Search

Background: Traditional cardiac rehabilitation (CR) improves cardiovascular outcomes and reduces mortality, but less is known about the relative benefit of intensive CR (ICR) which incorporates greater lifestyle education through 72 sessions (versus 36 in CR). Our objective was to determine whether ICR is associated with a mortality and cardiovascular benefit compared with CR., Methods: Retrospective cohort study of Medicare Fee-For-Service beneficiaries in a 100% sample, claims data set. Qualifying events were captured from May 1, 2016 to December 31, 2019 and ICR/CR utilization captured from May 1, 2016 to December 31, 2020. Among patients attending at least 1 day of either CR or ICR, Cox proportional hazards models using a 1 to 5 propensity score match were used to compare utilization and the association of ICR versus CR participation with (1) all-cause mortality and (2) cardiovascular-related hospitalizations or nonfatal cardiac events. Dose-response was assessed by the number of days attended., Results: From 2016 to 2019, 1 277 358 unique patients met at least one qualifying indication for ICR/CR from 2016 to 2019. Of these, 262 579 (20.6%) and 4452 (0.4%) attended at least one session of CR or ICR, respectively (mean [SD] age, 73.2 [7.8] years; 32.3% female). In the matched sample, including 26 659 total patients (median, 2.4-year follow-up), ICR was associated with 12% lower all-cause mortality (multivariable adjusted hazard ratio, 0.88 [95% CI, 0.78-0.99]; P =0.036) compared with CR but no significant difference for cardiovascular-related hospitalization or nonfatal cardiac events. The mortality benefit was seen for both ICR and CR per day strata, with each modality demonstrating a clear dose-response benefit., Conclusions: ICR is associated with lower mortality than traditional CR among Medicare beneficiaries but no difference in cardiovascular-related hospitalization or nonfatal cardiac events. Moreover, ICR and CR demonstrate a dose-response relationship for mortality. Additional studies are needed to confirm these observations and to better understand the mechanisms by which ICR may lead to a reduction in mortality., Competing Interests: Disclosures Dr Husaini received research support from Pritikin intensive cardiac rehabilitation (ICR), LLC through the Foundation for Barnes-Jewish Hospital and has received honoraria from Bristol Meyers Squibb. Dr Racette receives research support from the National Institutes of Health (NIH; R01 AG070717, R01 AG060499, R61 HL155858, R34 HL158947, R33 AG070455, R25 HL105400), and the Foundation for Barnes-Jewish Hospital. Dr Rich receives support from the NIH (R01 AG060499, R01 AG078153, R01 HL147862, R01 HL151431). Dr Joynt Maddox receives research support from the National Heart, Lung, and Blood Institute (R01HL143421 and R01HL164561), National Institute of Nursing research (U01NR020555), and National Institute on Aging (R01AG060935, R01AG063759, and R21AG065526), and from Humana. She also serves on the Health Policy Advisory Council for the Centene Corporation (St Louis, MO). Dr Peterson has stock holdings in Medtronic, Johnson and Johnson and receives research support from the NIH (R61/R33 HL155858, R01 AG060499-01, R01 HL 165238), the American Heart Association (No. 23SCISA1145192), the Children’s Discovery Institute, the Clinical and Translational Research Funding Program (CTRFP), and the Foundation for Barnes-Jewish Hospital in Saint Louis, MO. The other authors report no conflicts.

Tumors are comprised of a multitude of cell types spanning different microenvironments. Mass spectrometry imaging (MSI) has the potential to identify metabolic patterns within the tumor ecosystem and surrounding tissues, but conventional workflows have not yet fully integrated the breadth of experimental techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a spatial variant of Isotopologue Spectral Analysis to map distributions of metabolite abundances, nutrient contributions, and metabolic turnover fluxes across the brains of mice harboring GL261 glioma, a widely used model for glioblastoma. When integrated with MSI, the combination of ion mobility, desorption electrospray ionization, and matrix assisted laser desorption ionization reveals alterations in multiple anabolic pathways. De novo fatty acid synthesis flux is increased by approximately 3-fold in glioma relative to surrounding healthy tissue. Fatty acid elongation flux is elevated even higher at 8-fold relative to surrounding healthy tissue and highlights the importance of elongase activity in glioma., (© 2023. The Author(s).)

