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Objectives: To observe the effect of mild moxibustion on stem cell factor (SCF)/tyrosine kinase receptor (c-kit) signaling pathway and visceral hypersensitivity of diarrhea predominant irritable bowel syndrome (IBS-D) model rats with liver- qi stagnation and spleen deficiency syndrome, so as to explore its mechanisms underlying improvement of IBS-D., Methods: A total of 24 male Wistar rats were randomly divided into normal, IBS-D model, medication and mild moxibustion groups, with 6 rats in each group. The IBS-D model was established by glacial acetic acid (4%) enema plus restraint stress stimulation once daily for 14 days. Rats of the medication group were treated by gavage of pivamium bromide (15 mg/kg) once a day for 14 days. Mild moxibustion was applied to bilateral "Tianshu"(ST25), "Shangjuxu"(ST37) and "Taichong"(LR3) for 20 min, once daily for 14 consecutive days. After the intervention, the rats' general state of each group were observed. The rate of loose stools (LSR), and the minimum volume threshold for abdominal withdrawal reflex(AWR) were observed, and the open field test was used to assess the state of rats' motor activities (including rearing times, grooming times and total number of square-crossings in 5 min). Morphological changes of the colon tissue were observed by hematoxylin-eosin (H.E.) staining, The count of mast cells (MC) in the colon tissues was determined by toluidine blue staining. Contents of serum 5-hydroxytryptamine (5-HT) and substance P (SP) were determined by enzyme-linked immunosorbent assay (ELISA). The relative expression levels of SCF and c-kit mRNAs and proteins in the colon tissues were detected by real-time quantitative PCR and Western blot, respectively., Results: Compared with the normal group, the body weight, minimum volume threshold of AWR, total numbers of square-crossing, rearing times and grooming times were significantly decreased ( P <0.05), and the LSR, number of MC, contents of 5-HT and SP, and the expression levels of SCF and c-kit mRNAs and proteins were considerably increased in the model group ( P <0.01). In comparison with the model group after interventions, the body weight, minimum volume threshold of AWR, total numbers of square-crossing, rearing times and grooming times were apparently increased in both medication and moxibustion groups ( P <0.05, P <0.01), and the LSR, number of MC, 5-HT and SP contents in both medication and moxibustion groups, and the expression levels of SCF and c-kit mRNA and protein in the moxibustion group (not in the medication group) were obviously decreased ( P <0.05, P <0.01). H.E. staining showed that in the model group, a small amount of inflammatory cells in the mucosal layer of colon tissue could be seen. in the medication group, a small number of lymphocytes in colon tissue were observed, while in the mild moxibustion group, a small amount of neutrophils in colon tissue were observed., Conclusions: Mild moxibustion can reduce visceral hypersensitivity and improve abdominal pain, diarrhea and locomotion state in IBS-D rats with liver- qi stagnation and spleen deficiency syndrome, which may be associated with its functions in reducing the number of MC and the levels of 5-HT and SP and down-regulating the activities of SCF/c-kit signaling pathway.

A series of heterogeneous catalysts, designated as POP-n-Pd (where n = 1, 2, 3, or 4), were synthesized by polymerizing a six-membered N-heterocyclic compound with an alkyl substituted group monomer (S1), using divinylbenzene (DVB) as crosslinkers. This process was followed by the incorporation of palladium (Pd) nanoparticles. The impact of the substituted group and the S1:DVB ratio in the catalysts, together with the reaction conditions, was investigated to assess their influence on the catalytic performance in converting propylamine, carbon dioxide (CO 2 ) and 4-iodoanisole to oxazolidinones. The POP-1-Pd catalyst, featuring a methyl substituted group and a S1:DVB ratio of 1:4, exhibited remarkable efficiency, resulting in an excellent yield of 96 % under room temperature and ambient pressure conditions. Furthermore, it has demonstrated wide applicability across a variety of substrates and in the treatment of lime kiln exhaust gas. Additionally, POP-1-Pd can be used in a gram-scale reaction and maintains its performance after six recycles, with no significant decline in yield. The possible catalytic mechanism is proposed as follows: the catalyst's pores adsorb both CO 2 and substrates, creating a high concentration reactant enrichment microenvironment. This facilitates the activation of both CO 2 and substrates by the imidazole moiety and Pd nanoparticles in the catalyst, thereby generating oxazolidinones., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: To investigate causal associations between diabetes, insulin treatment and osteoporosis using LDSC analysis with a 2-way Mendelian randomization study., Methods: LDSC analysis was used to estimate the likelihood-scale heritability of the genome-wide association study used with genetic correlation between the 2 genome-wide association study used. Then a 2-sample Mendelian randomization study was performed using 3 methods including inverse variance weighted, MR Egger, and weighted median., Results: The genetic correlation between diabetes, insulin treatment (h2&#95;Z = 3.70, P = 2.16e-4), osteoporosis (h2&#95;Z = 4.93, h2&#95;p = 8.13e-7) and genes was significant. There was a significant genetic correlation (rg = 0.122, P = 0.0211). There was a causal association between diabetes, insulin treatment and osteoporosis [P = 0.003754, OR (95%CI) = 0.998876 (0.998116-0.999636)], while no causal association existed between osteoporosis and insulin use (P = 0.998116-0.999636) causal association existed (P = 0.333244)., Conclusion: There was a strong genetic correlation between diabetes, insulin treatment and osteoporosis, a causal association between diabetes, insulin treatment and osteoporosis, and no causal association between osteoporosis and diabetes, insulin treatment., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Abstract: The transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Knowing the genes involved in quantitative traits provides an entry point to understanding the biological bases of behavior, but there are very few examples where the pathway from genetic locus to behavioral change is known. To explore the role of specific genes in fear behavior, we mapped three fear-related traits, tested fourteen genes at six quantitative trait loci (QTLs) by quantitative complementation, and identified six genes. Four genes, Lamp, Ptprd, Nptx2, and Sh3gl, have known roles in synapse function; the fifth, Psip1, was not previously implicated in behavior; and the sixth is a long non-coding RNA, 4933413L06Rik, of unknown function. Variation in transcriptome and epigenetic modalities occurred preferentially in excitatory neurons, suggesting that genetic variation is more permissible in excitatory than inhibitory neuronal circuits. Our results relieve a bottleneck in using genetic mapping of QTLs to uncover biology underlying behavior and prompt a reconsideration of expected relationships between genetic and functional variation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the effects of physiotherapeutic scoliosis-specific exercises (PSSE) on coronal, horizontal, and sagittal deformities of the spine in adolescent idiopathic scoliosis (AIS) as well as how curve severity, intervention duration, and intervention type could modify these effects., Data Sources: Data sources included PubMed, Web of Science, Embase, Cochrane Library, and Scopus databases, which were searched from their inception to September 5, 2023., Study Selection: Clinical controlled trials reporting the effects of PSSE on the Cobb angle, angle of trunk rotation (ATR), thoracic kyphosis (TK), or lumbar lordosis in patients with AIS aged 10-18 years. The experimental groups received PSSE; the control groups received standard care (observation or bracing) or conventional exercise such as core stabilization exercise, Pilates, proprioceptive neuromuscular facilitation, and other nonspecific exercises., Data Extraction: Two researchers independently extracted key information from eligible studies. The quality of the studies was assessed using the Cochrane Handbook version 5.1.0 risk of bias assessment and the JBI Center for Evidence-Based Health Care (2016) of quasi-experimental research authenticity assessment tool. The level and certainty of evidence were rated according to the Grading of Recommendations, Assessment, Development, and Evaluation framework. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. The protocol for this study was registered in PROSPERO (CRD42023404996)., Data Synthesis: Twelve randomized controlled trials (RCTs) and 5 non-RCTs were meta-analyzed separately. The results indicated that compared with other nonsurgical management, PSSE significantly improved the Cobb angle, ATR, and TK, whereas the lumbar lordosis improvement was not statistically significant. Additionally, the efficacy of PSSE on Cobb angle was not significant in patients with curve severity ≥30° compared with controls. Nevertheless, the pooled effect of PSSE on Cobb angle was not significantly modified by intervention duration and intervention type and that on ATR was not significantly modified by intervention duration. The overall quality of evidence according to Grading of Recommendations, Assessment, Development, and Evaluation was moderate to low for RCT and very low for non-RCT., Conclusions: PSSE exhibited positive benefits on the Cobb angle, ATR, and TK in patients with AIS compared with other nonsurgical therapies. In addition, the effectiveness of PSSE may be independent of intervention duration and intervention type but may be influenced by the initial Cobb angle. However, more RCTs are needed in the future to validate the efficacy of PSSE in moderate AIS with a mean Cobb angle ≥30°. Current evidence is limited by inconsistent control group interventions and small sample size of the studies., (Copyright © 2024. Published by Elsevier Inc.)

