Chain mRNA Research Articles

Si-Miao-Yong-An Decoction Protects Against Cardiac Hypertrophy and Dysfunction by Inhibiting Platelet Aggregation and Activation.

Objective: The aim of this study was to determine whether Si-Miao-Yong-An decoction (SMYAD) could ameliorate pressure overload-induced heart hypertrophy and its mechanisms.Methods: C57BL/6 mice were subjected to either sham or transverse aortic constriction (TAC) surgery to induce heart hypertrophy. SMYAD (14.85 g/kg/day, ig) or captopril (16.5 mg/kg/day, ig) was administered to the mice for 4 weeks. Cardiac function was evaluated based on echocardiography. Heart hypertrophy was detected using hematoxylin and eosin or wheat germ agglutinin staining. Protein expression of CD41, CD61, and P-selectin were measured with Western blot and immunohistochemistry. The expression levels of atrial natriuretic peptide, brain natriuretic peptide, β-myosin heavy chain, β-thromboglobulin, and von Willebrand factor were evaluated by quantitative polymerase chain reaction.Results: Four weeks after TAC, mice developed exaggerated cardiac hypertrophy and demonstrated a strong decrease in left ventricular ejection fraction compared with sham (29.9 ± 9.3% versus 66.0 ± 9.9%; P < 0.001). Conversely, SMYAD improved cardiac dysfunction with preserved left ventricular ejection fraction (66.5 ± 17.2%; P < 0.001). Shortening fraction was increased by SMYAD, while the left ventricular internal diameter and left ventricular volume were decreased in SMYAD group. SMYAD treatment significantly attenuated cardiac hypertrophy as reflected by the inhibition of atrial natriuretic peptide, brain natriuretic peptide, β-myosin heavy chain mRNA expression, and by the decreasing of cardiac myocyte cross-sectional area. Furthermore, Western blot and immunohistochemistry indicated that the protein expression of platelet aggregation markers (CD41 and CD61) and platelet activation marker (P-selectin) were significantly higher in model mice compared with control. These pathological alterations in TAC-induced mice were significantly ameliorated or blocked by SMYAD administration.Conclusions: Our results suggested that SMYAD exerted its effect by inhibiting platelet aggregation and activation as revealed by CD41/CD61/P-selectin downregulation. Inhibition the activation of the platelets might contribute to the therapeutic effect of SMYAD in failing heart.

Ultrasensitive RNA In Situ Hybridization for Kappa and Lambda Light Chain mRNA in Marginal Zone Lymphoma Demonstrates Comparable Performance to Flow Cytometry

Abstract Objectives Evaluation of light chain restriction is critical to establish clonality in B-cell lymphoproliferative disorders (LPDs). Immunohistochemistry (IHC) and in situ hybridization (ISH) are commonly used to assess light chain restriction in formalin-fixed, paraffin-embedded (FFPE) tissues. However, except for cases with plasma cell differentiation, these techniques often fail to identify immunoglobulin light chains. An ultrasensitive technique, RNAscope, has been recently introduced that can identify light chains in cases of B-cell LPDs. We analyzed the utility of this ultrasensitive method in detection of clonality and correlated with flow cytometry results when available. Methods A tissue microarray was constructed using 1.6-mm diameter tissue punches of 31 FFPE tissue blocks from 27 cases that were previously characterized as marginal zone lymphoma (MZL) by a combination of morphology, IHC, and/or flow cytometry. Cases included 8 nodal and 19 extranodal MZLs. In two cases, additional blocks were included to assess reproducibility. For ultrasensitive ISH RNAscope assay, 4-µm thickness tissue sections were hybridized using kappa and lambda probes, incubated overnight, counterstained with hematoxylin, cover-slipped, and reviewed blindly without knowledge of prior flow cytometry results. Results Of 18 cases with evaluable staining, 15 were clonal and 3 were polytypic. Flow cytometry was available in 14 of these 18 cases with concordance in 13 of 14 (93%). The discordant case was polytypic by flow cytometry but kappa restricted by RNAscope. The false-negative flow results could be due to sampling issues. In six cases, staining failed and could not be evaluated. Conclusion Ultrasensitive RNAscope is a reliable assay in the detection of clonality in FFPE tissue, particularly where fresh tissue is not available for flow cytometry. In addition, our results confirm and further expand prior observations that RNAscope is a highly sensitive and specific assay with high concordance with flow cytometry.

