Angiogenesis-related immune response may be the prelude to the syndesmophyte formation in Ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic autoimmune arthritis that mainly affects spine joints. To date, the pathogenesis of AS remains unclear, although immune cells and innate immune response cytokines have been suggested to be crucial players. METHODS: By adopting a single-cell RNA sequencing approach in the AS cynomolgus model, we profiled and characterized PBMC proportions along disease progression. RESULTS: Here, our primary focus was on the activation of an immune cascade-initiating lymphocyte subtype known as CD4+CXCR5+ T follicular helper (Tfh) cells. These Tfhs demonstrated a localized residence in AS bone lesion as an ectopic lymphoid structure. Moreover, Tfhs would serve as an upstream initiator for a pro-angiogenic cascade. Then, an expansion in CD14+ monocytes and DC cells subsets resulted in enhanced expression of angiogenesis genes in these AS cynomolgus monkeys. With a confirmed higher abundance of TNF-α accompanying H-type vascular invasion in the osteophytic region, pronounced expansion of Tfhs at such lesion site signaling for monocytes and DCs intrusion is considered as the prelude to the characteristic angiogenic bony outgrowth in AS known as syndesmophytes. CONCLUSIONS: We explored the intimate relationship between local inflammation and bone formation in AS from the perspective of nascent vascularisation. Hence, our study lays the foundation for elucidating a unified AS pathogenesis through the immune-angiogenesis-osteogenesis axis.

Outlook of PINK1/Parkin signaling in molecular etiology of Parkinson's disease, with insights into Pink1 knockout models.

Parkinson's disease (PD) relates to defective mitochondrial quality control in the dopaminergic motor network. Genetic studies have revealed that PINK1 and Parkin mutations are indicative of a heightened propensity to PD onset, pinpointing mitophagy and inflammation as the culprit pathways involved in neuronal loss in the substantia nigra (SNpc). In a reciprocal manner, LRRK2 functions in the regulation of basal flux and inflammatory responses responsible for PINK1/Parkin-dependent mitophagy activation. Pharmacological intervention in these disease-modifying pathways may facilitate the development of novel PD therapeutics, despite the current lack of an established drug evaluation model. As such, we reviewed the feasibility of employing the versatile global Pink1 knockout (KO) rat model as a self-sufficient, spontaneous PD model for investigating both disease etiology and drug pharmacology. These rats retain clinical features encompassing basal mitophagic flux changes with PD progression. We demonstrate the versatility of this PD rat model based on the incorporation of additional experimental insults to recapitulate the proinflammatory responses observed in PD patients.

Machine Learning-Devised Immune-Related lncRNA Signature Panel Predicts the Prognosis and Immune Landscape in Breast Cancer Novel IRLP Signature in BRCA.

Long noncoding RNAs (lncRNAs) actively participate in breast cancer (BRCA) tumorigenesis via epigenetic mechanisms. Our study identified immune-related lncRNA (irlncRNA) pairs and compiled them into a set of noncoding gene signatures able to stratify subtypes of BRCA associated with variable degrees of survival and immune cell infiltration. A 40 immune-related lncRNA pair (IRLP) signature including 43 irlncRNAs was built, with high sensitivity and specificity for the prediction of survival in different molecular subtypes of BRCA. Results demonstrated that the low-risk group showed a significantly longer survival rate, and this novel IRLP signature was highly associated with survival status, T stage, metastatic disease, and overall stage in BRCA. Immune infiltrating analyses found that the low-risk group has a lower expression level of macrophage M2 and a higher expression level of immunosuppressed biomarkers than the high-risk group. DEirlncRNAs were further proven to be significantly related to the MAPK signaling, Jak-STAT signaling, and ErbB signaling pathways in BRCA. In conclusion, the 40 IRLP signature showed a promising clinical prediction value in the prognosis of different molecular subtypes and immunotherapy response in BRCA, and the underlying mechanism for these IRLPs warrants further investigations.

Single-Nucleotide Variations, Insertions/Deletions and Copy Number Variations in Myelodysplastic Syndrome during Disease Progression Revealed by a Single-Cell DNA Sequencing Platform.