Histone H3 lysine 4 mono-methylation (H3K4me1) marks poised or active enhancers. KMT2C (MLL3) and KMT2D (MLL4) catalyze H3K4me1, but their histone methyltransferase activities are largely dispensable for transcription during early embryogenesis in mammals. To better understand the role of H3K4me1 in enhancer function, we analyze dynamic enhancer-promoter (E-P) interactions and gene expression during neural differentiation of the mouse embryonic stem cells. We found that KMT2C/D catalytic activities were only required for H3K4me1 and E-P contacts at a subset of candidate enhancers, induced upon neural differentiation. By contrast, a majority of enhancers retained H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, H3K4me1 signals at these KMT2C/D-independent sites were reduced after acute depletion of KMT2B, resulting in aggravated transcriptional defects. Our observations therefore implicate KMT2B in the catalysis of H3K4me1 at enhancers and provide additional support for an active role of H3K4me1 in enhancer-promoter interactions and transcription in mammalian cells., Competing Interests: Declaration of interests B.R. is a co-founder of Arima Genomics, Inc. and Epigenome Technologies, Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: To observe the effect of catgut embedding at "Feishu"(BL13), "Dingchuan" (EX-B1) and "Danzhong" (CV17) on expression of phosphorylated p38 mitogen activated protein kinase (p-p38 MAPK), interleukin-4 (IL-4), interferon-γ (IFN-γ) and changes of airway epithelial cells (AEC) in the lung tissue of bronchial asthma (BA) rats, so as to explore its mechanisms underlying improvement of BA., Methods: Forty male Wistar rats were randomly and equally divided into blank control, model, dexamethasone (DEX) and catgut embedding groups. The BA model was established by intraperitoneal injection of suspension of ovalbumin and aluminum hydroxide. Rats of the DEX group received intraperitoneal injection of DEX (1.5 mg/kg), once daily for 2 weeks, and those of the catgut embedding group received catgut embedding at BL13, EX-B1 and CV17 only one time. The rats' sneezing times per miniute in each group were recorded. H.E. staining was used to observe the histopathological changes of the lung tissue under light microscope. A transmission electron microscope (TEM) was used to observe the ultrastructural changes of AEC in the lung tissue, including the thickness of bronchial wall and bronchial smooth muscle by using an image analysis software. The protein expressions of p-p38 MAPK, IL-4 and INF-γ in the lung tissue were determined using Western blot., Results: Morphological observation revealed that in the model group, light microscope showed deformed and swollen bronchial tube wall with increased folds and thickened bronchial smooth muscle；and TEM showed a large number of autophagy vesicles containing swollen and deformed organelles in the AEC, and apparent reduction of intracellular mitochondria, these situations were obviously milder in both DEX and catgut embedding groups. Compared with the blank control group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle in the model group were significantly increased ( P <0.01), and the expressions of p-p38 MAPK and IL-4 in lung tissue were significantly increased ( P <0.01), while the expression of IFN-γ was significantly decreased ( P <0.01) in the model group. In comparison with the model group, the sneezing times, thickness of bronchial wall and bronchial smooth muscle, protein expressions of p-p38 MAPK and IL-4 were significantly decreased ( P <0.01), while the expression of IFN-γ was obviously increased ( P <0.01) in both the DEX and catgut embedding groups., Conclusions: Acupoint catgut embedding can reduce the expression of IL-4 and increase the expression of IFN-γ by inhibiting p38 MAPK signal pathway of lung tissues in BA rats, which may contribute to its effect in alleviating the degree of airway epithelial cells damage.

Efficient utilizing CO 2 is crucial approaches in achieving carbon neutralization. One of the challenges lies in the in-situ conversion of low concentration CO 2 found in waste gases. This study introduces a novel heterogeneous catalyst known as silver nanoparticles in porous N-heterocyclic carbene polymer (Ag@POP-NL-3). The catalyst is synthesized via a streamlined pre-coordination method. Ag@POP-NL-3 exhibits uniform distribution of silver nanoparticles, a porous structure and nitrogen activation groups. It demonstrates high efficiency and selectivity in absorbing and activating CO 2 and enabling the conversion of low concentration CO 2 (30 vol%) from lime kiln waste gas into cyclic carbonate under mild conditions. This catalytic system achieves both CO 2 capture and resource utilization of CO 2 simultaneously, effectively fixing low-concentration CO 2 from waste gases into C 2+ valuable chemicals. This approach elegantly addresses two goals in one solution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Relating genetic variants to behavior remains a fundamental challenge. To assess the utility of DNA methylation marks in discovering causative variants, we examined their relationship to genetic variation by generating single-nucleus methylomes from the hippocampus of eight inbred mouse strains. At CpG sequence densities under 40 CpG/Kb, cells compensate for loss of methylated sites by methylating additional sites to maintain methylation levels. At higher CpG sequence densities, the exact location of a methylated site becomes more important, suggesting that variants affecting methylation will have a greater effect when occurring in higher CpG densities than in lower. We found this to be true for a variant's effect on transcript abundance, indicating that candidate variants can be prioritized based on CpG sequence density. Our findings imply that DNA methylation influences the likelihood that mutations occur at specific sites in the genome, supporting the view that the distribution of mutations is not random., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Objectives: To observe the effect of acupuncture on the expressions of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome(IBS-D), so as to explore the mechanism of acupuncture in the treatment of IBS-D., Methods: Male Wistar rats were randomly divided into blank group, model group, medication group, and acupuncture group, with 6 rats in each group. Except for the blank group, the other groups were subjected to 14-day "acetic acid enema + restraint stress" to establish the IBS-D rat model. After successful modeling, the medication group received gavage of pinaverium bromide(15 mg/kg) once a day, and the acupuncture group received acupuncture at "Baihui"(GV20) and bilateral "Tianshu"(ST25), "Shangjuxu"(ST37), "Zusanli"(ST36), and "Taichong"(LR3) for 20 min every day, both groups were treated continuously for 14 days. The general state of the rats in each group was observed, and the body weight of the rats was measured. The open-field experiment was conducted to measure the horizontal and vertical movements, and the number of fecal pellets of rats. The histopathological morphology of hypothalamus and colon of rats was observed by HE staining. Toluidine blue staining was used to observe and count the mast cells(MCs) in the colon tissue of rats. ELISA was used to detect the serum contents of tumor necrosis factor-α(TNF-α) and interleukin(IL)-10. Real-time fluorescence quantitative PCR was performed to detect the mRNA expressions of calcitonin gene-related peptide(CGRP) in the hypothalamus and colon tissue. Western blot was used to detect the expressions of corticotropin-releasing factor(CRF) in the hypothalamus and colon tissue., Results: HE staining showed that there was inflammatory cell infiltration in the lamina propria of colon in the model group, and it was reduced in the other groups. Compared with the blank group, the model group showed significantly decreased body weight, decreased walking distance and upright times in open field experiment, decreased serum IL-10 contents( P <0.05, P <0.01), increased fecal pellet number ( P <0.01), increased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions and CRF expressions in the hypothalamus and colon tissue( P <0.01). Compared with the model group, both medication and acupuncture groups showed significantly increased body weight, walking distance and upright times in the open-field experiment, and serum IL-10 contents( P <0.01, P <0.05), significantly decreased fecal pellet number ( P <0.05), significantly decreased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions in the hypothalamus and colon tissue( P <0.01)；at the same time, the acupuncture group showed significantly decreased CRF expressions in the hypothalamus and colon tissue( P <0.01, P <0.05). There was no significant difference in the above indicators between the medication group and the acupuncture group., Conclusions: Acupuncture can improve the general and emotional state, inflammatory response, and neuropeptide expression in rats with IBS-D, and alleviate the symptoms of IBS-D, which may be related to the regulation of neuropeptides and inflammatory factors levels.

