Myosin 1g as a high-risk biomarker in a pediatric patient with lineage switch from acute lymphoblastic leukemia to myeloid phenotype / Miosina 1g como un marcador de alto riesgo en una paciente pediátrica con cambio de linaje de leucemia linfoblástica aguda a fenotipo mieloide

Abstract Background: Myosin 1g (Myo1g) has recently been identified as a potential diagnostic biomarker in childhood acute lymphocytic leukemia (ALL). Case report: We describe the case of a 1-year-old Mexican female patient. Although initially studied for hepatomegaly, an infectious or genetic etiology was excluded. Liver biopsy showed infiltration by neoplastic B-cell precursors (BCPs), and bone marrow (BM) aspirate showed 14.5% of BCPs. In a joint session of the oncology, hematology, and pathology departments, low-risk (LR) BCP-ALL of hepatic origin with aberrant myeloid markers was diagnosed. Although treatment was initiated, the patient presented early with BM relapse. Modest overexpression of Myo1g was observed from the onset. However, at the end of the steroid window, expression increased significantly and remained elevated during this first relapse to BM. The parents refused hematopoietic stem cell transplantation, but she continued chemotherapy. After a second BM relapse at 5 years of age, the phenotype switched to myeloid. Her parents then opted for palliative care, and the patient died two months later at home. Conclusions: This case shows the potential use of Myo1g in clinical practice as a high-risk indicator. Myo1g monitoring may reveal a high risk and relapse trend, even when typical parameter values are not altered: Myo1g could be used to classify patients from low to high risk from diagnosis, allowing patients to promptly receive the best treatment and potentially modifying prognosis and survival.

Resumen Introducción: Recientemente se ha identificado a miosina 1g (Myo1g) como un potencial biomarcador de diagnóstico en la leucemia linfoblástica aguda (LLA) infantil. Caso clínico: Se describe el caso de una paciente mexicana de 1 año de edad. Aunque inicialmente se estudió por hepatomegalia, se descartó una etiología infecciosa o genética. La biopsia hepática mostró infiltración por precursores de células B neoplásicas (PCB) y un aspirado de médula ósea (MO) mostró 14.5% de PCB. En una sesión conjunta de los departamentos de oncología, hematología y patología, se diagnosticó PCB-LLA de bajo riesgo de origen hepático con marcadores mieloides aberrantes. Aunque se inició tratamiento, la paciente presentó tempranamente recaída de MO. Se observó una modesta sobreexpresión de Myo1g. Sin embargo, al final de la ventana de esteroides, la expresión aumentó considerablemente y permaneció elevada durante esta primera recaída a MO. El trasplante de células madre hematopoyéticas fue rechazado por los padres, pero se continuó con la quimioterapia. Tras una segunda recaída de MO a los 5 años, el fenotipo cambió a mieloide. Sus padres optaron entonces por cuidados paliativos y la paciente falleció dos meses después en su domicilio. Conclusiones: Este caso muestra el potencial uso de Myo1g como indicador de alto riesgo en la práctica clínica. El seguimiento de Myo1g puede revelar una tendencia de alto riesgo y recaídas, incluso cuando los valores de los parámetros rutinarios son aparentemente normales; Myo1g podría utilizarse para clasificar a los pacientes de bajo a alto riesgo desde el diagnóstico, lo que permitiría que los pacientes reciban el mejor tratamiento de manera oportuna, modificando potencialmente el pronóstico y la supervivencia.

Cardiovascular disease risk prediction in scleroderma

SUMMARY OBJECTIVE: Cardiovascular disease risk prediction in scleroderma is important. In this study of scleroderma patients, the aim was to investigate the relationship between cardiac myosin-binding protein-C, sensitive troponin T, and trimethylamine N-oxide and cardiovascular disease risk with the Systematic COronary Risk Evaluation 2 model of the European Society of Cardiology. METHODS: Systematic COronary Risk Evaluation 2 risk groups of 38 healthy controls and 52 women with scleroderma were evaluated. Cardiac myosin-binding protein-C, sensitive troponin T, and trimethylamine N-oxide levels were analyzed with commercial ELISA kits. RESULTS: In scleroderma patients, cardiac myosin-binding protein-C and trimethylamine N-oxide levels were higher than healthy controls but sensitive troponin T was not (p<0.001, p<0.001, and p=0.274, respectively). Out of 52 patients, 36 (69.2%) were at low risk, and the other 16 (30.8%) patients were at high-moderate risk with the Systematic COronary Risk Evaluation 2 model. At the optimal cutoff values, trimethylamine N-oxide could discriminate high-moderate risk with sensitivity 76%, specificity 86% and cardiac myosin-binding protein-C with sensitivity 75%, specificity 83%. Patients with high trimethylamine N-oxide levels (≥10.28 ng/mL) could predict high-moderate- Systematic COronary Risk Evaluation 2 risk 15 times higher than those with low trimethylamine N-oxide (<10.28 ng/mL) levels (odds ratio [OR]: 15.00, 95%CI 3.585-62.765, p<0.001). Similarly, high cardiac myosin-binding protein-C (≥8.29 ng/mL) levels could predict significantly higher Systematic COronary Risk Evaluation 2 risk than low cardiac myosin-binding protein-C (<8.29 ng/mL) levels (OR: 11.00, 95%CI 2.786-43.430). CONCLUSION: Noninvasive cardiovascular disease risk prediction indicators in scleroderma, cardiac myosin-binding protein-C, and trimethylamine N-oxide could be recommended to distinguish between high-moderate risk and low risk with the Systematic COronary Risk Evaluation 2 model.