Ribosome pausing, a dangerous necessity for co-translational events.

In recent years translation elongation has emerged as an important contributor to the regulation of gene expression. There are multiple quality control checkpoints along the way of producing mature proteins and targeting them to the right cellular compartment, or associating them correctly with their partners. Ribosomes pause to allow co-translational protein folding, protein targeting or protein interactions, and the pausing is dictated by a combination of the mRNA sequence and structure, the tRNA availability and the nascent peptide. However, ribosome pausing can also lead to ribosome collisions and co-translational degradation of both mRNA and nascent chain. Understanding how the translating ribosome tunes the different maturation steps that nascent proteins must undergo, what the timing of these maturation events is, and how degradation can be avoided when pausing is needed, is now possible by the emergence of methods to follow ribosome dynamics in vivo. This review summarizes some of the recent studies that have advanced our knowledge about co-translational events using the power of ribosome profiling, and some of the questions that have emerged from these studies.

A co-ultramicronized palmitoylethanolamide/luteolin composite mitigates clinical score and disease-relevant molecular markers in a mouse model of experimental autoimmune encephalomyelitis

BackgroundPersistent and/or recurrent inflammatory processes are the main factor leading to multiple sclerosis (MS) lesions. The composite ultramicronized palmitoylethanolamide, an endogenous N-acylethanolamine, combined with the flavonoid luteolin, PEALut, have been found to exert neuroprotective activities in experimental models of spinal and brain injury and Alzheimer disease, as well as a clinical improvement in human stroke patients. Furthermore, PEALut enhances the expression of different myelin proteins in oligodendrocyte progenitor cells suggesting that this composite might have protective effects in MS experimental models.MethodsThe mouse model of experimental autoimmune encephalomyelitis (EAE) based on active immunization with a fragment of myelin oligodendrocyte glycoprotein (MOG35-55) was used. The daily assessment of clinical score and the expression of serum amyloid A (SAA1), proinflammatory cytokines TNF-α, IL-1β, IFN-γ, and NLRP3 inflammasome, as well as TLR2, Fpr2, CD137, CD3-γ, and TCR-ζ chain, heterodimers that form T cell surface glycoprotein (TCR), and cannabinoid receptors CB1, CB2, and MBP, were evaluated in the brainstem and cerebellum at different postimmunization days (PIDs).ResultsVehicle-MOG35-55-immunized (MOG35-55) mice developed ascending paralysis which peaked several days later and persisted until the end of the experiment. PEALut, given intraperitoneally daily starting on day 11 post-immunization, dose-dependently improved clinical score over the range 0.1–5 mg/kg. The mRNA expression of SAA1, TNF-α, IL-1β, IFN-γ, and NLRP3 were significantly increased in MOG35-55 mice at 14 PID. In MOG35-55 mice treated with 5 mg /kg PEALut, the increase of SAA1, TNF- α, IL-1β, and IFN-γ transcripts at 14 PID was statistically downregulated as compared to vehicle-MOG35-55 mice (p < 0.05).The expression of TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors showed a significant upregulation in vehicle-MOG35-55 mice at 14 PID. Instead, CB1 and MBP transcripts have not changed in expression at any time. In MOG/PEALut-treated mice, TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 mRNAs were significantly downregulated as compared to vehicle MOG35-55 mice.ConclusionsThe present results demonstrate that the intraperitoneal administration of the composite PEALut significantly reduces the development of clinical signs in the MOG35-55 model of EAE. The dose-dependent improvement of clinical score induced by PEALut was associated with a reduction in transcript expression of the acute-phase protein SAA1, TNF-α, IL-1β, IFN-γ, and NLRP3 proinflammatory proteins and TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors.

Obesity and aging affects skeletal muscle renin-angiotensin system and myosin heavy chain proportions in pre-diabetic Zucker rats.