To investigate the causal relationship between metformin use and osteoporosis and different subtypes of osteoporosis using a 2-sample Mendelian randomization method. Data from genome-wide association studies were analyzed, with the exposure factor being metformin and the outcome variables being osteoporosis and different subtypes. Mendelian randomization was performed using Inverse Variance Weighted (IVW), MR-Egger, and weight median (WM) methods, and heterogeneity tests, horizontal multivariate analyses, and sensitivity analyses were performed. The IVW method analysis with metformin and osteoporosis showed P = 1.53E-04, OR (95%CI) = 1.81E-02 (2.27E-02-1.44E-01); the IVW method analysis with metformin and postmenopausal osteoporosis with pathologic fracture showed P = 2.22E-01, OR (95%CI) = 4.89E-02 (3. 83E-04-6.23E + 00); the IVW method using metformin with osteoporosis with pathological fracture showed that P = 2.14E-01, OR (95%CI) = 1.64E + 00(5.78E-02-6.44E-04); the IVW method using metformin with pharmacological osteoporosis with pathological fracture showed that P = 9. 83E- 01, OR (95%CI) = 1.11E + 00 (3.99E-05-3.11E + 04); IVW method of metformin use and pharmacological osteoporosis showed that P = 5.99E-01, OR (95%CI) = 2.27E + 01 (2.00E-04-2.57E + 06); there is a causal relationship between metformin use and osteoporosis, but there is no causal relationship between metformin use and postmenopausal osteoporosis with pathological fracture, osteoporosis with pathological fracture, pharmacological osteoporosis, and pharmacological osteoporosis with pathological fracture, and metformin use is a protective factor for osteoporosis., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: This study aims to evaluate the effectiveness and safety of low-dose (1.5 mg) fondaparinux for venous thromboembolism (VTE) prophylaxis in patients post-total knee arthroplasty (TKA)., Methods: We retrospectively identified 314 patients who carried out the primary TKAs and received fondaparinux for VTE chemoprophylaxis between July 2020 and December 2021. A total of 141 TKA patients were excluded according to the exclusion criteria. Two groups of patients were established: the low-dose group included 84 patients who injected 1.5 mg of fondaparinux, and the regular-dose group included 89 patients who injected 2.5 mg of fondaparinux. The pre-operative blood analysis and coagulation assays were performed. The surgical time, the incidence of symptomatic VET, blood loss, wound complication, bleeding, drainage, and mortality of patients were determined and assessed., Results: The pre-operative blood analysis, body mass index, sex, age, and coagulation assays of patients in both groups were comparable. In terms of symptomatic pulmonary embolism and deep vein thrombosis, there was no significant difference (variation) between the two groups. However, patients in both groups showed a substantial difference in terms of blood loss, drain volume, wound complication, and transfusion rate., Conclusion: In prevention of VET in patients post-TKA, low-dose fondaparin is as effective as conventional dose fondaparinux. A significant decrease in blood loss, post-surgical transfusion rates, and wound complications were detected in patients given low-dose fondaparinux compared to those receiving regular-dose fondaparinux., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Chen, Wang, Wang, Xiong, Wang, Yang, Li, Wang and Zhang.)

Industrial waste gas is one of the major sources of atmospheric CO 2 , yet the direct conversion of the low concentrations of CO 2 in waste gases into high value-added chemicals have been a great challenge. Herein, a copper-based N-heterocyclic carbene porous polymer catalyst (Cu@NHC-1) for the direct conversion of low concentration CO 2 into oxazolidinones was successfully fabricated via a facile copolymerization process followed by the complexation with Cu(OAc) 2 . A continuous flow device was designed to deliver a continuous and stable carbon source for the reaction. Due to the triple synergistic effect of its porous structure, nitrogen activation sites and catalytic Cu center, Cu@NHC-1 shows highly efficient and selective adsorption, activation, and conversion of the low concentration CO 2 (30 vol%). Its practical application potential is demonstrated by the ability to successfully convert the CO 2 in lime kiln waste gas into oxazolidinones in satisfactory yields under mild conditions., (© 2023 Wiley-VCH GmbH.)

Purpose: There have been many studies in the operative management of pyogenic spondylodiscitis with foreign materials. However, it still remains an issue of debate on whether the allografts may be used in pyogenic spondylodiscitis. This study sought to evaluate the safety and effectiveness of PEEK cages and the cadaveric allograft in transforaminal lumbar interbody fusion (TLIF) for treating lumbar pyogenic spondylodiscitis., Methods: From January 2012 to December 2019, 56 patients underwent surgery for lumbar pyogenic spondylodiscitis. The posterior debridement of all patients and their fusion with allografts, local bone grafts, and bone chip cages were performed before posterior pedicle screw fusion. An assessment of the residual pain, the grade of neurological injury, and the resolution of infection was conducted on 39 patients. The clinical outcome was evaluated using a visual analog scale (VAS) and the Oswestry Disability Index (ODI), and neurological outcomes were appraised based on Frankel grades. The radiological outcomes were evaluated via focal lordosis, lumbar lordosis, and the state of the fusion., Results: Staphylococcus aureus and Staphylococcus epidermidis were the most common causative organisms. The mean preoperative focal lordosis was -1.2° (-11.4° to 5.7°), and the mean postoperative focal lordosis increased to 10.3° (4.3°-17.2°). At the final follow-up, there were five cases with subsidence of the cage, no case of recurrence, and no case with cage and screw loosening or migration. The mean preoperative VAS and ODI scores were 8.9 and 74.6%, respectively, and improvements in VAS and ODI were 6.6 ± 2.2 and 50.4 ± 21.3%, respectively. The Frankel grade D was found in 10 patients and grade C in 7. Following the final follow-up, only one patient improved from Frankel grade C to grade D while the others recovered completely., Conclusion: The PEEK cage and cadaveric allograft combined with local bone grafts is a safe and effective choice for intervertebral fusion and restoring sagittal alignment without increased incidence of relapse for treating lumbar pyogenic spondylodiscitis., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2023 Huo-Liang Zheng et al.)

There have been an increasing number of patients with degenerative disc diseases due to the aging population. In light of this, studies on the pathogenesis of intervertebral disc degeneration have become a hot topic, and gene knockout mice have become a valuable tool in this field of research. With the development of science and technology, constitutive gene knockout mice can be constructed using homologous recombination, zinc finger nuclease, transcription activator-like effector nuclease technology and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, and conditional gene knockout mice can be constructed using the Cre/LoxP system. The gene-edited mice using these techniques have been widely used in the studies on disc degeneration. This paper reviews the development process and principles of these technologies, functions of the edited genes in disc degeneration, advantages, and disadvantages of different methods and possible targets of the specific Cre recombinase in intervertebral discs. Recommendations for the choice of suitable gene-edited model mice are presented. At the same time, possible technological improvements in the future are also discussed., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Purpose: The current study attempts to investigate the role of anterior cervical discectomy and fusion (ACDF) in alleviating symptoms in patients with cervical vertigo associated with cervical instability., Methods: The patients of cervical instability with vertigo and dizziness who underwent ACDF between January 2011 and December 2019 were followed-up for more than two years. Demographic data (age, sex, follow up period and levels of instable cervical segments) were assessed; Symptoms of vertigo and dizziness before and after surgery were assessed by the 15-item version of the Vertigo Symptom Scale (VSS) and the 25-item Dizziness Handicap Inventory (DHI). The severity and frequency of other symptoms like neck and occipital pain, gastrointestinal discomfort, nausea, vomiting, tinnitus, palpitations, headache, diplopia and blurring of vision before and after surgery were also assessed., Results: A total of 92 patients underwent ACDF for cervical instability with vertigo and dizziness between January 2011 and December 2019, of which 79 patients were included in the final analysis. The number of instable levels had no correlation with VSS and DHI scores before surgery ( p  > 0.05), while patients with C3/4 instability suffering a severer vertigo than other levels. Both DHI and VSS scores were significantly reduced after ACDF and this was sustained within two years after surgery ( p  < 0.001). Although there was no statistical difference in the ratio of patients with vertigo relief, patients with one-level cervical instability demonstrated a more rapid recovery than patients with multi-level cervical instability ( p  = 0.048). Also, there was improvement in other symptoms such as neck and occipital pain, gastrointestinal discomfort, nausea, vomiting, tinnitus, palpitations, headache and blurring of vision after surgery., Conclusions: Vertigo caused by C3/4 instability was severer than other levels such as C4/5 and C5/6. During 2 years' follow-up the significant relief of vertigo and dizziness was observed after anterior cervical surgery. Other accompanying symptoms except hypomnesia were also extenuated in follow-up period., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Zheng, Li, Song, Chen, Jiang and Jiang.)