Mechanism of Xiangsha Liujunzi Tang in Regulation of RhoA/ROCK2/MYPT1 Pathway to Improve Gastric Motility in Rats with Functional Dyspepsia / 中国实验方剂学杂志

ObjectiveTo explore the intervention mechanism of Xiangsha Liujunzi Tang in rats with functional dyspepsia （FD） based on the Ras homolog gene family member A （RhoA）/Rho-associated coiled-coil containing protein kinase 2 （ROCK2）/Myosin phosphatase target Subunit 1 （MYPT1） pathway. MethodSixty male SD suckling rats in SPF grades were randomly divided into blank group （n=10） and model group （n=50）. The comprehensive modeling method （gavage administration of iodoacetamide+exhaustion of swimming+disturbance of hunger and satiety） was used to replicate the rat model of FD. After successful replication of the model， the rats in the model group were randomly divided into model group， mosapride group， and high， middle， and low-dose Xiangsha Liujunzi Tang groups， with 10 rats in each group. Rats in the blank group and model group were given 10 mL kg-1·d-1 normal saline， those in the mosapride group were given 1.35 mg·kg-1·d-1 mosapride， and those in the high， middle， and low-dose Xiangsha Liujunzi Tang groups were given 12， 6， and 3 g·kg-1·d-1 Xiangsha Liujunzi Tang， respectively. The intervention lasted 14 days. The general living conditions of rats were observed before and after modeling and administration， and the 3-hour food intake and body mass of rats were measured. After intervention， the intestinal propulsion rate of rats was measured， and the pathological changes in the gastric tissue were observed by hematoxylin-eosin （HE） staining. The content of choline acetyl transferase （ChAT） and vasoactive intestinal peptide （VIP） in the medulla oblongata and gastric tissue homogenate was determined by enzyme-linked immunosorbent assay （ELISA）， the distribution of adenosine triphosphate （ATP） enzyme in gastric antrum smooth muscle was observed by frozen section staining， and the protein expression levels of RhoA， ROCK2， and phosphorylated-myosin phosphatase target subunit 1 （p-MYPT1） in the gastric tissue were detected by Western blot. ResultCompared with the blank group， the model group had withered hair， lazy movement， slow action， poor general living condition， lower 3-hour food intake， body mass， and lower intestinal propulsion rate （P<0.05）， whereas no obvious abnormality in gastric histopathology. In the model group， the content of ChAT in the medulla oblongata and gastric tissue decreased， the content of VIP in gastric tissue increased， the distribution of ATP enzyme in gastric antrum smooth muscle decreased significantly， and the protein expression levels of RhoA， ROCK2， and p-MYPT1 in the gastric tissue decreased significantly （P<0.05）. As compared with the model group， the general living condition of rats in each intervention group was significantly improved， and the 3-hour food intake， body mass， and intestinal propulsion rate were significantly increased （P<0.05）. There was no significant difference in gastric pathology in the intervention groups. The content of ChAT in the medulla oblongata and gastric tissue increased significantly， the content of VIP in the gastric tissue decreased， the distribution of ATP enzyme in gastric antrum smooth muscle increased significantly， and the protein expression levels of RhoA， ROCK2， and p-MYPT1 in the gastric tissue increased significantly （P<0.05）. The intervention effect of Xiangsha Liujunzi Tang group on the above indexes was dose-dependent. ConclusionXiangsha Liujunzi Tang can effectively improve the general living condition and gastric motility of rats with FD， and its specific mechanism may be related to the activation of the RhoA/ROCK2/MYPT1 pathway in the gastric tissue to regulate smooth muscle relaxation and contraction and promote gastric motility.

Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

Tipos de fibras de los músculos intrínsecos laríngeos de seres humanos: una revisión de la literatura / Fiber types in human intrinsic laryngeal muscles: a literature review

El estudio de las fibras musculares permite comprender con mejor detalle la composición de los músculos y sus características funcionales. Además, facilita la aplicación de programas de entrenamiento y rehabilitación basados en las vías energéticas que regulan la contracción muscular. Su estudio generalmente va unido al análisis de las cadenas pesadas de miosina (MyHC), las que informan sobre las características y propiedades funcionales del músculo. El objetivo de este trabajo fue sintetizar la evidencia científica disponible sobre la distribución de fibras musculares y de isoformas de cadenas pesadas de miosina de los músculos intrínsecos de la laringe de seres humanos. Se realizó una revisión sistemática de la literatura mediante el análisis de artículos encontrados en las bases de datos PubMed, EBSCOHost y SciELO. Los hallazgos informan sobre la existencia de fibras tónicas lentas y tipo I, II, IIA y IIX/IIB. Además, se reconoce la presencia de las isoformas MyHC-I, MyHC-IIA, MyHC-IIX, MyHC-Fetal, MyHC-L y MyHC-IIB. En conclusión, los músculos intrínsecos de la laringe presentan una mezcla de fibras y de isoformas de MyHC lentas y rápidas,la que obedece a adaptaciones y cambios evolutivos que han permitido, por ejemplo, las características fonatorias que presenta la voz del ser humano.