There is a gap in the knowledge regarding regulation of local renin-angiotensin system (RAS) in skeletal muscle during development of obesity and insulin resistance in vivo. This study evaluates the obesity- and age-related changes in the expression of local RAS components. Since RAS affects skeletal muscle remodelling, we also evaluated the muscle fibre type composition, defined by myosin heavy chain (MyHC) mRNAs and protein content. Gene expressions were determined by qPCR and/or Western blot analysis in musculus quadriceps of 3- and 8-month-old male obese Zucker rats and their lean controls. The enzymatic activity of aminopeptidase A (APA) was determined flourometrically. Activation of renin receptor (ReR)/promyelocytic leukaemia zinc finger (PLZF) negative feedback mechanism was observed in obesity. The expression of angiotensinogen and AT1 was downregulated by obesity, while neutral endopeptidase and AT2 expressions were upregulated in obese rats with aging. Skeletal muscle APA activity was decreased by obesity, which negatively correlated with the increased plasma APA activity and plasma cholesterol. The expression of angiotensin-converting enzyme (ACE) positively correlated with MyHC mRNAs characteristic for fast-twitch muscle fibres. The obesity- and age-related alterations in the expression of both classical and alternative RAS components suggest an onset of a new equilibrium between ACE/AngII/AT1 and ACE2/Ang1-7/Mas at lower level accompanied by increased renin/ReR/PLZF activation. Increased APA release from the skeletal muscle in obesity might contribute to increased plasma APA activity. There is a link between reduced ACE expression and altered muscle MyHC proportion in obesity and aging.

Calcimimetic R568 improved cardiac remodeling by classic and novel renin-angiotensin system in spontaneously hypertensive rats.

One major cause of cardiac mortality is heart disease caused by hypertension. The formation of cyclic adenosine monophosphate (cAMP) is inhibited by calcium-sensitive receptor (CaSR) activation which increases intracellular Ca2+ concentrations and suppresses renin release. As we know, renin-angiotensin system (RAS) is closely related to development of essential hypertension (EH). Therefore, we focused on exploring the roles of NPSR568 (R568)-activated CaSR in cardiac remodeling of spontaneously hypertensive rats (SHRs), as well as the activity of classic and novel RAS. Wistar-Kyoto rats (WKYs) and SHRs were treated by R568 for four and eight weeks, respectively, and their blood pressure (BP), echocardiographic values, heart-to-body weight ratio (HW/BW%), and left ventricle-to-body weight ratio (LVW/BW%) were evaluated. Then Masson’s trichrome staining and hematoxylin and eosin staining as well as RT-qPCR analysis of β-isoform of myosin heavy chain and brain natriuretic peptide mRNA expression were performed. A Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and analysis of apoptosis marker proteins were used to assess the extent of myocardial apoptosis. The CaSR expression and the activity of classic and novel RAS were examined by immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay. The present study revealed that the development of hypertension was accompanied by increased BP, apoptosis, hypertrophy, and fibrosis, along with decreased expression of CaSR, decreased novel RAS, and increased classic RAS in myocardial tissues. R568 administration for four and eight weeks reduced BP and myocardial remodeling and reversed the low expression of CaSR; moreover, classic RAS was suppressed and novel RAS was activated in the myocardium. Taken together, these data indicate that R568 may effectively inhibit EH myocardial remodeling by inhibiting classic RAS and activating novel RAS in SHRs. Impact statement Our study reveals that low calcium-sensitive receptor (CaSR) expression is associated with the occurrence and development of essential hypertension-mediated myocardial remodeling. The activation of CaSR can reverse adverse myocardia remodeling by inhibiting local classical renin-angiotensin system (RAS) and activating novel RAS in cardiac tissues. CaSR is closely related to many cardiovascular diseases, but its specific mechanism remains not to be elucidated. To date, CaSR has not been investigated with regard to cardiovascular treatment; however, given the important relationship between CaSR and cardiovascular disease, CaSR regulators can be potential drugs for the treatment of cardiovascular disease.

