Analysis of factors affecting stability of high concentration VHH-Fc fusion protein / 中国生物制品学杂志

@#Objective To explore the factors affecting the stability of high concentration variable domain of heavy-chain antibody-Fc(VHH-Fc) fusion protein.Methods Three groups of forced degradation experiments,shaking,light and 40℃ high temperature were set up.Differential scanning fluorimetry,dynamic light scattering(DLS) and ultra performance liquid chromatography-mass spectrometry(UPLC-MS) were used to detect the effects of the three forced degradation conditions on the conformational stability,colloidal stability,average hydrodynamic diameter and post-translational modifications of high concentration VHH-Fc fusion protein.Results Under the light condition,the onset temperature of unfolding(T_(onset)),melting temperature(T_m) and aggregation onset temperature(T_(agg)) of high concentration VHH-Fc fusion protein decreased the most,and the oxidation ratio of Met160 and Met266 increased significantly.Under the condition of shaking,the variation of the diffusion interaction parameter(k_D) and the average hydrodynamic diameter was the largest.Conclusion Light can significantly reduce the conformational stability of high concentration VHH-Fc fusion protein and induce methionine oxidation.Shaking has the most significant effect on its colloidal stability and promotes aggregation.

A Case Report of MYH9 Gene Mutation Associated with Glomerular Minor Lesion / 罕见病研究

Non-muscle myosin heavy chain 9-related disease (MYH9-RD) is an autosomal dominant disease caused by the mutations of the MYH9 gene encoding the non-muscle mysoin heavy chain ⅡA and leads to abnormal accumulation of myosin in cells. These further causes functional disorders of the blood, eye, ear, kidney, and liver systems. MYH9-RD displays heterogeneous kidney involvement and outcomes, but doctors still lack understandings of the mechanism and treatment strategies, owing to difficulty of conducting renal biopsies. Here, we report a case of MYH9-RD with tail fragments heterozygous mutation, which renal pathology is presented as glomerular minor lesion. Moreover, we reviewed related relevant to strengthen clinical diagnosis and understanding of MYH9-RD.

Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells / 中国生物制品学杂志

@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines

Heavy chain deposition disease in a case of clear cell renal cell carcinoma- A jack in the box

Renal cell carcinoma (RCC) is the most common subtype of adult renal tumors, and its detection rate in the early stages has been increased in the dawn of advanced imaging modalities. Nephrectomy is the mainstay of treatment; determination of tumor category and staging is the primary concern of oncopathologists. Non-neoplastic renal parenchyma is overlooked majority of times and thus misses the opportunity to detect concomitant medical renal diseases which also predict the renal outcome in the postoperative era. Although any kind of glomerular or extraglomerular pathology may be encountered, vascular changes in the form of arterionephrosclerosis are the commonest one. Here, we take the opportunity to report an unusual association of heavy chain deposition disease (HCDD) with clear cell subtypes of renal cell carcinoma in a 48-year-old male of Indian ethnicity.

Optimization of condition for prokaryotic expression of recombinant tetanus toxin heavy chain fragment C protein / 中国生物制品学杂志

@#Objective To optimize the condition for prokaryotic expression of recombinant tetanus toxin heavy chain fragment C(rTTHc) protein.Methods The rTTHc gene fragment after optimization of codon was inserted into prokaryotic expression vector pET30a,low temperature expression vector pCold Ⅱ and high temperature expression vector pBV220separately.The constructed recombinant plasmids were transformed to E.coli BL21(DE3).The expression levels and solubility of recombinant protein at various temperatures were compared.Results The expression level of pBV220-rTTHc after induction at 42 ℃ for 4 h was relatively low,and the protein solubility was poor.The expression level of pET30a-rTTHc after induction at 37 ℃ for 4 h was equivalent to that of pCold-rTTHc after induction at 15 ℃ for 8 h,while the solubility of the former was slightly lower than that of the latter.However,both the expression level and solubility of pET30a-rTTHc after induction at 28 ℃ for 4 h were high,while the expression time was short.Conclusion The pET30a-rTTHc induced by2 mg/mL IPTG at 28℃ is optimal for high expression of rTTHc protein.

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.

