Mammals have four tropomyosin (TPM) genes (TPM1, TPM2, TPM3, and TPM4) that produce various isoforms through alternative splicing. TPM1 generates both myofibrillar (Tpm1.1, Tpm1.2) and non-myofibrillar isoforms, including those with an exon 9d peptide or with an exon 9a peptide. These non-myofibrillar isoforms are involved in regulating actin-based structures and processes in non-muscle cells. This study focused on the expression of the non-myofibrillar HMW Tpm1 isoforms (Tpm1.3, Tpm1.4, Tpm1.5, Tpm1.6, Tpm1.7, and Tpm1.14) during human inducible pluripotent stem cells (hiPSC) differentiation into cardiomyocytes (CMs) at different time points (Days 0, 5, 10, 15, 20). We have determined the expression of various Tpm1 transcripts by qRT-PCR using isoform-specific primer-pairs. Western blotting with Tpm1-exon 6a and Tpm1-exon 9d antibodies and 2D Western blotting with Tpm1-exon 6a antibody followed by mass spectra analyses were used to evaluate protein expression. Transcripts of non-myofibrillar Tpm1 isoforms peaked at Day 15 and continued at a slightly lower level in mature hiPSC-CM until Day 20. However, no expression of Tpm1.3 or Tpm1.14 has been observed. Protein expression of Tpm1.4, Tpm1.5, Tpm1.6, and Tpm1.7 increases up to Day 15 but practically disappears by Day 20 hiPSC-CM, suggesting their production is decreased, or more likely they are degraded intracellularly. The results suggest that the proteins have the potential to be transiently involved in the early stages of CM differentiation. This study also demonstrates that Tpm1.5, when fused with YFP in an expression construct and transfected into embryonic chicken CM and Day 20 mature hiPSC-CMs, can be organized into cardiac myofibrils despite being previously characterized as a non-muscle isoform. Our observation is further substantiated by the fact that YFP-Tpm1.5 fusion protein can be ectopically expressed and incorporated into the myofibrils of chicken myotube skeletal muscle, which is known to be more stringent than cardiac muscle with regards to myofibril remodeling. Paradoxically, anti-6a antibody fails to recognize the organized YFP-Tpm1.5 fusion protein in embryonic chicken CMs, embryonic chicken skeletal muscle myotubes, or in Day 20 mature hiPSC-CMs. Interestingly, the antibody recognizes the denatured YFP-Tpm1 fusion protein in Western blot analyses. It is well-documented in the literature that an antibody epitope may fail to recognize its antigen when the antigen is in its native state in living cells, often because the epitope is buried, altered conformationally, or inaccessible. Conversely, while the Tpm1.Ex6a antibody stains Tpm1.5 in cell nuclei, the YFP-Tpm1.5 fusion protein does not localize to the cell nuclei. This suggests conformational differences between nuclear and cytoplasmic Tpm1 protein(s) and differential access of the fusion protein to different cellular locations. This also suggests that it is endogenous Tpm1 6a-containing protein identified in the nuclei.

Introduction: SMARCA4/SMARCA2 deficiencies have been linked to a variety of neoplasms and have been mainly described in cases of thoracic non-small cell lung cancers. Case Presentation: Here we present a case of SMARCA4/SMARCA2-deficient undifferentiated metastatic malignant tumor with carcinoma of unknown primary. The staining on pathology as well as the unremarkable size of the lung masses raises the possibility of dedifferentiated tumor of unknown primary. Conclusion: This case highlights the need for more routine testing of SMARCA4/SMARCA2 deficiencies as well as the development of more targeted therapies as these mutations are often associated with an aggressive course and poor outcomes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1μ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and μ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1μ non-muscle isoform protein expression before and during cardiac differentiation.