Sibjotang attenuates Doxorubicin‐Induced Cardiac Hypertrophy: Involvement of necroptosis

Doxorubicin is one for the most widely used anti‐neoplastic agents, however severely limited due to its cumulative cardiotoxicity, which develops over time into congestive heart failure. Sibjotang (Shizaotang), traditional herbal medicine formula, has been reported regulates the body fluid blood pressure homeostasis. The present study was aimed at determining the effect and regulatory mechanism of Sibjotang in doxorubicin‐induced cardiotoxicity in H9c2 cells. Immunofluorescence staining analysis showed that Sibjotang decreased doxorubicin‐induced increase in H9c2 cardiomyocytes size in does dependent manner. Pre‐treatment with Sibjotang significantly inhibited doxorubicin‐induced cardiac hypertrophic markers such as ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and β‐MHC (β‐myosin heavy chain) mRNA expression levels. Sibjotang reduced hypertrophy‐associated transcription factor GATA 4 (GATA binding factor 4) expression. Pre‐treatment with Sibjotang significantly inhibited doxorubicin‐induced necroptosis‐associated kinase such as receptor‐interacting serine/threonine‐protein kinase RIPK1 and RIPK3 without affecting cell viability. In conclusion, doxorubicin‐induced cardiac hypertrophy and necroptosis were attenuated by Sibjotang. Therefore, Sibjotang may be potential herbal therapies targeting cardiotoxicity resulting from anti‐cancer agents.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Celecoxib Impairs Differentiation of Primary Human Skeletal Myoblasts

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are medications commonly consumed for their anti‐inflammatory, analgesic, and antipyretic effects. NSAIDs act by inhibiting cyclooxygenase (COX) enzymes, which mediate the synthesis of pro‐inflammatory prostaglandins from substrates such as arachidonic acid. Despite the widespread use of NSAIDs, the risks of taking NSAIDs are only beginning to be defined; in particular, their effects on highly metabolic tissues such as skeletal muscle. We therefore sought to determine whether celecoxib, an NSAID selective for COX2, impaired the differentiation and hypertrophy of cultured primary human skeletal myoblasts. Myoblasts were exposed to celecoxib at concentrations from 50 μM – 0.08 μM and allowed to differentiate for 72 hours. Vehicle‐treated myotubes showed a robust fusion index and myotube area as determined by immunocytochemistry and confocal microscopy; however, the fusion indices and areas of myotubes exposed to 50 μM and 25 μM celecoxib were significantly reduced (90% reduced fusion index and 89% reduced myotube area in cells exposed to 50 μM celecoxib; P&lt;0.001, and 33% reduced fusion index and 25% reduced myotube area in cells exposed to 25 μM celecoxib; P&lt;0.001). The total nuclei present across all treatments did not differ. Myoblasts treated with NS‐398 (a COX2‐selective pharmacological inhibitor) showed no impaired differentiation at any concentration. These observations were associated with dose‐dependent decreases in expression of myosin heavy chain mRNA transcripts of slow (Myh7) and developmental (Myh3 and Myh8) myosins, and exposure to 50 μM celecoxib virtually eliminated expression of all myosin heavy chain transcripts (P&lt;0.001 for all). Phosphorylation of AKT (S473), p70S6K (T389), and S6 (S235/236) were dose‐dependently reduced in celecoxib‐treated myotubes (P&lt;0.05), whereas administration of NS‐398 had no effect on phosphorylation of any of these molecules at any concentration. These data reveal that celecoxib inhibits differentiation of primary human skeletal myoblasts, possibly through a mechanism that regulates the activation of molecules involved in protein synthesis. Celecoxib‐mediated impairment of human myoblast differentiation may be independent of its actions on COX2, because treatment with NS‐398 did not alter differentiation. The exact mechanism through which celecoxib acts to alter muscle cell differentiation and physiology remains to be established.The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This abstract has been approved for public release with unlimited distribution.Support or Funding InformationUS Army Military operational Medicine Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Leucine regulates slow-twitch muscle fibers expression and mitochondrial function by Sirt1/AMPK signaling in porcine skeletal muscle satellite cells.

A previous study demonstrated that leucine upregulates the slow myosin heavy chain mRNA expression in C2C12 cells. However, the role of leucine in slow-twitch muscle fibers expression and mitochondrial function of porcine skeletal muscle satellite cells as well as its mechanism remain unclear. In this study, porcine skeletal muscle satellite cells cultured in differentiation medium were treated with 2 mM leucine for 3days. Sirt1 inhibitor EX527, AMPK inhibitor compound C, and AMPKα1 siRNA were used to examine its underlying mechanism. Here we showed that leucine increased slow-twitch muscle fibers and mitochondrial function-related gene expression, as well as increased succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. Moreover, leucine increased the protein levels of Sirt1 and phospho-AMPK. We also found that AMPKα1 siRNA, AMPK inhibitor compound C, or Sirt1 inhibitor EX527 attenuated the positive effect of leucine on slow-twitch muscle fibers and mitochondrial function-related gene expression. Finally, we showed that Sirt1 was required for leucine-induced AMPK activation. Our results provide, for the first time, evidence that leucine induces slow-twitch muscle fibers expression and improves mitochondrial function through Sirt1/AMPK signaling pathway in porcine skeletal muscle satellite cells.