The study of muscle fibers allows the composition of muscles and their functional characteristics to be understood in greaterdetail. In addition, it makes it possible to applytraining and rehabilitation programs based on the energypathways that regulatemuscle contraction. Studying muscle fibers is generally associated withthe analysis of myosin heavy chains (MHC) which provide information on the functional characteristics and properties of muscles. The objective of this study was to synthesize the available scientific evidence onthe distribution of muscle fibers and myosin heavy chain isoforms present in the intrinsic laryngeal muscles of human beings. A systematic reviewof the literature was carried outand articles found on PubMed, EBSCOHost,and SciELOwere analyzed.The findings showthe presenceof slow-tonic, type I, type II, type IIA, and type IIX/IIB fibers. Additionally,isoforms MHC-I, MHC-IIA, MHC-IIX, MHC-Fetal, MHC-L, and MHC-IIB canbe found. In conclusion, intrinsic laryngeal muscles are composed ofa combination of slow and fast fibers and MHC isoforms, derived from evolutionary adaptations and changes which have given way, among other things, to the phonetic characteristics ofthe human voice.

Progress in New Therapies Targeting the Pathogenesis of Cardiomyopathywith Hypertrophic Phenotype / 罕见病研究

Hypertrophic cardiomyopathy (HCM) is cardiomyopathy with a clinical phenotype of cardiac hypertrophy. The etiology includes genetically defective encoding sarcomeres, congenital metabolic diseases such as lysosomal storage diseases, systemic amyloidosis such as transthyretin amyloidosis(ATTR), and Fabry disease. Previous therapies did not target the etiology and pathogenesis and therefore were less effective. In recent years, treatments targeting different mechanisms of myocardial hypertrophy have achieved good results. Mavacamten can reduce myocardial contractility by inhibiting ATP activity, thereby significantly improving left ventricular outflow tract(LVOT) obstruction, cardiac contractility, ventricular tension, and limitting myocardial damage. By inhibiting the dissociation of transthyretin(TTR) and subsequent formation and deposition of the amyloid fibril, tafamidis can reduce the mortality and morbidity of patients with transthyretin cardiac amyloidosis(ATTR-CA). Gene silencing and gene editing technology can reduce abnormal TTR levels. Synthesis of α-galactosidase A by gene recombination technology in vitro can effectively reduce left ventricular mass index(LVMi), improve cardiac function, reduce angina attacks and decrease mortality of Fabry disease.

Advances in the treatment of hypertrophic cardiomyopathy with cardiac myosin inhibitors / 中国药房

In recent years， with the increasing understanding of the genetic mechanisms of hypertrophic cardiomyopathy， novel molecular-targeted drugs Mavacamten and Aficamten are two cardiac myosin inhibitors currently approved by the FDA for the treatment of hypertrophic obstructive cardiomyopathy. Both of them have a similar mechanism of action and can selectively bind to different variable sites of cardiac myosin to inhibit cardiac myosin， thus reducing myocardial hypercontractility. Relevant clinical studies have also shown that both drugs can reduce patients’ left ventricular outflow tract pressure gradient， the levels of N-terminal pro-B-type natriuretic peptide and cardiac troponin I as cardiac markers， and improve New York Heart Association （NYHA） cardiac function class. They are safe， have mild adverse reactions， and can be tolerated by patients. Compared to Mavacamten， Aficamten， as a structurally optimized product， has a shorter half-life and fewer drug-drug interactions， which is more conducive to drug- targeted dose titration.

HBXIP blocks myosin-IIA assembly by phosphorylating and interacting with NMHC-IIA in breast cancer metastasis

Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCβII, by coactivating Sp1, and triggered PKCβII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCβII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.

Retinoic acid induces differentiation of cochlear neural progenitor cells into hair cells / O ácido retinoico induz a diferenciação das células progenitoras neurais cocleares em células ciliadas

Abstract Introduction: Inner ear progenitor cells have the potential for multi-directional differentiation. Retinoic acid is an important requirement for the development of the inner ear. Blocking the Curtyr's retinoic acid signaling pathway can significantly reduce the number of hair cells. Therefore, we believe that retinoic acid may induce the regeneration of inner ear hair cells. Objective: To investigate whether the cochlear neural progenitor cells maintain the characteristics of stem cells during recovery and subculture, whether retinoic acid can induce cochlear neural progenitor cells into hair cells in vitro, and whether retinoic acid promotes or inhibits the proliferation of cochlear neural progenitor cells during differentiation. Methods: Cochlear neural progenitor cells were cultured and induced in DMEM/F12 + RA (10−6M) and then detected the expressions of hair cell markers (Math1 and MyosinVIIa) by immunofluorescence cytochemistry and realtime-polymerase chain reaction, and the proliferation of cochlear neural progenitor cells was detected by Brdu. Results: The nestin of cochlear neural progenitor cells was positively expressed. The ratios of Math1-positive cells in the control group and experimental group were 1.5% and 63%, respectively; the ratios of MyosinVIIa-positive cells in the control group and experimental group were 0.96% and 56%, respectively (p <0.05). The ratios of Brdu+-labeled cells in retinoic acid group, group PBS, and group FBS were 20.6%, 29.9%, and 54.3%, respectively; however, the proliferation rate in the experimental group decreased. Conclusion: Retinoic acid can promote cochlear neural progenitor cells to differentiate into the hair cells.

