DNA demethylase activity maintains intestinal cells in an undifferentiated state following loss of APC.

Although genome-wide hypomethylation is a hallmark of many cancers, roles for active DNA demethylation during tumorigenesis are unknown. Here, loss of the APC tumor suppressor gene causes upregulation of a DNA demethylase system and the concomitant hypomethylation of key intestinal cell fating genes. Notably, this hypomethylation maintained zebrafish intestinal cells in an undifferentiated state that was released upon knockdown of demethylase components. Mechanistically, the demethylase genes are directly activated by Pou5f1 and Cebpß and are indirectly repressed by retinoic acid, which antagonizes Pou5f1 and Cebpß. Apc mutants lack retinoic acid as a result of the transcriptional repression of retinol dehydrogenase l1 via a complex that includes Lef1, Groucho2, Ctbp1, Lsd1, and Corest. Our findings imply a model wherein APC controls intestinal cell fating through a switch in DNA methylation dynamics. Wild-type APC and retinoic acid downregulate demethylase components, thereby promoting DNA methylation of key genes and helping progenitors commit to differentiation.

A two-step model for colon adenoma initiation and progression caused by APC loss.

Aberrant Wnt/beta-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Using zebrafish and human cells, we show that homozygous loss of APC causes failed intestinal cell differentiation but that this occurs in the absence of nuclear beta-catenin and increased intestinal cell proliferation. Therefore, loss of APC is insufficient for causing beta-catenin nuclear localization. APC mutation-induced intestinal differentiation defects instead depend on the transcriptional corepressor C-terminal binding protein-1 (CtBP1), whereas proliferation defects and nuclear accumulation of beta-catenin require the additional activation of KRAS. These findings suggest that, following APC loss, CtBP1 contributes to adenoma initiation as a first step, whereas KRAS activation and beta-catenin nuclear localization promote adenoma progression to carcinomas as a second step. Consistent with this model, human FAP adenomas showed robust upregulation of CtBP1 in the absence of detectable nuclear beta-catenin, whereas nuclear beta-catenin was detected in carcinomas.

Genetic variants associated with syncope implicate neural and autonomic processes.

AIMS: Syncope is a common and clinically challenging condition. In this study, the genetics of syncope were investigated to seek knowledge about its pathophysiology and prognostic implications. METHODS AND RESULTS: This genome-wide association meta-analysis included 56 071 syncope cases and 890 790 controls from deCODE genetics (Iceland), UK Biobank (United Kingdom), and Copenhagen Hospital Biobank Cardiovascular Study/Danish Blood Donor Study (Denmark), with a follow-up assessment of variants in 22 412 cases and 286 003 controls from Intermountain (Utah, USA) and FinnGen (Finland). The study yielded 18 independent syncope variants, 17 of which were novel. One of the variants, p.Ser140Thr in PTPRN2, affected syncope only when maternally inherited. Another variant associated with a vasovagal reaction during blood donation and five others with heart rate and/or blood pressure regulation, with variable directions of effects. None of the 18 associations could be attributed to cardiovascular or other disorders. Annotation with regard to regulatory elements indicated that the syncope variants were preferentially located in neural-specific regulatory regions. Mendelian randomization analysis supported a causal effect of coronary artery disease on syncope. A polygenic score (PGS) for syncope captured genetic correlation with cardiovascular disorders, diabetes, depression, and shortened lifespan. However, a score based solely on the 18 syncope variants performed similarly to the PGS in detecting syncope risk but did not associate with other disorders. CONCLUSION: The results demonstrate that syncope has a distinct genetic architecture that implicates neural regulatory processes and a complex relationship with heart rate and blood pressure regulation. A shared genetic background with poor cardiovascular health was observed, supporting the importance of a thorough assessment of individuals presenting with syncope.

A rust-fungus Nudix hydrolase effector decaps mRNA in vitro and interferes with plant immune pathways.

To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini). We completed extensive in vitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild-type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity. AvrM14 is an extremely selective enzyme capable of removing the protective 5' cap from mRNA transcripts in vitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage in vitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild-type AvrM14, but not the catalytically dead version, suppressed immune-related reactive oxygen species production, altered the abundance of some circadian-rhythm-associated mRNA transcripts and reduced the hypersensitive cell-death response triggered by the flax disease resistance protein M1. To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with in vitro mRNA-decapping activity capable of interfering with plant immunity.

DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45.

