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The cavitation effect is an important geochemical phenomenon, which generally exists under strong hydrodynamic conditions. Therefore, developing an economical and effective sonocatalyst becomes a vital method in capitalizing on the cavitation effect for energy generation. In this study, we first report a novel Fe3O4 sonocatalyst that can be easily separated using a magnetic field and does not require any additional cocatalysts for H2 production from H2O. When subjected to ultrasonic vibration, this catalyst achieves an impressive H2 production rate of up to 175 μmol/h/USD (where USD stands for dollars), surpassing most previously reported mechanical catalytic materials. Furthermore, the ease and efficiency of separating this catalyst using an external magnetic field, coupled with its effortless recovery, highlight its significant potential for practical applications. By addressing the key limitations of conventional sonocatalysts, our study not only demonstrates the feasibility of using Fe3O4 as a highly efficient sonocatalyst but also showcases the exciting possibility of using a new class of magnetically separable sonocatalysts to productively transform mechanical energy into chemical energy. [ABSTRACT FROM AUTHOR]

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

BackgroundThe ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase H- RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs.ResultsOur findings on cellular RNA and in vitro synthesized RNA suggest that these products are likely the results of RNA self-priming to generate random cDNA products, which contributes to the loss of strand specificity. The use of RNase H+ RT enzyme and carrying the RT reaction at high temperature (50°C) greatly improved the strand specificity of the RT-PCR detection.ConclusionWhile RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when both sense and antisense RNA are expressed. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls.

The role of calcineurin (Cn) in skeletal muscle fiber-type expression has been a subject of great interest because of reports indicating that it controls the slow muscle phenotype. To delineate the role of Cn in phenotype remodeling, particularly its role in driving expression of the type I myosin heavy chain (MHC) gene, we used a novel strategy whereby a profound transition from fast to slow fiber type is induced and examined in the absence and presence of cyclosporin A (CsA), a Cn inhibitor. To induce the fast-to-slow transition, we first subjected rats to 7 days of hindlimb suspension (HS) + thyroid hormone [triiodothyronine ([T.sub.3])] to suppress nearly all expression of type I MHC mRNA in the soleus muscle. HS + [T.sub.3] was then withdrawn, and rats resumed normal ambulation and thyroid state, during which vehicle or CsA (30 mg x [kg.sup.-1] x [day.sup.-1]) was administered for 7 or 14 days. The findings demonstrate that, despite significant inhibition of Cn, pre-mRNA, mRNA, and protein abundance of type I MHC increased markedly during reloading relative to HS + [T.sub.3] (P < 0.05). Type I MHC expression was, however, attenuated by CsA compared with vehicle treatment. In addition, type IIa and IIx MHC pre-mRNA, mRNA, and relative protein levels were increased in Cn-treated compared with vehicle-treated rats. These findings indicate that Cn has a modulatory role in MHC transcription, rather than a role as a primary regulator of slow MHC gene expression. hindlimb suspension; cyclosporin A; antisense RNA; myosin heavy chain SDS-PAGE; thyroid hormone doi: 10.1152/ajpregu.00349.2009

This study investigated the dynamic regulation of IIx-IIb MHC genes in the fast white medial gastrocnemius (WMG) muscle in response to intermittent resistance exercise training (RE), a model associated with a rapid shift from IIb to IIx expression (11). We investigated the effect of 4 days of RE on the transcriptional activity across the skeletal MHC gene locus in the WMG in female Sprague-Dawley rats. Our results show that RE resulted in significant shifts from lib to IIx observed at both the pre-mRNA and mRNA levels. An antisense RNA (xII NAT) was detected in the intergenic (IG) region between IIx and IIb, extending across the entire IIx gene and into its promoter. The expression of the xII NAT was positively correlated with IIb pre-mRNA (R = +0.8), and negatively correlated with IIx pre-mRNA (R = -0.8). Transcription mapping of the IIx-IIb IG region revealed the generation of sense IIb and xII NATs from a single promoter region. This bidirectional promoter is highly conserved among species and contains several regulatory elements that may be implicated in its regulation. These results suggest that the IIx and the lib genes are physically and functionally linked via the bidirectional promoter. In order for the IIx MHC gene to be regulated, a feedback mechanism from the IG xII NAT is needed. In conclusion, the IG bidirectional promoter generating antisense RNA appears to be essential for the coordinated regulation of the skeletal muscle MHC genes during dynamic pheno-type shifts. gene transcription; pre-mRNA; natural antisense RNA; comparative genomics

