulmonary bypass successfully resuscitate the second dog. In conclusion, clinicians should alert the incidence of severe hypotension even with slow protamine infusion following canine cardiac surgery. This study also provides two effective treatments for catastrophic hypotension during protamine infusion.

TGFß superfamily signaling regulates the state of human stem cell pluripotency and capacity to create well-structured telencephalic organoids.

Telencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor ß (TGFß) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFß-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

Ru/Me-BIPAM-catalyzed asymmetric addition of arylboronic acids to aliphatic aldehydes and α-ketoesters.

A ruthenium-catalyzed asymmetric arylation of aliphatic aldehydes and α-ketoesters with arylboronic acids has been developed, giving chiral alkyl(aryl)methanols and α-hydroxy esters in good yields. The use of a chiral bidentate phosphoramidite ligand (Me-BIPAM) achieved excellent enantioselectivities.

Restoration of the defect in radial glial fiber migration and cortical plate organization in a brain organoid model of Fukuyama muscular dystrophy.

Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis.