Structurally divergent and recurrently mutated regions of primate genomes.

We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or ∼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.

Disruption of Cdh23 exon 68 splicing leads to progressive hearing loss in mice by affecting tip-link stability.

Inner ear hair cells are characterized by the F-actin-based stereocilia that are arranged into a staircase-like pattern on the apical surface of each hair cell. The tips of shorter-row stereocilia are connected with the shafts of their neighboring taller-row stereocilia through extracellular links named tip links, which gate mechano-electrical transduction (MET) channels in hair cells. Cadherin 23 (CDH23) forms the upper part of tip links, and its cytoplasmic tail is inserted into the so-called upper tip-link density (UTLD) that contains other proteins such as harmonin. The Cdh23 gene is composed of 69 exons, and we show here that exon 68 is subjected to hair cell-specific alternative splicing. Tip-link formation is not affected in genetically modified mutant mice lacking Cdh23 exon 68. Instead, the stability of tip links is compromised in the mutants, which also suffer from progressive and noise-induced hearing loss. Moreover, we show that the cytoplasmic tail of CDH23(+68) but not CDH23(-68) cooperates with harmonin in phase separation-mediated condensate formation. In conclusion, our work provides evidence that inclusion of Cdh23 exon 68 is critical for the stability of tip links through regulating condensate formation of UTLD components.

Accumulation of extracellular elastin-derived peptides disturbed neuronal morphology and neuron-microglia crosstalk in aged brain.

Extracellular elastin-derived peptides (EDPs) accumulate in the aging brain and have been associated with vascular dementia and Alzheimer's disease (AD). The activation of inflammatory processes in glial cells with EDP treatment has received attention, but not in neurons. To properly understand EDPs' pathogenic significance, the impact on neuronal function and neuron-microglia crosstalk was explored further. Among the EDP molecules, Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is a typical repeating hexapeptide. Here, we observed that EDPs-VGVAPG influenced neuronal survival and morphology in a dose-dependent manner. High concentrations of VGVAPG induced synapse loss and microglia hyperactivation in vivo and in vitro. Following EDP incubation, galectin 3 (Gal-3) released by neurons served as a chemokine, attracting microglial engulfment. Blocking Gal-3 and EDP binding remedied synapse loss in neurons and phagocytosis in microglia. In response to the accumulation of EDPs, proteomics in matrix remodeling and cytoskeleton dynamics, such as a disintegrin and metalloproteinase (ADAM) family, were engaged. These findings in extracellular EDPs provided more evidence for the relationship between aging and neuron dysfunction, increasing the insight of neuroinflammatory responses and the development of new specialized extracellular matrix remolding-targeted therapy options for dementia or other neurodegenerative disease.

Efficacy and safety of botulinum neurotoxin in the treatment of hemifacial spasms: a systematic review and meta-analysis.

BACKGROUND: Hemifacial spasm (HFS) is a neuromuscular disorder characterized by unilateral facial muscle spasms, negatively impacts quality of life due to social embarrassment. Botulinum Neurotoxin (BoNT) injections have emerged as a viable therapeutic approach. This systematic review evaluated the efficacy and safety of BoNT injections for HFS management, along with effects on patients' quality of life and mental health. MATERIALS AND METHODS: A systematic search for studies on BoNT treatment for HFS published between January 1, 2000, and May 1, 2024, was performed across major databases. Study quality was evaluated using Cochrane and Joanna Briggs Institute (JBI) tools, with data management handled by EndNote X9 and statistical analyses conducted via Review Manager (RevMan 5.4) and STATA 14.0. RESULTS: Thirty-five studies met the inclusion criteria: 2 RCTs comprising 83 HFS patients compared the efficacy of perioral injections of botulinum toxin and placebo, while 33 single-arm studies reported outcomes for 2786 patients post-BoNT injection. The selection of 17 single-arm studies focused on the effectiveness rate as the key outcome metric. Pooled estimate signified a remarkably high effectiveness (ES: 0.882, 95% CI: 0.830, 0.926, P < 0.001). Analysis of depression scale (SMD: -0.85, 95% CI: -1.34, -0.35, P < 0.001), anxiety scale (SMD: -1.50, 95% CI: -2.19, -0.80, P < 0.001) and total scale of quality of life (SMD: -0.64, 95% CI: -0.87, -0.41, P = 0.766) showed that BoNT therapy worked well especially in improving mental state and quality of life. Ptosis was considered as the most common adverse reaction during BoNT injections (OR: 0.30, 95% CI: 0.11, 0.81, P = 0.843). CONCLUSION: BoNT injection showed validity and clinical safety in the treatment of HFS, particular for depression relief. Injections around the mouth were only effective for HFS cases with severe symptoms. A standardized strategy for BoNT injections in managing HFS, detailing parameters such as injection sites, doses, and frequencies, remained elusive. Additional RCTs are necessary to further elucidate the interplay between efficacy and these components.