Role of Adipokines in Development of Metabolic Syndrome

Visceral adipose tissue releases a variety of adipokines which together determine a comprehensive cardiometabolic risk profile. Estrogen deficiency leads to central fat deposition in postmenopausal women. However, premenopausal women are also running high risk of central obesity owing to unhealthy lifestyles, making them prone to development of metabolic syndrome which leads to infertility, polycystic ovary syndrome (PCOS), insulin resistance, and type 2 diabetes (T2D). Premenopausal women with (n=30) and without (n=30) central obesity were studied. Metabolic risk factors and circulatory adipokines were measured. Insulin resistance was calculated by homeostasis model assessment (HOMA-IR). Adipokines' gene polymorphisms were studied by polymerase chain reaction and mRNA expression of leptin, adiponectin, resistin, and interleukin-6 (IL-6). Tumour necrosis factor-α (TNF-α), acylation stimulating protein (ASP) receptor gene (C5L2) were done by real time-polymerase chain reaction in visceral (VAT) and subcutaneous (SAT) adipose tissues was also obtained. Significant high circulating leptin, IL-6, TNF-α, resistin and their VAT mRNA expression and significant low circulating adiponectin and VAT mRNA expression were found in women with metabolic syndrome, irrespective of their menopausal status. Carriers of mutant genotype of TNF-α 308 AA, IL-6 174 CC, resistin 420 GG, leptin 2549 AA, adiponectin 276 TT and C5L2 698 CT had significant association with metabolic syndrome. Conclusively changes in fat distribution modulate the secretion profile of adipokines, therefore elevated circulating leptin, IL-6, TNF-α, ASP, resistin, and low adiponectin may serve as surrogate markers for metabolic syndrome and related morbidities in women with central obesity.

Preoperative oral thyroid hormones to prevent euthyroid sick syndrome and attenuate myocardial ischemia-reperfusion injury after cardiac surgery with cardiopulmonary bypass in children: A randomized, double-blind, placebo-controlled trial.

Background:Both euthyroid sick syndrome and myocardial ischemia-reperfusion injury are common and have been significantly associated with morbidity and mortality after pediatric cardiac surgery with cardiopulmonary bypass. This single-center, prospective, double-blind, randomized placebo-controlled clinical pilot trial was designed to assess if preoperative oral thyroid hormone therapy could prevent the occurrence of euthyroid sick syndrome (ESS) and attenuate myocardial ischemia-reperfusion injury (IRI) after cardiac surgery with cardiopulmonary bypass (CPB) in children.Methods:Forty children aged 3 to 12 year, scheduled for elective congenital heart disease repair surgery with CPB, were randomized into 2 groups of equal size to receive the following treatments in a double-blind manner: placebo (control group) and thyroid tablet 0.4 mg/kg (trial group) taken orally once a day for 4 days before surgery. The perioperative serum thyroid hormone levels and hemodynamic variables were determined. The extubation time, duration of intensive care unit (ICU) stay, and use of inotropic drugs in the ICU were recorded. The myocardial expressions of heat shock protein 70 (HSP70), myosin heavy chain (MHC) mRNA, and thyroid hormone receptor (TR) mRNA were detected. The serum creatine kinase-MB (CK-MB) activity and troponin I (TnI) positive ratio at 24 hour after surgery were assessed.Results:There were no significant differences in hemodynamic variables at all observed points, extubation time, and duration of ICU stay between groups. As compared with baselines on administration, serum triiodothyronine (T3) and free T3 (FT3) levels on the first, second, and fourth postoperative day, and serum thyrotropic-stimulating hormone (TSH), tetraiodothyronine (T4), and free T4 (FT4) levels on the first postoperative day were significantly decreased in the 2 groups. Serum T3, FT3, and T4 levels on the first and second postoperative day, and serum FT4 level on the first postoperative day were significantly higher in the trial group than in control group. As compared with the control group, the number of patients requiring inotropic drugs in the ICU, serum CK-MB activity, serum positive TnI ratio, and myocardial expression of MHCβ mRNA were significantly decreased, and myocardial expressions of both HSP70 and MHCα mRNA were significantly increased in the trial group.Conclusions:In children undergoing cardiac surgery with CPB, preoperative oral small-dose thyroid hormone therapy reduces severity of postoperative ESS and provides a protection against myocardial IRI by increasing HSP70 and MHCα expression.