Evidence for active DNA demethylation in vertebrates is accumulating, but the mechanisms and enzymes remain unclear. Using zebrafish embryos we provide evidence for 5-methylcytosine (5-meC) removal in vivo via the coupling of a 5-meC deaminase (AID, which converts 5-meC to thymine) and a G:T mismatch-specific thymine glycosylase (Mbd4). The injection of methylated DNA into embryos induced a potent DNA demethylation activity, which was attenuated by depletion of AID or the non enzymatic factor Gadd45. Remarkably, overexpression of the deaminase/glycosylase pair AID/Mbd4 in vivo caused demethylation of the bulk genome and injected methylated DNA fragments, likely involving a G:T intermediate. Furthermore, AID or Mbd4 knockdown caused the remethylation of a set of common genes. Finally, Gadd45 promoted demethylation and enhanced functional interactions between deaminase/glycosylase pairs. Our results provide evidence for a coupled mechanism of 5-meC demethylation, whereby AID deaminates 5-meC, followed by thymine base excision by Mbd4, promoted by Gadd45.

Decrements of mobility and power in recreationally active septuagenarians is related to loss of force, but not slowing of the muscle: a 5-year longitudinal study.

A lesser 6-min walk distance (6MWD) and timed up-and-go (TUG) in old compared with young adults was previously linked to slowing of muscle contractile properties. The purpose of the present study was to determine whether any further reductions in 6MWD and TUG over a 5-year period in septuagenarians are associated with further slowing of muscle contractile properties. We measured muscle function by a countermovement jump, isometric maximal knee extensor strength (MVC) on a dynamometer and quadriceps muscle size by magnetic resonance imaging (MRI) in 17 older women (71.1 ± 2.8 y) and 17 older men (71.3 ± 4.1y). Performance in TUG and 6MWD were reduced over the 5-year period, irrespective of sex (P < 0.001), and both were correlated with power at both baseline and follow-up (R ≥ 0.53; P ≤ 0.001). Jump take-off velocity (VCMJ) was slower at follow-up (P < 0.01) and correlated with 6MWD and TUG at both baseline and follow-up in both sexes (R ≥ 0.54; P ≤ 0.001). However, the relationship between 'body mass: maximal muscle force ratio' with VCMJ was not significantly changed, indicating that the lower VCMJ was attributable to muscles working at a higher relative load, hence a lower part of the force-velocity relationship, due to a reduction in MVC (body mass had not changed significantly), rather than slowing of the muscle. The lower VCMJ in women than men (P < 0.001) was likewise attributable to a lower MVC rather than slower contractile properties in women. In conclusion, the decrement in 6MWD and TUG in septuagenarians is due to a loss of muscle mass, rather than further loss of muscle quality.

Optimized Production of Disulfide-Bonded Fungal Effectors in Escherichia coli Using CyDisCo and FunCyDisCo Coexpression Approaches.

Effectors are a key part of the arsenal of plant-pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognized by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterize effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a coexpression system within the heterologous host Escherichia coli, termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalized coexpression approach termed FunCyDisCo (Fungi CyDisCo) can significantly improve the production yields of numerous disulfide-bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallization of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide bond-containing effectors.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Did the Pope ban blood transfusion in 1678?

This paper concerns an alleged event in the history of haematology that disrupts the otherwise positive narrative of papal encouragement for blood transfusion. It is frequently stated, in popular websites and in the scholarly literature, that when blood transfusion was first developed it was banned by the Pope. However, careful analysis of the sources cited shows this claim to be without historical foundation. There was never any papal prohibition of blood transfusion. It is a myth that needs to be dispelled if the full extent of the Catholic Church's support for blood and organ donation is to be appreciated.

The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and provides insight into Snn3 recognition and pro-domain protease processing of fungal effectors.

Plant pathogens cause disease through secreted effector proteins, which act to promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilized a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3. We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination and functional studies. We show this approach can be successfully applied to study effectors from other pathogenic fungi. The ß-barrel fold of SnTox3 is a novel fold among fungal effectors. Structure-guided mutagenesis enabled the identification of residues required for Snn3 recognition. SnTox3 is a pre-pro-protein, and the pro-domain of SnTox3 can be cleaved in vitro by the protease Kex2. Complementing this, an in silico study uncovered the prevalence of a conserved motif (LxxR) in an expanded set of putative pro-domain-containing fungal effectors, some of which can be cleaved by Kex2 in vitro. Our in vitro and in silico study suggests that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi and this may have broad implications for the approaches used to study their functions.

