Most migraineurs develop cutaneous allodynia (CA) during migraine, and the underlying mechanism of CA in migraine is thought to be sensitization of the third-order trigeminovascular neurons in the posterior thalamic nuclei. This study aimed to investigate whether the ascending/descending pathway associated with the thalamus is disturbed in migraineurs with CA (MWCA) using effective connectivity analysis of resting-state functional magnetic resonance imaging.

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, and its expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 in vitro and in vivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.

Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency.

β-glucan (BG) and mulberry have received increasing attention for their benefits as natural sources of metabolic health. In the current study, we investigated the synergetic beneficial effects of BG and mulberry leaf extract (MLE) in mice fed a high-fat diet (HFD). Male C57BL6 mice were fed a HFD for twelve weeks to induce significant obesity and insulin resistance. BG and MLE were administrated orally throughout the feeding period. The administration of BG resulted in a significant reduction in body weight gain, perirenal fat mass, fasting insulin, serum lipids, serum inflammation markers, and fatty liver, showing systemic health improvement. Likewise, the administration of MLE showed benefits similar to BG, with the exception of body weight gain. In addition to the systemic benefits, the combination of BG and MLE resulted in a synergetic improvement in insulin sensitivity. Meanwhile, only the combination of BG and MLE significantly enhanced liver GST (Glutathione S-Transferase) activity and CuZn-SOD (Superoxide dismutase (Cu-Zn)) activity, resulting in a significant reduction in GSH/GSSG (Glutathione disulfide) and reactive oxygen species (ROS) levels in the liver. These results further confirm the beneficial effects of BG and MLE on metabolic disorders and show that the combination of BG and MLE has synergetic effects.

Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)-mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly, depletion or inhibition of CTSL with vitamin D or specific inhibitors stabilized 53BP1 and increased genomic instability in response to radiation and poly(adenosine diphosphate-ribose) polymerase inhibitors, compromising proliferation. Analysis of human breast tumors identified nuclear CTSL as a positive biomarker for TNBC, which correlated inversely with 53BP1. Importantly, nuclear levels of CTSL, vitamin D receptor, and 53BP1 emerged as a novel triple biomarker signature for stratification of patients with BRCA1-mutated tumors and TNBC, with potential predictive value for drug response. We identify here a novel pathway with prospective relevance for diagnosis and customization of breast cancer therapy.

High-grade serous carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP inhibitors (PARPi) have become the mainstay of HGSC-targeted therapy, given that these tumors are driven by a high degree of genomic instability (GI) and homologous recombination (HR) defects. Nonetheless, approximately 30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and an increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a small-molecule activator of protein phosphatase 2A (PP2A; SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in patient-derived HGSC cells and xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for cellular transformation (B56α, B56γ, and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061-induced stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and HR-deficient HGSC cells and PDX models. Our studies identify PP2A as a novel regulator of HR and indicate PP2A modulators as a therapeutic therapy for HGSC. In summary, our findings further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 presents benefits in overcoming PARPi resistance driven by BRCA1/2 mutation reversions.

Ionizing radiation (IR) can induce DNA double-strand breaks (DSBs) in tumor cells during radiotherapy (RT), but the efficiency of RT is limited because of the toxicity to normal cells. Locating an adjuvant treatment to alleviate damage in normal cells while sensitizing tumor cells to IR has attracted much attention. Here, using the 7,12-dimethylbenz[α]anthracene (DMBA)-induced malignant transformed MCF10A cells, we found that valproate (VPA), a histone deacetylase inhibitor (HDACi), radiosensitized transformed cells while alleviated IR-induced damage in normal cells at a safe dose (0.5 mM). We further demonstrated the decrease of homologous recombination (HR)-associated Rad51 in the transformed cells was related to the increase of its ubiquitination regulated by E3 ligase RFWD3 for the radiosensitization, which was opposite to normal cells, indicating that RFWD3-dependent ubiquitination on Rad51 was involved in the VPA-mediated radio-bidirectional effect. Through DMBA-transformed breast cancer rat model, VPA at 200 mg/kg radiosensitized tumor tissue cells by increasing RFWD3 and inhibited Rad51, while radioprotected normal tissue cells by decreasing RFWD3 and enhanced Rad51. In addition, we found high-level Rad51 was associated with tumorigenesis and poor prognosis in breast cancer patients. Our findings uncovered RFWD3-dependent Rad51 ubiquitination was the novel mechanism of VPA-mediated radio-bidirectional effect, VPA is a potential adjuvant treatment for tumor RT.