The mitochondria-targeted antioxidant MitoQ ameliorates inorganic arsenic-induced DCs/Th1/Th2/Th17/Treg differentiation partially by activating PINK1-mediated mitophagy in murine liver.

Inorganic arsenic is a well-established environmental toxicant linked to acute liver injury, fibrosis, and cancer. While oxidative stress, pyroptosis, and ferroptosis are known contributors, the role of PTEN-induced kinase 1 (PINK1)-mediated mitophagy in arsenic-induced hepatic immunotoxicity remains underexplored. Our study revealed that acute arsenic exposure prompts differentiation of hepatic dendritic cells (DCs) and T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cells, alongside increased transcription factors and cytokines. Inorganic arsenic triggered liver redox imbalance, leading to elevated alanine transaminase (ALT), hydrogen peroxide (H2O2), malondialdehyde (MDA), and activation of nuclear factor erythroid 2-related factor (Nrf2)/heme oxygenase-1 (HO-1) pathway. PINK1-mediated mitophagy was initiated, and its inhibition exacerbates H2O2 accumulation while promoting DCs/Th1/Th2/Treg differentiation in the liver of arsenic-exposed mice. Mitoquinone (MitoQ) pretreatment relieved arsenic-induced acute liver injury and immune imbalance by activating Nrf2/HO-1 and PINK1-mediated mitophagy. To our knowledge, this is the first report identifying PINK1-mediated mitophagy as a protective factor against inorganic arsenic-induced hepatic DCs/Th1/Th2 differentiation. This study has provided new insights on the immunotoxicity of inorganic arsenic and established a foundation for exploring preventive and therapeutic strategies targeting PINK1-mediated mitophagy in acute liver injury. Consequently, the application of mitochondrial antioxidant MitoQ may offer a promising treatment for the metalloid-induced acute liver injury.

A novel heterozygous missense variant of the ARID4A gene identified in Han Chinese families with schizophrenia-diagnosed siblings that interferes with DNA-binding activity.

ARID4A plays an important role in regulating gene expression and cell proliferation. ARID4A belongs to the AT-rich interaction domain (ARID)-containing family, and a PWWP domain immediately precedes its ARID region. The molecular mechanism and structural basis of ARID4A are largely unknown. Whole-exome sequencing (WES) revealed that a novel heterozygous missense variant, ARID4A c.1231 C > G (p.His411Asp), was associated with schizophrenia (SCZ) in this study. We determined the crystal structure of the PWWP-ARID tandem at 2.05 Å, revealing an unexpected mode in which ARID4A assembles with its PWWP and ARID from a structural and functional supramodule. Our results further showed that compared with the wild type, the p.His411Asp ARID mutant protein adopts a less compact conformation and exhibits a weaker dsDNA-binding ability. The p.His411Asp mutation decreased the number of cells that were arrested in the G0-G1 phase and caused more cells to progress to the G2-M phase. In addition, the missense mutation promoted the proliferation of HEK293T cells. In conclusion, our data provide evidence that ARID4A p.His411Asp could cause a conformational change in the ARID4A ARID domain, influence the DNA binding function, and subsequently disturb the cell cycle arrest in the G1 phase. ARID4A is likely a susceptibility gene for SCZ; thus, these findings provide new insight into the role of ARID4A in psychiatric disorders.

Association between serum uric acid, hyperuricemia and periodontitis: a cross-sectional study using NHANES data.

OBJECTIVES: Diabetes and other metabolic diseases have been linked to the development of periodontitis, but little research has been done to determine whether serum uric acid (SUA) levels and hyperuricemia play a role. This study aimed to investigate the relationship between SUA, hyperuricemia, and periodontitis. METHODS: Using data from the National Health and Nutrition Examination Survey (NHANES) 2011-2014, we created a nationally representative data set. We used multivariable logistic regression models to assess the relationship between SUA, hyperuricemia, and periodontitis and presented odds ratios (OR) in women and men, respectively. RESULTS: In women, adjusted multivariable regression models showed that SUA (4.1-4.3mg/dl) was associated with higher odds of periodontitis (OR = 1.43; 95% confidence interval (CI):1.0 ~ 2.03, p = 0.047) with SUA (≤ 3.3mg/dl) as reference. The risk of periodontitis tended to increase slightly but insignificantly with increasing SUA levels, and the adverse effects occurred only when SUA increased to a certain level, and then reached a plateau. In men, the adjusted OR values for SUA (4.9-5.2mg/dl), SUA (5.3-5.5mg/dl), SUA (5.9-6.2mg/dl), and SUA (6.3-6.5mg/dl) were 0.66 (95% CI: 0.45 ~ 0.96, p = 0.029), 0.58 (95% CI: 0.40 ~ 0.85, p = 0.006), 0.67(95% CI: 0.47 ~ 0.97, p = 0.035), and 0.67 (95% CI: 0.45 ~ 0.99, p = 0.043), respectively, with SUA (≤ 4.3mg/dl) as reference. The elevated SUA levels are protective against periodontitis, but there is a range within which the risk of periodontitis decreases, followed by a non-significant tendency to increase. CONCLUSIONS: The levels of SUA that are linked to the risk of periodontitis. Future prospective longitudinal studies and strategies are required to further confirm whether controlled SUA treatment is an effective adjunct to systematic periodontal therapy and whether SUA can be used as a diagnostic biomarker to assess the risk or progression of periodontitis.

Expression of Cre recombinase in chondrocytes causes abnormal craniofacial and skeletal development.

The cranial base synchondroses are growth centers that drive cranial and upper facial growth. The intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS) are two major synchondroses located in the middle of the cranial base and are maintained at early developmental stages to sustain cranial base elongation. In this study, we report unexpected premature ossification of ISS and SOS when Cre recombinase is activated in a chondrocyte-specific manner. We used a Cre transgenic line expressing Aggrecan enhancer-driven, Tetracycline-inducible Cre (ATC), of which expression is controlled by a Col2a1 promoter. Neonatal doxycycline injection or doxycycline diet fed to breeders was used to activate Cre recombinase. The premature ossification of ISS and/or SOS led to a reduction in cranial base length and subsequently a dome-shaped skull. Furthermore, the mice carrying either heterozygous or homozygous conditional deletion of Tsc1 or Fip200 using ATC mice developed similar craniofacial abnormalities, indicating that Cre activity itself but not conditional deletion of Tsc1 or Fip200 gene, is the major contributor of this phenotype. In contrast, the Col2a1-Cre mice carrying Cre expression in both perichondrium and chondrocytes and the mice carrying the conditional deletion of Tsc1 or Fip200 using Col2a1-Cre did not manifest the same skull abnormalities. In addition to the defective craniofacial bone development, our data also showed that the Cre activation in chondrocytes significantly compromised bone acquisition in femur. Our data calls for the consideration of the potential in vivo adverse effects caused by Cre expression in chondrocytes and reinforcement of the importance of including Cre-containing controls to facilitate accurate phenotype interpretation in transgenic research.

Autophagy Regulates Craniofacial Bone Acquisition.