There exists a dearth of supplementary programs to educate physician-scientist trainees on anti-racism and topics surrounding social justice in medicine and science. Education on these topics is critical to prevent the perpetuation of systemic racism within the institutions of academia and medicine. Students in the Washington University School of Medicine Medical Scientist Training Program and the Tri-Institutional MD-PhD Program developed journal clubs with curricula focused on social justice and anti-racism for the summer of 2020. In this article, we describe the impact of the Washington University journal club on the education of first year MD-PhD students and summarize the progress to date. The role of the journal club in the midst of the "double pandemic" of COVID-19 and generational systemic racism is discussed, highlighting the need for such supplemental curricula in MD-PhD programs nation-wide., (© 2021 The Authors. FASEB BioAdvances published by the Federation of American Societies for Experimental Biology.)

Differentiation of BC3H1 myoblasts to myocytes is accompanied by a 67% drop in the rate of rpL32 gene transcription. Addition of high concentrations of serum to resting myocyte populations stimulates cell growth and subsequent dedifferentiation to proliferating myoblasts with a return to the normal rate of rpL32 gene transcription. During these growth rate changes the binding activities of previously identified factors (beta, gamma, delta) which interact with the rpL32 gene promoter were examined by mobility shift assays. Binding of the beta factor (an Ets related protein) to an oligonucleotide containing the beta element was reduced significantly in myocyte nuclear extracts, but subsequent dedifferentiation increased binding within 30 min in either the presence or absence of the cycloheximide. Binding of the gamma and delta factors to their respective elements changed only slightly during these processes. Dephosphorylation of either myoblast or myocyte extracts resulted in increased binding of the beta factor suggesting that binding activity of the beta factor is modulated by phosphorylation during the changes in BC3H1 myoblasts growth rate. In addition, mobility shift assays with recombinant GABP alpha and beta proteins and their specific antibodies revealed that GABP proteins bind to the rpL32 gene promoter in a sequence dependent manner, and that similar proteins are present in BC3H1 myoblast/myocyte extracts. These results support the premise that the GABP heterodimer is the rpL32 beta factor. Furthermore, during BC3H1 myoblast differentiation and dedifferentiation neither the levels of the GABP alpha and beta proteins nor their respective mRNAs change. These results suggest that GABP is a constitutively expressed protein and is involved in regulating rpL32 gene by post-transcriptional modifications.

To extend our understanding of the mechanisms regulating ribosome biosynthesis during changes in cellular growth rate, the expression and subcellular distribution of U3 snRNA and one of its associated proteins, fibrillarin, were examined in mouse 3T6 fibroblasts. Altering serum concentrations produces changes in the ribosome content of the cell as reflected by total RNA levels. When exponentially growing 3T6 cells are induced to become quiescent by serum starvation, a significant downshift in U3 snRNA gene transcription occurs in parallel to a decrease in pre-rRNA synthesis. Serum stimulation results in an increase in the rate of synthesis of both U3 snRNA and pre-rRNA. However, U3 snRNA synthesis lags behind that of pre-rRNA. Furthermore, in serum-starved fibroblasts, a significant portion of the total cellular U3 snRNA appears in the cytoplasm. Following serum stimulation, a redistribution occurs and U3 snRNA is localized predominantly in the nucleolus at a level similar to that observed in exponentially growing cells. This redistribution is inhibited when RNA or protein synthesis is repressed in serum-stimulated fibroblasts by actinomycin D or cycloheximide. In contrast, the level and subcellular distribution of fibrillarin remain unchanged during serum starvation. These results suggest that during changes in ribosome production, distinct pools of U3 snRNPs exist within the cell.

The ribosomal protein L32 (rpL32) gene transcribed by RNA polymerase II lacks a canonical TATA element, that binds the transcription factor TFIID tau or TBP (TATA binding protein). Instead this promoter contains an element, termed gamma, located at -30 relative to the transcription initiation site. We previously reported that, despite the lack of a canonical TATA element the rpL32 gene utilizes yeast TFIID tau for its transcriptional initiation. Whether TFIID tau participates in rpL32 gene transcription by binding directly to a promoter element or through another protein has not been resolved. These studies reveal that proteins ranging in size from 20-40 kDa binds to the gamma-element. The 40 kDa protein(s) displays strong affinity for the canonical TATA element and may be related or equivalent to TFIID tau. Furthermore, cloned and purified yeast TFIID (TBP) binds directly to the gamma-element implying that the gamma-element directs RNA polymerase II-dependent transcription of the rpL32 gene.

