The double homeodomain protein DUX4c is associated with regenerating muscle fibers and RNA-binding proteins.

BACKGROUND:  We have previously demonstrated that double homeobox 4 centromeric (DUX4C) encoded for a functional DUX4c protein upregulated in dystrophic skeletal muscles. Based on gain- and loss-of-function studies we have proposed DUX4c involvement in muscle regeneration. Here, we provide further evidence for such a role in skeletal muscles from patients affected with facioscapulohumeral muscular dystrophy (FSHD). METHODS: DUX4c was studied at RNA and protein levels in FSHD muscle cell cultures and biopsies. Its protein partners were co-purified and identified by mass spectrometry. Endogenous DUX4c was detected in FSHD muscle sections with either its partners or regeneration markers using co-immunofluorescence or in situ proximity ligation assay. RESULTS: We identified new alternatively spliced DUX4C transcripts and confirmed DUX4c immunodetection in rare FSHD muscle cells in primary culture. DUX4c was detected in nuclei, cytoplasm or at cell-cell contacts between myocytes and interacted sporadically with specific RNA-binding proteins involved, a.o., in muscle differentiation, repair, and mass maintenance. In FSHD muscle sections, DUX4c was found in fibers with unusual shape or central/delocalized nuclei (a regeneration feature) staining for developmental myosin heavy chain, MYOD or presenting intense desmin labeling. Some couples of myocytes/fibers locally exhibited peripheral DUX4c-positive areas that were very close to each other, but in distinct cells. MYOD or intense desmin staining at these locations suggested an imminent muscle cell fusion. We further demonstrated DUX4c interaction with its major protein partner, C1qBP, inside myocytes/myofibers that presented features of regeneration. On adjacent muscle sections, we could unexpectedly detect DUX4 (the FSHD causal protein) and its interaction with C1qBP in fusing myocytes/fibers. CONCLUSIONS: DUX4c upregulation in FSHD muscles suggests it contributes not only to the pathology but also, based on its protein partners and specific markers, to attempts at muscle regeneration. The presence of both DUX4 and DUX4c in regenerating FSHD muscle cells suggests DUX4 could compete with normal DUX4c functions, thus explaining why skeletal muscle is particularly sensitive to DUX4 toxicity. Caution should be exerted with therapeutic agents aiming for DUX4 suppression because they might also repress the highly similar DUX4c and interfere with its physiological role.

[MicroRNA-22-3p Regulates the Expression of Kruppel-like Factor 6 to Affect the Cardiomyocyte-like Differentiation of Bone Marrow Mesenchymal Stem Cell].

Objective To explore the effect of microRNA-22-3p (miR-22-3p) regulating the expression of Kruppel-like factor 6 (KLF6) on the cardiomyocyte-like differentiation of bone marrow mesenchymal stem cell (BMSC). Methods Rat BMSC was isolated and cultured,and the third-generation BMSC was divided into a control group,a 5-azacytidine(5-AZA)group,a mimics-NC group,a miR-22-3p mimics group,a miR-22-3p mimics+pcDNA group,and a miR-22-3p mimics+pcDNA-KLF6 group.Real-time fluorescent quantitative PCR (qRT-PCR) was carried out to determine the expression of miR-22-3p and KLF6 in cells.Immunofluorescence staining was employed to detect the expression of Desmin,cardiac troponin T (cTnT),and connexin 43 (Cx43).Western blotting was employed to determine the protein levels of cTnT,Cx43,Desmin,and KLF6,and flow cytometry to detect the apoptosis of BMSC.The targeting relationship between miR-22-3p and KLF6 was analyzed by dual luciferase reporter gene assay. Results Compared with the control group,5-AZA up-regulated the expression of miR-22-3p (q=7.971,P<0.001),Desmin (q=7.876,P<0.001),cTnT (q=10.272,P<0.001),and Cx43 (q=6.256,P<0.001),increased the apoptosis rate of BMSC (q=12.708,P<0.001),and down-regulated the mRNA (q=20.850,P<0.001) and protein (q=11.080,P<0.001) levels of KLF6.Compared with the 5-AZA group and the mimics-NC group,miR-22-3p mimics up-regulated the expression of miR-22-3p (q=3.591,P<0.001;q=11.650,P<0.001),Desmin (q=5.975,P<0.001;q=13.579,P<0.001),cTnT (q=7.133,P<0.001;q=17.548,P<0.001),and Cx43 (q=4.571,P=0.037;q=11.068,P<0.001),and down-regulated the mRNA (q=7.384,P<0.001;q=28.234,P<0.001) and protein (q=4.594,P=0.036;q=15.945,P<0.001) levels of KLF6.The apoptosis rate of miR-22-3p mimics group was lower than that of 5-AZA group (q=8.216,P<0.001).Compared with the miR-22-3p mimics+pcDNA group,miR-22-3p mimics+pcDNA-KLF6 up-regulated the mRNA(q=23.891,P<0.001) and protein(q=13.378,P<0.001)levels of KLF6,down-regulated the expression of Desmin (q=9.505,P<0.001),cTnT (q=10.985,P<0.001),and Cx43 (q=8.301,P<0.001),and increased the apoptosis rate (q=4.713,P=0.029).The dual luciferase reporter gene experiment demonstrated that KLF6 was a potential target gene of miR-22-3p. Conclusion MiR-22-3p promotes cardiomyocyte-like differentiation of BMSC by inhibiting the expression of KLF6.