Increasing evidence has demonstrated the important role of autophagy in skeletal homeostasis; however, the role of autophagy in craniofacial bone development and acquisition is largely unknown. In this study, we investigated the effect of autophagy suppression on craniofacial bone acquisition by deleting Fip200 or Atg5, two essential autophagy genes, using Osterix-Cre (Osx-Cre). We found that the Osx-Cre transgene mildly decreased the bone mass of parietal bone but not frontal bone, and did not affect cranial base bone mass in adult mice. In the cranial vault, Fip200 or Atg5 deletion similarly decreased 50% bone mass of neural crest-derived frontal bone; Atg5 deletion decreased 50% and Fip200 deletion decreased 30% bone mass of mesoderm-derived parietal bone. In the cranial base, Fip200 or Atg5 deletion similarly decreased 30% bone mass of neural crest-derived presphenoid bone; Atg5 deletion decreased 30% and Fip200 deletion decreased 16% bone mass of mesoderm-derive basioccipital bone. Lastly, we used doxycycline treatment to inhibit the Osx-Cre expression until 2 months of age and showed that postnatal Fip200 deletion led to cranial vault bone mass decrease in association with a small increase in both bone volume/tissue volume and tissue mineral density. Altogether, this study demonstrated the important role of autophagy in craniofacial bone acquisition during development and postnatal growth.

Mid-facial developmental defects caused by the widely used LacZ reporter gene when expressed in neural crest-derived cells.

Reporter genes play important roles in transgenic research. LacZ is a widely used reporter gene that encodes Escherichia coli ß-galactosidase, an enzyme that is well known for its ability to hydrolyze X-gal into a blue product. It is unknown whether transgenic LacZ has any adverse effects. R26R reporter mice, containing a LacZ reporter gene, were generated to monitor the in vivo recombination activity of various transgenic Cre recombinase via X-gal staining. P0-Cre is expressed in neural crest-derived cells, which give rise to the majority of the craniofacial bones. Herein, we report that 12% of the R26R reporter mice harboring P0-Cre had unexpected mid-facial developmental defects manifested by the asymmetrical growth of some facial bones, thus resulting in tilted mid-facial structure, shorter skull length, and malocclusion. Histological examination showed a disorganization of the frontomaxillary suture, which may at least partly explain the morphological defect in affected transgenic mice. Our data calls for the consideration of the potential in vivo adverse effects caused by transgenic ß-galactosidase.

Persistent Organic Nanopores Amenable to Structural and Functional Tuning.

Rigid macrocycles 2, which share a hybrid backbone and the same set of side chains while having inner cavities with different inward-pointing functional groups, undergo similar nanotubular assembly as indicated by multiple techniques including (1)H NMR, fluorescence spectroscopy, and atomic force microscopy. The formation of tubular assemblies containing subnanometer pores is also attested by the different transmembrane ion-transport behavior observed for these macrocycles. Vesicle-based stopped-flow kinetic assay and single-channel electrophysiology with planar lipid bilayers show that the presence of an inward-pointing functional (X) group in the inner cavity of a macrocyclic building block exerts a major influence on the transmembrane ion-transporting preference of the corresponding self-assembling pore. Self-assembling pores with inward-pointing amino and methyl groups possess the surprising and remarkable capability of rejecting protons but are conducive to transporting larger ions. The inward-pointing groups also resulted in transmembrane pores with a different extent of positive electrostatic potentials, leading to channels having different preferences for transporting chloride ion. Results from this work demonstrate that synthetic modification at the molecular level can profoundly impact the property of otherwise structurally persistent supramolecular assemblies, with both expected tunability and suprisingly unusual behavior.

Decoding Missense Variants by Incorporating Phase Separation via Machine Learning.