Old world monkeys separated from the great apes, including the ancestor of humans, about 25 million years ago, but most of the genes in humans and various nonhuman primates are quite similar even though their anatomical appearances are quite different. Like other mammals, primates have four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing. Only TPM1 produces two sarcomeric isoforms (TPM1α and TPM1κ), and TPM2, TPM3, and TPM4 each generate one sarcomeric isoform. We have cloned and sequenced TPM1α, TPM1κ, TPM2α, TPM3α, and TPM4α with RNA from cynomolgus (Cyn) monkey hearts and skeletal muscle. We believe this is the first report of directly cloning and sequencing of these monkey transcripts. In the Cyn monkey heart, the rank order of TPM isoform expression is TPM1α > TPM2α > TPM1κ > TPM3α > TPM4α. In the Cyn monkey skeletal muscle, the rank order of expression is TPM1α > TPM2α > TPM3α > TPM1κ > TPM4α. The major differences in the human heart are the increased expression of TPM1κ, although TPM1α is still the dominant transcript. In the Cyn monkey heart, the only sarcomeric TPM isoform at the protein level is TPM1α. This is in contrast to human hearts where TPM1α is the major sarcomeric isoform but a lower quantity of TPM1κ, TPM2α, and TPM3α is also detected at the protein level. These differences of tropomyosin and/or other cardiac protein expression in human and Cyn monkey hearts may reflect the differences in physiological activities in daily life.

Details of sarcomeric protein assembly during de novo myofibril formation closely resemble myofibrillogenesis in skeletal and cardiac myocytes in birds, rodents, and zebrafish. The arrangement of proteins during myofibrillogenesis follows a three-step process: beginning with premyofibrils, followed by nascent myofibrils, and concluding with mature myofibrils (reviewed in Sanger et al., 2017). Assembly and maintenance of myofibrils in living muscle cells. In: Handbook of experimental pharmacology, 2017 [pp. 39-75]. Our aim is to determine if the same pathway is followed in human cardiomyocytes derived from human inducible pluripotent stem cells. We found that the human cardiomyocytes developed patterns of protein organization identical to the three-step series seen in the model organisms cited above. Further experiments showed that myofibril assembly can be blocked at the nascent myofibril by five different inhibitors of the Ubiquitin Proteasome System (UPS) stage in both avian and human cardiomyocytes. With the exception of Carfilzomib, removal of the UPS inhibitors allows nascent myofibrils to proceed to mature myofibrils. Some proteasomal inhibitors, such as Bortezomib and Carfilzomib, used to treat multiple myeloma patients, have off-target effects of damage to hearts in three to 6 % of these patients. These cardiovascular adverse events may result from prevention of mature myofibril formation in the cardiomyocytes. In summary, our results support a common three-step model for the formation of myofibrils ranging from avian to human cardiomyocytes. The Ubiquitin Proteasome System is required for progression from nascent myofibrils to mature myofibrils. Our experiments suggest a possible explanation for the cardiac and skeletal muscle off-target effects reported in multiple myeloma patients treated with proteasome inhibitors.

In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.

We describe a 64-year-old Caucasian female with a history of Raynaud's disease, hand arthritis, photosensitivity, Sjogren's syndrome and leukocytoclastic vasculitis who presented with progressively worsening fingertip necrosis that began three days after receiving a first dose of Pfizer–BioNTech COVID-19 RNA vaccine. Our workup revealed cryoglobulinemia, hypocomplementemia, elevated antinuclear antibodies (ANA) and IgM antiphospholipid autoantibodies (aPL) directed against phosphatidylserine (aPL-PS), suggesting a diagnosis of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). The patient failed to develop anti-spike IgG antibodies up to two months following vaccination. Disease progression was halted by plasmapheresis, anticoagulation, and immune suppression. We conclude that the vaccine RNA moiety may induce SLE manifesting in APS, cryoglobulinemia, hypocomplementemia, and digital necrosis.