Resumo Introdução: As células progenitoras da orelha interna têm potencial para diferenciação multidirecional. O ácido retinoico é uma condição importante para o desenvolvimento da orelha interna. O bloqueio da via de sinalização do ácido retinoico no órgão de Corti pode reduzir significativamente o número de células ciliadas. Portanto, acreditamos que o ácido retinoico pode induzir a regeneração das células ciliadas do ouvido interno. Objetivo: Investigar se as células progenitoras neurais cocleares mantêm as características das células-tronco durante a recuperação e subcultura, se o ácido retinoico pode induzir a transformação de células progenitoras neurais cocleares em células ciliadas in vitro e se o ácido retinoico promove ou inibe a proliferação das células progenitoras durante a diferenciação. Método: As células progenitoras neurais cocleares foram cultivadas e induzidas em DMEM/F12+AR (106M) e, então, foram detectadas as expressões de marcadores das células ciliadas (Math1 e Myosin?a) com o uso de citoquímica por imunofluorescência e real time -polymerase chain reaction e a proliferação de células progenitoras neurais cocleares foi detectada pelo teste Brdu. Resultados: A nestina das células progenitoras neurais cocleares foi expressa positivamente. As proporções de células positivas para Math1 no grupo controle e no grupo experimental foram 1,5% e 63%, respectivamente; as proporções de células positivas para Myosin?a no grupo controle e no grupo experimental foram de 0,96% e 56%, respectivamente (p <0,05). As proporções de células marcadas com Brdu+ no grupo ácido retinoico, grupo PBS e grupo FBS foram de 20,6%, 29,9% e 54,3%, respectivamente; no entanto, a taxa de proliferação no grupo experimental diminuiu. Conclusões: O ácido retinoico pode promover a diferenciação das células progenitoras neurais cocleares em células ciliadas.

mRNA expression of myosin heavy chain isoforms in the sphenomandibularis portion of the temporalis muscle / Expresión de ARNm de las isoformas de la cadena pesada de la miosina en la porción esfenomandibular del músculo temporal

SUMMARY: The main objective of this study was to analyze by real-time quantitative polymerase chain reaction (RT-qPCR) the expression patterns of the myosin heavy chain (MHC) isoforms (MHC-I, MHC-IIa, MHC-IIx) in the sphenomandibularis portion of the temporalis muscle. We expected to find differences between the sphenomandibularis and the other portions of the temporalis that could be related to the functional characteristics of the sphenomandibularis identified by electromyography. We dissected the right temporalis muscle of ten adult human individuals (five men and five women). Samples of the anterior and posterior temporalis and of the sphenomandibularis portion were obtained from each dissected muscle. These samples were analyzed by RT-qPCR to determine the percentages of expression of the MHC-I, MHC-IIa and MHC-IIx isoforms. No significant differences were identified between the anterior and the posterior temporalis in the expression patterns of the MHC-I, MHC-IIa and MHC-IIx isoforms. However, there were significant differences between the sphenomandibularis and the anterior temporalis. Specifically, the sphenomandibularis portion had a higher percentage of expression of the MHC-I isoform (P=0.04) and a lower percentage of expression of the MHC-IIx isoform (P=0.003). The pattern of expression that we observed in the sphenomandibularis reflects a greater resistance to fatigue, a lower contraction speed, and a lower capacity of force generation in the sphenomandibularis compared to the anterior temporalis. These characteristics are consistent with electromyographic findings on the functional differences between these two portions.

RESUMEN: El principal objetivo de este estudio fue analizar mediante real-time quantitative polymerase chain reaction (RT-qPCR) los patrones de expresión de las isoformas de la cadena pesada de la miosina (MHC-I, MHC-IIa y MHC-IIx) en la porción esfenomandibular del músculo temporal. Se esperó encontrar diferencias entre el esfenomandibular y las otras porciones del músculo temporal que se pudieran relacionar con las características funcionales del esfenomandibular, identificadas mediante electromiografía. Para obtener estos resultados, se diseccionó el músculo temporal derecho en diez humanos adultos (cinco hombres y cinco mujeres) y se obtuvieron muestras de la porción anterior y posterior del músculo temporal y de su porción esfenomandibular. Estas muestras fueron analizadas mediante RT-qPCR para determinar los porcentajes de expresión de las isoformas MHC-I, MHC- IIa y MHC-IIx. No se identificaron diferencias significativas de los patrones de expresión entre la porción anterior y la porción posterior del músculo temporal, pero sí que se observaron diferencias significativas entre la porción anterior del músculo temporal y su porción esfenomandibular. Concretamente, la porción esfenomandibular presentó un mayor porcentaje de expresión de la isoforma MHC-I (P=0.04) y un menor porcentaje de expresión de la isoforma MHC-IIx (P=0.003). El patrón de expresión que hemos observado en la porción esfenomandibular del músculo temporal refleja una mayor resistencia a la fatiga, una velocidad de contracción más lenta y una menor capacidad de generar fuerza si se compara esta porción con la porción anterior del músclo temporal. Estas características son consistentes con las diferencias funcionales que presentan estas dos porciones, que han sido descritas mediante electromiografía.

The effect and mechanism of astragaloside Ⅳ on lysophosphatidic acid-induced neurite retraction of N1E-115 cells / 国际中医中药杂志