The Aspergillus nidulans IQGAP orthologue SepG is required for constriction of the contractile actomyosin ring.

In this research we report that the sepG1 mutation in Aspergillus nidulans resides in gene AN9463, which is predicted to encode an IQGAP orthologue. The genetic lesion is predicted to result in a G-to-R substitution at residue 1637 of the 1737-residue protein in a highly conserved region of the RasGAP-C-terminal (RGCT) domain. When grown at restrictive temperature, strains expressing the sepGG1637R (sepG1) allele are aseptate, with reduced colony growth and aberrantly formed conidiophores. The aseptate condition can be replicated by deletion of AN9463 or by downregulating its expression via introduced promoters. The mutation does not prevent assembly of a cortical contractile actomyosin ring (CAR) at putative septation sites, but tight compaction of the rings is impaired and the rings fail to constrict. Both GFP::SepG wild type and the GFP-tagged product of the sepG1 allele localize to the CAR at both permissive and restrictive temperatures. Downregulation of myoB (encoding the A. nidulans type-II myosin heavy chain) does not prevent formation of SepG rings at septation sites, but filamentous actin is required for CAR localization of SepG and MyoB. We identify fourteen probable IQ-motifs (EF-hand protein binding sites) in the predicted SepG sequence. Two of the A. nidulans EF-hand proteins, myosin essential light chain (AnCdc4) and myosin regulatory light chain (MrlC), colocalize with SepG and MyoB at all stages of CAR formation and constriction. However, calmodulin (CamA) appears at septation sites only after the CAR has become fully compacted. When expression of sepG is downregulated, leaving MyoB as the sole IQ-motif protein in the pre-compaction CAR, both MrlC and AnCdc4 continue to associate with the forming CAR. When myoB expression is downregulated, leaving SepG as the sole IQ-motif protein in the CAR, AnCdc4 association with the forming CAR continues but MrlC fails to associate. This supports a model in which the IQ motifs of MyoB bind both MrlC and AnCdc4, while the IQ motifs of SepG bind only AnCdc4. Downregulation of either mrlC or Ancdc4 results in an aseptate phenotype, but has no effect on association of either SepG or MyoB with the CAR.

Low frequency fatigue and changes in muscle fascicle length following eccentric exercise of the knee extensors.

NEW FINDING: What is the central question of this study? Does low frequency muscle fatigue indicate a failure of excitation-contraction coupling after eccentric exercise, or is it simply due to a change in muscle length? What is the main finding and its importance? The low to high frequency muscle fatigue ratio was relatively insensitive to changes in muscle length, and any changes in length following eccentric exercise were far too small to account for the high degree of low frequency fatigue. The results strengthen the suggestion that the early loss of force following eccentric exercise is due to a deficit of excitation-contraction coupling. ABSTRACT: Development of long lasting fatigue (low frequency fatigue; LFF), assessed as the ratio of forces at 20 and 100 Hz stimulation, suggests the early phase of muscle damage caused by eccentric exercise is due to a deficit of excitation-contraction coupling. However, this could be caused by a change of muscle length. Eleven men (21.3 ± 2.0 years) performed 200 maximum eccentric knee extensions (30-110 deg flexion). Force generated by 20 and 100 Hz stimulation and maximum isometric force (MIF) were determined at knee angles 50, 70 and 90 deg before and immediately after the exercise. Vastus lateralis fascicle length (FL) was measured by ultrasound of resting and contracting muscle. Peak MIF (829 ± 119 N) was at 70 deg knee flexion, falling to 486 ± 180 N (P < 0.001) after exercise, but with no change in optimum angle. FLs at rest were unaffected by eccentric exercise, but during contraction they were on average 8.8% (95% CI: 4.1, 13.5%, P = 0.002) longer after exercise. Before exercise, the 20/100 ratio increased with muscle length, from 0.69 ± 0.09 at 50 deg, 0.72 ± 0.05 at 70 deg and 0.80 ± 0.08 at knee angle 90 deg (P < 0.001). After eccentric exercise the 20/100 ratio was reduced to 0.29 ± 0.08 at 50 deg, 0.27 ± 0.04 at 70 deg and 0.34 ± 0.04 at 90 deg (P < 0.001). The 20/100 ratio was relatively insensitive to changes in muscle length and the decrease following eccentric exercise was far greater than might be caused by any changes in muscle length after eccentric exercise. The results show that LFF following eccentric exercise is not due to change in muscle length and strengthen the suggestion that it represents a deficit in excitation-contraction coupling.