Tumor lysis syndrome (TLS) is the most common hematologic emergency encountered during the treatment of high-grade malignancies. While it can lead to death, the prognosis is typically excellent if caught early on in the course. Risk stratification prior to treatment initiation is paramount in deciding the utility of prophylaxis and ultimately in reducing morbidity and mortality. The following case describes the development of TLS in a patient categorized as low risk and highlights the need for further elucidation of a unified risk stratification system.

In mammals, there are four tropomyosin (TPM) genes (TPM1, TPM2, TPM3, and TPM4) each of which generate a multitude of alternatively spliced mRNAs. TPM isoform diversity in bovine unlike in humans are not well characterized. The purpose of this investigation is to perform an extensive analysis of the expression of both transcripts and corresponding protein of sarcomeric TPMs in bovine strated muscles. We have cloned and sequenced the transcripts of the sarcomeric isoform of the TPM4 gene designated as TPM4α as well as a new splice variant TPM4ε from bovine striated muscles. Additionally, we have determined the expression of various sarcomeric TPM isoforms and TPM4ε in bovine heart and skeletal muscles. Relative expression as well as absolute copy number determination by qRT-PCR suggests that TPM1α expression is significantly higher in bovine cardiac muscle, whereas TPM2α is higher in skeletal muscle. The relative expression of TPM3α in bovine heart and skeletal muscle is very similar. The relative expression of TPM4α and TPM4ε is higher in bovine heart and skeletal muscle, respectively. We have evaluated the protein expression levels of various TPM isoforms by 2D western blot analyses in commercially available protein extracts of heart and skeletal muscles with the CH1 monoclonal antibody against TPM. Protein from each CH1-positive spot was extracted for LC-MS/MS analyses, which show that bovine heart extract contains 91.66% TPM1 and 8.33% TPM2, whereas skeletal muscle extract contains 57% TPM1 and 42.87% TPM2. We have failed to detect the presence of unique peptide(s) for TPM3α, TPM4α, and TPM4ε.

In mammals, four tropomyosin genes TPM1 , TPM2 , TPM3 , and TPM4 are known. One isoform of the TPM3 gene, encoding 285 amino acid residues designated as TPM3α, has been reported. TPM3α protein expression in human hearts is not definitively established. We have cloned from human heart and skeletal muscle transcripts of TPM3α and three novel TPM3 isoforms, TPM3ν, TPM3ξ, and TPM3ο. TPM3ν and TPM3ο are alternatively spliced RNAs with different 3′‐UTRs encoding an identical novel protein with 285 amino acid differing from TPM3α and TPM3ξ in exon 6 only. TPM3α and TPM3ξ, which have different 3′UTRs, also encode an identical protein. qRT‐PCR data show that the transcripts of TPM3α, TPM3ν, TPM3ξ, and TPM3ο are expressed in both heart and skeletal muscle. We have evaluated the expression of various TPM proteins in fetal and adult human hearts, and also in skeletal muscle samples. Western blots using CG3 antibody show a stronger signal of TPM3 protein in fetal heart and adult skeletal muscle compared to adult heart. LC–MS/MS studies with the protein spots separated and identified by CH1 antibody after 2D Western blot analyses, confirm the expression of TPM3α/TPM3ξ in heart, but some peptides detected could be either TPM3α or TPM3ν. In heart samples, TPM1 protein was the dominant with varying amount of TPM2 and TPM3, while TPM4 expression was not observed. In skeletal muscles, TPM2 was the majority TPM protein expressed. The biological consequences of these varying expression of individual tropomyosin proteins are yet to be established.

T-cell large granular lymphocyte (T-LGL) leukaemia is characterized by a clonal proliferation of cytotoxic T cells and is frequently associated with rheumatoid arthritis. Sera from some LGL leukaemia patients react to a portion of the human T-cell leukaemia virus (HTLV-1/2) transmembrane envelope protein, BA21, although HTLV-1/2 infection is rare in LGL leukaemia patients. Here we show that family members, including spouses, of an LGL leukaemia patient had elevated LGL counts, BA21 reactivity and, additionally, recognition of HIV-1 gp41. Thus, both LGL leukaemia patients and clinically normal contacts sharing the same environment have evidence of exposure to a retrovirus.