Dental pulp inflammation is a common bacterially driven inflammation characterized by the local accumulation of inflammatory mediators in human dental pulp. DNA methylation is a crucial epigenetic modification that that plays a fundamental role in gene transcription, and its role in inflammation-related diseases has recently attracted attention. However, its role in dental pulp inflammation is poorly understood. This study is aimed to elucidate the role of DNA methylation in lipopolysaccharide (LPS)-induced inflammatory reaction in human dental pulp cells (hDPCs). hDPCs were pretreated with DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and a cytokine antibody array was used to detect LPS-induced cytokine expression. The results indicated that 5-Aza-CdR significantly increased the expression of several pro-inflammatory cytokines in LPS-treated cells, including IL-6, IL-8, GM-CSF, MCP-2 and RANTES. The increased expression levels of IL-6 and IL-8 were further verified by qRT-PCR and ELISA. Furthermore, pretreatment with 5-Aza-CdR resulted in upregulation of p-IKKα/β, p-IκBα, p-p65 and p-ERK in the NK-κB and MAPK pathways. In addition, the 5mC level of the TRAF6 promoter was significantly decreased following 5-Aza-CdR pretreatment in the LPS-stimulated hDPCs. The findings indicate that 5-Aza-CdR significantly enhances the expression of proinflammatory cytokines and activates the NF-κB and MAPK signaling pathways by eliciting a decline in the 5mc level in the TRAF6 promoter in hDPCs, suggesting that DNA methylation may play an important role in dental pulp inflammation. This study highlights the important role of DNA methylation in the immunity defense of dental pulp infection.

Osteoclast differentiation and function are crucial for maintaining bone homeostasis and preserving skeletal integrity. N6-methyladenosine (m6A) is an abundant mRNA modification that has recently been shown to be important in regulating cell lineage differentiation. Nevertheless, the effect of m6A on osteoclast differentiation remains unknown. In the present study, we observed that the m6A level and methyltransferase METTL3 expression increased during osteoclast differentiation. Mettl3 knockdown resulted in an increased size but a decreased bone-resorbing ability of osteoclasts. The expression of osteoclast-specific genes (Nfatc1, c-Fos, Ctsk, Acp5 and Dcstamp) was inhibited by Mettl3 depletion, while the expression of the cellular fusion-specific gene Atp6v0d2 was upregulated. Mechanistically, Mettl3 knockdown elevated the mRNA stability of Atp6v0d2 and the same result was obtained when the m6A-binding protein YTHDF2 was silenced. Moreover, the phosphorylation levels of key molecules in the MAPK, NF-κB and PI3K-AKT signaling pathways were reduced upon Mettl3 deficiency. Depletion of Mettl3 maintained the retention of Traf6 mRNA in the nucleus and reduced the protein levels of TRAF6. Taken together, our data suggest that METTL3 regulates osteoclast differentiation and function through different mechanisms involving Atp6v0d2 mRNA degradation mediated by YTHDF2 and Traf6 mRNA nuclear export. These findings elucidate the molecular basis of RNA epigenetic regulation in osteoclast development.

DNA lesion bypass facilitates DNA synthesis across bulky DNA lesions, playing a critical role in DNA damage tolerance and cell survival after DNA damage. Assessing lesion bypass efficiency in the cell is important to better understanding of the mechanism of carcinogenesis and chemoresistance. Here we developed a chromatin immunoprecipitation (ChIP)-based method to measure lesion bypass activity across cisplatin-induced intrastrand crosslinks in cancer cells. DNA lesion bypass enables the replication to continue in the presence of replication blocks. Thus, the successful lesion bypass should result in the coexistence of DNA lesions and the newly synthesized DNA fragment opposite to this lesion. Using ChIP, we precipitated the cisplatin-induced intrastrand crosslinks, and quantitated the precipitated newly synthesized DNA that was labeled with BrdU. We validated this method on ovarian cancer cells with inhibited TLS activity. We then applied this method to show that ovarian cancer stem cells exhibit high lesion bypass activity relative to bulk cancer cells from the same cell line. In conclusion, this novel ChIP-based lesion bypass assay can detect the extent to which cisplatin-induced DNA lesions are bypassed in live cells. Our study may be applied more broadly to the study of other DNA lesions, as specific antibodies to these specific lesions are available.