Pulmonary arterial hypertension induced by a novel method: Twice-intraperitoneal injection of monocrotaline.

Pulmonary arterial hypertension (PAH) in humans manifests as a chronic process. However, PAH induced by high-dose monocrotaline (MCT) in animals occurs as a subacute process. To establish a chronic PAH model, rats were randomly divided into three groups, control (ctrl), single injection (SI), and twice injection (TI) groups. Rats in the SI group received a single intraperitoneal injection of 40 mg/kg MCT on day 0. Rats in the TI group received twice injections of 20 mg/kg MCT on days 0 and 7. Survival percentage, characteristic changes of pulmonary arterial variables, and right ventricular features were evaluated. Thirty-five days after the first MCT injection, survival percentage in TI group was higher than that in the SI group. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), pulmonary vascular remodeling, serum tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) were higher either in SI or in TI 28 and 35 days after the first MCT injection. The rats in the SI and TI groups exhibited higher right ventricle end diastolic diameter (RVEDD) and lower adjusted pulmonary artery acceleration time (PAAT/HR), tricuspid annular plane systolic excursion (TAPSE), cardiac output (CO) and right ventricle fractional shortening (RVFS) when compared with controls. However, mPAP, RVHI, TAPSE, PAAT/HR, CO, TNFα, and IL-6 were lower and RVEDD were higher in the TI group than in the SI group. Pulmonary macrophage infiltration and right ventricle (RV) fibrosis were lower in TI than SI groups. The cardiomyocyte cross-sectional area and the beta myosin heavy chain (MYH7) mRNA level of RV were lower in TI than SI, whereas alpha myosin heavy chain (MYH6) was increased. These results show that two intraperitoneal injections of 20 mg/kg MCT with seven days interval could induce a model similar to chronic PAH with increased survival percentage in rats. Impact statement We demonstrated previously that a single intraperitoneal injection of 40 mg/kg MCT produced a subacute, not chronic, PAH model in rats, and the short survival periods of these rats did not represent adequately the chronic PAH seen in humans. To overcome this limitation, in this study, the single dose of 40 mg/kg MCT was divided into twice injections of 20 mg/kg with an interval of seven days. This modified administration of MCT produced an animal model much more similar to chronic PAH with prolonged survival and characteristic changes of structures and function in pulmonary arteries and right ventricles.

Pregnancy late in rodent life has detrimental effects on the heart.

During pregnancy, the heart undergoes significant and numerous changes, including hypertrophy, that are usually described as physiological and reversible. Two aspects of the cardiac response to pregnancy are relatively understudied: advanced maternal age and multiple pregnancies (multiparity). Repeated breeder (RB) mice that have undergone five to seven consecutive pregnancies were euthanized 21 days after the weaning of their last pups and compared with age-matched primiparous, one-time pregnant (O1P) mice. The ages of the older mouse groups were similar (12 ± 1 mo). Pregnancy at a later age resulted in reduced fertility (40%); resorption was 29%, maternal mortality was 10%, and mortality of the pups was 17%. Contractile function as indicated by percent fractional shortening was significantly decreased in O1P and RB groups compared with the old nonpregnant control (ONP) group. There was no pathological induction of the fetal program of gene expression, with the exception of β-myosin heavy chain mRNA, which was induced in O1P compared with ONP mice ( P < 0.05) but not in RB mice. MicroRNA-208a was significantly increased in O1P compared with ONP mice ( P < 0.05) but significantly decreased in RB compared with ONP mice ( P < 0.05). mRNA of genes regulating angiogenesis (i.e., vascular endothelial growth factor-A) were significantly downregulated, whereas proinflammatory genes [i.e., interleukin-6, chemokine (C-C motif) ligand 2, and Cd36] were significantly upregulated in O1P ( P < 0.05) but not in RB mice. Overall, our results suggest that rather than multiparity, pregnancy in advanced age is a much more stressful event in both pregnant dams and fetuses, as evidenced by increased mortality, lower fertility, downregulation of angiogenesis, upregulation of inflammation, and cardiac dysfunction. NEW & NOTEWORTHY Pregnancy in older mice significantly decreases cardiac function, although repeated breeder mice demonstrated increased wall hypertrophy and dilated chamber size compared with one-time pregnant mice. Interestingly, many of the molecular changes were altered in one-time pregnant mice but not in repeated breeder mice, which may contribute to adverse pregnancy outcomes in a first pregnancy at a later age.

