Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids.

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

Reduction rate of monoclonal protein as a useful prognostic factor in standard-risk group of newly diagnosed multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a common hematologic malignancy that originates from a malignant clone of plasma cells. Solitary plasmacytoma, history of diabetes, and platelet count are considered as prognostic factors for MM. But some patients are still associated with much worse outcomes without any prognostic predictors. This study aimed to observe the reduction rate of monoclonal protein (M protein) after the first and fourth chemotherapy cycles, which is considered as a new prognostic factor for progression-free survival (PFS) in standard-risk group of newly diagnosed MM patients. AIM: To investigate the reduction rate of M protein after first and fourth cycle chemotherapy as a useful prognostic factor. METHODS: A total of 316 patients diagnosed with MM for the first time between 2010 and 2019 at the Lishui Municipal Central Hospital were included. All patients were diagnosed according to the National Comprehensive Cancer Network (NCCN) 2020.V1 diagnostic criteria. The risk assessment was performed by the Mayo Stratification for Macroglobulinemia and Risk-Adapted Therapy guidelines. After diagnosis, 164 patients were evaluated and underwent treatment with four to eight courses of continuous induction chemotherapy. The patients with no response after induction treatment were administered additional therapy following the NCCN 2020.V1 criteria. The following baseline data from the patients were collected: Gender, age at diagnosis, Durie-Salmon stage, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, catabolite activator protein, albumin/globulin ratio, lactate dehydrogenase, translocation (t)(6;14), t(11;14), maintenance regimen, total cholesterol (TC), triglyceride, and phosphorous. All baseline data and the reduction rate of M protein after each chemotherapy cycle from the first to fourth were assessed by univariate analysis. The factors influencing the overall survival and PFS were then assessed by multivariate analysis. We found the first cycle (C1) reduction rate and the fourth cycle (C4) reduction rate as predictors of PFS. Then, PFS was compared between patients with a C1 reduction rate of M protein of ≥ 25% vs < 25% and ≥ 50% vs < 50%, and between patients with a C4 reduction rate of ≥ 25% vs < 25%, ≥ 50% vs < 50%, and ≥ 75% vs < 75%. RESULTS: Multivariate analysis revealed age [hazard ratio (HR): 1.059, 95% confidence interval (95%CI): 1.033-1.085, P ≤ 0.001], International Staging System stage (HR: 2.136, 95%CI: 1.500-3.041, P ≤ 0.001), autotransplantion (HR: 0.201, 95%CI: 0.069-0.583, P = 0.019), TC (HR: 0.689, 95%CI: 0.533-0.891, P = 0.019), C1 reduction rate (HR: 0474, 95%CI: 0.293-0.767, P = 0.019), and C4 reduction rate (HR: 0.254, 95%CI: 0.139-0.463, P = 0.019) as predictors of PFS. The Kaplan-Meier survival analysis and the log-rank tests revealed that a higher reduction rate of M protein after first cycle (≥ 50%) and fourth cycle (≥ 75%) chemotherapy was associated with a longer PFS than the lower one. CONCLUSION: Higher reduction rates of M protein after the first and fourth chemotherapy cycles can act as advantageous prognostic factors for PFS in standard-risk group of MM patients during initial diagnosis.

[Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning. METHODS: Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively. RESULTS: Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group. CONCLUSION: EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.

[Effect of Tongdu Tiaoshen electroacupuncture pretreatment on PPARγ-mediated pyroptosis of cerebral cortex in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI. METHODS: A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1ß and IL-18 in the cerebral cortex of rats were determined by ELISA. RESULTS: Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1ß and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1ß and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1ß and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1ß and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1ß and IL-18 were higher (P<0.01). CONCLUSION: Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.

[Tongdu Tiaoshen acupuncture combined with carotid endarterectomy for carotid artery stenosis: a randomized controlled trial].