The chicken has been used since the 1980s as an animal model for developmental studies regarding tropomyosin isoform diversity in striated muscles, however, the pattern of expression of transcripts as well as the corresponding TPM proteins of various tropomyosin isoforms in avian hearts are not well documented. In this study, using conventional and qRT-PCR, we report the expression of transcripts for various sarcomeric TPM isoforms in striated muscles through development. Transcripts of both TPM1α and TPM1κ, the two sarcomeric isoforms of the TPM1 gene, are expressed in embryonic chicken hearts but disappear in post hatch stages. TPM1α transcripts are expressed in embryonic and adult skeletal muscle. The sarcomeric isoform of the TPM2 gene is expressed mostly in embryonic skeletal muscles. As reported earlier, TPM3α is expressed in embryonic heart and skeletal muscle but significantly lower in adult striated muscle. TPM4α transcripts are expressed from embryonic to adult chicken hearts but not in skeletal muscle. Our 2D Western blot analyses using CH1 monoclonal antibody followed by mass spectra evaluations found TPM4α protein is the major sarcomeric tropomysin expressed in embryonic chicken hearts. However, in 7-day-old embryonic hearts, a minute quantity of TPM1α or TPM1κ is also expressed. This finding suggests that sarcomeric TPM1 protein may play some important role in cardiac contractility and/or cardiac morphogenesis during embryogenesis. Since only the transcripts of TPM4α are expressed in adult chicken hearts, it is logical to presume that TPM4α is the only sarcomeric TPM protein produced in adult cardiac tissues.

Tropomyosins, a family of actin-binding, coiled-coil dimeric proteins, are found in all eukaryotic organisms from yeast to man. Tropomyosin (TPM) is best known for its role in muscle contraction. It is a component of thin filaments in muscle cells and also a component of microfilaments in non-muscle cells The protein was discovered in the 1940s as a component of the actin filaments of striated muscle [1]. To date, there are more than 40 TPM isoforms known in humans as well asinother vertebrates. These isoforms are encoded in vertebrates by four TPM genes (TPM1, TPM2, TPM3, and TPM4) [2-4] except infish where six TPM genes are present [5]. The TPM1 gene was previously known to produce nine isoforms only one of which, TPM1α is specific for striated muscle (i.e., termed asarcomeric isoform). In 2004 we reported a second sarcomeric isoform called TPM1κ (or Tpm 1.2) (Figure1) [6,7]. Since then, we have reported 4 more high molecular weight isoforms from humans, which are not sarcomeric isoforms (Figure 1 as shown in the box).

In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin.Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles.In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters.Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited.Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle.We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively.By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart.On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart.Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart.Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle.Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle.To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.

Tropomyosin is a component of thin filaments that constitute myofibrils, the contractile apparatus of striated muscles. In vertebrates, except for fish, four TPM genes TPM1, TPM2, TPM3, and TPM4 are known. In zebrafish, there are six TPM genes that include the paralogs of the TPM1 (TPM1‐1 and TPM1‐2), the paralogs of the TPM4 gene (TPM4‐1 and TPM4‐2), and the two single copy genes TPM2 and TPM3. In this study, we have identified, cloned, and sequenced the TPM1‐1κ isoform of the TPM1‐1 gene and also discovered a new isoform TPM1‐2ν of the TPM1‐2. Further, we have cloned and sequenced the sarcomeric isoform of the TPM4‐2 gene designated as TPM4‐2α. Using conventional RT‐PCR, we have shown the expression of the sarcomeric isoforms of TPM1‐1, TPM1‐2, TPM2, TPM3, TPM4‐1, and TPM4‐2 in heart and skeletal muscles. By qRT‐PCR using both relative expression as well as the absolute copy number, we have shown that TPM1‐1α, TPM1‐2α, and TPM1‐2ν are expressed mostly in skeletal muscle; the level of expression of TPM1‐1κ is significantly lower compared to TPM1‐1α in skeletal muscle. In addition, both TPM4‐1α and TPM4‐2α are predominantly expressed in heart. 2D Western blot analyses using anti‐TPM antibody followed by Mass Spectrometry of the proteins from the antibody‐stained spots show that TPM1‐1α and TPM3α are expressed in skeletal muscle whereas TPM4‐1α and TPM3α are expressed in zebrafish heart. To the best of our knowledge, this is by far the most comprehensive analysis of tropomyosin expression in zebrafish, one of the most popular animal models for gene expression study.