Poly (ADP-ribose) Polymerase (PARP) inhibitors (PARPi) have been approved for both frontline and recurrent setting in ovarian cancer with homologous recombination (HR) repair deficiency. However, more than 40% of BRCA1/2-mutated ovarian cancer lack the initial response to PARPi treatment, and the majority of those that initially respond eventually develop resistance. Our previous study has demonstrated that increased expression of aldehyde dehydrogenase 1A1 (ALDH1A1) contributes to PARPi resistance in BRCA2-mutated ovarian cancer cells by enhancing microhomology-mediated end joining (MMEJ) but the mechanism remains unknown. Here, we find that ALDH1A1 enhances the expression of DNA polymerase θ (Polθ, encoded by the POLQ gene) in ovarian cancer cells. Furthermore, we demonstrate that the retinoic acid (RA) pathway is involved in the transcription activation of the POLQ gene. The RA receptor (RAR) can bind to the retinoic acid response element (RARE) located in the promoter of the POLQ gene, promoting transcription activation-related histone modification in the presence of RA. Given that ALDH1A1 catalyzes the biosynthesis of RA, we conclude that ALDH1A1 promotes POLQ expression via the activation of the RA signaling pathway. Finally, using a clinically-relevant patient-derived organoid (PDO) model, we find that ALDH1A1 inhibition by the pharmacological inhibitor NCT-505 in combination with the PARP inhibitor olaparib synergistically reduce the cell viability of PDOs carrying BRCA1/2 mutation and positive ALDH1A1 expression. In summary, our study elucidates a new mechanism contributing to PARPi resistance in HR-deficient ovarian cancer and shows the therapeutic potential of combining PARPi and ALDH1A1 inhibition in treating these patients.

Physical exercise is considered to exert a positive effect on health, whereas strenuous or excessive exercise (Exe) causes fatigue and damage to muscle and immune functions. The underlying molecular mechanisms are still unclear. We designed a protocol to mimic Exe and explore the ensuing cellular damage and involvement of mitochondrial dynamics. We found that Exe was prone to decrease endurance capacity and induce damage to renal function and the immune system. Muscle atrophy markers atrogin-1 and MuRF1 mRNA were increased by Exe, accompanied by increased autophagy and mitochondrial fission in skeletal muscle. Exe caused a decrease in PGC-1α and complex I expression; it also activated JNK and Erk1/2 pathways and consequently induced p53, p21, and MnSOD expression in skeletal muscle. The involvement of oxidant-induced autophagy and mitochondrial dysfunction was confirmed in C2C12 myoblasts. Hydroxytyrosol (HT), a natural olive polyphenol, efficiently enhanced endurance capacity and prevented Exe-induced renal and immune system damage. Also, HT treatment inhibited both the Exe-induced increase in autophagy and mitochondrial fission and the decrease in PGC-1α expression. In addition, HT enhanced mitochondrial fusion and mitochondrial complex I and II activities in muscle of Exe rats. These results demonstrate that Exe-induced fatigue and damage to muscle and immune functions may be mediated via the regulation of mitochondrial dynamic remodeling, including the downregulation of mitochondrial biogenesis and upregulation of autophagy. HT supplementation may regulate mitochondrial dynamic remodeling and enhance antioxidant defenses and thus improve exercise capacity under Exe conditions.

Prenatal stress induces cognitive functional impairment in offspring, an eventuality in which mitochondrial dysfunction and oxidative stress are believed to be closely involved. In this study, the involvement of the AMP-activated protein kinase (AMPK) pathway was investigated. A well-known activator, resveratrol (Res), was used to induce AMPK activation in SH-SY-5Y cells. Significant mitochondrial biogenesis and phase II enzyme activation, accompanied by decreased protein oxidation and GSSG content, were observed after Res treatment, and inhibition of AMPK with Compound c abolished the induction effects of Res. Further study utilizing a prenatal restraint stress (PRS) animal model indicated that maternal supplementation of Res may activate AMPK in the hippocampi of both male and female offspring, and that PRS-induced mitochondrial loss in the offspring hippocampus was inhibited by Res maternal supplementation. In addition, Res activated Nrf2-mediated phase II enzymes and reduced PRS-induced oxidative damage in both male and female offspring. Moreover, PRS markedly decreased mRNA levels of various neuron markers, as well as resultant offspring cognitive function, based on spontaneous alternation performance and Morris water maze tests, the results of which were significantly improved by maternal Res supplementation. Our results provide evidence indicating that AMPK may modulate mitochondrial content and phase II enzymes in neuronal cells, a process which may play an essential role in preventing PRS-induced cognitive impairment. Through the coupling of mitochondrial biogenesis and the Nrf2 pathway, AMPK may modulate oxidative stress and be a promising target against neurological disorders.