A scarcity of functionally validated enhancers in the human genome presents a significant hurdle to understanding how these cis-regulatory elements contribute to human diseases. We carry out highly multiplexed CRISPR-based perturbation and sequencing to identify enhancers required for cell proliferation and fitness in 10 human cancer cell lines. Our results suggest that the cell fitness enhancers, unlike their target genes, display high cell-type specificity of chromatin features. They typically adopt a modular structure, comprised of activating elements enriched for motifs of oncogenic transcription factors, surrounded by repressive elements enriched for motifs recognized by transcription factors with tumor suppressor functions. We further identify cell fitness enhancers that are selectively accessible in clinical tumor samples, and the levels of chromatin accessibility are associated with patient survival. These results reveal functional enhancers across multiple cancer cell lines, characterize their context-dependent chromatin organization, and yield insights into altered transcription programs in cancer cells., Competing Interests: Declaration of interests B.R. is a co-founder and consultant of Arima Genomics, Inc. and co-founder of Epigenome Technologies, Inc.; and P.S.M. is a co-founder and consultant of Boundless Bio, Inc. He has equity in the company and chairs the scientific advisory board, for which he is compensated. S.W. is a member of the scientific advisory board of Dimension Genomics Inc., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the effects of acupuncture on phosphorylated P38 mitogen-activated protein kinase (p-P38MAPK), intercellular adhesion molecule-1 (ICAM-1), interferon γ (IFN-γ), and eosinophilic granulocytes (EOS) in lung tissue of asthmatic rats, and to explore the mechanism of acupuncture in regulating the apoptosis of EOS., Methods: Clean-grade male Wistar rats were randomly divided into normal, model, dexamethasone and acupuncture groups, 8 rats in each group. The asthmatic model was established by intraperitoneal injection of mixture suspension (1 mL) of 10% ovalbumin and 10% Al(OH)&#95;3+ normal saline, followed by inhalation of atomized 1% ovalbumin solution for 30 min, once daily for 2 weeks to trigger occurrence of asthmatic symptoms. The rats in dexamethasone group were intraperitoneally injected with 0.9 mg/kg dexamethasone since day 15 once a day for two consecutive weeks. In the acupuncture group, bilateral "Feishu" (BL13), "Pishu" (BL20), "Shenshu" (BL23), "Dingchuan" (EX-B1), and "Danzhong" (CV17) were selected for acupuncture treatment once every other day since day 15 for two consecutive weeks. Uniform reinforcing and reducing manipulation was carried out, and the needles were retained for 30 min. The pathological changes of the lung tissue were observed by hematoxylin-eosin (HE) staining. The apoptosis of EOS in the lung tissue of rats was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The expression of p-P38MAPK in the lung tissue was detected by Western blot. The protein and mRNA levels of ICAM-1 and IFN-γ in the lung tissue were determined by immunohistochemistry and real-time PCR, respectively., Results: The results of HE staining showed that the pulmonary alveoli and surrounding tissues were intact with no inflammatory cell infiltration in the normal group. The model group showed massive exudation of inflammatory materials and thickened pulmonary interstitium. The dexamethasone group and acupuncture group showed less damage of the alveolar structure and only a small number of inflammatory cells around the airway. Compared with the normal group, the apoptosis rate of EOS in lung tissue was decreased ( P <0.01), the expression levels of p-P38MAPK and ICAM-1 proteins and mRNAs in the lung tissue were up-regulated ( P <0.01), while the expression levels of IFN-γ protein and mRNA in the lung tissue were down-regulated ( P <0.01) in the model group. Compared with the model group, the apoptosis rate of EOS in the lung tissue was increased ( P <0.05), the expression levels of p-P38MAPK and ICAM-1 proteins and mRNAs in the lung tissue were down-regulated ( P <0.01, P <0.05), while the expression levels of IFN-γ protein and mRNA were up-regulated ( P <0.05, P <0.01) in the dexamethasone and acupuncture groups., Conclusion: Acupuncture may inhibit the P38MAPK signaling pathway, down-regulate ICAM-1 expression, and up-regulate IFN-γ expression to promote the apoptosis of EOS and reduce EOS aggregation, thus alleviating the inflammatory response of airway in asthma.

A healthy microenvironment of the intervertebral disc tissue is characterized by hypoxia owing to its sparse vascular distribution. Oxidative stress plays a pivotal role in the pathological development of intervertebral disc degeneration (IVDD). We found that the expression of prolyl endopeptidase (PREP) increased in degenerative nucleus pulposus (NP) tissues. The purpose of this study was to determine whether PREP is involved in oxidative-stress-induced IVDD. Tertbutyl hydroperoxide can inhibit the expression of PREP by activating the PI3K/AKT signaling pathway at low concentrations in NP cells. Knockdown of PREP protected NP cells from apoptosis induced by oxidative stress, whereas overexpression of PREP exacerbated the apoptosis of NP cells. We also investigated the connection between the PI3K/AKT signaling pathway and PREP and found that the activation of the PI3K/AKT signaling pathway downregulated the expression of PREP by inhibiting p53. As a crucial transcription factor, p53 binds to the PREP promoter region and promotes its transcription. Overexpression of PREP also impairs protein secretion in the extracellular matrix of NP cells. Furthermore, the in vivo knockout of PREP could attenuate puncture-induced IVDD. These findings suggested that the downregulation of PREP might maintain the viability of NP cells and attenuate IVDD under oxidative stress., Competing Interests: The authors confirm no conflicts of interest., (Copyright © 2022 Huo-Liang Zheng et al.)

Objective: To observe the effect of acupoint catgut embedment(ACE) on expression of p38 mitogen activated protein kinase (p38MAPK), intercellular adhesion molecule-1(ICAM-1), interleukin-4 (IL-4) and eosinophils (EOS) in lung tissue of asthmatic rats, so as to explore its mechanism underlying improvement of asthma., Methods: Forty male Wistar rats were randomly divided into control, model, ACE and dexamethasone (DEX) groups, with 10 rats in each group. The asthmatic model was established by intraperitoneal injection of mixture suspension (1 mL) of ovalbumin (OVA,10%) and 10% Al (OH) 3 + normal saline, followed by inhalation of atomized 1% OVA solution for 30 min, once daily for 2 weeks to trigger occurrence of asthmatic symptoms. The ACE was applied once to "Feishu" (BL13), "Dingchuan" (EX-B1) and "Danzhong" (CV17). Rats of the DEX group were given intraperitoneal injection of DEX once a day for 2 weeks. H.E. staining was used to evaluate histopathological changes of the lung tissue. The relative number of EOS in the bronchoalveolar lavage fluid (BALF) was detected by Wright Giemsa staining. The apoptosis level of EOS in the lung tissue was detected by TUNEL staining. The ultrastructural changes of EOS in the lung tissues were observed by transmission electron microscope (TEM). The expression of p38MAPK, ICAM-1 and IL-4 mRNAs in the lung tissue was detected by quantitative real-time PCR., Results: Findings of optical microscope and TEM showed obvious bronchial deformation and inflammatory cell infiltration, rupture of EOS cell membrane, uneven cytoplasm with swelling and uneven density of eosinophilic granules in EOS of the model group, which was relatively milder in the ACE and DEX groups. Compared with the control group, the EOS number in BALF and the expressions of p38MAPK, ICAM-1 and IL-4 mRNAs in the lung tissue were significantly increased ( P <0.01), and the apoptosis index of EOS in the lung tissue was significantly decreased ( P <0.01) in the model group. After intervention, the EOS number in BALF and expression levels of p38MAPK, ICAM-1 and IL-4 mRNAs in the lung tissue of ACE and DEX groups were significantly decreased ( P <0.01, P <0.05), as well as the apoptosis index of EOS in the lung tissue was significantly increased in both ACE and DEX groups ( P <0.01) in comparison with the model group. The EOS number in BALF and expression of ICAM-1 mRNA were significantly lower in the DEX group than those in the ACE group ( P <0.05)., Conclusion: Catgut embedding at acupoints may alleviate the airway inflammatory response in asthma rats, which may be related with its effects in down-regulating p38MAPK signaling, ICAM-1 and IL-4 mRNA expression, reducing the aggregation of EOS, and promoting the apoptosis of EOS.