Host Desmin Interacts with RABV Matrix Protein and Facilitates Virus Propagation.

Microfilaments and microtubules, two crucial structures of cytoskeletal networks, are usurped by various viruses for their entry, egress, and/or intracellular trafficking, including the Rabies virus (RABV). Intermediate filaments (IFs) are the third major component of cytoskeletal filaments; however, little is known about the role of IFs during the RABV infection. Here, we identified the IF protein desmin as a novel host interactor with the RABV matrix protein, and we show that this physical interaction has a functional impact on the virus lifecycle. We found that the overexpression of desmin facilitates the RABV infection by increasing the progeny virus yield, and the suppression of endogenous desmin inhibits virus replication. Furthermore, we used confocal microscopy to observe that the RABV-M co-localizes with desmin in IF bundles in the BHK-21 cells. Lastly, we found that mice challenged with RABV displayed an enhanced expression of desmin in the brains of infected animals. These findings reveal a desmin/RABV-M interaction that positively regulates the virus infection and suggests that the RABV may utilize cellular IFs as tracks for the intracellular transport of viral components and efficient budding.

Differential expression of desmin in the uterine myometrium and cervix as a possible mechanism for successful parturition in rats.

Expression of desmin, an intermediate filament, in the myometrium and cervix were investigated in peripartum rats (full term day 22 of pregnancy (DP22)). Des mRNA was expressed in lesser amounts in the cervix at peripartum (DP17 and 21, and day of birth 1 (DB1)), compared to those in the cervixes of ovariectomized rats. Immunohistochemical analysis revealed that desmin protein was diffusely present in the myometrium, and locally in the epithelium of the cervix. Western blot analysis showed that desmin protein levels in the myometrium increased 4- to 6-fold at DP17, 21 and DB1, and decreased rapidly at DB2 to the basal level observed in ovariectomized or non-pregnant rats. In contrast, cervical desmin protein levels increased approximately 10-fold at DP21 compared to those in ovariectomized rats, but decreased rapidly at DB1, indicating its decrease at parturition and an inconsistency between mRNA and protein expression. The administration of 17ß-estradiol to ovariectomized rats increased desmin protein levels in the myometrium and cervix after 24 h. S-nitrosylated desmin protein was detected in the myometrium and cervix at DP21. The mRNA expression of inducible nitric oxide synthase was consistent with the expression of desmin protein. Thus, desmin, which is regulated by estradiol, is differentially expressed in the myometrium and cervix at peripartum possibly for successful pregnancy and parturition. In the cervix, desmin protein expression seems to be regulated by estradiol at the translational level. S-nitrosylation of desmin may have a potential role in the peripartum uterus.

Identification of immunocell infiltrates and effective diagnostic biomarkers in laryngeal carcinoma.