Cardiac myosin heavy chain (MHC) gene expression undergoes a rapid transition from [beta]- to [alpha]-MHC during early rodent neonatal development (0-21 days of age). Thyroid hormone (3,5,3'-triiodothyronine, [T.sub.3]) is a major player in this developmental shift; however, the exact mechanism underlying this transition is poorly understood. The goal of this study was to conduct a more thorough analysis of transcriptional activity of the cardiac MHC gene locus during the early postnatal period in the rodent, in order to gain further insight on the regulation of cardiac MHC genes. We analyzed the expression of ct- and [beta]-MHC at protein, mRNA, and pre-mRNA levels at birth and 7, 10, 15, and 21 days after birth in euthyroid and hypothyroid rodents. Using novel technology, we also analyzed RNA expression across the cardiac gene locus, and we discovered that the intergenic (IG) region between the two cardiac genes possesses bidirectional transcriptional activity. This IG transcription results in an antisense RNA product as described previously, which is thought to exert an inhibitory effect on [beta]-MHC gene transcription. On the second half of the IG region, sense transcription occurs, resulting in expression of a sense IG RNA that merges with the [alpha]-MHC pre-mRNA. This sense IG RNA transcription was detected in the [alpha]-MHC gene promoter, approximately -1.8 kb relative to the [alpha]-MHC transcription start site. Both sense and antisense IG RNAs were developmentally regulated and responsive to a hypothyroid state (11, 14). This novel observation provides more complexity to the cooperative regulation of the two genes, suggesting the involvement of epigenetic processes in the regulation of cardiac MHC gene locus. gene transcription; antisense RNA; pre-mRNA; thyroid hormone; reverse transcriptase-polymerase chain reaction; bidirectional promoter; epigenetic regulation; Sprague-Dawley rats

In skeletal muscle of the adult mammal IIx is a pivotal myosin heavy chain (MHC) isoform that can be either up- or downregulated depending on both the fiber type of the target muscle and the type of external stimulus imposed. Since little is known about promoter elements of the IIx MHC gene that are important for its transcriptional regulation in vivo, the main goal of this study was to characterize IIx MHC promoter activity and identify potential regulatory elements on the IIx MHC promoter. A direct gene transfer approach was used, and this approach involved transfection of promoter-reporter constructs into intact rat soleus and plantaris muscle under control and denervated conditions, as well as hindlimb suspension (i.e., models to upregulate IIx MHC transcription). Fast-twitch (plantaris) muscle fibers were confirmed to have significantly greater IIx MHC transcriptional products (pre-mRNA and mRNA) than slow-twitch (soleus) muscle fibers. However, promoter sequences corresponding to -2671 to + 1720, -1000 to +392, and -605/+392 relative to the IIx MHC transcription start site, plus an additional construct ligated to a putative embryonic MHC enhancer, failed to produce a fiber type-specific response that is characteristic of the endogenous IIx MHC promoter. Furthermore, the activity of these promoter constructs did not demonstrate the expected response to denervation or hindlimb suspension (i.e., marked upregulation), despite normal uptake and activity of a coinjected [alpha]-actin reference promoter. On the basis of these findings with IIx MHC promoter-reporters we conclude that the loss of the native chromatin environment as well as other necessary cis elements may preclude use of the gene transfer approach, thereby suggesting that there are hidden layers of regulation for the IIx MHC gene. soleus; plantaris; reverse transcription-polymerase chain reaction: pre-messenger RNA; messenger RNA; skeletal muscle