Myelodysplastic syndrome (MDS) is a clonal myeloid neoplasm characterized by ineffective hematopoiesis, cytopenia, dysplasia, and clonal instability, leading to leukemic transformation. Hypomethylating agents are the mainstay of treatment in higher-risk MDS. However, treatment resistance and disease transformation into acute myeloid leukemia (AML) is observed in the majority of patients and is indicative of a dismal outcome. The residual cell clones resistant to therapy or cell clones acquiring new genetic aberrations are two of the key events responsible for drug resistance. Bulk tumor sequencing often fails to detect these rare subclones that confer resistance to therapy. In this study, we employed a single-cell DNA (sc-DNA) sequencing approach to study the clonal heterogeneity and clonal evolution in two MDS patients refractory to HMA. In both patients, different single nucleotide variations (SNVs) or insertions and deletions (INDELs) were detected with bulk tumor sequencing. Rare cell clones with mutations that are undetectable by bulk tumor sequencing were detected by sc-DNA sequencing. In addition to SNVs and short INDELs, this study also revealed the presence of a clonal copy number loss of DNMT3A, TET2, and GATA2 as standalone events or in association with the small SNVs or INDELs detected during HMA resistance and disease progression.

Epigenetic Silencing of PTEN and Epi-Transcriptional Silencing of MDM2 Underlied Progression to Secondary Acute Myeloid Leukemia in Myelodysplastic Syndrome Treated with Hypomethylating Agents.

In myelodysplastic syndrome (MDS), resistance to hypomethylating agents (HMA) portends a poor prognosis, underscoring the importance of understanding the molecular mechanisms leading to HMA-resistance. In this study, P39 and Kasumi-1 cells and their azacitidine-resistant and decitabine-resistant sublines were evaluated comparatively with transcriptomic and methylomic analyses. Expression profiling and genome-wide methylation microarray showed downregulation of PTEN associated with DNA hypermethylation in P39 cell lines resistant to azacitidine and decitabine. This pattern of PTEN dysregulation was also confirmed in a cohort of patients failing treatment with HMA. DNA hypomethylation of MDM2 was detected with downregulation of MDM2 in HMA resistant cell lines. Long-read sequencing revealed significant RNA hypomethylation of MDM2 resulting in alternative splicing and production of a truncated MDM2 transcript in azacitidine-resistant P39 cells. The expression of this MDM2 truncated transcript was also significantly increased in HMA-resistant patients compared with HMA-responsive patients. In conclusion, epigenetic and epi-transcriptomic dysregulation of PTEN and MDM2 were associated with resistance to hypomethylating agents.

Nrf2-mediated liver protection by 18ß-glycyrrhetinic acid against pyrrolizidine alkaloid-induced toxicity through PI3K/Akt/GSK3ß pathway.

BACKGROUND: Misusage of pyrrolizidine alkaloid (PA)-containing plants or unaware intake of PA-contaminated foodstuffs causes thousands of PA poisoning cases in humans. PA intoxication is accompanied by oxidative stress and subsequent extensive hepatocellular damage. Our previous study has demonstrated that 18ß-glycyrrhetinic acid (GA), a bioactive constituent of liquorice, prevented PA-induced hepatotoxicity in rats, however the underlying mechanisms remain unclear. OBJECTIVE: This study aims to explore the mechanisms underlying the hepato-protective effect of GA in combating retrorsine (RTS, a representative toxic PA)-induced liver injury. METHODS: Histological and biochemical assessments were employed to evaluate the protective effect of GA on RTS-induced hepatotoxicity in rats. Sulforhodamine B assay, real-time PCR, western blotting, and immunostaining were used to explore the underlying mechanisms in human hepatocytes and rats. RESULTS: Our findings demonstrated that GA alleviated RTS-induced elevation of serum ALT and bilirubin levels, as well as hepatocytes necrosis and sinusoidal endothelial cells (SECs) damage in rats. GA also enhanced the activities and expressions of several antioxidant enzymes through upregulating nuclear factor-erythroid 2-related factor2 (Nrf2). Moreover, inhibition of Nrf2 blocked the hepatoprotective effect of GA against RTS intoxication. Mechanistically, GA increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and enhanced glycogen synthase kinase 3 beta (GSK3ß) inhibitory phosphorylation at serine 9, thus promoting the nuclear accumulation of Nrf2 and activating its downstream targets. CONCLUSION: This study for the first time demonstrated that GA exerted protective effects against RTS-induced liver injury by potentiating the Nrf2-mediated antioxidant system through PI3K/Akt/GSK3ß pathway. The findings indicated that GA may serve as a potential candidate drug for the treatment of PA intoxication.