Laryngeal cancer (LC) is a malignant tumor that occurs in the head and neck. Laryngeal cancer is one of the most common cancers of the neck and head, and its prognosis has always been poor. The incidence of LC increased gradually and showed an early rising trend. Laryngeal cancer is rarely studied in relation to immunity, Malignant tumors will change the state of the human body in various ways to adapt to their own survival and avoid the immune system. This study aims to explore the immune molecular mechanism of laryngeal cancer through bioinformatics analysis. The gene expression data was downloaded for 3 microarray datasets: GSE27020, GSE59102, and GSE51985. CIBERSORT algorithm was performed to evaluate immune cell infiltration in tissues between LC and healthy control (HC). Differentially expressed genes (DEGs) were screened. Functional correlation of DEGs were analyzed by Gene Ontology, Gene Set Enrichment Analysis and Kyoto encyclopedia of genes and genomes. Candidate biomarkers were identified by cytoHubba of Cytoscape. Spearman correlations between the above biomarkers and infiltrating immune cells were explored using R software analysis. The immune cell types of LC and HC were significantly different. Twenty-one DEGs were obtained by cross-screening. The function of DEGs is closely related to the number of immune cells. Five central genes (TNNT3, TNNI2, Desmin, matrix metallopeptidase 9 and cytotoxic T lymphocyte antigen 4) were screened. The HUB gene was demonstrated to have the ability to diagnose LC and HC with good specificity and sensitivity. The correlation between immune cells and biomarkers showed that hub gene was positively correlated with macrophages and dendritic cells, and negatively correlated with CD4 + T cell. TNNT3, TNNI2, Desmin, matrix metallopeptidase 9 and cytotoxic T lymphocyte antigen 4 can be used as diagnostic biomarker for LC. Macrophages, dendritic cells and CD4 + T cell may participate in the occurrence and development of LC.

Early postmortem muscle proteome and metabolome of beef longissimus thoracis muscle classified by pH at 6 hours postmortem.

The objective was to identify metabolome and proteome differences at 1 h and 1 d postmortem between longissimus thoracis (LT) muscle classified based on 6 h pH values. Twenty beef LT rib sections were sorted based on 6 h postmortem pH values into low (LpH; pH < 5.55; n = 9) and high (HpH; pH > 5.84; n = 8) pH classifications. Warner-Bratzler shear force (WBSF), desmin degradation, and calpain-1 autolysis were measured. Two-dimensional difference in gel electrophoresis (3-10, 4-7, and 6-9 pH range) and Tandem mass tagging (TMT) protein analyses were employed to determine how the sarcoplasmic protein profile varied across pH classification. Non-targeted metabolomic analyses were conducted on extracts prepared at 1 h and 1 d postmortem. The LpH classification had a lower WBSF value at 1 d postmortem, which was explained by greater calpain-1 autolysis and desmin degradation at 1 d postmortem. Proteome and metabolome analysis revealed a phenotype that promotes more rapid energy metabolism in the LpH group. Proteome and metabolome analyses identified energy production, apoptotic, calcium homeostasis, and proteasome systems influencing pH classifications that could explain the observed pH, proteolysis, and beef tenderness differences. SIGNIFICANCE: This study is the first to identify proteomic and metabolomic variations early (1 h and 1 day) postmortem that are linked to differences in early (6 h) postmortem pH values and to tenderness differences at 1 day postmortem. This study integrates postmortem biochemical features (protein degradation, proteome, and metabolome variations) to postmortem pH decline and eating quality of beef steaks. Potential biomarkers of more rapid postmortem metabolism linked to earlier tenderization in beef are suggested. Identification of these biochemical features will assist in predicting the eating quality of beef products.

Bi-Allelic DES Gene Variants Causing Autosomal Recessive Myofibrillar Myopathies Affecting Both Skeletal Muscles and Cardiac Function.

Mutations in the human desmin gene (DES) may cause both autosomal dominant and recessive cardiomyopathies leading to heart failure, arrhythmias and atrio-ventricular blocks, or progressive myopathies. Cardiac conduction disorders, arrhythmias and cardiomyopathies usually associated with progressive myopathy are the main manifestations of autosomal dominant desminopathies, due to mono-allelic pathogenic variants. The recessive forms, due to bi-allelic variants, are very rare and exhibit variable phenotypes in which premature sudden cardiac death could also occur in the first or second decade of life. We describe a further case of autosomal recessive desminopathy in an Italian boy born of consanguineous parents, who developed progressive myopathy at age 12, and dilated cardiomyopathy four years later and died of intractable heart failure at age 17. Next Generation Sequencing (NGS) analysis identified the homozygous loss-of-function variant c.634C>T; p.Arg212*, which was likely inherited from both parents. Furthermore, we performed a comparison of clinical and genetic results observed in our patient with those of cases so far reported in the literature.