OBJECTIVE: To observe the clinical therapeutic effect of Tongdu Tiaoshen acupuncture combined with carotid endarterectomy (CEA) and simple CEA on carotid artery stenosis (CAS). METHODS: A total of 60 patients with CAS were randomized into an observation group (30 cases, 2 cases dropped off) and a control group (30 cases, 3 cases dropped off). Both groups were treated with eversion CEA (eCEA). The conventional treatment of internal medicine and antiplatelet drugs i.e. aspirin enteric-coated tablet and clopidogrel hydrogen sulfate tablet were given in the control group for 4 weeks. On the basis of the treatment in the control group, Tongdu Tiaoshen acupuncture was applied at Baihui (GV 20), Fengfu (GV 16), Yamen (GV 15), cervical Jiaji (EX-B 2), Dazhui (GV 14), etc. in the observation group, once a day, 1-day rest was taken after 6-day treatment, 2 weeks were as one course and totally 2 courses were required. The carotid intima-media thickness (IMT) before and after treatment was detected by ultrasonic diagnostic apparatus, the TCM symptom score was compared before and after treatment and in the follow-up of 6 months after treatment, the clinical efficacy was evaluated in the two groups. The occurrence of endpoints within 1 year was recorded. RESULTS: After treatment, the carotid IMT and TCM symptom scores were decreased compared before treatment in the both groups (P<0.05), and the changes in the observation group were greater than the control group (P<0.05). In the follow-up, the TCM symptom scores were decreased compared before treatment in the both groups (P<0.05). The total effective rate was 96.4% (27/28) in the observation group, which was superior to 88.9% (24/27) in the control group (P<0.05). There were 1 case of stoke in the observation group and 2 cases of stroke in the control group within 1-year follow-up, and there was no significant difference in the number of endpoints between the two groups within 1 year (P>0.05). CONCLUSION: Tongdu Tiaoshen acupuncture combined with CEA can effectively reduce the IMT in patients with CAS, improve the TCM symptom score, the efficacy is superior to simple CEA treatment.

[Moxibustion at acpoints of governor vessel on regulating PI3K/Akt/mTOR signaling pathway and enhancing autophagy process in APP/PS1 double-transgenic Alzheimer's disease mice].

OBJECTIVE: To observe the eliminating effects of moxibustion at "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) on amyloid ß-peptide (Aß) in brain of the amyloid precursor protein/presenili1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD) by regulating the phosphoinositide 3-kinases/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. METHODS: A total of 60 APP/PS1 double-transgenic mice with AD were randomly divided into a model group, a moxibustion group, a rapamycin group and a combination group (treated with moxibustion and inhibitor), 15 mice in each group, another 15 male C57BL/6J mice with same age and background were selected as the control group. In the moxibustion group, pressing moxibustion was applied at "Baihui" (GV 20) while the mild moxibustion was applied at "Fengfu" (GV 16) and "Dazhui" (GV 14). The treatment was manipulated for 20 min each time, once a day for 2 weeks. In the rapamycin group, rapamycin (2 mg/kg) was given by intraperitoneal injection once a day for 2 weeks. On the basis of the treatment in the moxibustion group, 3-methyladenine (1.5 mg/kg) was given by intraperitoneal injection once a day for 2 weeks. The mice in the control and the model group received normal diet and no intervention was given for 2 weeks. Immunohistochemica method was used to measure the levels of Aß1-42 in the cerebral cortex and hippocampal, transmission electron microscopy was used to observe the formation of autophagosome in hippocampus, and Western blot method was used to observe the levels of PI3K, Akt, p-Akt, mTOR and p-mTOR in hippocampus. RESULTS: Compared with the control group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the model group (P<0.01). Compared with the model group, the levels of Aß1-42 in the cerebral cortex and hippocampal were decreased in the moxibustion group, the rapamycin group and the combination group (all P<0.01), compared with the moxibustion group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the combination group (P<0.01), while there was no significant difference between the moxibustion group and the rapamycin group in the levels of Aß1-42（P>0.05）. Compared with the rapamycin group, the levels of Aß1-42 in the cerebral cortex and hippocampal were increased in the combination group (P<0.01). In the model group, the cytoplasmic utophagic vacuoles and organelles of neuron were reduced. In the moxibustion group, the utophagic vacuoles were increased, and the organelles showed deformation and atrophy. In the rapamycin group, the utophagic vacuoles were widely disturbed and few deformed organelles were found. In the combination group, few utophagic vacuoles were found and additional organelles showed deformation and atrophy. Compared with the control group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the model group (all P<0.01). Compared with the model group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were reduced in the moxibustion group, the rapamycin group and the combination group (all P<0.01). Compared with the moxibustion group, the levels of PI3K、Akt、and p-mTOR were increased in the rapamycin group and the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the combination group (all P<0.01). Compared with the rapamycin group, the levels of PI3K、Akt、p-Akt、mTOR and p-mTOR were increased in the combination group (P<0.01). CONCLUSION: Moxibustion at acupoints of governor vessel can enhance the autophagy process on Aß1-42 in brain of the APP/PS1 double-transgenic AD mice, which may be associated with its effects on inhibiting the abnormal activation of PI3K/Akt/mTOR signaling pathway.