The process of intervertebral disc degeneration (IVDD) is complex, and its mechanism is considered multifactorial. Apoptosis of oxidative stressed nucleus pulposus cells (NPCs) should be a fundamental element in the pathogenesis of IVDD. In our pilot study, we found that the expression of MAT2A decreased, and METTL16 increased in the degenerative nucleus pulposus tissues. Previous studies have shown that the balance of splicing, maturation, and degradation of MAT2A pre-mRNA is regulated by METTL16 m 6 A modification. In the current study, we aimed to figure out whether this mechanism was involved in the aberrant apoptosis of NPCs and IVDD. Human NPCs were isolated and cultured under oxidative stress. An IVDD animal model was established. It showed that significantly higher METTL16 expression and lower MAT2A expression were seen in either the NPCs under oxidative stress or the degenerative discs of the animal model. MAT2A was inhibited with siRNA in vitro or cycloleucine in vivo . METTL16 was overexpressed with lentivirus in vitro or in vivo . Downregulation of MAT2A or upregulation of METTL16 aggravated nucleus pulposus cell apoptosis and disc disorganization. The balance of splicing, maturation, and degradation of MAT2A pre-mRNA was significantly inclined to degradation in the NPCs with the overexpression of METTL16. Increased apoptosis of NPCs under oxidative stress could be rescued by reducing the expression of METTL16 using siRNA with more maturation of MAT2A pre-mRNA. Collectively, oxidative stress aggravates apoptosis of NPCs through disrupting the balance of splicing, maturation, and degradation of MAT2A pre-mRNA, which is m 6 A modified by METTL16., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2022 Peng-Bo Chen et al.)

Competing Interests: This work was partially supported by National Institutes of Health grants (R01MH115979 (JF, PBC). Publication charges for this article have been funded by 1R01MH115979. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Bone health requires adequate bone mass, which is maintained by a critical balance between bone resorption and formation. In our study, we identified beraprost as a pivotal regulator of bone formation and resorption. The administration of beraprost promoted differentiation of mouse bone mesenchymal stem cells (M-BMSCs) through the PI3K-AKT pathway. In co-culture, osteoblasts stimulated with beraprost inhibited osteoclastogenesis in a rankl-dependent manner. Bone mass of p53 knockout mice remained stable, regardless of the administration of beraprost, indicating that p53 plays a vital role in the bone mass regulation by beraprost. Mechanistic in vitro studies showed that p53 binds to the promoter region of neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) to promote its transcription. As a ubiquitinating enzyme, Nedd4 binds to runt-related transcription factor 2 (Runx2), which results in its ubiquitination and subsequent degradation. These data indicate that the p53-Nedd4-Runx2 axis is an effective regulator of bone formation and highlight the potential of beraprost as a therapeutic drug for postmenopausal osteoporosis.

Objective: To observe the effect of proximal and distal acupoint catgut-embedding on the contents of prostaglandin E 2 (PGE 2 ) and prostaglandin F 2α (PGF 2α ) in the uterus, serum Interleukin-2 (IL-2) contents and splenic natural killer (NK) cell activity in primary dysmenorrhea (PD) rats，so as to explore the different effects of proximal and distal acupoint catgut-embedding on PD rats., Methods: Female Wistar rats were randomly divided into control, model, proximal catgut-embedding and distal catgut-embedding groups, with 8 rats in each group. The PD model was established by subcutaneous injection of estradiol ben-zoate (0.5 mg/rat on the first day and 10th day, and 0.2 mg/rat from the 2nd to the 9th day) and intraperitoneal injection of oxytocin (2 U/rat, on the 11th day). Catgut-embedding was applied at bilateral "Guanyuan"(CV4) and "Ciliao"(BL32) in the proximal catgut-embedding group, and bilateral "Sanyinjiao" (SP6) and "Diji"(SP8) in the distal catgut-embedding group. After the treatment, the body writhing times in 30 min and uterine mass were recorded, PGE2 and PGF2αin uterus and IL-2 in serum were assayed by using ELISA, the activity of NK cell in the spleen was detected using MTT colorimetry., Results: Following modeling, the body writhing times of the model group was increased than that of the control group ( P <0.01); After interventions, the body writhing times were decreased in the proximal and distal catgut-embedding groups than those of the model group ( P <0.01). Compared with the control group, the uterine mass and uterus PGF 2α content were increased ( P <0.01), while uterus PGE 2 , serum IL-2 and splenic NK cell activity were decreased ( P <0.01) in the model group. After interventions, the uterine mass and uterus PGF 2α were down-regulated ( P <0.01, P <0.05), while the contents of uterus PGE 2 , serum IL-2 and splenic NK cell activity up-regulated ( P <0.01) in the proximal and distal catgut-embedding groups in contrast to the model group. The content of uterus PGF 2α was down-regulated ( P <0.05) and splenic NK cell activity was up-regulated ( P <0.01) in the proximal catgut-embedding group than those of the distal catgut-embedding group., Conclusion: Both proximal and distal acupoint catgut-embeding can increase PGE 2 and decrease PGF 2α in the uterus, increase the level of serum IL-2 and the activity of NK cells in the spleen in PD model rats, thereby achieving analgesic effect. The effect of proximal catgut-embedding is better than that of distal catgut-embedding.

CRISPR is a revolutionary genome-editing tool that has been broadly used and integrated within novel biotechnologies. A major component of existing CRISPR design tools is the search engines that find the off-targets up to a predefined number of mismatches. Many CRISPR design tools adapted sequence alignment tools as the search engines to speed up the process. These commonly used alignment tools include BLAST, BLAT, Bowtie, Bowtie2 and BWA. Alignment tools use heuristic algorithm to align large amount of sequences with high performance. However, due to the seed-and-extend algorithms implemented in the sequence alignment tools, these methods are likely to provide incomplete off-targets information for ultra-short sequences, such as 20-bp guide RNAs (gRNA). An incomplete list of off-targets sites may lead to erroneous CRISPR design. To address this problem, we derived four sets of gRNAs to evaluate the accuracy of existing search engines; further, we introduce a search engine, namely CRISPR-SE. CRISPR-SE is an accurate and fast search engine using a brute force approach. In CRISPR-SE, all gRNAs are virtually compared with query gRNA, therefore, the accuracies are guaranteed. We performed the accuracy benchmark with multiple search engines. The results show that as expected, alignment tools reported an incomplete and varied list of off-target sites. CRISPR-SE performs well in both accuracy and speed. CRISPR-SE will improve the quality of CRISPR design as an accurate high-performance search engine., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Substantial studies have shown that ω-3 polyunsaturated fatty acids (PUFAs) have various health-promoting effects; however, there are inconsistent results from animal studies that showed that ω-3 PUFAs have no effects or even detrimental effects. Emerging research suggests that oxidized ω-3 PUFAs have different effects compared to unoxidized ω-3 PUFAs; therefore, lipid oxidation of dietary ω-3 PUFAs could contribute to the mixed results of ω-3 PUFAs in animal studies. Here, we prepared an AIN-93G-based, semi-purified, powder diet, which is one of the most commonly used rodent diets in animal studies, to study the oxidative stability of fortified ω-3 PUFAs in animal feed. We found that lowering the storage temperature or the addition of a certain antioxidant, notably tert -butylhydroquinone (TBHQ), helps to stabilize ω-3 PUFAs and suppress ω-3 oxidation in the animal diet, while reducing the level of oxygen in the storage atmosphere is not very effective. The addition of 50 ppm of TBHQ in the diet inhibited 99.5 ± 0.1% formation of primary oxidation products and inhibited 96.1 ± 0.7% formation of secondary oxidation products, after 10 days of storage of the prepared diet at a typical animal-feeding experiment condition. Overall, our results highlight that ω-3 PUFAs are highly prone to lipid oxidation in a typical animal-feeding experiment, emphasizing the critical importance to stabilize ω-3 PUFAs in animal studies.

Inhibition of neuropeptide Y1 receptor stimulates osteogenesis in vitro and in vivo . However, the underlying mechanisms involved in these effects remain poorly understood. Here we identify the effects of Y1 receptor deficiency on osteogenic differentiation in human bone marrow stromal cells (BMSCs) by using genetic and pharmacological regulation, and to explore the pathways mediating these effects. In BMSCs, inhibition of Y1 receptor stimulates osteogenesis and upregulates the expression levels of the master transcriptional factor RUNX2. Mechanistically, Y1 receptor deficiency increases the levels of intracellular cAMP, which via protein kinase A (PKA) mediated pathways results in activation of phospho-CREB (p-CREB). We find RUNX2 activation induced by Y1 receptor deficiency is reversed by H-89, a PKA inhibitor. These results indicate Y1 receptor deficiency activates PKA-mediated phosphorylation of CREB, leading to activation of RUNX2 and enhances osteogenic differentiation in BMSCs. In conclusion, these data indicate that Y1 receptor deficiency promotes osteogenic differentiation by RUNX2 stimulation through cAMP/PKA/CREB pathway., (Copyright © 2020 Yu, Chen, Xu, Ding, Chen, Yang, Jiang and Pan.)