The N-Terminal Part of the 1A Domain of Desmin Is a Hot Spot Region for Putative Pathogenic DES Mutations Affecting Filament Assembly.

Desmin is the major intermediate filament protein of all three muscle cell types, and connects different cell organelles and multi-protein complexes such as the cardiac desmosomes. Several pathogenic mutations in the DES gene cause different skeletal and cardiac myopathies. However, the significance of the majority of DES missense variants is currently unknown, since functional data are lacking. To determine whether desmin missense mutations within the highly conserved 1A coil domain cause a filament assembly defect, we generated a set of variants with unknown significance and systematically analyzed the filament assembly using confocal microscopy in transfected SW-13, H9c2 cells and cardiomyocytes derived from induced pluripotent stem cells. We found that mutations in the N-terminal part of the 1A coil domain affect filament assembly, leading to cytoplasmic desmin aggregation. In contrast, mutant desmin in the C-terminal part of the 1A coil domain forms filamentous structures comparable to wild-type desmin. Our findings suggest that the N-terminal part of the 1A coil domain is a hot spot for pathogenic desmin mutations, which affect desmin filament assembly. This study may have relevance for the genetic counselling of patients carrying variants in the 1A coil domain of the DES gene.

Desmin intermediate filaments and tubulin detyrosination stabilize growing microtubules in the cardiomyocyte.

In heart failure, an increased abundance of post-translationally detyrosinated microtubules stiffens the cardiomyocyte and impedes its contractile function. Detyrosination promotes interactions between microtubules, desmin intermediate filaments, and the sarcomere to increase cytoskeletal stiffness, yet the mechanism by which this occurs is unknown. We hypothesized that detyrosination may regulate the growth and shrinkage of dynamic microtubules to facilitate interactions with desmin and the sarcomere. Through a combination of biochemical assays and direct observation of growing microtubule plus-ends in adult cardiomyocytes, we find that desmin is required to stabilize growing microtubules at the level of the sarcomere Z-disk, where desmin also rescues shrinking microtubules from continued depolymerization. Further, reducing detyrosination (i.e. tyrosination) below basal levels promotes frequent depolymerization and less efficient growth of microtubules. This is concomitant with tyrosination promoting the interaction of microtubules with the depolymerizing protein complex of end-binding protein 1 (EB1) and CAP-Gly domain-containing linker protein 1 (CLIP1/CLIP170). The dynamic growth and shrinkage of tyrosinated microtubules reduce their opportunity for stabilizing interactions at the Z-disk region, coincident with tyrosination globally reducing microtubule stability. These data provide a model for how intermediate filaments and tubulin detyrosination establish long-lived and physically reinforced microtubules that stiffen the cardiomyocyte and inform both the mechanism of action and therapeutic index for strategies aimed at restoring tyrosination for the treatment of cardiac disease.

Desmin Knock-Out Cardiomyopathy: A Heart on the Verge of Metabolic Crisis.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

Clinicopathological Features and Prognostic Factors of Sinonasal Rhabdomyosarcoma.

BACKGROUND/AIM: Sinonasal rhabdomyosarcoma (RMS) is a rare soft tissue malignancy. Due to the limited cases, the clinicopathological features and prognostic factors are still not well understood. PATIENTS AND METHODS: This retrospective review included eight patients with sinonasal RMS at our institution between 2004 and 2020. Patient demographics, tumor features, Intergroup Rhabdomyosarcoma Study Group (IRSG) stage and clinical group, treatment strategy, and survival rates were evaluated. Kaplan-Meier analysis and log-rank tests were performed to analyze the possible prognostic factors. RESULTS: We observed a predominance of male sex and alveolar-type tumor in sinonasal RMS. Nasal obstructions and neck masses were the most common symptoms. Patients with pretreatment lactate dehydrogenase (LDH) levels >400 U/l and negative immunohistochemical staining for desmin or MyoD1 had lower survival rates. CONCLUSION: In patients with sinonasal RMS, pretreatment LDH levels >400 U/l and negative immunohistochemical staining for desmin or MyoD1 may suggest a poor prognosis. These factors can not only contribute to the prediction of prognosis in patients with sinonasal RMS but also facilitate the design of more appropriate treatment strategies.