Two genes encoding cardiac myosin heavy chain (MHC) isoforms, [beta] and [alpha], are arranged in tandem 4.5 kb apart. We examined pre-mRNA and mature mRNA levels of [beta] and ct genes in control, diabetic (streptozotocin), hypothyroid (propylthiouracil), and hyperthyroid rat hearts and analyzed the naturally occurring antisense (AS) [beta] RNA species that starts in the middle of the 4.5-kb intergenic region and extends upstream to the [beta]-gene promoter. The [beta] and [alpha] genes are expressed antithetically in control, diabetic, hypothyroid, and hyperthyroid hearts. Expression of AS [beta]-RNA was positively correlated with [alpha]-mRNA and negatively correlated with sense [beta] mRNA. These results support the novel idea of common promoter-regulatory elements situated in the intergenic region that likely control transcription of both sense [alpha] and AS [beta] genes and that AS [beta] transcription negatively regulates [beta]-MHC gene expression. To test whether an intergenic promoter drives transcription of AS [beta] RNA, a 1340-bp sequence of the intergenic region was inserted into a luciferase plasmid in the 3'-to-5' AS direction and was injected into rat ventricle. This promoter was activated in control heart and decreased greatly in response to propylthiouracil and streptozotocin and increased in hyperthyroid rats, similar in pattern to the endogenous AS [beta] RNA. When a putative retinoic acid receptor (RAR) site (a known thyroid hormone receptor cofactor) in this promoter was mutated, the reporter activity was almost abolished in control, propylthiouracil, and streptozotocin hearts. We conclude that there is an intergenic promoter that is active in the AS direction and that the putative RAR element is a vital regulatory site. mRNA; hyperthyroidism; in vivo gene injection; retinoic acid receptor; peroxisome proliferator-activated receptor doi: 10.1152/ajpheart.01224.2006.

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, [beta]-MHC isoform. Different length rat [beta]-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of -3,500, -914, -408, and -215 bp promoters increased in response to 1 wk of OL. The smallest -171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the -171 and -408 bp region were performed. The -408 bp promoters containing mutations of the [beta]e1, distal muscle CAT (MCAT; [beta]e2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT ([beta]e3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the [beta]e3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the [beta]e3 site functions as a putative OL-responsive element in the rat [beta]-MHC gene promoter. gel mobility shift assay; plantaris muscle; direct gene transfer; dual luciferase; [beta]-myosin heavy chain

Research was conducted to examine nuclear 3,,5,3'-triodothyronine (T3) binding capacity and the pattern of TR mRNA expression in the rodent heart and skeletal muscles of different fiber type as a function of the thyroid state. The possible existence of differential regulation of TR mRNA expression by T3 in the different types of striated muscle was also examined. Results indicate that divergent patterns of TR mRNA expression in various muscle types go through similar qualitative changes under altered thyroid state.

Abstract Background The ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase H- RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs. Results Our findings on cellular RNA and in vitro synthesized RNA suggest that these products are likely the results of RNA self-priming to generate random cDNA products, which contributes to the loss of strand specificity. The use of RNase H+ RT enzyme and carrying the RT reaction at high temperature (50°C) greatly improved the strand specificity of the RT-PCR detection. Conclusion While RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when both sense and antisense RNA are expressed. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls.

Giger, Julia M., Fadia Haddad, Anqi X. Qin, and Kenneth M. Baldwin. In vivo regulation of the [Beta]-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats. Am J Physiol Cell Physiol 278: C1153-C1161, 2000.--In the weight-bearing hindlimb soleus muscle of the rat, ~90% of muscle fibers express the [Beta]-myosin heavy chain ([Beta]-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from [Beta] toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the [Beta]-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length [Beta]-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest [Beta]-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced (~40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter. [Beta]-myosin heavy chain promoter; direct gene transfer; hindlimb suspension; dual luciferase

This study investigated the dynamic regulation of lIx-IIb MHC genes in the fast white medial gastrocnemius (WMG) muscle in response to intermittent resistance exercise training (RE), a model associated with a rapid shift from IIb to IIx expression (11). We investigated the effect of 4 days of RE on the transcriptional activity across the skeletal MHC gene locus in the WMG in female Sprague-Dawley rats. Our results show that RE resulted in significant shifts from lIb to lIx observed at both the pre-mRNA and mRNA levels. An antisense RNA (xlI NAT) was detected in the intergenic (10) region between lIx and IIb, extending across the entire lIx gene and into its promoter. The expression of the xlI NAT was positively correlated with lIb pre-mRNA (R = +0.8), and negatively correlated with lIx pre-mRNA (R = -0.8). Transcription mapping of the lIx-lIb 10 region revealed the generation of sense IIb and xlI NATs from a single promoter region. This bidirectional promoter is highly conserved among species and contains several regulatory elements that may be implicated in its regulation. These results suggest that the lIx and the IIb genes are physically and functionally linked via the bidirectional promoter. In order for the lIx MHC gene to be regulated, a feedback mechanism from the 10 xII NAT is needed. In conclusion, the IG bidirectional promoter generating antisense RNA appears to be essential for the coordinated regulation of the skeletal muscle MHC genes during dynamic phenotype shifts. [ABSTRACT FROM AUTHOR]