The gut microbiome is a malleable microbial community that can remodel in response to various factors, including diet, and contribute to the development of several chronic diseases, including atherosclerosis. We devised an in vitro screening protocol of the mouse gut microbiome to discover molecules that can selectively modify bacterial growth. This approach was used to identify cyclic D,L-α-peptides that remodeled the Western diet (WD) gut microbiome toward the low-fat-diet microbiome state. Daily oral administration of the peptides in WD-fed LDLr -/- mice reduced plasma total cholesterol levels and atherosclerotic plaques. Depletion of the microbiome with antibiotics abrogated these effects. Peptide treatment reprogrammed the microbiome transcriptome, suppressed the production of pro-inflammatory cytokines (including interleukin-6, tumor necrosis factor-α and interleukin-1β), rebalanced levels of short-chain fatty acids and bile acids, improved gut barrier integrity and increased intestinal T regulatory cells. Directed chemical manipulation provides an additional tool for deciphering the chemical biology of the gut microbiome and might advance microbiome-targeted therapeutics.

Many studies have revealed the function of long noncoding RNA (LncRNA) in regulating tumorigenesis of osteosarcoma (OS). As a subgroup of LncRNA, small nucleolar RNA host genes (SNHGs) have emerged as potentially important in OS. According to our recent findings, small nucleolar RNA host gene 22 (SNHG22) plays an important role in inhibiting the growth and metastasis of OS. However, the underlying mechanism of SNHG22 in regulating OS progression remains unknown. In this study, we confirmed that SNHG22 was downregulated in OS, and the overexpression of SNHG22 significantly inhibited OS progression in vivo and in vitro . Meanwhile, overexpression of SNHG22 also inhibited the migration and proliferation of human umbilical vein endothelial cells (HUVECs) and prevented the epithelial-to-mesenchymal transition (EMT) in OS. Furthermore, the interaction between miR-4492 and SNHG22 we previously predicted was validated by RNA pull-down assays and RNA immunoprecipitation assays. Dual-luciferase reporter assays showed that SNHG22 could directly interact with miR-4492 and upregulate the expression of NK-κB inhibitor-interacting Ras-like 2 (NKIRAS2) by its competing endogenous RNA (ceRNA) activity on miR-4492. In conclusion, our study has clarified the function of SNHG22 in OS progression and suggests a novel therapeutic target for OS.

CRISPR screens are a powerful technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By targeting non-coding sequences for perturbation, CRISPR screens have the potential to systematically discover novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from CRISPR screens. RELICS specifically addresses many of the challenges of non-coding CRISPR screens such as the unknown locations of functional sequences, overdispersion in the observed single guide RNA counts, and the need to combine information across multiple pools in an experiment. RELICS outperforms existing methods with higher precision, higher recall, and finer-resolution predictions on simulated datasets. We apply RELICS to published CRISPR interference and CRISPR activation screens to predict and experimentally validate novel regulatory sequences that are missed by other analysis methods. In summary, RELICS is a powerful new analysis method for CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy., Competing Interests: The authors have declared that no competing interests exist.

MicroRNAs (miRNAs/miRs) are non-coding RNAs that regulate protein synthesis by targeting mRNAs for translational repression or degradation. Previous studies have reported that aberrant expression of miR‑744 may be involved in human osteosarcoma; however, the underlying mechanisms remain elusive. In the present study, the expression levels of miR‑744 and its downstream signals were determined by reverse transcription‑quantitative PCR and western blotting. Cell proliferation was assessed using the bromodeoxyuridine assay, and the target of miR‑744 was investigated using a dual‑luciferase activity assay. The present study identified a significant upregulation of miR‑744 in osteosarcoma tissues compared with adjacent non‑tumor tissues. Furthermore, it was demonstrated that ectopic overexpression of miR‑744 induced by a miR‑744 precursor significantly enhanced proliferation of the osteosarcoma cell line MG63, whereas opposite results were observed following suppression of miR‑744 with its inhibitor. Moreover, as a unique anti‑oncogene, PTEN was identified as a direct target of miR‑744. It was confirmed that miR‑744 downregulated PTEN expression in MG63 cells by targeting the PTEN 3'untranslated region, and that the downstream AKT signal was also regulated by miR‑744. Collectively, the present results suggested that miR‑744 promoted proliferation of human osteosarcoma cells by directly regulating the PTEN/AKT signaling pathway.

There is increasing interest in the potential role of epigallocatechin gallate (EGCG) in changing body composition to lower body fat with increased lean mass. In this study, we examined the sex-dependent effect of EGCG on body composition, locomotion, feeding behaviour, sugar levels, and transcription levels of key regulators in lipid, carbohydrate, and energy metabolisms in Drosophila melanogaster . EGCG had no effects on body weights in both females and males, but decreased fat accumulation in females compared to the control, accompanied by a reduction in food intake. EGCG treatments increased lean mass and locomotor activity, and downregulated transcription levels of brummer ( bmm ), adipokinetic hormone ( akh ), and Drosophila insulin-like peptide 2 ( dilp2 ), and upregulated spargel ( srl ) in males. In addition, EGCG decreased sugar levels in both females and males. In conclusion, EGCG promotes lean phenotype in D. melanogaster via sex-specific metabolic regulations.

Objective: To observe the effect of herbal-cake-partitioned moxibustion (HCPM) of "Shenque" (CV8) and "Daheng" (SP15) on abdominal pain, plasma β-endorphin (β-EP), uterine prostaglandin E 2 (PGE 2 ) and prostaglandin F 2α (PGF 2α ) levels, as well as splenetic natural killer cell (NK cell) activity in primary dysmenorrhea (PD) rats, so as to explore the specificity of acupoint function and the underlying mechanisms of moxibustion in relieving dysmenorrhea., Methods: A total of 40 female rats were randomized into blank control, model, CV8-direct moxibustion, CV8-HCPM and SP15-HCPM groups ( n ＝8 rats in each). The PD model was established by subcutaneous injection of estradiol benzoate injection (0.2-0.5 mg/rat) for 10 consecutive days and intraperitoneal injection of oxytocin (2 U) 24 h after the last subcutaneous injection. Moxibustion or herbal-cake (composed of Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix Paeoniae Rubra, Cortex Cinnamomi , etc.)-partitioned moxibustion was applied to CV8, SP15 or umbilicus respectively for 7 moxa-cones every time, once daily for 10 successive days. The rats of the control and model groups were also restrained as those in the moxibustion groups. The writhing times within 30 minutes was recorded and the contents of plasma β-EP, uterine PGE 2 and PGF 2α were detected by ELISA, and NK cell activity was detected using MTT., Results: Compared with the control group, the writhing times and the content of PGF 2α in the uterus tissue were significantly increased in the model group ( P <0.01), while the contents of plasma β-EP, uterine PGE 2 and splenetic NK cell activity were significantly decreased ( P <0.01). In comparison with the model group, the writhing times and uterine PGF 2α content were obviously down-regulated in the SP15-HCPM, CV8-direct moxibustion and CV8-HCPM groups ( P <0.05, P <0.01), and the contents of plasma β-EP and uterine PGE 2, and splenetic NK cell activity were significantly increased ( P <0.05, P <0.01). The therapeutic effects of CV8-HCPM group were significantly superior to those of SP15-HCPM and CV8-direct moxibustion groups in lowering writhing times and PGF 2α level, and in up-regulating β-EP, PGE 2 ( P <0.05, P <0.01). The NK cell activity of CV8-HCPM group was significantly increased compared with that in the SP15-HCPM group( P <0.05). No significant differences were found between the SP15-HCPM and CV8-direct moxibustion groups in the levels of writhing times, plasma β-EP and uterine PGE 2, PGF 2α contents and splenetic NK cell activity ( P >0.05)., Conclusion: Moxibustion of both CV8 and SP15 can relieve abdominal pain in PD rats, which may be closely associated with its effect in suppressing PD-induced decrease of plasma β-EP and uterine PGE 2 levels and splenetic NK cell activity and increase of uterine PGF 2α . The therapeutic effect of CV8-HCPM is obviously better than that of SP15-HCPM and CV8-direct moxibustion.