Effect of High-altitude Hypoxia Environment on Uric Acid Excretion, Desmin Protein Level in Podocytes, and Na+-K+- ATPase Activity.

To investigate the oxidative stress and adaptive compensation of kidneys in rats in high-altitude hypoxia environments, 20 Wistar rats (3 months) were randomly and equally grouped. The rats in the test group were fed in a low-pressure oxygen chamber, and those in the control group (controls) were fed in a normal environment. On the 5th, 10th, 20th, and 30th day, the excretion of uric acid in rats was detected by a biochemical analyzer, the level of desmin protein in rat podocytes was detected by immunohistochemistry, and the activity of Na+-K+- ATPase in rat proximal tubular epithelial cells was measured by liquid scintillation method. The results showed that with the increased time, the level of uric acid in the blood of rats in the test group increased dramatically (P<0.05). On the 30th day, the blood uric acid content of the test group was 52.33µmol/L, and that of the control group was 38.43µmol/L. The blood uric acid content in the test group was dramatically increased relative to the control group. Immunohistochemistry showed that the desmin protein in podocytes of the test group (0.14) was considerably higher than that in the control group (P<0.05). The Na+-K+- ATPase activity of proximal renal tubular epithelial cells in the test group was 611.2 pmol pi/mg protein/h, which was considerably lower than the versus control group (P<0.05). In summary, in high altitude hypoxia environment, uric acid accumulated in the body, and renal filtration and excretion ability was limited.

Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway.

BACKGROUND: Danshen injection (DSI) is an agent extracted from the Salvia miltiorrhiza Bunge, a natural drug commonly used to alleviate kidney diseases. However, the material basis and therapeutic effects of DSI on nephrotic syndrome (NS) remain unclear. PURPOSE: To investigate the material basis of DSI and the therapeutic effects and underlying mechanisms of NS. METHODS: NS models were established using adriamycin-induced BALB/c mice and lipopolysaccharide-induced mouse podocytes (MPC-5). Following DSI and prednisone administration, kidney coefficients, 24 h urine protein, blood urea nitrogen, and serum creatinine levels were tested. Histomorphology was observed by periodic acid-Schiff staining and hematoxylin and eosin staining of the kidney sections. The glomerular basement membrane and autophagosomes of the kidneys were observed using transmission electron microscopy. Nephrin and desmin levels in the glomeruli were tested using immunohistochemistry. The viability of MPC-5 cells was tested using cell counting kit-8 after chloroquine and rapamycin administration in combination with DSI. The in vivo and in vitro protein levels of phosphatidylinositol 3-kinase (PI3K), AKT, phosphorylated AKT (Ser473), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), beclin1, cleaved caspase-3, and caspase-3 were detected using western blotting. RESULTS: Our results showed that DSI contained nine main components: caffeic acid, danshensu, lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, and 3, 4-Dihydroxybenzaldehyde. In in vivo studies, the NS mice showed renal function and pathological impairment. Podocytes were damaged, with decreased levels of autophagy and apoptosis, accompanied by inhibition of the PI3K/AKT/mTOR signaling. DSI administration resulted in improved renal function and pathology in NS mice, with the activation of autophagy and PI3K/AKT/mTOR signaling in the kidneys. Additionally, podocytes were less damaged and intracellular autophagosomes were markedly increased. In vitro studies have shown that DSI activated MPC-5 autophagy and reduced apoptosis via the PI3K/AKT/mTOR pathway. CONCLUSION: Collectively, this study demonstrated that DSI activated podocyte autophagy and reduced apoptosis via the PI3K/AKT/mTOR signaling, ultimately attenuating NS. Our study clarified the main components of DSI and elucidated its therapeutic effects and potential mechanisms for NS, providing new targets and agents for the clinical treatment of NS.

[Embryonal botryoid rhabdomyosarcoma of the uterus]. / Rabdomiosarcoma embrionario botrioide de útero.