The beta factor, which interacts with the rpL32 promoter, binds to the sequence 5'-GAGCCGGAAGTG and trans-activates this gene. Comparison of the DNA sequences bound by the beta factor with those bound by other known DNA-binding proteins revealed that the ETS proteins interact with similar DNA sequences. Consequently we have examined the relationship of the beta factor to the several ETS proteins so far reported. Antibody and oligonucleotide competition experiments, performed by using electrophoretic shift analysis, revealed that the beta factor contains ETS epitopes and that it is immunologically related to both of the GA-binding proteins (GABPs), implying that the beta factor may consist of two separate protein subunits.

Translational efficiency of a minor group of mRNAs is regulated by serum levels in 3T6 fibroblasts. Included within this group is the poly(A)-binding protein (PABP) mRNA. We analyzed the distribution of PABP mRNA in polysome profiles and found a large percentage of this mRNA to be translationally repressed in both actively growing (approximately 60%) and resting cells (approximately 70%). Elevated serum levels induced a distinct bimodal distribution of this mRNA between actively translated and repressed fractions. Similarly, treatment of cells with low doses of cycloheximide also generated a partial shift of repressed PABP mRNA into the actively translated fraction. In an attempt to characterize the factors which regulate PABP mRNA translation we have identified the proteins which bind to this mRNA in vitro. Sequences within the 5' untranslated region were found to be sufficient for binding of all proteins to this mRNA. We suggest that this region and the proteins associated with it may be essential for translation control of PABP mRNA.

Differentiation of proliferating rat L6 myoblasts to syncytial multinucleated myotubes results in a significant downshift in the rate of U3 snRNA gene transcription, paralleling the decrease in rRNA synthesis previously documented. Coordinate production of U3 snRNA and rRNA during the differentiation process adds further support for a role of U3 snRNA in ribosome biogenesis. Despite the dramatic decrease in U3 snRNA transcription during differentiation, a corresponding drop in the cellular level of U3 snRNA does not occur. In myotubes, the amount of U3 snRNA is regulated at the post-transcriptional level in which there is a significant accumulation of U3 snRNA in the cytoplasm of myotubes. This intracellular redistribution of U3 snRNA may significantly affect the entire process of rRNA maturation or result from the decrease in ribosome production accompanying terminal differentiation of myoblasts.

The murine ribosomal protein (rp) L32 gene contains essential promoter sequences located both upstream and downstream of the cap site. A combination of gel mobility shift, UV cross-linking, and cell-free transcription assays were used to analyze the interaction of factors binding to a downstream element (located at position +25 to +37). The rpL32 downstream element identified polypeptides (transcription factors) ranging in size from 45 to 25 kilodaltons (kDa). Four base pair changes in the wild-type sequence of the downstream element eliminated binding. An oligonucleotide containing the glucocorticoid responsive element sequence competed specifically for the 45-kDa protein in both the gel mobility shift assay and in the UV cross-linking studies. Our data also indicate that the downstream binding factors contribute to cell-free transcription of the rpL32 gene.

The mouse-ribosomal-protein-L32-gene promoter contains a 12-bp sequence motif within the 5'-upstream region termed the beta element which shows significant similarity with the consensus sequence of the polyoma-virus-enhancer PEA3. A cloned PEA3 DNA-binding protein, expressed in Escherichia coli and purified, activates the expression of the ribosomal-protein-L32 gene in a cell-free system. Moreover, the PEA3 protein participates in the formation of the ribosomal-protein-L32-promoter-preinitiation-transcription complex. The preinitiation complex formed with PEA3 is resistant to competition by oligonucleotides containing the beta element. In addition anti-PEA3 serum interacts with a factor in mouse L1210 nuclear extract that binds to the beta element, causing a supershift in a mobility-shift assay. Our study demonstrates for the first time that the PEA3 protein can transactivate a cellular gene in a cell-free transcription system.

Following serum stimulation of quiescent 3T6 cells, an elevated in vivo rate of translation was observed. These studies were designed to identify the proteins associated with polysomal mRNA under different growth conditions in an attempt to establish a relationship between translational rate and the mRNA-associated proteins. Ultraviolet cross-linking of proteins to mRNA was employed to ensure that only genuine mRNA-associated proteins were investigated. Our results revealed little change in the population of mRNA-binding proteins, although minor variations in the synthesis of several proteins, most notably a 32 kilodalton species, were observed during growth transitions. These investigations demonstrate further that most of the mRNA-binding proteins were phosphorylated with the degree of phosphorylation of several proteins influenced by growth conditions.