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 mechanism of itaconic acid alleviating acute lung injury induced by lipopolysaccharide / 中华急诊医学杂志

Objective:To investigate the role and mechanism of exogenous derivative 4-octyl itaconate (4-OI) in lipopolysaccharide (LPS)-induced acute lung injury (ALI).Methods:C57BL/6 male mice were randomly divided into the control group, 4-OI group, LPS group, 4-OI+LPS group and deferiprone (DFP)+LPS group, with 6 mice in each group. LPS-induced ALI model was established by intraperitoneal injection of LPS. For the 4-OI+LPS group, mice were pretreated with 4-OI for 2 h before stimulation with LPS. The mice were sacrificed 12 h later and lung tissues were collected for pathological and molecular biological examination. Hematoxylin-eosin and Masson staining were used to detect the level of lung injury and collagen deposition. The expression levels of inflammatory cytokines and ferroptosis associated genes were detected by real-time quantitative PCR, and ferroptosis associated proteins were detected by Western blotting. The chi-square test was performed before the measurement data were counted. One-way analysis of variance was used to compare differences between multiple groups.Results:Compared with the control group, the histopathological damage was aggravated, and collagen deposition and lung injury score and lung wet-dry ratio were significantly increased in the LPS group (all P<0.05), and 4-OI pre-treatment significantly alleviated LPS-induced ALI. 4-OI treatment also significantly reduced the mRNA level of inflammatory cytokines, including IL-1β [(4.38±0.47) vs. (32.65±4.49)], IL-6 [(3.97±0.64) vs. (12.22±0.91)] and TNF-α [(15.06±2.26) vs. (38.53±2.31)]. At the same time, compared with the control group, the levels of lipid peroxidation metabolite 4-hydroxynonenal and malondialdehyde, iron level of lung tissue were significantly increased in the LPS group, and the mRNA level of ferroptosis marker prostaglandin-endoperoxide synthase 2 was also significantly increased (all P<0.05), but these indicators were significantly lower in the 4-OI+LPS group than in the LPS group. The results of immunofluorescence, Western blotting and PCR showed that the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH1) significantly decreased in the LPS group, while 4-OI treatment significantly increased the Nrf2, GPX4 and FTH1 levels, and showed similar inhibition of ferroptosis with DFP (all P<0.05). Conclusions:4-OI attenuates LPS-induced ALI by increasing Nrf2 and upregulating FTH1 and GPX4, providing a potential drug and its theoretical mechanism for the prevention and treatment of ALI.

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.

Silencing of Myh3 Gene by siRNA Inhibits Glycolysis in C2C12 Cells / 中国生物化学与分子生物学报

The Myh3 (myosin heavy chain 3) gene is a marker gene of muscle cell differentiation and regulates the utilization of energy in muscle cells, but whether it affects the glycolysis process of muscle cells in different states is rarely reported. In this paper, the expression patterns of Myh3 and glycolysis-related genes Pkm (M-type pyruvate kinse), Prkag3 (protein kinase adenosine monophosphate-activated γ3-subunit), and Gsk3β (glycogen synthase kinase-3β) were studied by the qRT-PCR (quantitative-Real-Time-PCR) method using C2C12 cells at different stages of myoblast and adipogenic differentiation as models. It was found that in the process of myoblast differentiation of C2C12 cells, the relative expression trends of Myh3 and glycolysis genes Prkag3 and Pkm were basically the same, and the relative expression levels first increased, reached the peak on the second day of differentiation, and then decreased; glycogen synthase the expression trend of the inhibitory gene Gsk3β was relatively stable. In the process of adipogenic differentiation of C2C12 cells, the relative expression trend of Myh3 and glycolysis genes Prkag3 and Pkm remained basically the same, and the relative expression gradually increased, reaching the highest value on the 8th day of differentiation; glycogen synthase inhibitory gene Gsk3β expression remained stable. In the myogenic differentiation state of C2C12 cells, qRT-PCR and Western blotting were used to detect the effects of interfering Myh3 on the mRNA and protein expressions of glycolysis-related genes Pkm, Prkag3, and Gsk3β. The results showed that after interfering with Myh3, the mRNA expressions of glycolysis genes Pkm and Prkag3 were significantly decreased (P 0.05). The protein levels of Myh3 and Pkm were significantly lower than those in the blank group and NC group. Under the adipogenic differentiation state of C2C12 cells, after interfering with Myh3, the mRNA levels of glycogen synthase inhibitor Gsk3β and glycolysis gene Prkag3 were significantly increased (P<0.01), and the mRNA level of glycolysis gene Pkm was decreased; the protein levels of Myh3 and Pkm in the Myh3 interference group were also lower than those in the blank group and NC group. Based on the above studies, there are significant differences in the levels of glycolysis in C2C12 cells in the myogenic and adipogenic states, and the expression patterns of Myh3 and glycolysis genes are similar. Further results showed that Myh3 suppression could inhibit the glycolysis of C2C12 cells in the myogenic state without affecting the glycogen synthesis. Unlike in the myogenic state, interfering expression of Myh3 in the adipogenic state of C2C12 cells inhibited both glycogen synthesis and glycolysis.

