Cardiolipin is a specific mitochondrial phospholipid that has a high affinity for proteins and that stabilizes the assembly of supercomplexes involved in oxidative phosphorylation. We found that sequestration of cardiolipin in protein complexes is critical to protect it from degradation. The turnover of cardiolipin is slower by almost an order of magnitude than the turnover of other phospholipids. However, in subjects with Barth syndrome, cardiolipin is rapidly degraded via the intermediate monolyso-cardiolipin. Treatments that induce supercomplex assembly decrease the turnover of cardiolipin and the concentration of monolyso-cardiolipin, whereas dissociation of supercomplexes has the opposite effect. Our data suggest that cardiolipin is uniquely protected from normal lipid turnover by its association with proteins, but this association is compromised in subjects with Barth syndrome, leading cardiolipin to become unstable, which in turn causes the accumulation of monolyso-cardiolipin.

Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival, proliferation, differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2, previously shown to promote Cx32 expression in mature hepatocytes, up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation, resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast, negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast, the p38 MAPK activator, anisomycin, blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation.

Long noncoding RNAs (lncRNAs) mediate important epigenetic regulation in a wide range of biological processes and diseases. We applied comprehensive analyses of RNA-seq and CAGE-seq (cap analysis of gene expression and sequencing) to characterize the dynamic changes in lncRNA expression in rhesus macaque (Macaca mulatta) brain in four representative age groups. We identified 18 anatomically diverse lncRNA modules and 14 mRNA modules representing spatial, age, and sex specificities. Spatiotemporal- and sex-biased changes in lncRNA expression were generally higher than those observed in mRNA expression. A negative correlation between lncRNA and mRNA expression in cerebral cortex was observed and functionally validated. Our findings offer a fresh insight into spatial-, age-, and sex-biased changes in lncRNA expression in macaque brain and suggest that the changes represent a previously unappreciated regulatory system that potentially contributes to brain development and aging.

Both environmental cues and intracellular bioenergetic states profoundly affect intracellular pH (pHi). How a cell responds to pHi changes to maintain bioenergetic homeostasis remains elusive. Here we show that Smad5, a well-characterized downstream component of bone morphogenetic protein (BMP) signaling responds to pHi changes. Cold, basic or hypertonic conditions increase pHi, which in turn dissociates protons from the charged amino acid clusters within the MH1 domain of Smad5, prompting its relocation from the nucleus to the cytoplasm. On the other hand, heat, acidic or hypotonic conditions decrease pHi, blocking the nuclear export of Smad5, and thus causing its nuclear accumulation. Active nucleocytoplasmic shuttling of Smad5 induced by environmental changes and pHi fluctuation is independent of BMP signaling, carboxyl terminus phosphorylation and Smad4. In addition, ablation of Smad5 causes chronic and irreversible dysregulation of cellular bioenergetic homeostasis and disrupted normal neural developmental processes as identified in a differentiation model of human pluripotent stem cells. Importantly, these metabolic and developmental deficits in Smad5-deficient cells could be rescued only by cytoplasmic Smad5. Cytoplasmic Smad5 physically interacts with hexokinase 1 and accelerates glycolysis. Together, our findings indicate that Smad5 acts as a pHi messenger and maintains the bioenergetic homeostasis of cells by regulating cytoplasmic metabolic machinery.

There are three quantitative phosphoproteomic strategies most commonly used to study receptor tyrosine kinase (RTK) signaling. These strategies quantify changes in: (1) all three forms of phosphosites (phosphoserine, phosphothreonine and phosphotyrosine) following enrichment of phosphopeptides by titanium dioxide or immobilized metal affinity chromatography; (2) phosphotyrosine sites following anti- phosphotyrosine antibody enrichment of phosphotyrosine peptides; or (3) phosphotyrosine proteins and their binding partners following anti-phosphotyrosine protein immunoprecipitation. However, it is not clear from literature which strategy is more effective. In this study, we assessed the utility of these three phosphoproteomic strategies in RTK signaling studies by using EphB receptor signaling as an example. We used all three strategies with stable isotope labeling with amino acids in cell culture (SILAC) to compare changes in phosphoproteomes upon EphB receptor activation. We used bioinformatic analysis to compare results from the three analyses. Our results show that the three strategies provide complementary information about RTK pathways.