The capacity for 3' processing of the histone H4 pre-mRNA is lost following differentiation of rat L6 myoblasts to myotubes. Nuclear extracts prepared from proliferating myoblasts, but not differentiated myotubes, actively process histone H4 pre-mRNA in vitro. The activity of two factors required for 3' processing, the heat-labile factor and U7 snRNP, also changes during the differentiation period, concurrent with the loss of 3' processing activity. During myotube formation, the activity of the heat-labile factor decreases significantly while the 5' sequences of the U7 snRNA become progressively resistant to micrococcal nuclease digestion. Thus, the dramatic down-shift in histone H4 mRNA levels which occurs during myoblast differentiation is controlled at both the transcriptional and posttranscriptional level.

Synthesis of poly(A)-binding protein is regulated at the translational level. We have investigated the binding of proteins to this mRNA on the premise that the protein(s) of the mRNP complex may be involved in regulating the expression of the mRNA. We found the first 243 nucleotides of the 5' untranslated region to contain sequences essential for RNP formation. A large, single-stranded bulge structure encompassing stretches rich in adenine nucleotides and a potential stem-loop domain appear to be the primary sites for protein binding. Removal of the 243-nucleotide segment results in a drastic reduction in protein binding and a concomitant increase in translational efficiency in vitro. We suggest that proteins binding to this region, including poly(A)-binding protein itself, may be essential for regulating translation of this mRNA.

Studies have been initiated to identify the protein component(s) which interact with the beta regulatory region of the mouse ribosomal protein L32 gene promoter. By the combined use of the mobility shift assay and UV cross-linking, a factor specific for the upstream transcriptional control sequence of the beta region of the ribosomal protein L32 promoter has been detected in mouse L1210 nuclear extracts. A mutation (GT----TC at -71 to -70) in this sequence eliminates the binding. Beta factor is identified as a 55 kDa polypeptide by UV cross-linking. Addition of excess beta element (double-stranded oligonucleotide) to a cell-free transcription system reduces transcription of the ribosomal protein L32 gene. Our results provide evidence that the interaction between the beta element and the beta factor is involved in ribosomal protein L32 transcription.

The rate of ribosome biogenesis is closely coupled with cell proliferation, representing a unique model system for studying gene regulation. Terminal differentiation of rat L6 myoblasts, an example of a rapidly proliferating population of cells being converted into a non-dividing syncytial population, results in an 80% decline in the rate of ribosome accumulation. Ribosome production during myogenesis is regulated by a down-shift in the rate of rRNA accumulation, controlled at the level of transcription by specific trans-acting factors. The synthesis of both ribosomal proteins and 5S rRNA remains unchanged in myotubes, however, resulting in an over-production of these precursors. The excess molecules are rapidly degraded, preventing the accumulation of a static pool of ribosome components.

The presence of specific promoter elements, notably the TATA and GC boxes, has been useful for categorizing genes transcribed by RNA polymerase II. The gene for the murine ribosomal protein (r-protein) L32 lacks both of these elements, although it has GC-rich regions. The conditions required for its optimal synthesis in vitro, however, resemble the properties of promoters containing TATA (adenovirus major late promoter) rather than GC boxes (dihydrofolate reductase). To further investigate the relationship of the r-protein gene to different promoter elements, transcription competition analyses were used to distinguish the presence of common protein-binding sequences. The low levels of competition observed by either the adenovirus major late promoter or dihydrofolate reductase promoter with the r-protein gene promoter resulted from general transcription factors present in each initiation complex. Competition by factors binding to common sequence elements was not observed, indicating the r-protein L32 gene possesses elements distinct from those present in the other genes examined.

These studies were designed to identify the proteins associated with specific mRNAs. L6 myoblasts contain a unique poly(A)-rich H4 mRNA as well as poly(A)-minus H4 mRNA subspecies. We have characterized the proteins present in both poly(A)-rich and poly(A)-minus histone H4 mRNP complexes following ultraviolet cross-linking in vivo. In addition, the muscle-specific myosin heavy chain (MHC) mRNP complex was characterized in myoblasts. [35S]Methionine-labelled poly(A)-rich and poly(A)-minus RNP complexes were prepared from both the polysomal and free (post-polysomal) RNP compartments. From each fraction the mRNP encoding histone H4 or MHC was purified by hybrid selection to a cloned human histone H4 gene or MHC cDNA. A unique set of 6-16 proteins was found bound to each of the specific mRNP complexes. These proteins were a subset of the total population of either polysomal or free RNP proteins and some proteins appeared common among the different hybrid-selected RNP fractions. The results demonstrate that (a) mRNAs bind a different set of proteins depending upon whether they are present in the polysomal or free mRNP fraction; (b) the presence of poly(A) sequences affects the proteins which bind to H4 mRNA in the free RNP compartment.