Five-year longitudinal changes in thigh muscle mass of septuagenarian men and women assessed with DXA and MRI.

Magnetic resonance imaging (MRI) and dual-energy X-ray absorptiometry (DXA) were used to assess changes in thigh lean mass in septuagenarian men and women during a 5-year longitudinal study. Twenty-four older individuals participated in the study (10 men: 71.6 ± 4.1 years; 14 women: 71.3 ± 3.2 years at baseline). Thigh MRI and whole-body DXA scans were used to estimate changes in thigh lean mass. Both MRI and DXA showed that thigh lean mass was reduced by approximately 5% (P = 0.001) over the 5-year period in both men and women. The percentage loss of muscle mass determined with MRI and DXA showed moderate correlation (R2 = 0.466; P < 0.001). Bland-Altman analysis showed that the average change over 5 years of follow-up measured by DXA was only 0.18% greater than MRI, where the limits of agreement between DXA and MRI were ± 10.4%. Baseline thigh lean mass did not predict the percentage loss of thigh lean mass over the 5-year period (R2 = 0.003; P = 0.397), but a higher baseline body fat percentage was associated with a larger loss of thigh muscle mass in men (R2 = 0.677; P < 0.003) but not in women (R2 = 0.073; P < 0.176). In conclusion, (1) DXA and MRI showed a similar percentage loss of muscle mass over a 5-year period in septuagenarian men and women that (2) was independent of baseline muscle mass, but (3) increased with higher baseline body fat percentage in men.

Are Force Enhancement after Stretch and Muscle Fatigue Due to Effects of Elevated Inorganic Phosphate and Low Calcium on Cross Bridge Kinetics?

Background and Objectives: Muscle fatigue is characterised by (1) loss of force, (2) decreased maximal shortening velocity and (3) a greater resistance to stretch that could be due to reduced intracellular Ca2+ and increased Pi, which alter cross bridge kinetics. Materials and Methods: To investigate this, we used (1) 2,3-butanedione monoxime (BDM), believed to increase the proportion of attached but non-force-generating cross bridges; (2) Pi that increases the proportion of attached cross bridges, but with Pi still attached; and (3) reduced activating Ca2+. We used permeabilised rat soleus fibres, activated with pCa 4.5 at 15 °C. Results: The addition of 1 mM BDM or 15 mM Pi, or the lowering of the Ca2+ to pCa 5.5, all reduced the isometric force by around 50%. Stiffness decreased in proportion to isometric force when the fibres were activated at pCa 5.5, but was well maintained in the presence of Pi and BDM. Force enhancement after a stretch increased with the length of stretch and Pi, suggesting a role for titin. Maximum shortening velocity was reduced by about 50% in the presence of BDM and pCa 5.5, but was slightly increased by Pi. Neither decreasing Ca2+ nor increasing Pi alone mimicked the effects of fatigue on muscle contractile characteristics entirely. Only BDM elicited a decrease of force and slowing with maintained stiffness, similar to the situation in fatigued muscle. Conclusions: This suggests that in fatigue, there is an accumulation of attached but low-force cross bridges that cannot be the result of the combined action of reduced Ca2+ or increased Pi alone, but is probably due to a combination of factors that change during fatigue.

The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana.

The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans.

Frailty phenotype and frailty index are associated with distinct neuromuscular electrophysiological characteristics in men.

NEW FINDINGS: What is the central question of this study? Human frailty is characterized by accumulated health complaints, including medical conditions, low physical and psychological function and social components. It is currently unknown whether the condition is associated with neuromuscular changes detectable by electrophysiology obtained from voluntary and involuntary muscle contractions. What is the main finding and its importance? A higher likelihood of frailty was significantly associated with a smaller size of vastus lateralis motor unit potentials during voluntary contractions and smaller compound muscle action potentials generated by electrical stimulation. Importantly, these associations were independent of age and body mass index. ABSTRACT: The purpose of this study was to determine whether neuromuscular electrophysiological characteristics that are known to underlie sarcopenia are also associated with the more complex frailty syndrome. Eighty-six men [mean (SD) age, 74 (8) years] were classed as non-frail (robust), prefrail or frail using criteria from the frailty phenotype (FP) and the frailty index (FI). The femoral nerve was stimulated maximally and the resulting compound muscle action potential amplitude (CMAP) measured over the vastus lateralis. Motor unit potential (MUP) size was assessed during voluntary contractions using intramuscular electromyography (iEMG). Logistic and negative binomial regression models determined relationships between FP and FI with CMAP and MUP sizes before and after adjustments for age and body mass index. Larger CMAP size was associated with a lower likelihood of frailty in fully adjusted models: a 1SD higher level in vastus lateralis CMAP size was associated with a 0.4 (95% confidence interval: 0.2, 0.6; P < 0.01) unit lower FI (40% of the FI range) and more than halving of the odds [odds ratio: 0.43 (95% confidence interval: 0.21, 0.90)] of having a frail/prefrail phenotype. Greater MUP size was also related to lower FI values using unadjusted and fully adjusted models. However, MUP size was not significantly related to FP in any model. Smaller MUPs and a smaller CMAP were significantly associated with a higher likelihood of frailty, independent of age and body mass index. These results relate neuromuscular electrophysiological characteristics to the complex frailty syndrome and identify motor unit remodelling as a possible contributing factor.