Background: Conflicting results regarding the association of MMTV with human breast cancer have been reported. Published sequence data have indicated unique MMTV strains in some human samples. However, concerns regarding contamination as a cause of false positive results have persisted. Methods: We performed PCR assays for MMTV on human breast cancer cell lines and fresh frozen and formalin fixed normal and malignant human breast epithelial samples. Assays were also performed on peripheral blood mononuclear cells from volunteer blood donors and subjects at risk for human retroviral infections. In addition, assays were performed on DNA samples from wild and laboratory mice. Sequencing of MMTV positive samples from both humans and mice were performed and phylogenetically compared. Results: Using PCR under rigorous conditions to prevent and detect "carryover" contamination, we did detect MMTV DNA in human samples, including breast cancer. However, the results were not consistent and seemed to be an artifact. Further, experiments indicated that the probable source of false positives was murine DNA, containing endogenous MMTV, present in our building. However, comparison of published and, herein, newly described MMTV sequences with published data, indicates that there are some very unique human MMTV sequences in the literature. Conclusion: While we could not confirm the true presence of MMTV in our human breast cancer subjects, the data indicate that further, perhaps more traditional, retroviral studies are warranted to ascertain whether MMTV might rarely be the cause of human breast cancer.

Cloning and sequencing of various tropomyosin isoforms expressed in chickens have been described since the early 1980s. However, to the best of our knowledge, this is the first report on the molecular characterization and the expression of the sarcomeric isoform of the TPM3 gene in cardiac and skeletal muscles from developing as well as adult chickens. Expression of TPM3α was performed by conventional RT‐PCR as well as qRT‐PCR using relative expression (by ΔCT as well as ΔΔCT methods) and by determining absolute copy number. The results employing all these methods show that the expression level of TPM3α is maximum in embryonic (10‐day/15‐day old) skeletal muscle and can barely be detected in both cardiac and skeletal muscles from the adult chicken. Our various RT‐PCR analyses suggest that the expression of high molecular weight TPM3 isoforms are regulated at the transcription level from the proximal promoter at the 5′‐end of the TPM3 gene.

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

Tropomyosin, a coiled-coil dimeric protein, is a component of thin filaments that constitute myofibrils, the contractile apparatus of striated muscles. It is also a component of the actin filament network in non-muscle cells. In vertebrates, except for fish, there are four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, except for TPM4, the sarcomeric TPM isoform (s) for each TPM gene have been known for long time. Recently, we have cloned and sequenced the sarcomric isoform of the TPM4 gene designated as TPM4a. In addition, using qRT-PCR we have reported the expression of TPM4a in human striated muscles. Also, using 2D Western blot analyses followed by Mass spectra, we have reported the potential TPM4a protein expression in human cardiac tissues. However, much of the role of TPM4a in muscle contraction in human is yet to be elucidated. Although the role of the TPM4 gene in human diseases in not well documented, new information is emerging in this regard. For example, of the TPM4 isoforms TPM4g has been reported as a non-invasive biomarker in prenatal diagnosis of congenital heart defects; mutations in TPM4 have been implicated in Macrothombocytopenia in humans; differential expression of two TPM isoforms TPM4b and TPM4g in human breast cancer cells. However, the role of TPM4-ALK oncogenes in inflammatory myofibroblastic tumors in humans is well documented.