Efforts at altering the dismal prognosis of pediatric midline gliomas focus on direct delivery strategies like convection-enhanced delivery (CED), where a cannula is implanted into tumor. Successful CED treatments require confirmation of tumor coverage, dosimetry, and longitudinal in vivo pharmacokinetic monitoring. These properties would be best determined clinically with image-guided dosimetry using theranostic agents. In this study, we combine CED with novel, molecular-grade positron emission tomography (PET) imaging and show how PETobinostat, a novel PET-imageable HDAC inhibitor, is effective against DIPG models. PET data reveal that CED has significant mouse-to-mouse variability; imaging is used to modulate CED infusions to maximize tumor saturation. The use of PET-guided CED results in survival prolongation in mouse models; imaging shows the need of CED to achieve high brain concentrations. This work demonstrates how personalized image-guided drug delivery may be useful in potentiating CED-based treatment algorithms and supports a foundation for clinical translation of PETobinostat.

In Schizosaccharomyces pombe, the spore wall confers strong resistance against external stress. During meiosis II, the double-layered intracellular forespore membrane (FSM) forms de novo and encapsulates the nucleus. Eventually, the inner FSM layer becomes the plasma membrane of the spore, while the outer layer breaks down. However, the molecular mechanism and biological significance of this membrane breakdown remain unknown. Here, by genetic investigation of an S. pombe mutant (E22) with normal prespore formation but abnormal spores, we showed that Meu5, an RNA-binding protein known to bind to and stabilize more than 80 transcripts, is involved in this process. We confirmed that the E22 mutant does not produce Meu5 protein, while overexpression of meu5+ in E22 restores the sporulation defect. Furthermore, electron microscopy revealed that the outer membrane of the FSM persisted in meu5∆ spores. Investigation of the target genes of meu5+ showed that a mutant of cyc1+ encoding cytochrome c also showed a severe defect in outer FSM breakdown. Lastly, we determined that outer FSM breakdown occurs coincident with or after formation of the outermost Isp3 layer of the spore wall. Collectively, our data provide novel insights into the molecular mechanism of spore formation.

Cord blood (CB) is an attractive source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation. However, its application remains limited due to the low number of HSCs/progenitors in a single CB unit and its notoriously difficulty in expanding ex vivo. Here, we demonstrated that the human fetal liver sinusoidal endothelial cells engineered to constitutively express the adenoviral E4orf1 gene (hFLSECs-E4orf1) is capable of efficient expansion ex vivo for human CB hematopoietic stem and progenitor cells (HSPCs). Coculture of CD34+ hCB cells with hFLSECs-E4orf1 resulted in generation of substantially more total nucleated cells, CD34+CD38- and CD34+ CD38-CD90+ HSPCs in comparison with that of cytokines alone after 14 days. The multilineage differentiation potential of the expanded hematopoietic cells in coculture condition, as assessed by in vitro colony formation, was also significantly heightened. The CD34+ hCB cells amplified on hFLSECs-E4orf1 were capable of engraftment in vivo. Furthermore, hFLSECs-E4orf1 highly expressed hematopoiesis related growth factor and Notch receptors. Accordingly, the CD34+ hCB cells amplified on hFLSECs-E4orf1 exhibited Notch signaling activation. Taken together, our findings indicated that FLSECs may potentially be the crucial component of the microenvironment to support recapitulation of embryonic HSC amplification in vitro and allow identification of new growth factors responsible for collective regulation of hematopoiesis.

Delivering ions and molecules into living cells has become an important challenge in medical and biological fields. Conventional molecular delivery, however, has several issues such as physical and chemical damage to biological cells. Here, we present a method of directly delivering molecules into adhesive cells with an Au-based nanostraw membrane stamp that can physically inject a target molecule into the cytoplasm through a nanostraw duct. We successfully delivered calcein target molecules into adhesive cells with high efficiency (85%) and viability (90%). Furthermore, we modeled the molecular flow through Au nanostraws and then demonstrated the control of calcein flow by changing the concentration and geometry of Au nanostraws. Our Au membrane stamping provides a new way of accessing the cytoplasm to modulate cellular functions via injected molecules.

Long noncoding RNAs (lncRNAs) represent a group of regulatory RNAs that play critical roles in numerous cellular events, but their functional importance in development remains largely unexplored. Here, we discovered a series of previously unidentified gene clusters harboring conserved lncRNAs at the nonimprinting regions in brain (CNIBs). Among the seven identified CNIBs, human CNIB1 locus is located at Chr 9q33.3 and conserved from Danio rerio to Homo sapiens. Chr 9q33.3-9q34.11 microdeletion has previously been linked to human nail-patella syndrome (NPS) which is frequently accompanied by developmental and visual deficiencies. By generating CNIB1 deletion alleles in zebrafish, we demonstrated the requirement of CNIB1 for proper growth and development, and visual activities. Furthermore, we found that the role of CNIB1 on visual activity is mediated through a regulator of ocular development-lmx1bb. Collectively, our study shows that CNIB1 lncRNAs are important for zebrafish development and provides an lncRNA cluster-mediated pathophysiological mechanism for human Chr 9q33.3-9q34.11 microdeletion syndrome.