The contribution of low-frequency fatigue to the loss of quadriceps contractile function following repeated drop jumps.

NEW FINDINGS: What is the central question of this study? Why do some subjects recover slowly following a bout of eccentric exercise and why is recovery faster following a repeated bout? What is the main finding and its importance? The results are consistent with two major causes of the reduction of quadriceps torque, the onset of low-frequency fatigue which recovered relatively fast and a second, delayed form of damage. Differences in the delayed damage process largely accounted for the differences in the rate of torque recovery between subjects after a first bout and it was suppression of the delayed damage which accounted for the faster recovery following a repeated bout of eccentric exercise. ABSTRACT: The purpose of this study was to determine the extent to which low-frequency fatigue (LFF) accounts for the loss of quadriceps strength and time course of recovery following a series of drop jumps (DJs). Seventeen female subjects (20.8 ± 1.4 years) undertook 100 DJs, which were repeated 4 weeks later. Maximum isometric torque (MIT) and the ratio of torque generated by 20 and 100 Hz electrical stimulation (20/100), as a measure of LFF, were measured over 7 days following each series of DJs. After the first series the 20/100 ratio fell to a greater extent than MIT (to 35 ± 8.7% and 69 ± 11%, respectively) but recovered over 2-3 days, while MIT showed little recovery over this time. Changes of the 20/100 ratio were similar between subjects with fast or slow MIT recovery. Following the second series of DJs, changes in the 20/100 ratio were similar to those of the first bout and there were no differences between fast and slow recovering subjects. MIT, however, recovered more rapidly than after the first bout; the faster recovery was confined to the subjects who recovered slowly following the first bout. The results are consistent with two major causes of the reduction of quadriceps torque, the onset of low-frequency fatigue which recovered relatively fast and a second, delayed, form of damage. The latter largely accounted for the differences in MIT recovery between subjects after the first bout, while suppression of the delayed damage accounted for the faster recovery following the repeated bout.

Cholinesterase Inhibitory Arisugacins L-Q from a Penicillium sp. Isolate Obtained through a Citizen Science Initiative and Their Activities in a Phenotype-Based Zebrafish Assay.

Phenotype-based screening of a fungal extract library yielded an active sample from a Penicillium sp. isolate that impaired zebrafish motility. Bioassay-guided purification led to the identification of 14 meroterpenoids including six new metabolites, arisugacins L-Q (4, 5, 8, and 12-14), seven known arisugacins (1-3, 6, 7, 9, and 10), and one known terreulactone (11). Their structures were determined using a combination of NMR and HRESIMS data, evidence secured from theoretical and experimental ECD spectra, and the modified Mosher's method. The purified compounds were tested in zebrafish embryos, as well as in vitro for cholinesterase inhibition activities. Compound 12 produced defects in myotome structure (metameric muscle, which is critical for locomotion) in vivo and showed the most potent and selective acetylcholinesterase inhibitory activity with an IC50 of 191 nM in vitro. The phenotype assay was also used to reveal bioactivities for several previously reported arisugacins, which had failed to show activity in prior cell-based and in vitro testing. This study demonstrates that utilization of the zebrafish phenotype assay is an effective approach for the identification of bioactive extracts, is compatible with the bioassay-guided compound purification strategies, and offers a valuable tool for probing complex natural product sources to detect bioactive small molecules with potential therapeutic or other commercial applications.

Slow torque recovery after eccentric exercise and the repeated bout effect; the role of primary and secondary muscle damage.