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.

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.

SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation

Neointimal hyperplasia after vascular injury is a representative complication of restenosis. Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) is involved in the pathogenesis of vascular intimal hyperplasia. PARP16, a member of the poly(ADP-ribose) polymerases family, is correlated with the nuclear envelope and the ER. Here, we found that PERK and IRE1

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.

The role of three-dimensional speckle tracking imaging derived parameters on predicting outcome of hypertrophic cardiomyopathy patients with MYH7 mutations / 中华心血管病杂志

Objective: To evaluate the cardiac functional changes in hypertrophic cardiomyopathy(HCM) patients with β-myosin heavy chain gene (MYH7) mutations by three-dimensional (3D) speckle tracking imaging(3D-STI) and conventional echocardiography modalities, and then to explore the potential predictors of adverse cardiovascular events in these patients. Methods: A consecutive series of 192 HCM patients admitted in our center from October 2014 to October 2016 were genetically screened to identify MYH7 mutations in this retrospective study. A total of 43 HCM patients with MYH7 mutations were enrolled. The patients were divided into events group(n=13) and no event group(n=30) according to the presence or absence of adverse cardiovascular events(primary and secondary endpoints). All patients were followed up to January 2019 after comprehensive evaluation of 3D-STI, two-dimensional and Doppler echocardiography. The adverse cardiovascular events were recorded. Results: The median follow up time was 1 012 (812, 1 330) days. During follow-up, 13 patients (30.2%) reached endpoints: 6 cases of the primary endpoints(2 cases of sudden cardiac death(SCD), 3 cases of survival after defibrillation, and 1 case of appropriate implantable cardioverter-defibrillator(ICD) discharge); 7 cases of the second endpoints(5 cases of heart failure hospitalization, 1 case of syncope and cardioversion due to supraventricular tachycardia, and 1 case of end-stage HCM). Patients with adverse cardiovascular events had higher prevalence of syncope and risk of SCD, enlarged left atrial volume index(LAVI) and reduced 3D left ventricular global longitudinal train (3D-GLS), as compared to those without adverse events(all P<0.05). The multivariate Cox regression analysis showed that reduced 3D-GLS(HR=0.814, 95%CI 0.663-0.999, P=0.049) was an independent predictor for adverse cardiovascular events. The cutoff value of 3D-GLS≤13.67% was linked with significantly increased risk of adverse cardiovascular events in this patient cohort(AUC=0.753, 95%CI 0.558-0.948, sensitivity 86%, specificity 69%, P<0.05). The Kaplan-Meier analysis indicated that the patients with the 3D-GLS≤ 13.67% faced higher risk of death than those with 3D-GLS>13.67%. Conclusion: 3D-GLS is useful on predicting adverse cardiovascular events in HCM patients with MYH7 mutations.

Platelet-derived growth factor-bb promotes proliferation, differentiation and migration of skeletal muscle myoblast / 中国组织工程研究