In this article, we discuss our attempts to establish the existence in the cytoplasm of regulatory molecules involved in translational control. Our studies have revealed the presence of cAMP independent protein kinase in the free mRNP complex capable of phosphorylating a Mr = 38 000 polypeptide, also part of the same complex. Both the kinase and the acceptor protein were found also as free proteins in the cytoplasmic pool. This kinase has been shown to be distinct from the heme regulated enzyme that phosphorylates the small subunit of eIF-2. Other regulatory molecules include small molecular weight RNAs found as part of an RNP complex. A 4S fraction isolated from this complex inhibited the translation of both capped and uncapped mRNAs in a cell-free protein synthesizing system. The biological role of the protein kinase and the 4S RNA fraction is considered.

Studies were undertaken to determine the component(s) responsible for the temperature optimum characteristic of the protein-synthesizing system from skate and rat. 1. The macromolecular constituents of rat and skate liver ribosomes are compared. The number of ribosomal proteins is similar in the two species, although most proteins display different electrophoretic mobilities on polyacrylamide gels. The RNAs from the small subunit of skate and rat have similar sedimentation coefficients; however, the RNA from the large subunit of skate ribosomes appeared to be slight smaller than the comparable RNA from the rat. 2. Ribosomes from either rat or skate were capable of supporting poly(U)dependent polyphenylalanine synthesis with soluble factors from either species. 3. Maximal leucine incorporation directed by endogenous mRNA occurred at 35--40 degrees C with post-mitochondrial supernatant from the rat liver and at 20--30 degrees C with that from skate liver. 4. The characteristic temperature sensitivity of protein synthesis was dependent upon the source of cell sap and independent of the source of ribosomes. 5. Elongation factor 1 from both the rat and skate exhibited maximum activity at approx. 30 degrees C. 6. Phenylalanyl-tRNA synthetase from skate liver showed maximum activity at 30 degrees C while that from rat was maximally active at 37 degrees C. The rat enzyme, however, was active at 0--10 degrees C, at which temperature protein synthesis in the reconstructed rat system is virtually absent. 7. The protein-synthesizing capacity of the reconstituted system at various temperatures was closely correlated with the activity of Elongation factor 2 (translocase). Elongation factor 2 from rat liver displayed an optimum at 30 degrees C and lost all activity below 10 degrees C, while this same factor from skate liver showed an optimum at 20 degrees C and significant activity below 10 degrees C. At this low temperature the reconstituted skate liver system continued to exhibit the ability to synthesize protein. These studies suggest that Elongation factor 2 is the component responsible for determining the temperature at which the protein-synthesizing system displays its characteristic maximum activity.

Lactoperoxidase-catalyzed iodination was employed as a probe to monitor conformational change in 40-S ribosomal subunits from rat liver. Using this probe, it was observed that phosphorylation of protein S6 resulted in no detectable change in the iodination pattern of 40-S subunit proteins. These results suggest that the conformation of the small subunit remains unaltered following phosphorylation. On the other hand, the differences noted in the iodination pattern between 40-S ribosomal proteins derived from isolated subunits and those from 80-S monosomes, suggest that the 40-S subunit undergoes a conformational change during association with the 60-S subunit. Following 40-S and 60-S subunit association, proteins S2, S3, S5, S6, S8, S10 and S14 became less accessible to iodination. It is suggested that these proteins may be located at the interface between the 40-S and 60-S subunits.

The regulation of ribosomal proteins (r-proteins) and their mRNAs (rp-mRNAs) was studied in the L6 myoblast, a mammalian cell line which can undergo myogenesis. Upon terminal differentiation, the rate of accumulation of mature ribosomes dropped to approximately 25% of the rate found in undifferentiated myoblasts. Despite the drop in the rate of ribosome accumulation and the rate of rRNA synthesis following terminal differentiation, the rate of r-protein synthesis remained constant. The excess r-protein synthesized in myotubes was quickly degraded. The levels of rp-mRNAs were assessed before and after differentiation. Over 90% of the rp-mRNAs were found on polysomes in both myoblasts and myotubes and represented similar fractions of total poly(A)-rich mRNA. The half-lives of the rp-mRNAs averaged approximately 11 h in both myoblasts and myotubes. In vitro nuclear transcription measurements of a representative rp-mRNA (L32 mRNA) revealed that following differentiation, its rate of synthesis relative to the overall transcription rate dropped by approximately 26% in myotubes while the rate of transcription of rRNA dropped by approximately 77%. These results indicate that the coordination of r-protein and rRNA synthesis observed in myoblasts was uncoupled in myotubes at the level of transcription.