B- and T-cell Immune Repertoire Characterization using Anchored Multiplex PCR and Next-Generation Sequencing

Abstract Introduction NGS-based analysis of the immune repertoire (IR) is a powerful tool to monitor disease, adaptive immune responses to disease, vaccination and therapeutic interventions. IR characterization by NGS usually requires large primer panels, due to the extensive combinatorial diversity, and a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Anchored Multiplex PCR (AMP™) uses molecular barcoded (MBC) adapters and gene-specific primers (GSPs), enabling NGS-based immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Here, we describe AMP-based NGS assays for IR characterization, Immunoverse™ BCR and TCR, which utilize a minimal set of unidirectional GSPs and MBC adapters that reduce amplification bias. Methods The quantitative reproducibility and sensitivity of our assays was validated using mRNA isolated from PBMCs of healthy donors, B-cell chronic lymphocytic leukemia donors and formalin-fixed paraffin-embedded (FFPE) tissue. Results Both assays demonstrated high reproducibility between replicates with quantitative clone tracking down to 0.01%. The ability to determine isotype, clonotype and IGHV mutational status in a single assay was demonstrated. Preliminary TCR assay data indicates that CDR3 sequence capture is possible from FFPE tissue with clonotype calling being driven by input quantity, T-cell content, and, to a lesser degree, mRNA quality. Conclusions AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.

MicroRNA-214-5p/TGF-β/Smad2 signaling alters adipogenic differentiation of bone marrow stem cells in postmenopausal osteoporosis

Postmenopausal osteoporosis (OPM) is a common type of osteoporosis in females. It is a systemic, chronic bone disease that presents as microstructure degradation of osseous tissue, decreased bone mineral density and increased osteopsathyrosis caused by hypoovarianism and reduced estrogen levels in the body following menopause. In the present study, the role of microRNA (miR)-214-5p in the regulation of the expression of bone marrow stem cells (BMSCs) was investigated, and its molecular mechanism of osteogenic induction in vitro was assessed. When dexamethasone-induced adipogenic differentiation was performed, miR-214-5p expression was increased compared with the control group, as determined by RT-qPCR. Furthermore, oil red O staining, RT-qPCR and western blot analysis demonstrated that overexpression of miR-214-5p promoted adipogenic differentiation, inhibited alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OC) and collagen α-1 (I) chain (COL1A1) mRNA expression, and suppressed transforming growth factor (TGF)-β, phosphorylated (p)-Smad2 and collagen type IV α1 chain (COL4A1) protein expression in BMSCs. Additionally, downregulation of miR-214-5p increased the ALP, Runx2, OC and COL1 mRNA expression and increased TGF-β, Smad2 and COL4A1 protein expression in BMSCs. Furthermore, a TGF-β inhibitor was employed to inhibit TGF-β expression in BMSCs following miR-214-5p downregulation, which led to reduced Smad2, TGF-β and COL4A1 protein expression, and ALP, Runx2, OC and COL1 mRNA expression was also reduced, compared with the miR-214-5p downregulation only group. It was demonstrated that miR-214-5p may weaken osteogenic differentiation of BMSCs through regulating COL4A1. In conclusion, the results of the present study indicated that miR-214-5p may promote the adipogenic differentiation of BMSCs through regulation of the TGF-β/Smad2/COL4A1 signaling pathway, and potentially may be used to develop a novel drug for postmenopausal osteoporosis.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