Objective:To observe the effect of astragaloside Ⅳ on lysophosphatidic acid(LPA)- induced neurite retraction of N1E-115 cells and its potential mechanism.Methods:N1E-115 cells were divided into blank group, model group, the low, medium and high dose groups of astragaloside Ⅳ. The blank group and model group was not intervened by astragaloside; while the low, medium and high dose groups were treated with 20,40 and 80 μg/ml astragaloside Ⅳ for 24 h. Each group was cultured with serum-free medium for 12 h. The model group and astragaloside Ⅳ groups were intervened by 40 μmol/L LPA for 10 min. Each group was observed and photographed with the inverted microscope, and the number of neurites in N1E-115 cells was counted by Image J software. The fluorescence expression of recombinant ras homolog gene family member A (RhoA), rho associated coiledcoil protein kinase 2 (ROCK2), phospho-rho associated coiledcoil protein kinase 2 (p-ROCK2) and phospho-myosin light chain 2 (p-MLC2) proteins was detected by immunohistochemistry. Real-time fluorescent quantitative polymerase chain reaction was used to detect the mRNA expression levels of RhoA and ROCK2 ; the protein expression levels of RhoA, ROCK2, p-MLC2 and myosin light chain 2 (MLC2) were detected by Western blotting.Results:Compared with 20 μg/ml astragaloside Ⅳ group, the inhibition rate of neurite retraction in 40 and 80 μg/ml astragalosideⅣ groups increased ( P<0.05). Compared with model group, the average fluorescence intensity of RhoA, p-ROCK2, p-MLC2 in 20, 40, 80 μg/ml astragaloside Ⅳ groups and the ROCK2 average fluorescence intensity in 40 μg/ml astragaloside Ⅳ group were decreased ( P<0.05, P<0.01); the expression of RhoA mRNA (0.89±0.09, 0.41±0.01, 0.09±0.03 vs. 1.50±0.01) and ROCK2 mRNA (0.89±0.09, 0.14±0.01, 0.20±0.01 vs. 1.62±0.17) decreased in 20, 40, 80 μg/ml astragaloside Ⅳ groups ( P<0.05, P<0.01); the ROCK2 protein (0.75±0.06, 0.57±0.02, 0.66±0.01 vs. 1.08±0.02), p-MLC2 protein (1.72±0.03, 1.40±0.04, 1.29±0.03 vs. 2.19±0.11), MLC2 protein (1.13±0.02, 0.68±0.03, 0.75±0.03 vs. 1.60±0.03) in 20, 40, 80 μg/ml astragaloside Ⅳ groups and the RhoA protein (0.35±0.01, 0.40±0.03 vs. 0.57±0.08) in 20, 40 μg/ml astragaloside Ⅳ groups were decreased ( P<0.05, P<0.01). Conclusion:Astragaloside Ⅳ can prevent LPA-induced neurite retraction and promote damaged nerve regeneration. The mechanism may down-regulae the protein expression levels of RhoA, ROCK2, p-ROCK2, p-MLC2 and MLC2 in RhoA-ROCK2 signaling pathway, and inhibite nerve growth cone collapse.

MicroRNA-499 improved septic myocardial dysfunction by regulating myosin heavy chain gene axis / 中华危重病急救医学

Objective:To investigate the role and mechanism of microRNA-499 (miR-499) regulating α-myosin heavy chain (α-MHC) and β-myosin heavy chain (β-MHC) gene axis in septic myocardial dysfunction (SMD) and its significance.Methods:Sixty healthy adult male Sprague-Dawley (SD) rats were divided into phosphate buffered saline (PBS) control group (PBS group), lipopolysaccharide (LPS) induced SMD model group (LPS group), miR-499 agonist pretreatment group (agomir+LPS group), and miR-499 inhibitor pretreatment group (antagomir+LPS group) by random number table, with 15 rats in each group. SMD rat model was reproduced by intraperitoneal injection of LPS 10 mg/kg. The PBS group was intraperitoneally injected with the same amount of PBS. The two pretreatment groups were injected with agomir 30 mg/kg or antagomir 80 mg/kg through the caudal vein for 3 days, once a day. PBS group and LPS group were not pretreated. Echocardiography was detected 5 hours after LPS injection, and relevant indexes were recorded. The expression of miR-499 in plasma and myocardial tissue was detected by real-time quantitative polymerase chain reaction (qPCR). Western blotting was used to detect the protein expressions of α-MHC and β-MHC in myocardial tissue. Plasma N-terminal pro-brain natriuretic peptide (NT-proBNP), a marker of heart failure, was measured by electrochemiluminescence.Results:Compared with the PBS group, the rats in LPS group were depressed. Additionally, LPS down-regulated the level of miR-499 in plasma and myocardial tissue, decreased α-MHC expression in myocardial tissue and up-regulated the expression of β-MHC. Echocardiography showed that left ventricular ejection fraction (LVEF), left ventricle fractional shortening (LVFS), cardiac output (CO), stroke volume (SV) and heart rate (HR) decreased by 49.1%, 59.2%, 48.8%, 39.4% and 15.9%, respectively, and the level of plasma NT-proBNP increased significantly in LPS group, indicating that LPS could induce cardiac dysfunction in rats. Compared with the LPS group, after pretreatment with agomir to overexpress the miR-499, LVEF and LVFS were significantly increased [LVEF: 0.662±0.020 vs. 0.323±0.024, LVFS: (36.16±1.43)% vs. (20.20±1.32)%, both P < 0.01], which suggested that the cardiac function of rats was improved in agomir+LPS group. At the same time, pretreatment with agomir significantly down-regulated the β-MHC protein expression (β-MHC/GAPDH: 0.74±0.04 vs. 2.97±0.34, P < 0.01), significantly up-regulated α-MHC protein expression (α-MHC/GAPDH: 1.59±0.05 vs. 0.74±0.14, P < 0.01), and significantly decreased the plasma NT-proBNP level (ng/L: 114.49±6.85 vs. 334.13±4.36, P < 0.01). After pretreatment with antagomir to inhibit the expression of miR-499, echocardiography showed that LVEF and LVFS were significantly lower than those in the LPS group [LVEF: 0.297±0.021 vs. 0.323±0.024, LVFS: (19.38±1.52)% vs. (21.20±1.32)%, both P < 0.01], which suggested that the cardiac function of rats was significantly inhibited. At the same time, pretreatment with antagomir significantly down-regulated α-MHC protein expression in myocardial tissue (α-MHC/GAPDH: 0.63±0.03 vs. 0.74±0.14, P < 0.01), significantly up-regulated β-MHC protein expression (β-MHC/GAPDH: 3.03±0.47 vs. 2.97±0.34, P < 0.01), and significantly increased the level of plasma NT-proBNP (ng/L: 373.91±4.23 vs. 334.13±4.36, P < 0.05). Conclusions:miR-499 could regulate the expression of α-MHC and β-MHC which improved cardiac dysfunction caused by sepsis. Targeted regulation of miR-499 expression may be an effective way to treat SMD.