Rhabdomyosarcoma is a malignant neoplasm of mesenchymal origin with skeletal striated muscular differentiation. It is the most common sarcoma of childhood and has four subtypes: embryonal, alveolar, pleomorphic and spindle cell/sclerosing. Of all of them, the embryonal one is the most prevalent and presents a variant, botryoid, which usually involves hollow organs in the form of a multilobed polypoid mass. We present the case of a 27-year-old woman who consulted for vaginal bleeding and in whom colposcopy revealed a whitish polypoid lesion that was externalized through the external cervical os. Histological examination revealed cellular sectors alternated by lax, myxoid areas, together with typical isthmic-endometrial glands. The atypical spindle cell proliferation was arranged in nests, made up of ce lls with large eosinophilic cytoplasm with transverse cytoplasmic striations and eccentric nuclei with homogeneous chromatin. Areas of densely packed cells were exhibited immediately, but separated from, the intact epithelial lining by a thin layer of loose stroma (cambium layer). The immunostaining profile was positive for desmin, muscle-specific actin and myogenin, and negative for smooth muscle actin. A diagnosis of embryonal botryoid rhabdomyosarcoma of the uterine corpus was made.

El rabdomiosarcoma es una neoplasia maligna de origen mesenquimal con diferenciación muscular estriada esquelética. Es el sarcoma más común de la infancia y presenta cuatro subtipos: embrionario, alveolar, pleomórfico y de células ahusadas/esclerosante. De todos ellos el embrionario es el de mayor prevalencia y presenta una variante, botrioide, que suele comprometer órganos huecos en forma de una masa polipoide multilobulada. Presentamos el caso de una mujer de 27 años que consultó por sangrado vaginal y en quien se evidenció en la colposcopia, una lesión polipoide blanquecina que se exteriorizaba a través del orificio cervical externo. El estudio histológico reveló sectores celulares alternados por áreas laxas, mixoides, junto a glándulas ístmico-endometriales típicas. La proliferación fusocelular atípica, se disponía en nidos, constituidos por células de amplio citoplasma eosinófilo con estriaciones citoplasmáticas transversales y núcleos excéntricos con cromatina homogénea. Se exhibían áreas de células densamente condensadas inmediatas y próximas al revestimiento epitelial intacto, pero separadas de él, por una fina capa de estroma laxo (capa cambial). El perfil de inmunomarcación resultó positivo para desmina, actina músculo específico y miogenina, y negativo para actina músculo liso. Se realizó diagnóstico de rabdomiosarcoma embrionario botroide de cuerpo uterino.

The gene variant for desmin rs1058261 may protect against combined cancer and cardiovascular death, the Tampere adult population cardiovascular risk study.

Desmin-containing intermediate filaments are a part of muscle cytoskeleton. We have previously reported that the wild-type cytosine/cytosine genotype of a common Desmin synonymous single nucleotide polymorphism (C > T) (rs1058261) associated with cardiovascular diseases in a cohort of subjects from the Tampere adult population cardiovascular risk study. We now examined whether rs1058261 also associates with early death by following the cohort of 801 subjects from the age of 50 up to the age of 65. Outcomes for death were collected from the National Statistics Centre. Linkage disequilibrium analysis and gene expression correlations for rs1058261 were done in silico. With follow-up, subjects with wild-type cytosine/cytosine genotype had higher incidence of cancer deaths (odds ratio [OR] 5.27, confidence interval [CI] 1.160-23.946, P = .031), combined deaths from cardiovascular diseases or cancers (OR 3.92, CI 1.453-10.596, P = .007), and "hard" acute cardiovascular disease events (early myocardial infarction and/or death) (OR 3.90, CI 1.287-11.855, P = .016) compared to subjects with the T-allele. The in silico results of linkage disequilibrium and gene expression analyses showed negative gene expression sizes associated with rs1058261, which theoretically decreases desmin expression. Our findings suggest that variation rs1058261 in Desmin may serve as a surrogate marker for other variations involved in decrease of deaths from combined cancer and cardiovascular disease.

[The role of clock gene BMAL1 in exercise-induced skeletal muscle injury recovery].