Computational models have made significant progress in predicting the effect of protein variants. However, deciphering numerous variants of uncertain significance (VUS) located within intrinsically disordered regions (IDRs) remains challenging. To address this issue, we introduce phase separation, which is tightly linked to IDRs, into the investigation of missense variants. Phase separation is vital for multiple physiological processes. By leveraging missense variants that alter phase separation propensity, we develop a machine learning approach named PSMutPred to predict the impact of missense mutations on phase separation. PSMutPred demonstrates robust performance in predicting missense variants that affect natural phase separation. In vitro experiments further underscore its validity. By applying PSMutPred on over 522,000 ClinVar missense variants, it significantly contributes to decoding the pathogenesis of disease variants, especially those in IDRs. Our work provides insights into the understanding of a vast number of VUSs in IDRs, expediting clinical interpretation and diagnosis.

EEG decoding for datasets with heterogenous electrode configurations using transfer learning graph neural networks.

Objective. Brain-machine interfacing (BMI) has greatly benefited from adopting machine learning methods for feature learning that require extensive data for training, which are often unavailable from a single dataset. Yet, it is difficult to combine data across labs or even data within the same lab collected over the years due to the variation in recording equipment and electrode layouts resulting in shifts in data distribution, changes in data dimensionality, and altered identity of data dimensions. Our objective is to overcome this limitation and learn from many different and diverse datasets across labs with different experimental protocols.Approach. To tackle the domain adaptation problem, we developed a novel machine learning framework combining graph neural networks (GNNs) and transfer learning methodologies for non-invasive motor imagery (MI) EEG decoding, as an example of BMI. Empirically, we focus on the challenges of learning from EEG data with different electrode layouts and varying numbers of electrodes. We utilize three MI EEG databases collected using very different numbers of EEG sensors (from 22 channels to 64) and layouts (from custom layouts to 10-20).Main results. Our model achieved the highest accuracy with lower standard deviations on the testing datasets. This indicates that the GNN-based transfer learning framework can effectively aggregate knowledge from multiple datasets with different electrode layouts, leading to improved generalization in subject-independent MI EEG classification.Significance. The findings of this study have important implications for brain-computer-interface research, as they highlight a promising method for overcoming the limitations posed by non-unified experimental setups. By enabling the integration of diverse datasets with varying electrode layouts, our proposed approach can help advance the development and application of BMI technologies.

The role of retinoic acid receptor-related orphan receptors in skeletal diseases.

Bone homeostasis, depending on the balance between bone formation and bone resorption, is responsible for maintaining the proper structure and function of the skeletal system. As an important group of transcription factors, retinoic acid receptor-related orphan receptors (RORs) have been reported to play important roles in bone homeostasis by regulating the transcription of target genes in skeletal cells. On the other hand, the dysregulation of RORs often leads to various skeletal diseases such as osteoporosis, rheumatoid arthritis (RA), and osteoarthritis (OA). Herein, we summarized the roles and mechanisms of RORs in skeletal diseases, aiming to provide evidence for potential therapeutic strategies.

Cardiac biophysical detailed synergetic modality rendering and visible correlation.

The heart is a vital organ in the human body. Research and treatment for the heart have made remarkable progress, and the functional mechanisms of the heart have been simulated and rendered through the construction of relevant models. The current methods for rendering cardiac functional mechanisms only consider one type of modality, which means they cannot show how different types of modality, such as physical and physiological, work together. To realistically represent the three-dimensional synergetic biological modality of the heart, this paper proposes a WebGL-based cardiac synergetic modality rendering framework to visualize the cardiac physical volume data and present synergetic correspondence rendering of the cardiac electrophysiological modality. By constructing the biological detailed interactive histogram, users can implement local details rendering for the heart, which could reveal the cardiac biology details more clearly. We also present cardiac physical-physiological correlation visualization to explore cardiac biological association characteristics. Experimental results show that the proposed framework can provide favorable cardiac biological detailed synergetic modality rendering results in terms of both effectiveness and efficiency. Compared with existing methods, the framework can facilitate the study of the internal mechanism of the heart and subsequently deduce the process of initiation, development, and transformation from a healthy heart to an ill one, and thereby improve the diagnosis and treatment of cardiac disorders.

Temporal and spatial assembly of inner ear hair cell ankle link condensate through phase separation.