Liquorice Extract and 18ß-Glycyrrhetinic Acid Protect Against Experimental Pyrrolizidine Alkaloid-Induced Hepatotoxicity in Rats Through Inhibiting Cytochrome P450-Mediated Metabolic Activation.

Misuse of pyrrolizidine alkaloid (PA)-containing plants or consumption of PA-contaminated foodstuffs causes numerous poisoning cases in humans yearly, while effective therapeutic strategies are still limited. PA-induced liver injury was initiated by cytochrome P450 (CYP)-mediated metabolic activation and subsequent formation of adducts with cellular proteins. Liquorice, a hepato-protective herbal medicine, is commonly used concurrently with PA-containing herbs in many compound traditional Chinese medicine formulas, and no PA-poisoning cases have been reported with this combination. The present study aimed to investigate hepato-protective effects of liquorice aqueous extract (EX) and 18ß-glycyrrhetinic acid (GA, the primary bioactive constituent of liquorice) against PA-induced hepatotoxicity and the underlying mechanism. Histopathological and biochemical analysis demonstrated that both single- and multiple-treatment of EX (500 mg/kg) or GA (50 mg/kg) significantly attenuated liver damage caused by retrorsine (RTS, a representative hepatotoxic PA). The formation of pyrrole-protein adducts was significantly reduced by single- (30.3% reduction in liver; 50.8% reduction in plasma) and multiple- (32.5% reduction in liver; 56.5% reduction in plasma) treatment of GA in rats. Single- and multiple-treatment of EX also decreased the formation of pyrrole-protein adducts, with 30.2 and 31.1% reduction in rat liver and 51.8 and 53.1% reduction in rat plasma, respectively. In addition, in vitro metabolism assay with rat liver microsomes demonstrated that GA reduced the formation of metabolic activation-derived pyrrole-glutathione conjugate in a dose-dependent manner with the estimated IC50 value of 5.07 µM. Further mechanism study showed that GA inhibited activities of CYPs, especially CYP3A1, the major CYP isoform responsible for the metabolic activation of RTS in rats. Enzymatic kinetic study revealed a competitive inhibition of rat CYP3A1 by GA. In conclusion, our findings demonstrated that both EX and GA exhibited significant hepato-protective effects against RTS-induced hepatotoxicity, mainly through the competitive inhibition of CYP-mediated metabolic activation of RTS.

A new and spontaneous animal model for ankylosing spondylitis is found in cynomolgus monkeys.

BACKGROUND: Ankylosing spondylitis is a progressive, disabling joint disease that affects millions worldwide. Given its unclear etiology, studies of ankylosing spondylitis relied heavily on drug-induced or transgenic rodent models which retain only partial clinical features. There is obviously a lack of a useful disease model to conduct comprehensive mechanistic studies. METHODS: We followed a group of cynomolgus monkeys having joint lesions reported of spinal stiffness for 2 years by conducting hematological testing, radiographic examination, family aggregation analysis, pathological analysis, and genetic testing. RESULTS: The results confirmed that these diseased animals suffered from spontaneous ankylosing spondylitis with clinical features recapitulating human ankylosing spondylitis disease progression, manifested by pathological changes and biochemical indicators similar to that of ankylosing spondylitis patients. CONCLUSION: The study offers a promising non-human primate model for spontaneous ankylosing spondylitis which may serve as an excellent substitute for its pre-clinical research.

Genome-wide 5-hydroxymethylcytosine (5hmC) reassigned in Pten-depleted mESCs along neural differentiation.