BACKGROUND: Skeletal muscle myoblasts differentiate and fuse to form polynuclear muscle tubes and muscle fibers to complete the repair of muscle injury when skeletal muscles were injured. However, the repair process is slow and incomplete. Platelet-derived growth factor-BB can stimulate the proliferation, differentiation and migration of a variety of tissue cells, and plays an important role in the process of tissue repair after various injuries. OBJECTIVE: To explore the effects of different concentrations of platelet-derived growth factor-BB on proliferation, differentiation and migration of skeletal muscle myoblast C2C12 cells and the action mechanism. METHODS: The mouse skeletal muscle myoblast C2C12 cells were cultured with platelet-derived growth factor-BB 0, 5, 10, 20 and 40 μg/L and platelet-derived growth factor receptor inhibitor imatinib. The expression of platelet-derived growth factor receptor in C2C12 cells was detected by immunocytochemistry and western blot assay. After 1, 2, 3, 4, and 5 days of culture, CCK8 method was used to detect cell proliferation. After 4 days of induction and differentiation culture, the formation of myotubes in each group was observed by light microscope. The expression of myosin heavy chain and MyoG Gene was observed by immunofluorescence and western blot assay. After 48 hours of culture, Transwell method was used to detect cell migration. RESULTS AND CONCLUSION: (1) Immunofluorescence chemistry and western blot assay indicated that platelet-derived growth factor receptor expression could be detected in C2C12 cells, and semi-quantitative statistical analysis showed no significant difference in platelet-derived growth factor receptor expression between groups with different platelet-derived growth factor-BB concentrations (P > 0.05). (2) CCK8 assay demonstrated that the proliferation of C2C12 cells showed no significant change in platelet-derived growth factor-BB groups compared with the control group (platelet-derived growth factor-BB 0 μg/L group). (3) Immunofluorescence chemistry showed that compared with control group, number of myosin heavy chain positive cells increased in platelet-derived growth factor-BB groups; 40 μg/L platelet-derived growth factor-BB concentration got the highest; the mature muscle tubes up to (27.00±0.76) per field of vision, and MyoG expression populations was most obviously compared with the control group (P < 0.05). (4) Transwell results showed that compared with the control group, the migration number of C2C12 cells in the platelet-derived growth factor-BB group increased, and the migration number in the 40 μg/L group was up to 144.00±13.03 (P < 0.05). (5) It is concluded that platelet-derived growth factor-BB promoted the migration, differentiation and myotube formation of C2C12 cells, and the pro-differentiation mechanism is related to its enhanced binding with platelet-derived growth factor receptor, so as to improve the expression of differentiation related gene MyoG.

MiRNA-148a promotes the differentiation of human induced pluripotent stem cells into cardiomyocyte-like cells / 中国组织工程研究

BACKGROUND: Studies have shown that miRNA-148a can promote human bone marrow mesenchymal stem cells to differentiate into mature cardiomyocyte-like cells, but the effect of miRNA-148a on the differentiation of human induced pluripotent stem cells into cardiomyocyte-like cells has not been reported. OBJECTIVE: To investigate the effect of miRNA-148a on the differentiation of human induced pluripotent stem cells into cardiomyocyte-like cells. METHODS: Human induced pluripotent stem cells differentiating into cardiomyocyte-like cells were divided into three groups. Cells in the control group were not treated. Cells in the low expression group were treated with miRNA-148a for 28 days, and those in the high expression group were treated with mimics of miRNA-148a for 28 days. In addition, human induced pluripotent stem cells cultured for 28 days were taken as the blank control group. CCK-8 was used to detect cell proliferation activity. qRT-PCR was used to detect the expression of miRNA-148a. Immunofluorescence staining and western blot analysis were performed to detect the expression of MHC and cTnT protein. RESULTS AND CONCLUSION: The expression of intracellular miR-148a mRNA and cell proliferation activity in the low expression group were lower than those in the blank control and control groups, while those in the high expression group were significantly higher than those in the other three groups (P < 0.01). There were no positive expression of MHC and cTnT in the blank control group. There were positive expressions of MHC and cTnT in the control, low expression and high expression groups. The expression of MHC and cTnT protein in the low expression group was significantly lower than that in the control group, and that in the high expression group was significantly higher than that in the other three groups (P < 0.01). These results suggest that miRNA-148a can promote the differentiation of human induced pluripotent stem cells into cardiomyocyte-like cells.

Cardiac Myosin Heavy Chains

Myosin is defined as a mechano-enzyme molecule which converts the chemical energy stored as adenosine triphosphate (ATP) into mechanical energy (muscle contraction). Moreover, the cardiac muscle has different types of myosin heavy chain when it separated with the one dimensional electrophoresis; in addition to their structural difference cardiac myosin isozymes have different contractile functions.