Electrochemical Luminescent DNA Sensor Based on Polymerase-assisted Signal Amplification

A novel polymerase-based electrochemical luminescence (ECL) DNA sensor was constructed for messenger RNA (mRNA) detection by cyclic chain displacement polymerization, assisted by target mRNA cycle and quantum dots signal amplification. Firstly, the mercapto-modified capture-type probe DNA (CP) was immobilized on the surface of a magneto-controlled glassy carbon electrode via Au-S bond. After the addition of target mRNA, CP was opened and hybridized with mRNA to form double-stranded DNA (dsDNA). Then polymerase, primer chain (DNA1) and bases were added, which made the primer chain extend to replace the target mRNA. After one amplification cycle, the mRNA chain could open another hairpin in order to carry out next cycle of amplification. Finally, the ECL detection was carried out by adding DNA2 labeled thioglycolic acid-CdTe quantum dots. The amplification of the target mRNA by the addition of polymerase and the signal combined with the quantum dots label greatly improved the sensitivity of the sensor. The results showed that corresponding ECL signal had a good linear relationship with logarithm of target mRNA concentration in the range of 1 × 10−15 to 1 × 10−11 M, with a detection limit of 3.4 × 10−16 M (S/N = 3). Under the optimal conditions, the recoveries of mRNA spiked in human serum sample were from 97.2 % to 102.3 %. This sensor exhibited good selectivity, stability and reproducibility.

Evolutionarily-Encoded Translation Kinetics Coordinate Co-Translational SSB Chaperone Binding in Yeast

Chaperones can bind to the ribosomes and nascent polypeptides co-translationally to assist protein folding. It was not known previously whether there is any interplay between chaperone binding and translation kinetics. To study this effect, we utilize the high-throughput transcriptome-wide quantitative data for translation kinetics and chaperone binding from ribosome profiling and selective ribosome profiling methods respectively. In vivo selective ribosome profiling has shown that yeast Hsp70 chaperone Ssb associates broadly with a major fraction of nascent proteins. Using the ribosome footprint densities as a measure of local translation rate, we find that mRNA segments within the ribosome are translated faster during periods of Ssb binding to the nascent polypeptides compared to when Ssb is not bound. The acceleration of translation is maintained even when Ssb is knocked out thus implying an inherent encoding of faster translation within the mRNA sequence. Testing for mRNA features that can slow translation, we find that mRNA segments translated by Ssb-engaged ribosomes are enriched for fast-translated codons (having higher cognate tRNA concentrations), are depleted for slowly translated codons, and contain fewer proline codons. In addition, mRNA segments located 1-15 nucleotides downstream of Ssb-bound ribosomes have reduced mRNA secondary structure. Finally, nascent chain segments located in the ribosome tunnel of Ssb-bound ribosomes have average numbers of positively charged residues but are enriched in negatively charged residues. Taken together, these evolutionarily-encoded mRNA and nascent chain features cause faster translation during Ssb binding. This finding is significant as any alteration of translation kinetics due to synonymous mutations can potentially disrupt efficient binding of Ssb leading to possible misfolding of the protein without any change in its sequence.

Intersubunit Mobility of the Ribosome

Ribosomes are ribonucleoprotein nanoparticles synthesizing all proteins in living cells. The function of the ribosome is to translate the genetic information encoded in a nucleotide sequence of mRNA into the amino acid sequence of a protein. Each translation step (occurring after the codon-dependent binding of the aminoacyl-tRNA with the ribosome and mRNA) includes (i) the transpeptidation reaction and (ii) the translocation that unidirectionally drives the mRNA chain and mRNA-bound tRNA molecules through the ribosomal intersubunit space; the latter process is driven by the free energy of the chemical reaction of transpeptidation. Thus, the translating ribosome can be considered a conveying protein-synthesizing molecular machine. In this review we analyze the role of ribosomal intersubunit mobility in the process of translocation.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally, processes with the optimized media maintained an oxidizing intracellular environment, important for correct disulfide bond pairing, which likely contributed to reduced aggregate formation. These findings shed important understanding into how cells respond to process changes and can be useful to guide future development efforts to enhance productivity and improve product quality.