Social behaviors, including behaviors directed toward young offspring, exhibit striking sex differences. Understanding how these sexually dimorphic behaviors are regulated at the level of circuits and transcriptomes will provide insights into neural mechanisms of sex-specific behaviors. Here, we uncover a sexually dimorphic role of the medial amygdala (MeA) in governing parental and infanticidal behaviors. Contrary to traditional views, activation of GABAergic neurons in the MeA promotes parental behavior in females, while activation of this population in males differentially promotes parental versus infanticidal behavior in an activity-level-dependent manner. Through single-cell transcriptomic analysis, we found that molecular sex differences in the MeA are specifically represented in GABAergic neurons. Collectively, these results establish crucial roles for the MeA as a key node in the neural circuitry underlying pup-directed behaviors and provide important insight into the connection between sex differences across transcriptomes, cells, and circuits in regulating sexually dimorphic behavior., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Upper extremity pain among manual wheelchair users induces functional decline and reduces quality of life. Research has identified chronic overuse due to wheelchair propulsion as one of the factors associated with upper limb injuries. Lack of a feasible tool to track wheelchair propulsion in the community precludes testing validity of wheelchair propulsion performed in the laboratory. Recent studies have shown that wheelchair propulsion can be tracked through machine learning methods and wearable accelerometers. Better results were found in subject-specific machine learning method. To further develop this technique, we conducted a pilot study examining the feasibility of measuring wheelchair propulsion patterns., Methods: Two participants, an experienced manual wheelchair user and an able-bodied individual, wore two accelerometers on their arms. The manual wheelchair user performed wheelchair propulsion patterns on a wheelchair roller system and overground. The able-bodied participant performed common daily activities such as cooking, cleaning, and eating., Results: The support vector machine built from the wrist and arm acceleration of wheelchair propulsion pattern recorded on the wheelchair roller system predicted the wheelchair propulsion patterns performed overground with 99.7% accuracy. The support vector machine built from additional rotation data recorded overground predicted wheelchair propulsion patterns (F1 = 0.968)., Conclusions: These results further demonstrate the possibility of tracking wheelchair propulsion in the community., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Conjugated linoleic acid (CLA) has been reported to be a bioactive food component. However, there is limited knowledge on the sex-dependent effects of CLA on energy metabolism. In the present study, Drosophila melanogaster was used to investigate the sex-dependent effects of CLA with respect to body fat, muscle, locomotion, and a key metabolic regulator, AMP-activated protein kinase α (AMPKα). Adult flies were fed a cornmeal-based fly food with 0.5% of CLA oil (50:50 of cis-9,trans-11 and trans-10,cis-12 CLA isomers in triacylglycerol (TAG) form), 0.5% safflower oil (high in linoleic acid [LNA] as control), or 0.5% water (as blank) for 5 days. Accumulation of CLA in tissue was verified using gas chromatography-mass spectrometry. CLA-fed flies had reduced TAG and increased locomotor activity when compared to LNA-fed control flies. In addition, CLA increased the muscle content when compared to the blank. Moreover, following CLA supplementation, increased AMPKα activity was observed in females, but not in males. These sex-dependent metabolic effects of CLA may be due to physiological differences in lipid metabolism and nutrient requirements. In conclusion, CLA promoted the body composition and locomotion behavior in D. melanogaster and regulated the sex-specific metabolism in part via AMPKα. As key physiological processes are conserved between fly and human, information obtained from this research could provide valuable insights into sex-dependent responses to CLA in humans., (© 2018 AOCS.)

Mitochondria intricately modulate their energy production through the control of mitochondrial adaptation (mitochondrial biogenesis, fusion, and/or fission) to meet energy demands. Nutrient overload may result in dysregulated mitochondrial biogenesis, morphology toward mitochondrial fragmentation, and oxidative stress in the skeletal muscle. In addition, physical activity and diet components influence mitochondrial function. Exercise may stimulate mitochondrial biogenesis and promote mitochondrial fusion/fission in the skeletal muscle. Moreover, some dietary fatty acids, such as n-3 polyunsaturated fatty acids and conjugated linoleic acid, have been identified to positively regulate mitochondrial adaptation in the skeletal muscle. This review discusses the association of mitochondrial impairments and obesity, and presents an overview of various mechanisms of which exercise training and mitochondrial nutrients promote mitochondrial function in the skeletal muscle., (© 2018 AOCS.)

Objective: To explore the underlying mechanism of acupoint catgut embedding in improving primary dysme-norrhea (PD) in rats based on functional activities of the neuro-endocrine-immune (NEI) network., Methods: Forty female rats were equally randomized into blank control, PD model, medication, and acupoint catgut embedding groups. The PD model was established by subcutaneous injection of estradiol benzoate (0.5 mg/rat on the 1 st and 10 th d, and 0.2 mg/rat from 2 nd to 9 th d) and oxytocin (2 U/rat, i．p.). Rats of the medication group were treated by intragastric perfusion of fenbid (0.8 mL/rat, 125 mg/100 mL), once daily for 10 days. The catgut embedding was applied to bilateral "Ciliao" (BL 32), "Sanyinjiao" (SP 6) and "Guanyuan" (CV 4) before modeling. The body writhing times in 30 minutes were recorded, plasma β-endorphin(β-EP) content, and prostaglandin E 2 (PGE 2 ) and prostaglandin F 2 α (PGF 2α ) contents in the uterus tissue were assayed using ELISA, and the activity of natural killer cell (NK cell) in the spleen tissue was detected using 3-(4，5-dimethyl- 2 -thiazolyl)- 2 ，5-diphenyl- 2 -H-tetrazolium bromide (MTT) method after isolation and co-culture with K 562 cells., Results: The body writhing times were no-tably more in the model group than in the control group ( P <0.01), and obviously fewer in both medication and catgut embedding groups than in the model group ( P <0.01). After modeling, the plasma β-EP and uterus PGE 2 contents and splenic NK cell activity were significantly decreased ( P <0.01), while the uterus PGF 2α content was evidently increased in the model group relevant to the control group ( P <0.01). Following the treatment, plasma β-EP and uterus PGE 2 contents and splenic NK cell activity were considerably up-regulated ( P <0.01), and uterus PGF 2α content was markedly down-regulated in both medication and acupoint catgut embedding groups ( P <0.01), suggesting an involvement of the NEI network in catgut embedding-induced improvement of PD. The therapeutic effect of catgut embedment was markedly superior to that of medication in up-regulating splenic NK cell activity ( P <0.01). No significant differences were found between the medication and catgut embedding groups in the body writhing times within 30 min, and in the levels of plasma β-EP and uterus PGE 2 and PGF 2α ( P >0.05)., Conclusion: The acupoint catgut embedding has a significant efficacy in relieving PD in rats, which may be related to its effect in up-regulating plasma β-EP, uterus PGE 2 contents and splenic NK cell activity and in down-regulating uterus PGF 2α level.

Long-lasting forms of synaptic plasticity that underlie learning and memory require new transcription and translation for their persistence. The remarkable polarity and compartmentalization of neurons raises questions about the spatial and temporal regulation of gene expression within neurons. Alternative cleavage and polyadenylation (APA) generates mRNA isoforms with different 3' untranslated regions (3'UTRs) and/or coding sequences. Changes in the 3'UTR composition of mRNAs can alter gene expression by regulating transcript localization, stability and/or translation, while changes in the coding sequences lead to mRNAs encoding distinct proteins. Using specialized 3' end deep sequencing methods, we undertook a comprehensive analysis of APA following induction of long-term potentiation (LTP) of mouse hippocampal CA3-CA1 synapses. We identified extensive LTP-induced APA changes, including a general trend of 3'UTR shortening and activation of intronic APA isoforms. Comparison with transcriptome profiling indicated that most APA regulatory events were uncoupled from changes in transcript abundance. We further show that specific APA regulatory events can impact expression of two molecules with known functions during LTP, including 3'UTR APA of Notch1 and intronic APA of Creb1. Together, our results reveal that activity-dependent APA provides an important layer of gene regulation during learning and memory.