This paper proposed a prewhitening invariance of noise space (PW-INN) as a new magnetoencephalography (MEG) source analysis method, which is particularly suitable for localizing closely spaced and highly correlated cortical sources under real MEG noise. Conventional source localization methods, such as sLORETA and beamformer, cannot distinguish closely spaced cortical sources, especially under strong intersource correlation. Our previous work proposed an invariance of noise space (INN) method to resolve closely spaced sources, but its performance is seriously degraded under correlated noise between MEG sensors. The proposed PW-INN method largely mitigates the adverse influence of correlated MEG noise by projecting MEG data to a new space defined by the orthogonal complement of dominant eigenvectors of correlated MEG noise. Simulation results showed that PW-INN is superior to INN, sLORETA, and beamformer in terms of localization accuracy for closely spaced and highly correlated sources. Lastly, source connectivity between closely spaced sources can be satisfactorily constructed from source time courses estimated by PW-INN but not from results of other conventional methods. Therefore, the proposed PW-INN method is a promising MEG source analysis to provide a high spatial-temporal characterization of cortical activity and connectivity, which is crucial for basic and clinical research of neural plasticity.

We showed in this study that cells deficient of the BRCA1-associated BACH1 helicase, also known as BRIP1, failed to elicit homologous recombination (HR) after DNA double-stranded breaks (DSBs). BACH1-deficient cells were also sensitive to mitomycin C (MMC) and underwent MMC-induced chromosome instability. Moreover, we identified a homozygous nonsense mutation in BACH1 in a FA-J patient-derived cell line and could not detect BACH1 protein in this cell line. Expression of wild-type BACH1 in this cell line reduced the accumulation of cells at G2/M phases following exposure to DNA crosslinkers, a characteristic of Fanconi anemia (FA) cells. These results support the conclusion that BACH1 is FANCJ.

Bone mesenchymal stem cells (BMSCs) can be a useful cell resource for developing biological treatment strategies for bone repair and regeneration, and their therapeutic applications hinge on an understanding of their physiological characteristics. N⁶-methyl-adenosine (m⁶A) is the most prevalent internal chemical modification of mRNAs and has recently been reported to play important roles in cell lineage differentiation and development. However, little is known about the role of m⁶A modification in the cell differentiation of BMSCs. To address this issue, we investigated the expression of N⁶-adenosine methyltransferases (Mettl3 and Mettl14) and demethylases (Fto and Alkbh5) and found that Mettl3 was upregulated in BMSCs undergoing osteogenic induction. Furthermore, we knocked down Mettl3 and demonstrated that Mettl3 knockdown decreased the expression of bone formation-related genes, such as Runx2 and Osterix. The alkaline phosphatase (ALP) activity and the formation of mineralized nodules also decreased after Mettl3 knockdown. RNA sequencing analysis revealed that a vast number of genes affected by Mettl3 knockdown were associated with osteogenic differentiation and bone mineralization. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that the phosphatidylinositol 3-kinase/AKT (PI3K-Akt) signaling pathway appeared to be one of the most enriched pathways, and Western blotting results showed that Akt phosphorylation was significantly reduced after Mettl3 knockdown. Mettl3 has been reported to play an important role in regulating alternative splicing of mRNA in previous research. In this study, we found that Mettl3 knockdown not only reduced the expression of Vegfa but also decreased the level of its splice variants, vegfa-164 and vegfa-188, in Mettl3-deficient BMSCs. These findings might contribute to novel progress in understanding the role of epitranscriptomic regulation in the osteogenic differentiation of BMSCs and provide a promising perspective for new therapeutic strategies for bone regeneration.