DNA methylation and hydroxymethylation have been implicated in the regulatory dynamics of gene expression in normal development and differentiation. 5-Hydroxymethylcytosine (5hmC), created by the ten-eleven translocation (TET) protein-catalyzed oxidation of 5-methylcytosine (5mC), is abundant in the brain, but the genome-wide distribution and impact of 5hmC during diverse neuronal differentiation remain unknown. Here, we used an in vitro model to differentiate mouse embryonic stem cells (mESCs) into ventral midbrain and hindbrain neural progenitors, followed by characterizing global 5hmC distribution using a nano-5hmC-seal approach. The 5hmC pattern was dynamic in promoter, exon, and enhancer regions, associated with gene activation and repression. For example, ventral midbrain markers (Lmx1a, Otx2, and Th) and hindbrain markers (Hoxa1, Zic1, and Tph1) acquire 5hmC and are upregulated during differentiation. Among the differentially expressed genes involved in both midbrain and hindbrain lineage commitment, phosphatase and tensin homolog (Pten) was identified as a key regulator for neuronal development. We confirmed that Pten knockout disrupted the normal differentiation of midbrain/hindbrain neural progenitors, resulting in immature neurons. In addition, 5421 and 4624 differentially hydroxymethylated regions (DhMRs) were identified in the differentiation of Pten-/- mESC into ventral midbrain and hindbrain progenitors, respectively. Gene ontology analysis showed that the majority of these DhMRs were associated with neurogenesis, ectoderm development, and signal transduction. Moreover, further combinational analysis of the 5hmC pattern and transcriptomic profile in the midbrain progenitor cells demonstrated Pten as a toggle to modulate mitochondrial associated pathways. Therefore, our findings elucidated the molecular mechanisms underlying lineage-specific differentiation of pluripotent stem cells to the midbrain/hindbrain progenitors, where Pten participates as one key regulator.

A Core Omnigenic Non-coding Trait Governing Dex-Induced Osteoporotic Effects Identified Without DEXA.

Iatrogenic glucocorticoid (GC)-induced osteoporosis (GIO) is an idiosyncratic form of secondary osteoporosis. Genetic predisposition among individuals may give rise to variant degree of phenotypic changes but there has yet been a documented unified pathway to explain the idiosyncrasy. In this study, we argue that the susceptibility to epigenetic changes governing molecular cross talks along the BMP and PI3K/Akt pathway may underline how genetic background dictate GC-induced bone loss. Concordantly, osteoblasts from BALB/c or C57BL/6 neonatal mice were treated with dexamethasone for transcriptome profiling. Furthermore, we also confirmed that GC-pre-conditioned mesenchymal stem cells (MSCs) would give rise to defective osteogenesis by instigating epigenetic changes which affected the accessibility of enhancer marks. In line with these epigenetic changes, we propose that GC modulates a key regulatory network involving the scavenger receptor Cd36 in osteoblasts pre-conditioning pharmacological idiosyncrasy in GIO.

Hepatic miR-192-3p reactivation alleviates steatosis by targeting glucocorticoid receptor.

BACKGROUND & AIMS: The paradox of hepatic insulin resistance describes the inability for liver to respond to bioenergetics hormones in suppressing gluconeogenesis whilst maintaining lipid synthesis. Here, we report the deficiency of miR-192-3p in the livers of mice with diabetes and its role in alleviating hepatic steatosis. METHODS: As conventional pre-microRNA (miRNA) stem-loop overexpression only boosts guiding strand (i.e. miR-192-5p) expression, we adopted an artificial AAV(DJ)-directed, RNA Pol III promoter-driven miRNA hairpin construct for star-strand-specific overexpression in the liver. Liver steatosis and insulin resistance markers were evaluated in primary hepatocytes, mice with diabetes, and mice with excessive carbohydrate consumption. RESULTS: Functional loss of miR-192-3p in liver exacerbated hepatic micro-vesicular steatosis and insulin resistance in either mice with diabetes or wild-type mice with excessive fructose consumption. Liver-specific overexpression of miR-192-3p effectively halted hepatic steatosis and ameliorated insulin resistance in these mice models. Likewise, hepatocytes overexpressing miR-192-3p exhibited improved lipid accumulation, accompanied with decreases in lipogenesis and lipid-accumulation-related transcripts. Mechanistically, glucocorticoid receptor (GCR, also known as nuclear receptor subfamily 3, group C, member 1 [NR3C1]) was demonstrated to be negatively regulated by miR-192-3p. The effect of miR-192-3p on mitigating micro-vesicular steatosis was ablated by the reactivation of NR3C1. CONCLUSIONS: The star strand miR-192-3p was an undermined glycerolipid regulator involved in controlling fat accumulation and insulin sensitivity in liver through blockade of hepatic GCR signalling; this miRNA may serve as a potential therapeutic option for the common co-mobility of diabetic mellitus and fatty liver disease. LAY SUMMARY: The potential regulatory activity of star strand microRNA (miRNA) species has been substantially underestimated. In this study, we investigate the role and mechanism of an overlooked star strand miRNA (miR-192-3p) in regulating hepatic steatosis and insulin signalling in the livers of mice with diabetes and mice under excessive carbohydrate consumption.

