A common nonsense polymorphism (R577X) in the ACTN3 gene results in complete deficiency of the fast skeletal muscle fiber protein α-actinin-3 in an estimated one billion humans worldwide. The XX null ...genotype is under-represented in elite sprint athletes, associated with reduced muscle strength and sprint performance in non-athletes, and is over-represented in endurance athletes, suggesting that α-actinin-3 deficiency increases muscle endurance at the cost of power generation. Here we report that muscle from Actn3 knockout mice displays reduced force generation, consistent with results from human association studies. Detailed analysis of knockout mouse muscle reveals reduced fast fiber diameter, increased activity of multiple enzymes in the aerobic metabolic pathway, altered contractile properties, and enhanced recovery from fatigue, suggesting a shift in the properties of fast fibers towards those characteristic of slow fibers. These findings provide the first mechanistic explanation for the reported associations between R577X and human athletic performance and muscle function.
There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We ...sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 ‘pure’ acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.
The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specific actin filament populations. We ...have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was significantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 β-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confirming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs affect glucose metabolism in vivo by inhibiting Tpm3.1's function with few off-target effects.
Fusarium head blight (FHB), caused mainly by Fusarium graminearum Schwabe telomorph: Gibberella zeae Schwein.(Petch) in the US, is one of the most destructive diseases of wheat (Triticum aestivum L. ...and T. durum L.). Infected grain is usually contaminated with deoxynivalenol (DON), a serious mycotoxin. The challenge in FHB resistance breeding is combining resistance with superior agronomic and quality characteristics. Exotic QTL are widely used to improve FHB resistance. Success depends on the genetic background into which the QTL are introgressed, whether through backcrossing or forward crossing; QTL expression is impossible to predict. In this study four high-yielding soft red winter wheat breeding lines with little or no scab resistance were each crossed to a donor parent (VA01W-476) with resistance alleles at two QTL: Fhb1 (chromosome 3BS) and QFhs.nau-2DL (chromosome 2DL) to generate backcross and F2 progeny. F2 individuals were genotyped and assigned to 4 groups according to presence/ absence of resistance alleles at one or both QTL. The effectiveness of these QTL in reducing FHB rating, incidence, index, severity, Fusarium-damaged kernels (FDK) and DON, in F2-derived lines was assessed over 2 years. Fhb1 showed an average reduction in DON of 17.5%, and conferred significant resistance in 3 of 4 populations. QFhs.nau-2DL reduced DON 6.7% on average and conferred significant resistance in 2 of 4 populations. The combination of Fhb1 and QFhs.nau-2DL resistance reduced DON 25.5% across all populations. Double resistant lines had significantly reduced DON compared to double susceptible lines in 3 populations. Backcross derived progeny were planted in replicated yield trials (2011 and 2012) and in a scab nursery in 2012. Several top yielding lines performed well in the scab nursery, with acceptable DON concentrations, even though the average effect of either QTL in this population was not significant. Population selection is often viewed as an "all or nothing" process: if the average resistance level is insufficient, the population is discarded. These results indicate that it may be possible to find rare segregants which combine scab resistance, superior agronomic performance and acceptable quality even in populations in which the average effect of the QTL is muted or negligible.
Background Postthrombotic syndrome is a common complication of deep vein thrombosis, with limited treatment options. Methods and Results ACCESS PTS (Accelerated Thrombolysis for Post-Thrombotic ...Syndrome Using the Acoustic Pulse Thrombolysis Ekosonic Endovascular System) is a multicenter, single-arm, prospective study evaluating patients with chronic deep vein thrombosis and postthrombotic syndrome (Villalta score ≥8) who received minimum 3 months of anticoagulation. Patients underwent percutaneous transluminal venoplasty and ultrasound-accelerated thrombolysis, with data collected on clinical characteristics, postthrombotic syndrome, imaging, and quality of life to 1 year. The primary efficacy outcome was a reduction of ≥4 points in the Villalta score 30 days after procedure. The primary safety outcomes were major bleeding episodes within 72 hours and symptomatic pulmonary embolism during the index hospitalization. A total of 82 limbs (78 patients) were treated (age, 54.6±12.7 years; 32.1% women; mean Villalta score, 15.5±5.2). The primary end point was met in 64.6% (51/79). At 1 year, 77.3% (51/66) of limbs continued with a Villalta reduction ≥4. At 365 days, >90% of segments had patency with ultrasound flow present. Baseline to 1-year Physical Component Summary mean score of the Short Form-36 increased from 38.9±9.5 to 45.2±9.8 (
≤0.0001), and mean VEINES-QOL (Venous Insufficiency Epidemiological and Economic Study-Quality of Life) increased from 61.9±19.7 to 82.6±20.8 at 1 year (
<0.0001). Iliofemoral venous stenting was performed in 42 patients, with similar improvements seen in all outcomes, regardless of stenting status. One patient developed severe bleeding within 72 hours of the intervention and died at 32 days after procedure (1.3% mortality rate). Conclusions Percutaneous transluminal venoplasty and ultrasound-accelerated thrombolysis resulted in successful recanalization of chronic venous obstruction with improved postthrombotic syndrome severity and quality of life. Results were sustained at 1-year after procedure. Clinical Trial Registration URL: https://www.clinicaltrials.gov/. Unique identifier: NCT02159521.