Effect of electroacupuncture of acupoints on the healthy limb (opposing needling) on acute skeletal muscle contusion in rats / 针刺研究

OBJECTIVE: To observe the therapeutic effect of electroacupuncture (EA) of "Zusanli" (ST36) and "Ashi"-point on the healthy side (opposing needling) on muscular injury and expression of myogenin (myoG) and fast myosin skeletal heavy chain (Fast MyHC) proteins in the gastrocnemius muscle (GM) tissues in skeletal muscle contusion rats，so as to explore its mechanism underlying improvement of skeletal muscle injury. METHODS: A total of 54 male SD rats were divided into normal control (n ＝ 6)，model (n＝24) and opposing needling (EA, n＝24) groups. The latter two groups were further randomized into 3, 5, 7 and 14 d subgroups (n＝6 per subgroup). The skeletal muscle contusion model of the hind-limb was established by using a self-made striking device. EA (1 Hz/3 Hz，1－2 mA) was applied to ST36 and "Ashi"-point on the uninjured side of the hind-limb for 15 min every time, once a day for 3, 5, 7 and 14 days, respectively. The injured GM was harvested on the 3rd, 5th, 7th and 14th day after muscular contusion. The morphological changes of the injured GM and the mean cross-sectional areas (CSAs) of the neonatal muscle cells were observed by microscope after H．E. staining. The immunoactivity of desmin protein (myogenic marker protein of myoblast cell) of GM was detected by immunofluorescence stain on the 7th day after injury, and the expression levels of myoG (on the 3rd and 5th day after injury) and fast MyHC protein of GM tissues (on the 7thand 14th day after injury) were detected by Western blot. RESULTS: H．E. staining of GS tissue showed fewer neuronal myocytes with disordered arrangement at different sizes, and appearance of some collagenous fibers among the mesenchyme on day 7 and 14 after muscular contusion, which was relatively milder in the EA group. In the EA group, the CSA values of the neonatal muscle cells were significantly larger than those in the model group on the day 7th (P0.05). On the 3rd and 5th day after muscular contusion, the expression level of myoG protein was significantly up-regulated in the model group compared with the normal control group (P<0.001), and significantly up-regulated in the EA group than that in the model group (P<0.001). On the 7th and14th day after contusion, the expression level of fast MyHC protein was significantly down-regulated in the model group relevant to the normal control group (P<0.001), and markedly up-regulated in the EA group relevant to the model group (P<0.01)．. CONCLUSION: EA of ST36 and "Ashi"-point on the contralateral limb can up-regulate the expression of myoG and fast MyHC proteins of GM in acute skeletal muscle contusion rats, which may contribute to its effect in promoting the repair of skeletal muscle injury.

Preparation and identification of the nano antibodies of anti-programmed death receptor-1 / 国际生物医学工程杂志

Objective To prepare camelid-derived nano antibodies with high affinity binding to programmed death receptor-1 (PD-1) antigen,and to provide experimental basis for subsequent functional studies.Methods The PD-1-Fc recombinant protein expressed in eukaryotic expression was used to immunize Xinjiang Bactrian camel 6 times.The peripheral blood was collected and the lymphocytes were isolated.Nested PCR amplification was performed to obtain the genes in variable region of camelid heavy chain antibody (VHH),and to construct a phage display library.The phage display library was screened by solid phase enzyme-linked immunosorbent assay (ELISA).The PD-1 antigen,which was sequentially reduced in mass concentration (5.00、2.50、1.00 μg/ml),was coated in an ELISA plate,and the phage display library was subjected to 3 rounds of affinity selection.Individual clones that bind to PD-1 were further screened by soluble monoclonal ELISA.According to the results of DNA sequencing,three VHH monoclonals with multiple repeats were selected and ligated into pET22b vector,and transformed into E.coli BL21 (DE3) competent cells,and then induced by isopropyl-β3-D-thiogalactoside.The recombinant VHH antibody protein was purified by nickel column affinity chromatography,and its binding activity and affinity to PD-1 antigen were detected by Western Blot and ELISA.Results After immunization of Bactrian camel 6 times with recombinant protein PD-1-Fc,high titer specific antibody was stimulated,and the immune serum titer reached 1∶32 000.A VHH phage display library with a reservoir size of 2.6×108 cfu/ml was constructed from the immunized camel lymphocytes.After 3 rounds of affinity selection,46 VHH monoclonals with absorbance (A600) values above 0.6 were obtained by soluble monoclonal ELISA.Among them,three clones of VHH-B7,VHH-H5 and VHH-H12 had higher repeats,indicating that significant enrichment was obtained.The results of Western Blot and ELISA showed that the purified B7,H5 and H12 nanobodies had good binding activity to PD-1 antigen and had high affinity.Their affinity constants were 1.19×1011 and 1.63×1011,1.59×1011 L/mol,respectively.Conclusion The anti-PD-1 camelid-derived nanobodies were obtained by affinity selection of VHH phage display library,which can bind to the PD-1 antigen with high affinity.This study can provide an experimental basis for subsequent functional studies.