Microvillus inclusion disease: a case report and literature review / 中华围产医学杂志

Objective:To investigate the clinical features and genetic mutations of microvillus inclusion disease (MVID).Methods:Clinical features and gene sequencing results of a neonate with MVID in Children's Hospital of Chongqing Medical University in August 2019 were retrospectively analyzed. Literature was retrieved up to October 2021, with the terms of microvillus inclusion disease, congenital microvilli atrophy, MVID, MYO5B, STX3, and STXBP2 in China National Knowledge Infrastructure, Wanfang Database, VIP database, and PubMed. Clinical features, diagnosis, and treatment of the reported MVID cases were reviewed. Results:(1) Case report: A male infant presented with jaundice two days after birth and was admitted to our hospital. Clinical features included intractable diarrhea, intermittent abdominal distension, uncorrectable dehydration, and weight loss. Laboratory test results indicated metabolic acidosis, electrolyte disorder, and cholestasis. Whole exome sequencing confirmed the diagnosis of MVID in this baby boy with compound heterozygous mutations of c.1021C>T(p.Q341*) and c.1125G>A(p.W375*) in the MYO5B gene, which were inherited from the father and the mother, respectively. (2) Literature review: Except for the present case, 31 patients from 20 articles were reviewed, and the typical clinical manifestations were intractable diarrhea, accompanied by dehydration, metabolic acidosis, electrolyte disorder, etc. Some patients also developed extra-gastrointestinal symptoms, including feeding difficulties and malnutrition (8/18), respiratory distress syndrome (4/18) and jaundice/cholestasis (4/18) in patients with MYO5B mutations; feeding difficulties and malnutrition (2/5), respiratory distress syndrome (1/5), and sepsis (1/5) in patients with STX3 mutations; feeding difficulties (2/9), respiratory distress syndrome (1/9), jaundice/cholestasis (1/9), sepsis (1/9), and hypoglycemia (1/9) in patients with STXBP2 mutations. In terms of the demographic data and prenatal examination, preterm birth (8/18), fetal bowel dilatation (5/18), polyhydramnios (5/18), parental consanguinity (2/18), and meconium-stained amniotic fluid (2/18) occurred among patients with MYO5B mutations. In those with STX3 mutations, parental consanguinity (3/5), fetal bowel dilatation (1/5), polyhydramnios (1/5), and meconium-stained amniotic fluid (1/5) occurred. Of nine patients with STXBP2 mutations, parental consanguinity (3/9), preterm birth (2/9), and polyhydramnios (2/9) occurred. Conclusions:MVID has atypical clinical features and a high mortality, resulting in difficulty in the diagnosis and treatment. The possibility of MVID should be considered when an infant presents with intractable diarrhea, dehydration, metabolic acidosis, and electrolyte disorder accompanied by multiple extra-gastrointestinal symptoms. Early identification of MYO5B, STX3, and STXBP2 mutations will benefit prompt intervention, prognosis evaluation, and genetic counseling.

Transcriptome analysis of primary human oral keratinocytes stimulated with Prevotella melaninogenica / 口腔疾病防治

Objective@# To investigate the transcriptomic changes in primary human oral keratinocytes (pHOKs) after coculture with Prevotella melaninogenica (P.m) and to verify the changes in human oral keratinocyte (HOK) cell lines.@*Methods@# pHOK was isolated and cocultured with P.m for 0, 4 and 24 h. Total RNA was extracted, a gene library was constructed, transcriptional sequencing was performed, differentially expressed genes (DEGs) were analyzed, gene ontology (GO) pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed, and the validation of DEGs was performed by qRT-PCR and Western Blot in the HOK and P.m coculture cell model. @*Results @#1 788 DEGs were detected between the 4 h group and control group, including upregulated DEGs such as lymphocyte cytosolic protein 1(LCP1), keratin 7 (KRT7) and Cilia and flagella associated protein 251(CFAP251) and downregulated DEGs such as FERM, ARH/RhoGEF and Pleckstrin domain protein 1 (FARP1), WW domain containing transcription regulator 1(WWTR1) and Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2). 1 832 DEGs were detected between the 24 h group and control group, including upregulated DEGs such as LCP1, complement C1s(C1S), kynureninase (KYNU) and downregulated DEGs such as phosphoserine aminotransferase 1 (PSAT1), FARP1 and FKBP prolyl isomerase 10 (FKBP10). There were 1 090 common differentially expressed genes (cDEGs) in the 4 h and 24 h groups, including LCP1, KYNU and long intergenic nonprotein coding RNA 958 (LINC00958). The GO pathways were mainly enriched in response to lipopolysaccharide and the molecules of bacterial origin and apical part of the cell. KEGG pathway analysis revealed enrichment in the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, Toll-like receptor (TLR) pathway, etc. We verified the expression of a cDEG, Myosin1B (MYO1B), and qRT-PCR and Western Blot analysis showed that MYO1B expression was significantly upregulated between the control group and the P.m cocultured group (P<0.001), and its expression followed a time-dependent and concentration-dependent manner. @*Conclusion@#P.m played an important role in the transcriptome of oral keratinocytes.