We have identified novel actin filaments defined by tropomyosin Tpm4.2 at the ER. EM analysis of mouse embryo fibroblasts (MEFs) isolated from mice expressing a mutant Tpm4.2 (Tpm4Plt53/Plt53), ...incapable of incorporating into actin filaments, revealed swollen ER structures compared with wild‐type (WT) MEFs (Tpm4+/+). ER‐to‐Golgi, but not Golgi‐to‐ER trafficking was altered in the Tpm4Plt53/Plt53 MEFs following the transfection of the temperature sensitive ER‐associated ts045‐VSVg construct. Exogenous Tpm4.2 was able to rescue the ER‐to‐Golgi trafficking defect in the Tpm4Plt53/Plt53 cells. The treatment of WT MEFs with the myosin II inhibitor, blebbistatin, blocked the Tpm4.2‐dependent ER‐to‐Golgi trafficking. The lack of an effect on ER‐to‐Golgi trafficking following treatment of MEFs with CK666 indicates that branched Arp2/3‐containing actin filaments are not involved in anterograde vesicle trafficking. We propose that unbranched, Tpm4.2‐containing filaments have an important role in maintaining ER/Golgi structure and that these structures, in conjunction with myosin II motors, mediate ER‐to‐Golgi trafficking.
Pulsed field gradient nuclear magnetic resonance (PFG-NMR) measurements were successfully applied to the 27Al (I = 5/2) nucleus in concentrated electrolytes to investigate the diffusion of aluminate ...ions Al(OH)4 – in simulant high-level nuclear waste (3 M NaOH) between 25 and 85 °C. The temperature-dependent diffusion coefficients obtained from 1H, 23Na, and 27Al PFG-NMR were well fit by a Vogel–Fulcher–Tammann model and a power law equation. Comparison of 27Al diffusion coefficients of 0.1 M Al(OH)4 – in ∼3 M MOH (where M = Na+, K+, (CH3)4N+) at room temperature varied in agreement with the expected changes in solution viscosity via Stokes–Einstein relationship, confirming that the dominant Al species at these conditions are Al(OH)4 – monomers. This 27Al PFG-NMR study extends an established methodology to a previously unexplored nucleus enabling this experimental technique to be leverage for exploring ion transport, speciation, and solution structure in concentrated electrolytes.
In addition to the highly specialized contractile apparatus, it is becoming increasingly clear that there is an extensive actin cytoskeleton which underpins a wide range of functions in striated ...muscle. Isoforms of cytoskeletal actin and actin-associated proteins (non-muscle myosins, cytoskeletal tropomyosins, and cytoskeletal α-actinins) have been detected in a number of regions of striated muscle: the sub-sarcolemmal costamere, the Z-disc and the T-tubule/sarcoplasmic reticulum membranes. As the only known function of these proteins is through association with actin filaments, their presence in striated muscles indicates that there are spatially and functionally distinct cytoskeletal actin filament systems in these tissues. These filaments are likely to have important roles in mechanical support, ion channel function, myofibrillogenenous and vesicle trafficking.
Precise orchestration of actin polymer into filaments with distinct characteristics of stability, bundling, and branching underpins cell migration. A key regulator of actin filament specialization is ...the tropomyosin family of actin-associating proteins. This multi-isoform family of proteins assemble into polymers that lie in the major groove of polymerized actin filaments, which in turn determine the association of molecules that control actin filament organization. This suggests that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, such as wound healing. We have therefore analyzed the requirement for tropomyosin isoform expression in a mouse model of cutaneous wound healing. We find that mice in which the 9D exon from the TPM3/γTm tropomyosin gene is deleted (γ9D -/-) exhibit a more rapid wound-healing response 7 days after wounding compared with wild-type mice. Accelerated wound healing was not associated with increased cell proliferation, matrix remodeling, or epidermal abnormalities, but with increased cell migration. Rac GTPase activity and paxillin phosphorylation are elevated in cells from γ9D -/- mice, suggesting the activation of paxillin/Rac signaling. Collectively, our data reveal that tropomyosin isoform expression has an important role in temporal regulation of cell migration during wound healing.
Nemaline myopathy, the most common congenital myopathy, is caused by mutations in genes encoding thin filament and thin filament-associated proteins in skeletal muscles. Severely affected patients ...fail to survive beyond the first year of life due to severe muscle weakness. There are no specific therapies to combat this muscle weakness. We have generated the first knock-in mouse model for severe nemaline myopathy by replacing a normal allele of the α-skeletal actin gene with a mutated form (H40Y), which causes severe nemaline myopathy in humans. The Acta1(H40Y) mouse has severe muscle weakness manifested as shortened lifespan, significant forearm and isolated muscle weakness and decreased mobility. Muscle pathologies present in the human patients (e.g. nemaline rods, fibre atrophy and increase in slow fibres) were detected in the Acta1(H40Y) mouse, indicating that it is an excellent model for severe nemaline myopathy. Mating of the Acta1(H40Y) mouse with hypertrophic four and a half LIM domains protein 1 and insulin-like growth factor-1 transgenic mice models increased forearm strength and mobility, and decreased nemaline pathologies. Dietary l-tyrosine supplements also alleviated the mobility deficit and decreased the chronic repair and nemaline rod pathologies. These results suggest that l-tyrosine may be an effective treatment for muscle weakness and immobility in nemaline myopathy.
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