OBJECTIVES: To determine the role of primary and secondary damage in the variation between people of maximum voluntary contraction (MVC) torque recovery following eccentric exercise and the faster recovery following a repeated bout of exercise. METHODS: Twenty-one healthy, active but untrained young female subjects undertook eccentric exercise of the elbow flexors and 11 repeated the exercise 28 days later. Changes of MVC torque and creatine kinase (CK) were followed for 7 days after each bout of exercise. RESULTS: Following the first bout, 45% of subjects showed a continuing decline in MVC torque, suggesting secondary damage, which was correlated with a large delayed CK release (R2=0.54, p<0.001). After the second bout of exercise, the initial MVC torque loss was similar to that after the first bout while torque recovery was faster, but only for the previously slow recovering subjects. Comparing the time course of MVC torque recovery of first and second bouts suggests secondary damage develops over 4 days. CONCLUSIONS: The data are consistent with primary damage being similar between subjects and unaffected by the repeated bout while it is secondary damage which accounts for differences in MVC torque recovery and is suppressed following a repeated bout of exercise.

The relationship between stiffness and pain following unaccustomed eccentric exercise: the effects of gentle stretch and repeated bout.

PURPOSE: To determine how muscle stiffness and pain which develop after eccentric exercise are affected by gentle stretching and repeated exercise. METHODS: Twenty-one healthy female participants undertook eccentric exercise of the elbow flexors and changes in resting elbow flexion angle (REFA; a measure of muscle stiffness), pain on stretch scale, pain elicited by pressure (PPT pain, a measure of mechanoreceptor hypersensitivity), and upper arm girth were followed for 7 days after exercise. The effects of gentle passive stretching on pain and muscle stiffness were investigated 2 and 4 days after exercise. Eleven participants also repeated the exercise with the same arm 6 weeks after the first bout. RESULTS: There was a significant relationship between the pain on stretch scale and increased REFA (day 4; R2 = 0.65, p < 0.001), whereas there was no relationship between REFA and PPT pain. REFA was reduced by passive stretching and pain on stretch scale was also reduced from 3.0 (1.4, 5.1) to 0.75 (0.0, 2.0) [median (IQR), p = 0.01]. PPT pain was unaffected by the passive stretching, as was muscle swelling. Following the repeated bout, increases in REFA were much reduced, as was pain on stretch scale (p = 0.02). However, PPT pain was not significantly different between the two bouts of exercise. CONCLUSIONS: The results indicate that reductions in pain on stretch scale, either by gentle passive stretching or as the result of repeated exercise, are primarily due to reductions in muscle stiffness which develops after eccentric exercise, whereas mechanoreceptor hypersensitivity is relatively unaffected.

The immediate effects of breathing with oscillated inspiratory and expiratory airflows on secretion clearance in intubated patients with cervical spinal cord injury.

STUDY DESIGN: A prospective, randomized crossover trial. OBJECTIVES: To evaluate the efficacy of the combination of incentive spirometry with oscillation (OIS) and positive expiratory pressure with oscillation (OPEP) to promote secretion clearance in intubated patients with cervical spinal cord injury. SETTING: Spinal cord unit, tertiary care hospital, North East Thailand. METHODS: Thirteen intubated patients (C4-7, AIS score C) with secretion retention performed three interventions randomly allocated on consecutive days, a Sham deep breathing, OPEP and OPEP + OIS breathing exercise. Secretions were collected by sterile suction for 3 h before, and 3 h after, each intervention and wet weight recorded. Cardiopulmonary parameters were measured before and after each intervention. RESULTS: The median (IQR) secretion wet weight pre-intervention was 2.61 g (2.21, 3.85) and in the 3 h after Sham there was an increase of 1.97 g (0.6, 3.6). The increase after OPEP was 2.67 g (1.7, 3.9) and after OPEP + OIS, 4.28 g (2.4, 6.7); all the increases being significant (p ≤ 0.007). The clearance after OPEP and OPEP + OIS were both greater than Sham while OPEP + OIS was greater than OPEP (p ≤ 0.019). There were no significant changes in cardiopulmonary measures following any intervention or when compared between interventions. CONCLUSIONS: Deep breathing with an oscillated and humidified air flow in a combination of OIS + OPEP more than doubled secretion clearance and was more effective than OPEP or Sham deep breathing. There were no adverse effects of the procedures which were well tolerated by the patients and may be used to complement existing methods for secretion clearance.