Stereocilia are actin-based cell protrusions of inner ear hair cells and are indispensable for mechanotransduction. Ankle links connect the ankle region of developing stereocilia, playing an essential role in stereocilia development. WHRN, PDZD7, ADGRV1 and USH2A have been identified to form the so-called ankle link complex (ALC); however, the detailed mechanism underlying the temporal emergence and degeneration of ankle links remains elusive. Here we show that WHRN and PDZD7 orchestrate ADGRV1 and USH2A to assemble the ALC through liquid-liquid phase separation (LLPS). Disruption of the ALC multivalency for LLPS largely abolishes the distribution of WHRN at the ankle region of stereocilia. Interestingly, high concentration of ADGRV1 inhibits LLPS, providing a potential mechanism for ALC disassembly. Moreover, certain deafness mutations of ALC genes weaken the multivalent interactions of ALC and impair LLPS. In conclusion, our study demonstrates that LLPS mediates ALC formation, providing essential clues for understanding the pathogenesis of deafness.

Structurally divergent and recurrently mutated regions of primate genomes.

To better understand the pattern of primate genome structural variation, we sequenced and assembled using multiple long-read sequencing technologies the genomes of eight nonhuman primate species, including New World monkeys (owl monkey and marmoset), Old World monkey (macaque), Asian apes (orangutan and gibbon), and African ape lineages (gorilla, bonobo, and chimpanzee). Compared to the human genome, we identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. Across 50 million years of primate evolution, we estimate that 819.47 Mbp or ~27% of the genome has been affected by SVs based on analysis of these primate lineages. We identify 1,607 structurally divergent regions (SDRs) wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (CARDs, ABCD7, OLAH) and new lineage-specific genes are generated (e.g., CKAP2, NEK5) and have become targets of rapid chromosomal diversification and positive selection (e.g., RGPDs). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species for the first time.

Proline 107 is a major determinant in maintaining the structure of the distal pocket and reactivity of the high-spin heme of MauG.

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro107 was converted to Cys, Val, and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive, and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to those of wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change that resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glu113. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with greatly increased rhombicity as compared to that of the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H(2)O(2) and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, allowing exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus minimizing the oxidation of other residues of MauG.

Chondrocyte Tsc1 controls cranial base bone development by restraining the premature differentiation of synchondroses.

Cranial base bones are formed through endochondral ossification. Synchondroses are growth plates located between cranial base bones that facilitate anterior-posterior growth of the skull. Coordinated proliferation and differentiation of chondrocytes in cranial base synchondroses is essential for cranial base bone growth. Herein, we report that constitutive activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling via Tsc1 (Tuberous sclerosis 1) deletion in chondrocytes causes abnormal skull development with decreased size and rounded shape. In contrast to decreased anterior-posterior growth of the cranial base, mutant mice also exhibited significant expansion of cranial base synchondroses including the intersphenoid synchondrosis (ISS) and the spheno-occipital synchondrosis (SOS). Cranial base synchondrosis expansion in TSC1-deficient mice was accounted for by an expansion of the resting zone due to increased cell number and size without alteration in cell proliferation. Furthermore, our data showed that mTORC1 activity is inhibited in the resting and proliferating zone chondrocytes of wild type mice, and Tsc1 deletion activated mTORC1 signaling of the chondrocytes in the resting zone area. Consequently, the chondrocytes in the resting zone of TSC1-deficient mice acquired characteristics generally attributed to pre-hypertrophic chondrocytes including high mTORC1 activity, increased cell size, and increased expression level of PTH1R (Parathyroid hormone 1 receptor) and IHH (Indian hedgehog). Lastly, treatment with rapamycin, an inhibitor of mTORC1, rescued the abnormality in synchondroses. Our results established an important role for TSC1-mTORC1 signaling in regulating cranial base bone development and showed that chondrocytes in the resting zone of synchondroses are maintained in an mTORC1-inhibitory environment.

A bioinspired and chemically defined alternative to dimethyl sulfoxide for the cryopreservation of human hematopoietic stem cells.

The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34+ progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.