Recently, deep learning technologies have rapidly expanded into medical image analysis, including both disease detection and classification. As far as we know, migraine is a disabling and common neurological disorder, typically characterized by unilateral, throbbing and pulsating headaches. Unfortunately, a large number of migraineurs do not receive the accurate diagnosis when using traditional diagnostic criteria based on the guidelines of the International Headache Society. As such, there is substantial interest in developing automated methods to assist in the diagnosis of migraine.

Breast carcinoma is one of the most common malignancies in women. Previous studies have reported that 500 μM valproic acid can sensitize breast tumor cells to the anti-neoplastic agent hydroxyurea. However, the dose requirements for valproic acid is highly variable due to the wide inter-individuals clinical characteristics. High therapeutic dose of valproic acid required to induce anti-tumor activity in solid tumor was associated with increased adverse effects. There are attempts to locate suitably high-efficient low-toxicity valproic acid derivatives. We demonstrated that lower dose of 2-hexyl-4-pentynoic acid (HPTA; 15 μM) has similar effects as 500 μM VPA in inhibiting breast cancer cell growth and sensitizing the tumor cells to hydroxyurea on MCF7 cells, EUFA423 cells, MCF7 cells with defective RPA2-p gene and primary culture cells derived from tissue-transformed breast tumor cells. We discovered HPTA resulted in more DNA double-strand breaks, the homologous recombination was inhibited through the interference of the hyperphosphorylation of replication protein A2 and recombinase Rad51. Our data postulate that HPTA may be a potential novel sensitizer to hydroxyurea in the treatment of breast carcinoma.

Hyperlipidemia is associated with metabolic disorders, but the detailed mechanisms and related interventions remain largely unclear. As a functional food in Asian diets, Herba houttuyniae has been reported to have beneficial effects on health. The present research was to investigate the protective effects of Herba houttuyniae aqueous extract (HAE) on hyperlipidemia-induced liver and heart impairments and its potential mechanisms. Male C57BL/6J mice were administered with 200 or 400 mg/kg/day HAE for 9 days, followed by intraperitoneal injection with 0.5 g/kg poloxamer 407 to induce acute hyperlipidemia. HAE treatment significantly attenuated excessive serum lipids and tissue damage markers, prevented hepatic lipid deposition, improved cardiac remodeling, and ameliorated hepatic and cardiac oxidative stress induced by hyperlipidemia. More importantly, NF-E2 related factor (Nrf2)-mediated antioxidant and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α)-mediated mitochondrial biogenesis pathways as well as mitochondrial complex activities were downregulated in the hyperlipidemic mouse livers and hearts, which may be attributable to the loss of adenosine monophosphate (AMP)-activated protein kinase (AMPK) activity: all of these changes were reversed by HAE supplementation. Our findings link the AMPK/PGC-1α/Nrf2 cascade to hyperlipidemia-induced liver and heart impairments and demonstrate the protective effect of HAE as an AMPK activator in the prevention of hyperlipidemia-related diseases.

The aims are to investigate and compare the activated cerebral regions of Uygur-speaking and Chinese-speaking participants during verb generation task.A total of 31 cases of Uygur and 28 cases of Han healthy volunteers were enrolled. They were requested to take verb generation tasks. Blood oxygenation level-dependent functional magnetic resonance imaging (BOLD-fMRI) was performed. The fMRI images were collected and activated brain regions were analyzed.In Chinese-speaking participants, the main activated cerebral regions were as follows: the left caudate nucleus, the left occipital gyrus, the left fusiform gyrus, bilateral supplementary motor area (BA8/ 6), the left BA32, left precuneus, the left superior parietal lobule, the left inferior parietal lobule (BA7), the left angular gyrus, the right side of the central gyrus (BA9), the left inferior frontal gyrus triangular section, the right pars opercularis gyri frontalis inferiorista, and bilateral cerebellum. In Uygur-speaking subjects, the main activated cerebral regions included left precentral gyrus (BA9 region), inferior frontal gyrus of left opercular part, inferior frontal gyrus of left triangle part, and left cerebellum. Left caudate nucleus, left orbital frontal gyrus, right caudate nucleus, and bilateral anterior cingulate gyrus (BA32 region) of Chinese group were significantly activated compared with Uygur group. By contrast, Uygur group showed no region that was more activated than Chinese group.The present study demonstrates that activated brain regions in verb generation tasks are different between Uygur and Chinese languages. Processing of Uygur characters is mainly in the left hemisphere of the brain, while the processing of Chinese characters needs more participation by the right hemisphere of the brain.