Objective: To investigate the role of clock gene BMAL1 in exercise-induced skeletal muscle injury recovery. Methods: Two hundred and eight 8-week-old SD rats were randomly divided into the control group (Group C, n=104) and the exercise group (Group E, n=104). Group E performed a 90-minute downhill run on the treadmill. After exercise, the gastrocnemius muscle of 8 rats in Group C and Group E were collected at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, 60 h, 66 h and 72 h. The expression of skeletal muscle core clock gene, BMAL1 was measured by real-time fluorescence quantitative PCR. The parameters of fitting cosine curve were obtained by cosine analysis software circacompare (R package), and the change trend of rhythmic oscillation was analyzed. The ultrastructure of skeletal muscle fibers was observed by transmission electron microscope. The expressions of skeletal muscle BMAL1 and DESMIN were detected by Western blot; Immunofluorescence was used to observe the localization and contents of BMAL1 and DESMIN. Results: In Group C, three complete circadian rhythm cycles of mRNA BMAL1 were observed within 72 hours; in Group E, the circadian rhythm of BMAL1 mRNA disappeared at 0 h～24 h. Compared with Group C, the expression level of BMAL1 mRNA was significantly increased at 0 h, 6 h, 12 h, and 18 h after exercise in Group E (P＜0.05), and the expression of BMAL1 protein was significantly increased at 0 h and 12 h after exercise(P＜0.05), and recovered to the level of that in Group C from 24 h to 72 h(P＞0.05). The expression of DESMIN protein was decreased at 0 h and 12 h after exercise(P＜0.05), gradually increased at 24 h, increased significantly at 48 h(P＜0.01), and recovered to the control level at 72 h (P＞0.05). In Group E, BMAL1 and DESMIN were co-localized at 0 h, 12 h, and 24 h after exercise; the colocalization at 0 h～24 h showed a trend of first decreasing and then increasing, and the fluorescence intensity at 24 h reached the highest value. Conclusion: The post-exercise clock gene BMAL1 may be involved in the enhanced synergy of regulating the cytoskeletal protein DESMIN, it is thus related to the promotion of muscle fiber structure recovery.

Incidental detection of gastrointestinal stromal tumors during laparoscopic sleeve gastrectomy. What to do?

INTRODUCTION: We evaluated and characterized the incidental GISTs during laparoscopic sleeve gastrectomy in our clinic. METHODS: All GISTs identified during laparoscopic sleeve gastrectomy between January 2015 and December 2017 were evaluated. Typical demographic, clinicopathologic, treatment, location, resection margins, immunohistochemistry (CD 34, CD 117, ASMA, desmin and S100) and criteria for oncological aggressiveness (tumor size, number of mitoses, presence or absence of tumor necrosis) data were recorded. RESULTS: Within the 800 bariatric surgeries at our institution, 7 GISTs were identified (0.87%). The median age of the patients was 32 years (age range: 24-42 years). The mean BMI was found to be 40.66 kg/m2 (range: 35-44 kg/m2). All GIST cases were found in the stomach samples. All tumors were not larger than 20 mm. All tumors were found close to the greater curvature of the stomach; in five cases, tumors were located in a single focus, while in 1 case, it was located both in the corpus and fundus. CD117 and CD34 were found to be positive in the pathological examination of all parts. In addition, desmin, smooth muscle actin (SMA) and S-100 were also positively stained. No complications or mortality were observed in this series. CONCLUSION: Tumor resection with a negative surgical margin may be considered complete oncologic treatment in case of presence of very low or low risk classification of postoperative GIST recurrence. After GIST resection, all patients should remain under long-term postoperative care. KEY WORDS: Bariatric surgery, Incidental gastrointestinal stromal tumors, Obesity, Sleeve gastrectomy.

Cardiac alterations following experimental hip fracture - inflammaging as independent risk factor.

Background: Cardiac injuries following trauma are associated with a worse clinical outcome. So-called trauma-induced secondary cardiac injuries have been recently described after experimental long bone fracture even in absence of direct heart damage. With the progressive aging of our society, the number of elderly trauma victims rises and therefore the incidence of hip fractures increases. Hip fractures were previously shown to be associated with adverse cardiac events in elderly individuals, which have mainly been attributed to pre-conditioned cardiac diseases. The aim of the present study was to investigate the effect of hip fractures on the heart in healthy young and middle-aged mice. Materials and Methods: Young (12-week-old) and middle-aged (52-week-old) female C57BL/6 mice either received an intramedullary stabilized proximal femur fracture or sham treatment. The observation time points included 6 and 24 h. Systemic levels of pro-inflammatory mediators as well as local inflammation and alterations in myocardial structure, metabolism and calcium homeostasis in left ventricular tissue was analyzed following hip fracture by multiplex analysis, RT-qPCR and immunohistochemistry. Results: After hip fracture young and middle-aged mice showed increased systemic IL-6 and KC levels, which were significantly elevated in the middle-aged animals. Furthermore, the middle-aged mice showed enhanced myocardial expression of HMGB1, TLR2/4, TNF, IL1ß and NLRP3 as well as considerable alterations in the myocardial expression of glucose- and fatty acid transporters (HFABP, GLUT4), calcium homeostasis proteins (SERCA) and cardiac structure proteins (desmin, troponin I) compared to the young animals following hip fracture. Conclusion: Young and middle-aged mice showed local myocardial alterations, which might predispose for the development of secondary cardiac injury following hip fracture. Age and the age-associated phenomenon of 'inflammaging' seemed to be an independent risk factor aggravating and accelerating cardiac alterations following hip fracture.