Chronic stress hormone norepinephrine (NE) has been previously reported to play a role in the development of cancer, but the correlation between NE and oral squamous cell carcinoma (OSCC) progression is not well understood.

Extracellular vesicles (EVs) are cell-derived membrane structures enclosing proteins, lipids, RNAs, metabolites, growth factors, and cytokines. EVs have emerged as essential intercellular communication regulators in multiple physiological and pathological processes. Previous studies revealed that mesenchymal stem cells (MSCs) could either support or suppress tumor progression in different cancers by paracrine signaling via MSC-derived EVs. Evidence suggested that MSC-derived EVs could mimic their parental cells, possessing pro-tumor and anti-tumor effects, and inherent tumor tropism. Therefore, MSC-derived EVs can be a cell-free cancer treatment alternative. This review discusses different insights regarding MSC-derived EVs' roles in cancer treatment and summarizes bioengineered MSC-derived EVs' applications as safe and versatile anti-tumor agent delivery platforms. Meanwhile, current hurdles of moving MSC-derived EVs from bench to bedside are also discussed.

Psoriasis is a chronic and inflammatory skin disorder characterized by inflammation and epidermal hyperplasia. Punicalagin (PUN) is a main active ingredient of pomegranate (Punica granatum L.) peel with multiple biological activities, such as antibacterial, antioxidant and anti-tumor effects. However, the potential effect of PUN on psoriasis remains unknown. In this study, we want to investigate the pharmacological effect of PUN on psoriasis by using imiquimod (IMQ)-induced psoriatic mice model in vivo and tumor necrosis factor a (TNF-α) and interleukin-17A (IL-17A)-stimulated HaCaT cells in vitro. Our results showed that PUN can effectively alleviate the severity of psoriasis-like symptoms. Mechanistically, PUN potently suppresses the aberrant upregulation of interleukin-1β (IL-1β) and subsequent IL-1β-mediated inflammatory cascade in keratinocytes by inhibiting the nuclear factor kappa B (NF-κB) activation and cleaved caspase-1 expression in vitro and in vivo. Taken together, our findings indicate that PUN can relieve psoriasis by repressing NF-κB-mediated IL-1β transcription and caspase-1-regulated IL-1β secretion, which provide evidence that PUN might represent a novel and promising candidate for the treatment of psoriasis.

Trained immunity refers to the long-lasting memory traits of innate immunity. Recent studies have shown that trained immunity is orchestrated by sustained changes in epigenetic marks and metabolic pathways, leading to an altered transcriptional response to a second challenge. However, the potential heterogeneity of trained-immunity induction in innate immune cells has not been explored. In this study, we demonstrate cellular transcriptional programs in response to 4 different inducers of trained immunity in monocyte populations at single-cell resolution. Specifically, we identified 3 monocyte subpopulations upon the induction of trained immunity, and replicated these findings in an in vivo study. In addition, we found gene signatures consistent with these functional programs in patients with ulcerative colitis, sepsis, and COVID-19, suggesting the impact of trained-immunity programs in immune-mediated diseases.

Tumor heterogeneity has been associated with immunotherapy and targeted drug resistance in hepatocellular carcinoma (HCC). However, communications between tumor and cytotoxic cells are poorly understood to date. In the present study, thirty-one clusters of cells were discovered in the tumor tissues and adjacent tissues through single-cell sequencing. Moreover, the quantity and function exhaustion of cytotoxic cells was observed to be induced in tumors by the TCR and apoptosis signal pathways. Furthermore, granzyme failure of cytotoxic cells was observed in HCC patients. Importantly, the GZMA secreted by cytotoxic cells was demonstrated to interact with the F2R expressed by the tumor cells both in vivo and in vitro. This interaction induced tumor suppression and T cell-mediated killing of tumor cells via the activation of the JAK2/STAT1 signaling pathway. Mechanistically, the activation of JAK2/STAT1 signaling promoted apoptosis under the mediating effect of the LDPRSFLL motif at the N-terminus of F2R, which interacted with GZMA. In addition, GZMA and F2R were positively correlated with PD-1 and PD-L1 in tumor tissues, while the expressions of F2R and GZMA promoted PD-1 mAb-induced tumor suppression in both mouse model and HCC patients. Finally, in HCC patients, a low expression of GZMA and F2R in the tumor tissues was correlated with aggressive clinicopathological characteristics and poor prognosis. Collectively, GZMA-F2R communication inefficient induces deficient PD-1 mAb therapy and provide a completely novel immunotherapy strategy for tumor suppression in HCC patients.