Purpose: In head and neck squamous cell carcinoma (HNSCC), the incidence of RAS mutation, which is the major cause of cetuximab resistance, is relatively rare compared with the other types of cancers, and the mechanism mediating acquired resistance is unclear compared with the driver gene mutation-mediated de novo resistance. Here, we investigated the driver gene mutation-independent mechanism for cetuximab resistance in HNSCC. Experimental Design: We used the in vitro -selected and in vivo -selected cetuximab-resistant sublines of HNSCC cell lines for investigating the mechanism of acquired resistance to cetuximab. Zebrafish model was applied for evaluating the synergistic effect of combinatory drugs for overcoming cetuximab resistance. Results: The cetuximab-resistant HNSCC cells undergo a Snail-induced epithelial-mesenchymal transition. Mechanistically, Snail induces the expression of lymphotoxin-β (LTβ), a TNF superfamily protein that activates NF-κB, and protein arginine methyltransferase 1 (PRMT1), an arginine methyltransferase that methylates EGFR. LTβ interacts with methylated EGFR to promote its ligand-binding ability and dimerization. Furthermore, LTβ activates the NF-κB pathway through a LTβ receptor-independent mechanism. Combination of an EGFR tyrosine kinase inhibitor and a NF-κB inhibitor effectively suppressed cetuximab-resistant HNSCC and interfering with the EGFR-LTβ interaction reverses resistance. Conclusions: Our findings elucidate the mechanism of driver gene mutations-independent mechanism of acquired resistance to cetuximab in HNSCC and also provide potential strategies for combating cetuximab resistance. Clin Cancer Res; 23(15); 4388-401. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Plasmodium falciparum HKMTs (PfHKMTs) play a key role in controlling Plasmodium gene expression and represent exciting new anti-malarial epigenetic targets. Using an inhibitor series derived from the diaminoquinazoline HKMT inhibitory chemotype, we have previously identified compounds with highly promising antimalarial activity, including irreversible asexual cycle blood stage-independent cytotoxic activity at nM concentrations, oral efficacy in in vivo models of disease, and the unprecedented ability to reactivate dormant liver stage parasites (hypnozoites). However, future development of this series will need to address host versus parasite selectivity, where inhibitory activity against human G9a is removed from the lead compounds, while maintaining potent anti- Plasmodium activity. Herein, we report an extensive study of the SAR of this series against both G9a and P. falciparum . We have identified key SAR features which demonstrate that high parasite vs. G9a selectivity can be achieved by selecting appropriate substituents at position 2, 4 and 7 of the quinazoline ring. We have also, in turn, discovered that potent G9a inhibitors can be identified by employing a 6-carbon 'Nle mimic' at position 7. Together, this data suggests that while broadly similar, the G9a and potential PfHKMT target(s) binding pockets and/or binding modes of the diaminoquinazoline analogues exhibit clear and exploitable differences. Based on this, we believe this scaffold to have clear potential for development into a novel anti-malarial therapeutic.

The persistence of long-lasting changes in synaptic connectivity that underlie long-term memory require new RNA and protein synthesis. To elucidate the temporal pattern of gene expression that gives rise to long-lasting neuronal plasticity, we analyzed differentially-expressed (DE) RNAs in mouse hippocampal slices following induction of late phase long-term potentiation (L-LTP) specifically within pyramidal excitatory neurons using Translating Ribosome Affinity Purification RNA sequencing (TRAP-seq). We detected time-dependent changes in up- and down-regulated ribosome-associated mRNAs over 2 h following L-LTP induction, with minimal overlap of DE transcripts between time points. TRAP-seq revealed greater numbers of DE transcripts and magnitudes of LTP-induced changes than RNA-seq of all cell types in the hippocampus. Neuron-enriched transcripts had greater changes at the ribosome-loading level than the total RNA level, while RNA-seq identified many non-neuronal DE mRNAs. Our results highlight the importance of considering both time course and cell-type specificity in activity-dependent gene expression during memory formation.

Behavioral, physiological, and anatomical evidence indicates that the dorsal and ventral zones of the hippocampus have distinct roles in cognition. How the unique functions of these zones might depend on differences in synaptic and neuronal function arising from the strikingly different gene expression profiles exhibited by dorsal and ventral CA1 pyramidal cells is unclear. To begin to address this question, we investigated the mechanisms underlying differences in synaptic transmission and plasticity at dorsal and ventral Schaffer collateral (SC) synapses in the mouse hippocampus. We find that, although basal synaptic transmission is similar, SC synapses in the dorsal and ventral hippocampus exhibit markedly different responses to θ frequency patterns of stimulation. In contrast to dorsal hippocampus, θ frequency stimulation fails to elicit postsynaptic complex-spike bursting and does not induce LTP at ventral SC synapses. Moreover, EPSP-spike coupling, a process that strongly influences information transfer at synapses, is weaker in ventral pyramidal cells. Our results indicate that all these differences in postsynaptic function are due to an enhanced activation of SK-type K + channels that suppresses NMDAR-dependent EPSP amplification at ventral SC synapses. Consistent with this, mRNA levels for the SK3 subunit of SK channels are significantly higher in ventral CA1 pyramidal cells. Together, our findings indicate that a dorsal-ventral difference in SK channel regulation of NMDAR activation has a profound effect on the transmission, processing, and storage of information at SC synapses and thus likely contributes to the distinct roles of the dorsal and ventral hippocampus in different behaviors. SIGNIFICANCE STATEMENT Differences in short- and long-term plasticity at Schaffer collateral (SC) synapses in the dorsal and ventral hippocampus likely contribute importantly to the distinct roles of these regions in cognition and behavior. Although dorsal and ventral CA1 pyramidal cells exhibit markedly different gene expression profiles, how these differences influence plasticity at SC synapses is unclear. Here we report that increased mRNA levels for the SK3 subunit of SK-type K + channels in ventral pyramidal cells is associated with an enhanced activation of SK channels that strongly suppresses NMDAR activation at ventral SC synapses. This leads to striking differences in multiple aspects of synaptic transmission at dorsal and ventral SC synapses and underlies the reduced ability of ventral SC synapses to undergo LTP., (Copyright © 2017 the authors 0270-6474/17/371951-15$15.00/0.)

Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes.

Numerous chromatin-remodeling factors are regulated by interactions with RNA, although the contexts and functions of RNA binding are poorly understood. Here we show that R loops, RNA-DNA hybrids consisting of nascent transcripts hybridized to template DNA, modulate the binding of two key chromatin-regulatory complexes, Tip60-p400 and polycomb repressive complex 2 (PRC2) in mouse embryonic stem cells (ESCs). Like PRC2, the Tip60-p400 histone acetyltransferase complex binds to nascent transcripts; however, transcription promotes chromatin binding of Tip60-p400 but not PRC2. Interestingly, we observed higher Tip60-p400 and lower PRC2 levels at genes marked by promoter-proximal R loops. Furthermore, disruption of R loops broadly decreased Tip60-p400 occupancy and increased PRC2 occupancy genome wide. In agreement with these alterations, ESCs partially depleted of R loops exhibited impaired differentiation. These results show that R loops act both positively and negatively in modulating the recruitment of key pluripotency regulators.

Nerve growth factor (NGF) is elevated in certain chronic pain conditions and is a sufficient stimulus to cause lasting pain in humans, but the actual mechanisms underlying the persistent effects of NGF remain incompletely understood. We developed a rat model of NGF-induced persistent thermal hyperalgesia and mechanical allodynia to determine the role of transient receptor potential vanilloid 1 (TRPV1) and oxidative mechanisms in the persistent effects of NGF. Persistent thermal hypersensitivity and mechanical allodynia require de novo protein translation and are mediated by TRPV1 and oxidative mechanisms. By comparing effects after systemic (subcutaneous), spinal (intrathecal) or hindpaw (intraplantar) injections of test compounds, we determined that TRPV1 and oxidation mediate persistent thermal hypersensitivity via peripheral and spinal sites of action and mechanical allodynia via only a spinal site of action. Therefore, NGF-evoked thermal and mechanical allodynia are mediated by spatially distinct mechanisms. NGF treatment evoked sustained increases in peripheral and central TRPV1 activity, as demonstrated by increased capsaicin-evoked nocifensive responses, increased calcitonin gene-related peptide release from hindpaw skin biopsies, and increased capsaicin-evoked inward current and membrane expression of TRPV1 protein in dorsal root ganglia neurons. Finally, we showed that NGF treatment increased concentrations of linoleic and arachidonic-acid-derived oxidized TRPV1 agonists in spinal cord and skin biopsies. Furthermore, increases in oxidized TRPV1-active lipids were reduced by peripheral and spinal injections of compounds that completely blocked persistent nociception. Collectively, these data indicate that NGF evokes a persistent nociceptive state mediated by increased TRPV1 activity and oxidative mechanisms, including increased production of oxidized lipid TRPV1 agonists., (Copyright © 2015 the authors 0270-6474/15/358593-11$15.00/0.)