Expression patterns and correlation analyses of muscle-specific genes in the process of sheep myoblast differentiation.

The purpose of this study was to establish a system for the isolation, culture, and differentiation of sheep myoblasts, and to explore the expression patterns as well as mutual relationships of muscle-specific genes. Sheep fetal myoblasts (SFMs) were isolated by two-step enzymatic digestion, purified by differential adhesion and identified using immunofluorescence techniques. Two percent horse serum was used to induce differentiation in SFMs. Real-time quantitative and Western blot analyses were respectively used to detect the mRNA and protein expressions of muscle-specific genes including MyoD, MyoG, Myf5, Myf6, PAX3, PAX7, myomaker, desmin, MYH1, MYH2, MYH4, MYH7, and MSTN during the differentiation of SFMs. Finally, the correlation between muscle-specific genes was analyzed by the Pearson correlation coefficient method. The results showed that the isolated and purified SFMs could form myotubes after the induction for differentiation. The marker factors including MyoD, MyoG, myomaker, desmin, and MyHC were positively stained in SFMs. The mRNA expressions of MyoD, MyoG, and myomaker increased and then decreased, while Myf5, PAX3, and PAX7 decreased; Myf6, desmin, MYH1, MYH2, MYH4, and MYH7 increased; and MSTN fluctuated up and down during the differentiation of SFMs. The expression patterns of protein were basically consistent with those of mRNA except MSTN. There existed significant or highly significant correlations at mRNA or protein level among some genes. Some transcription factor proteins (MyoD, Myf5, Myf6, PAX3, PAX7) showed significant or highly significant correlations with the mRNA level of some other genes and/or themselves. In conclusion, SFMs with good myogenic differentiation ability were successfully isolated, and the expression patterns and correlations of muscle-specific genes during SFM differentiation were revealed, which laid an important foundation for elucidating the gene regulation mechanism of sheep myogenesis.

Malignant gastrointestinal neuroectodermal tumor: A case-based review of literature.

Malignant gastrointestinal (GI) neuroectodermal tumor is an extremely rare entity that was first described by Zambrano et al. in 2003 as "clear cell sarcoma (CCS)-like tumor of the GI tract." It shares some of the histopathological features of CCS but lacks the immunohistochemical (IHC) reactivity for melanocytic markers. Most mesenchymal neoplasms of the GI tract belong to the category of GI stromal tumors and are characterized by the IHC expression of c-KIT. In cases, without detectable KIT receptor expression, several differential diagnoses have to be taken into consideration. In this article, we describe such a case and present a review of all the reported cases till date. We also present the current available knowledge on its pathology and molecular genetics along with the limitations in its diagnosis. Here, we report a case of a 32-year-old man with a tumor of the small bowel composed of polygonal tumor cells arranged in solid nests, alveolar pattern, and pseudopapillary and admixed with numerous osteoclast-like multinucleated giant cells. Immunohistochemically, the tumor cells strongly expressed S-100 protein only. HMB-45, melan-A, CD117, cytokeratin, desmin, smooth muscle actin, and CD-34 were absent. Ki-67 index was 15%. The diagnosis was further confirmed by fluorescence in situ hybridization (FISH) demonstrating the presence of EWSR1 (22q12) translocation. A final diagnosis of malignant gastroneuroectodermal tumor was rendered. The patient is disease-free for 20 months of postsurgery. The diagnosis of this entity should be considered in the presence of S-100-positivity and multinucleated osteoclastic giant cells and the absence of melanocytic differentiation in a tumor arising from GI tract. Further confirmation can be done by performing FISH analysis.