Slow-twitch soleus, a weight-bearing hindlimb muscle, predominantly expresses the type l myosin heavy chain (MHC) isoform. However, under unloading conditions, a transition in MHC expression occurs from slow type I toward the fast-type isoforms. Transcriptional processes are believed to be involved in this adaptation. To test the hypothesis that the downregulation of MHC1 in soleus muscle following unloading is controlled through cis element(s) in the proximal region of the promoter, the MHC1 promoter was injected into soleus muscles of control rats and those subjected to 7 days of hindlimb suspension. Mutation analyses of six putative regulatory elements within the -408-bp region demonstrated that three elements, an A/T-rich, the proximal muscle-type CAT (βe3), and an E-box (-63 bp), play an important role in the basal level of MHC1 gene activity in the control soleus and function as unloading- responsive elements. Gel mobility shift assays revealed a diminished level of complex formation of the βe3 and E-box probes with nuclear extract from hindlimb suspension soleus compared with control soleus. Supershift assays indicated that transcriptional enhancer factor 1 and myogenin factors bind the βe3 and E-box elements, respectively, in the control soleus. Western blots showed that the relative concentrations of the transcriptional enhancer factor 1 and myogenin factors were significantly attenuated in the unloaded soleus compared with the control muscle. We conclude that the downregulation of MHC1 in response to unloading is due, in part, to a significant decrease in the concentration of these transcription factors available for binding the positive regulatory elements. [ABSTRACT FROM AUTHOR]

Mechanical energy driven piezocatalytic hydrogen (H 2 ) production is a promising way to solve the energy crisis . But limited by the slow separation and transfer efficiency of piezoelectric charges generated on the surface of piezocatalysts , the piezocatalytic performance is still not satisfactory. Here, defect engineering is first used to optimize the piezocatalytic performance of microcrystalline cellulose (MCC). The piezocatalytic H 2 production rate of MCC with the optimal defect concentration can reach up to 84.47 µmol g -1 h -1 under ultrasonic vibration without any co-catalyst, which is ≈3.74 times higher than that of the pure MCC (22.65 µmol g -1 h -1 ). The enhanced H 2 production rate by piezoelectric catalysis is mainly due to the introduction of defect engineering on MCC, which disorders the symmetry of MCC crystal structure, improves the electrical conductivity of the material, and accelerates the separation and transfer efficiency of piezoelectric charges. Moreover, the piezocatalytic H 2 production rate of MCC with the optimal defect concentration can still reach up to 93.61 µmol g -1 h -1 in natural seawater, showingits commendable practicability. This study presents a novel view for designing marvelous-performance biomass piezocatalysts through defect engineering, which can efficiently convert mechanical energy into chemical energy ., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

The estimation of PM 2.5 -related mortality is becoming increasingly important. The accuracy of results is largely dependent on the selection of methods for PM 2.5 exposure assessment and Concentration-Response (C-R) function. In this study, PM 2.5 observed data from the China National Environmental Monitoring Center, satellite-derived estimation, widely collected geographic and socioeconomic information variables were applied to develop a national satellite-based Land Use Regression model and evaluate PM 2.5 exposure concentrations within 2013-2015 with the resolution of 1 km × 1 km. Population weighted concentration declined from 72.52 μg/m 3 in 2013 to 57.18 μg/m 3 in 2015. C-R function is another important section of health effect assessment, but most previous studies used the Integrated Exposure Regression (IER) function which may currently underestimate the excess relative risk of exceeding the exposure range in China. A new Shape Constrained Health Impact Function (SCHIF) method, which was developed from a national cohort of 189,793 Chinese men, was adopted to estimate the PM 2.5 -related premature deaths in China. Results showed that 2.19 million (2013), 1.94 million (2014), 1.65 million (2015) premature deaths were attributed to PM 2.5 long-term exposure, different from previous understanding around 1.1-1.7 million. The top three provinces of the highest premature deaths were Henan, Shandong, Sichuan, while the least ones were Tibet, Hainan, Qinghai. The proportions of premature deaths caused by specific diseases were 53.2% for stroke, 20.5% for ischemic heart disease, 16.8% for chronic obstructive pulmonary disease and 9.5% for lung cancer. IER function was also used to calculate PM 2.5 -related premature deaths with the same exposed level used in SCHIF method, and the comparison of results indicated that IER had made a much lower estimation with less annual amounts around 0.15-0.5 million premature deaths within 2013-2015., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.