Inhibited Maternal Bone Resorption Suppress Fetal Rat Bone Development During Pregnancy.

OBJECTIVE: To determine the relationship between maternal bone resorption and bone development in fetuses. METHODS: Female SD rats were injected with either fluorescent calcium indicator calcein alone or together with tetracycline 1 week before pregnancy, followed by fluorescence detection in fetal tibias 21 days post-treatment. Alendronate was subsequently administered to pregnant rats to inhibit maternal bone resorption, while maternal bone turnover and fetal bone development were both examined. RESULTS: The maternal fluorescent labeled calcium before pregnancy was found in the fetal tibia. This indicated that the calcium of maternal bones may be released into the maternal circulation through high bone resorption during pregnancy, thereby participating in the fetal bone development. Bone histomorphometry and serum biomarker results showed that Alendronate significantly inhibited maternal bone resorption in pregnant rats when compared to normal pregnant rats. Moreover, the body weight, bone mass, and bone length of the fetuses in the Alendronate group were significantly decreased; while no apparent abnormality in placental morphology was observed. The above results implied that when maternal bone resorption is suppressed, the development of the fetal bone shall also be suppressed. CONCLUSION: Calcium in the maternal bone is released into the maternal circulation through bone resorption during pregnancy which represents an important material source in fetal bone development. Therefore, high bone turnover during pregnancy is essential for mammalian embryonic bone development.

Comparison of multi-lineage differentiation of hiPSCs reveals novel miRNAs that regulate lineage specification.

MicroRNAs (miRNAs) are known to be crucial players in governing the differentiation of human induced pluripotent stem cells (hiPSCs). Despite their utter importance, identifying key lineage specifiers among the myriads of expressed miRNAs remains challenging. We believe that the current practice in mining miRNA specifiers via delineating dynamic fold-changes only is inadequate. Our study, therefore, provides evidence to pronounce "lineage specificity" as another important attribute to qualify for these lineage specifiers. Adopted hiPSCs were differentiated into representative lineages (hepatic, nephric and neuronal) over all three germ layers whilst the depicted miRNA expression changes compiled into an integrated atlas. We demonstrated inter-lineage analysis shall aid in the identification of key miRNAs with lineage-specificity, while these shortlisted candidates were collectively known as "lineage-specific miRNAs". Subsequently, we followed through the fold-changes along differentiation via computational analysis to identify miR-192 and miR-372-3p, respectively, as representative candidate key miRNAs for the hepatic and nephric lineages. Indeed, functional characterization validated that miR-192 and miR-372-3p regulate lineage differentiation via modulation of the expressions of lineage-specific genes. In summary, our presented miRNA atlas is a resourceful ore for the mining of key miRNAs responsible for lineage specification.

New insights into the unfolded protein response in stem cells.

The unfolded protein response (UPR) is an evolutionarily conserved adaptive mechanism to increase cell survival under endoplasmic reticulum (ER) stress conditions. The UPR is critical for maintaining cell homeostasis under physiological and pathological conditions. The vital functions of the UPR in development, metabolism and immunity have been demonstrated in several cell types. UPR dysfunction activates a variety of pathologies, including cancer, inflammation, neurodegenerative disease, metabolic disease and immune disease. Stem cells with the special ability to self-renew and differentiate into various somatic cells have been demonstrated to be present in multiple tissues. These cells are involved in development, tissue renewal and certain disease processes. Although the role and regulation of the UPR in somatic cells has been widely reported, the function of the UPR in stem cells is not fully known, and the roles and functions of the UPR are dependent on the stem cell type. Therefore, in this article, the potential significances of the UPR in stem cells, including embryonic stem cells, tissue stem cells, cancer stem cells and induced pluripotent cells, are comprehensively reviewed. This review aims to provide novel insights regarding the mechanisms associated with stem cell differentiation and cancer pathology.