Single-stage type I hybrid total aortic arch repair is a surgical treatment for extensive aortic arch disease, but the clinical outcomes were distinguishing. The purposes of this study were to share our experience and evaluate the perioperative safety and long-term durability.

In this study, rod-like hydroxyapatite (HA) with uniform morphology and controllable particle size modified by doping with two different amino acids (alanine and threonine) was synthesized by a microwave hydrothermal method. The physical and chemical properties of the composites were tested by utilizing X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), general thermogravimetric analysis (TG) and scanning electron microscopy (SEM). The SEM and XRD results show that the presence of amino acids (especially threonine) can significantly reduce the aspect ratio and crystallinity of hydroxyapatite. Pure hydroxyapatite and modified hydroxyapatite doped with two different proportions of amino acids were cultured with mouse osteoblasts (MC3T3-E1) for 1, 3 and 5 days, respectively, nanohydroxyapatite modified by threonine has better biocompatibility compared with pure hydroxyapatite. The amino acid-modified hydroxyapatite samples were co-cultured with osteosarcoma cells (MG63) for 1, 4 and 7 days, respectively, and showed better inhibitory effects on osteosarcoma cells. The nanohydroxyapatite doped with amino acids could be used as a potential drug that promotes bone repair and inhibits the growth of osteosarcoma cells.

This study presents a chromosome-level reference genome assembly of a traditional Tibetan medicinal plant, Herpetospermum pedunculosum belonging to the Cucurbitaceae family. Following a combined PacBio high-fidelity sequencing and Hi-C analysis, a final H. pedunculosum genome assembly, 804.11 Mb in length was obtained, 90.45% of which was anchored into ten pseudochromosomes with a contig N50 of 24.39 Mb. In addition, 579.55 Mb repetitive sequences and 23,924 high-confidence protein-coding genes were annotated. Phylogenetic analysis revealed that H. pedunculosum was sister to a clade formed by cucumber, zucchini, and wax gourd. Further whole-genome duplication analysis revealed no recent polyploidization event in the H. pedunculosum genome. The high-quality H. pedunculosum genome presented here will be highly useful in investigating the molecular mechanisms underlying the biosynthesis of its active compounds and adaptation strategies to the extreme environment. It will also provide great insights into comparative genomic studies of Cucurbitaceae and flowering plants.

Osteosarcoma (OS) patients have a poor response to immunotherapy due to the sheer complexity of the immune system and the nuances of the tumor-immune microenvironment. Methodology. To gain insights into the immune heterogeneity of OS, we identified robust clusters of patients based on the immune gene expression profiles of OS patients in the TARGET database and assessed their reproducibility in an independent cohort collected from the GEO database. The association of comprehensive molecular characterization with reproducible immune subtypes was accessed with ANOVA. Furthermore, we visualized the distribution of individual patients in a tree structure by the graph structure learning-based dimensionality reduction algorithm.

Background: Stanford type A aortic dissection (AAD) is a catastrophic disease. An immune infiltrate has been found within the aortic wall of dissected aortic specimens. The recall and activation of macrophages are key events in the early phases of AAD. Herein, the immune filtration profile of AAD was uncovered. Methods: Gene expression data from the GSE52093, GSE98770 and GSE153434 datasets were downloaded from the Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) of each dataset were calculated and then integrated. A protein-protein interaction (PPI) network was established with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), and the hub genes were identified in Cytoscape. Furthermore, gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of hub genes were performed. Finally, we set GSE52093 and GSE98770 as the training set and GSE153434 as the validation set to assess immune infiltration in AAD using CIBERSORTx and analyzed the correlations between immune cells and hub genes in both the training and validation sets. Results: Sixty-one integrated DEGs were identified. The top 10 hub genes were selected from the PPI network, and 140 biological process (BP) terms and 12 pathways were enriched among the top 10 hub genes. The proportions of monocytes and macrophages were significantly higher in AAD tissues than in normal tissues. Notably, this result was consistent in the training set and the validation set. In addition, we found that among the hub genes, CA9, CXCL5, GDF15, VEGFA, CCL20, HMOX1, and SPP1 were positively correlated with CD14, a cell marker of monocytes, while CA9, CXCL5, GDF15, and VEGFA were positively correlated with CD68, a cell marker of macrophages in the training set. Finally, according to the results of the GO and KEGG analysis of hub genes, we found that the monocyte/macrophage-related genes were involved in immune-inflammatory responses through degradation of the extracellular matrix, endothelial cell apoptosis, hypoxia and the interaction of cytokines and chemokines. Conclusion: The monocyte-macrophage system plays a major role in immune-inflammatory responses in the development of AAD. Several hub genes are involved in this process via diverse mechanisms.