Ruscogenin alleviates LPS-triggered pulmonary endothelial barrier dysfunction through targeting NMMHC IIA to modulate TLR4 signaling

Pulmonary endothelial barrier dysfunction is a hallmark of clinical pulmonary edema and contributes to the development of acute lung injury (ALI). Here we reported that ruscogenin (RUS), an effective steroidal sapogenin of Radix Ophiopogon japonicus, attenuated lipopolysaccharides (LPS)-induced pulmonary endothelial barrier disruption through mediating non-muscle myosin heavy chain IIA (NMMHC IIA)‒Toll-like receptor 4 (TLR4) interactions. By in vivo and in vitro experiments, we observed that RUS administration significantly ameliorated LPS-triggered pulmonary endothelial barrier dysfunction and ALI. Moreover, we identified that RUS directly targeted NMMHC IIA on its N-terminal and head domain by serial affinity chromatography, molecular docking, biolayer interferometry, and microscale thermophoresis analyses. Downregulation of endothelial NMMHC IIA expression in vivo and in vitro abolished the protective effect of RUS. It was also observed that NMMHC IIA was dissociated from TLR4 and then activating TLR4 downstream Src/vascular endothelial cadherin (VE-cadherin) signaling in pulmonary vascular endothelial cells after LPS treatment, which could be restored by RUS. Collectively, these findings provide pharmacological evidence showing that RUS attenuates LPS-induced pulmonary endothelial barrier dysfunction by inhibiting TLR4/Src/VE-cadherin pathway through targeting NMMHC IIA and mediating NMMHC IIA‒TLR4 interactions.

RhoGEF Trio Regulates Radial Migration of Projection Neurons via Its Distinct Domains / 神经科学通报·英文版

The radial migration of cortical pyramidal neurons (PNs) during corticogenesis is necessary for establishing a multilayered cerebral cortex. Neuronal migration defects are considered a critical etiology of neurodevelopmental disorders, including autism spectrum disorders (ASDs), schizophrenia, epilepsy, and intellectual disability (ID). TRIO is a high-risk candidate gene for ASDs and ID. However, its role in embryonic radial migration and the etiology of ASDs and ID are not fully understood. In this study, we found that the in vivo conditional knockout or in utero knockout of Trio in excitatory precursors in the neocortex caused aberrant polarity and halted the migration of late-born PNs. Further investigation of the underlying mechanism revealed that the interaction of the Trio N-terminal SH3 domain with Myosin X mediated the adherence of migrating neurons to radial glial fibers through regulating the membrane location of neuronal cadherin (N-cadherin). Also, independent or synergistic overexpression of RAC1 and RHOA showed different phenotypic recoveries of the abnormal neuronal migration by affecting the morphological transition and/or the glial fiber-dependent locomotion. Taken together, our findings clarify a novel mechanism of Trio in regulating N-cadherin cell surface expression via the interaction of Myosin X with its N-terminal SH3 domain. These results suggest the vital roles of the guanine nucleotide exchange factor 1 (GEF1) and GEF2 domains in regulating radial migration by activating their Rho GTPase effectors in both distinct and cooperative manners, which might be associated with the abnormal phenotypes in neurodevelopmental disorders.

Expression of myosin heavy chain isoform mRNA transcripts in the masseter and medial pterygoid muscles / Expresión de transcripciones de ARNm de isoforma de cadena pesada de miosina en los músculos masetero y pterigoideo medial

SUMMARY: Both the masseter and medial pterygoid muscles elevate the mandible, raising the lower jaw by acting simultaneously on the lateral and medial surfaces of the mandibular ramus. Nevertheless, electromyographic studies indicate that these muscles, as well as the superficial and deep heads of the masseter, act in a different way during mastication. We have analyzed by real time quantitative polymerase chain reaction (RT-qPCR) the expression of myosin heavy chain (MHC) isoforms in the masseter and medial pterygoid muscles in humans in order to identify possible differences in the expression patterns that may be related to functional differences identified with electromyography. Our findings indicate that the expression pattern of MHC isoforms in the two muscles is characteristic of fast and powerful phasic muscles. We have also observed a high percentage of expression of the MHC-IIx isoform and the expression of the MHC-M isoform at the mRNA level in both muscles, an isoform that does not translate into protein in the masticatory muscles of humans. The high percentage of expression of the MHC-IIx isoform in humans can be related to a high contractile speed of the masseter and medial pterygoid in humans. On the other hand, the low percentage of expression of the MHC-M isoform at the mRNA level in both muscles can be related to the complex evolutionary process that has reduced the size and force of the masticatory muscles in humans.

RESUMEN: Los músculos masetero y pterigoideo medial elevan la mandíbula actuando de forma simultánea sobre las caras lateral y medial de su rama. Sin embargo, los estudios electromiográficos indican que estos dos músculos actúan de forma diferente durante la masticación, de la misma forma que lo hacen las porciones superficial y profunda del músculo masetero. En el presente estudio hemos analizado mediante PCR en tiempo real la expresión de las isoformas de la cadena pesada de la miosina o myosin heavy chain (MHC) en los músculos masetero y pterigoideo medial en humanos, con la finalidad de identificar diferencias en los patrones de expresión que se puedan relacionar con las diferencias funcionales identificadas con la electromiografía. Nuestros resultados indican que el patrón de expresión de las isoformas de la MHC en los dos músculos es la característica de los músculos rápidos y potentes. También hemos observado un elevado porcentaje de expresión de la isoforma MHC-IIx y la expresión a nivel de ARNm de la isoforma MHC-M en los dos músculos, una isoforma que no se detecta a nivel de proteína en los músculos masticadores humanos. El elevado porcentaje de expresión de la isoforma MHC-IIx que hemos observado se puede relacionar con una elevada velocidad de contracción de los músculos masetero y pterigoideo medial en los humanos. Por otro lado, el bajo porcentaje de expresión de la isoforma MHC-M a nivel de ARNm en ambos músculos se puede relacionar con los procesos evolutivos complejos que han reducido el tamaño y la fuerza de los músculos masticadores en los humanos.

Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro / 中华放射肿瘤学杂志

Objective:To investigate the effect and mechanism of Myosin X on the radiosensitivity of non-small cell lung cancer (NSCLC) cell line H1975 in vitro. Methods:Western blot was applied to detect the expression level of Myosin X expression. The H1975 cell line with stable knockout of Myosin X (KO group) and infected with control virus (NC group) were constucted by using CRISPR/Cas9 technique. The knockout efficiency was validated. The radiosensitivity of two cell lines was measured by colony formation assay and single-hit multi-target model. γ-H 2AX focus formation test and RNA sequencing (RNAseq) analysis were employed to identify the regulatory mechanism of the radiosensitivity of lung cancer cell lines mediated by Myosin X. Results:The expression level of Myosin X in the H1975 cells was significantly up-regulated than those in other NSCLC cell lines (all P<0.01). The lentiviral vector of Myosin X sgRNA-Lenti-CRISPR v2 was successfully constructed. After the puromycin screening, H1975 cell lines with complete knockout of Myosin X and control cell lines (NC group) were obtained. Colony formation assay demonstrated that compared with the NC group, the radiosensitivity in the KO group was significantly higher (The D 0 value was decreased from 1.28 Gy to 1.03 Gy, SF 2 decreased from 0.29 to 0.21, and the sensitization ratio was 1.24). The γ-H 2AX focus formation test showed that the number of damage focus formed at 1 h and 6 h after irradiation in the KO group was significantly larger than that in the NC group ( P<0.05. RNAseq analysis indicated that the expression level of ISLR in the KO group was significantly down-regulated than that IN the NC group ( P<0.05). Conclusion:Knockout of Myosin X can increase the radiosensitivity of H1975 cells probably by interfering the repair of DNA double-strand damage and down-regulating the expression level of ISLR.

Analyzing the clinical phenotype of heart disease caused by the double mutation of p.Gly743Arg and p.Glu1389Lys carrying the myosin heavy chain gene / 中华危重病急救医学

Objective:To investigate the relationship between double mutations of myosin heavy chain gene (MYH6) p.Gly743Arg and p.Glu1389Lys and the cardiac phenotype.Methods:Patients carrying double mutations in the MYH6 gene p.Gly743Arg and p.Glu1389Lys were screened from 52 unrelated left ventricular hypertrophy (LVH) who were admitted to the Second Hospital of Chongqing Medical University from 2015 to 2020, and the genetic testing of peripheral blood of patients by second-generation whole-exome sequencing assay technology and genomic DNA of their family members Sanger sequencing was performed to validate the genomic DNA of the family members. The cardiac phenotype was evaluated by electrocardiogram, coronary computed tomography angiography (CTA), echocardiography, and cardiac magnetic resonance imaging (MRI) as adjuncts.Results:All whole-exome gene were detected in 52 unrelated patients with LVH, of which 1 patient (1.9%) had double mutations in MYH6 gene p.Gly743Arg and p.Glu1389Lys (proband). Two members of the maternal line of this patient carried p.Glu1389Lys mutation, but there was no obvious clinical phenotype. Two members of the paternal line carried p.Gly743Arg mutation and had obvious clinical phenotype of bradycardia, but there was no LVH. The male proband, aged 21 years old, presented with LVH and sinus bradycardia but no coronary artery stenosis on CTA before treatment, MRI showed that the left ventricular end diastolic diameter was 58 mm. After treatment with angiotensin receptor-enkephalinase inhibitor (ARNI), electrocardiogram showed that the heart rate increased significantly (from 43 bpm to 72 bpm). Echocardiography showed that the left ventricular end diastolic diameter decreased significantly (from 60 mm to 49 mm).Conclusions:The p.Glu1389Lys mutation of the MYH6 gene may not manifest the phenotype of heart disease. MYH6 gene p.Gly743Arg mutation may be manifested asymptomatic sinus bradycardia, but there is no LVH phenotype. The cardiac disease phenotype caused by the double mutations of p.Gly743Arg and p.Glu1389Lys in the MYH6 gene is more obvious. Asymptomatic LVH and sinus bradycardia can appear in adolescence, but the LVH phenotype can be reversed in a short period of time after ARNI treatment.

Effect of acupoint thread-embedding on tight junction of intestinal mucosa epithelium in rats with ulcerative colitis / 中国针灸

OBJECTIVE@#To observe the effect of acupoint thread-embedding on tight junction of intestinal mucosal epithelial barrier in rats with ulcerative colitis (UC) under the state of "deficiency and stasis", and to explore its mechanism.@*METHODS@#Sixty male SD rats were randomly divided into a control group (@*RESULTS@#Compared with the control group, in the model group the body weight was decreased (@*CONCLUSION@#The thread-embedding could repair the tight junction of intestinal mucosa epithelium and reduce the permeability of intestinal mucosa epithelium, which may be related to the decrease of the expression of CaMKⅡ, MLCK and other protein kinases.