Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs.

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.

A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics.

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious, requiring 2 h of mass spectrometry time per single muscle fiber; 50 fibers would take approximately 4 days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 min total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 h. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Ninety-four proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, oxidative phosphorylation, and muscle structure and contractile function. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs.

RNA modifications, such as méthylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered.

Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics.

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious requiring two hours of mass spectrometry time per single muscle fiber; 50 fibers would take approximately four days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 minutes total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 hours. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Sixty-five proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, muscle structure and regulation. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.

Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease; however, its etiology remains largely unknown. We previously demonstrated that genetic variants in the MYH6 gene are significantly associated with HLHS. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from an HLHS-affected family trio (affected parent, unaffected parent, affected proband) carrying an MYH6-R443P head domain variant demonstrated dysmorphic sarcomere structure and increased compensatory MYH7 expression. Analysis of iPSC-CMs derived from the HLHS trio revealed that only beta myosin heavy chain expression was observed in CMs carrying the MYH6-R443P variant after differentiation day 15 (D15). Functional assessments performed between D20-D23 revealed that MYH6-R443P variant CMs contracted more slowly (40 ± 2 vs. 47 ± 2 contractions/min, P < 0.05), shortened less (5.6 ± 0.5 vs. 8.1 ± 0.7% of cell length, P < 0.05), and exhibited slower shortening rates (19.9 ± 1.7 vs. 28.1 ± 2.5 µm/s, P < 0.05) and relaxation rates (11.0 ± 0.9 vs. 19.7 ± 2.0 µm/s, P < 0.05). Treatment with isoproterenol had no effect on iPSC-CM mechanics. Using CRISPR/Cas9 gene editing technology, introduction of the R443P variant into the unaffected parent's iPSCs recapitulated the phenotype of the proband's iPSC-CMs, and conversely, correction of the R443P variant in the proband's iPSCs rescued the cardiomyogenic differentiation, sarcomere organization, slower contraction (P < 0.05) and decreased velocity phenotypes (P < 0.0001). This is the first report to identify that cardiac tissues from HLHS patients with MYH6 variants can exhibit sarcomere disorganization in atrial but not ventricular tissues. This new discovery was not unexpected, since MYH6 is expressed predominantly in the postnatal atria in humans. These findings demonstrate the feasibility of employing patient-derived iPSC-CMs, in combination with patient cardiac tissues, to gain mechanistic insight into how genetic variants can lead to HLHS. Results from this study suggest that decreased contractility of CMs due to sarcomere disorganization in the atria may effect hemodynamic changes preventing development of a normal left ventricle.

Ca2+ dependency of limb muscle fiber contractile mechanics in young and older adults.

Age-induced declines in skeletal muscle contractile function have been attributed to multiple cellular factors, including lower peak force (Po), decreased Ca2+ sensitivity, and reduced shortening velocity (Vo). However, changes in these cellular properties with aging remain unresolved, especially in older women, and the effect of submaximal Ca2+ on contractile function is unknown. Thus, we compared contractile properties of muscle fibers from 19 young (24 ± 3 yr; 8 women) and 21 older adults (77 ± 7 yr; 7 women) under maximal and submaximal Ca2+ and assessed the abundance of three proteins thought to influence Ca2+ sensitivity. Fast fiber cross-sectional area was ~44% larger in young (6,479 ± 2,487 µm2) compared with older adults (4,503 ± 2,071 µm2, P < 0.001), which corresponded with a greater absolute Po (young = 1.12 ± 0.43 mN; old = 0.79 ± 0.33 mN, P < 0.001). There were no differences in fast fiber size-specific Po, indicating the age-related decline in force was explained by differences in fiber size. Except for fast fiber size and absolute Po, no age or sex differences were observed in Ca2+ sensitivity, rate of force development (ktr), or Vo in either slow or fast fibers. Submaximal Ca2+ depressed ktr and Vo, but the effects were not altered by age in either sex. Contrary to rodent studies, regulatory light chain (RLC) and myosin binding protein-C abundance and RLC phosphorylation were unaltered by age or sex. These data suggest the age-associated reductions in contractile function are primarily due to the atrophy of fast fibers and that caution is warranted when extending results from rodent studies to humans.

Drug Repurposing of Bromodomain Inhibitors as Potential Novel Therapeutic Leads for Lymphatic Filariasis Guided by Multispecies Transcriptomics.

To better understand the transcriptomic interplay of organisms associated with lymphatic filariasis, we conducted multispecies transcriptome sequencing (RNA-Seq) on the filarial nematode Brugia malayi, its Wolbachia endosymbiont wBm, and its laboratory vector Aedes aegypti across the entire B. malayi life cycle. In wBm, transcription of the noncoding 6S RNA suggests that it may be a regulator of bacterial cell growth, as its transcript levels correlate with bacterial replication rates. For A. aegypti, the transcriptional response reflects the stress that B. malayi infection exerts on the mosquito with indicators of increased energy demand. In B. malayi, expression modules associated with adult female samples consistently contained an overrepresentation of genes involved in chromatin remodeling, such as the bromodomain-containing proteins. All bromodomain-containing proteins encoded by B. malayi were observed to be upregulated in the adult female, embryo, and microfilaria life stages, including 2 members of the bromodomain and extraterminal (BET) protein family. The BET inhibitor JQ1(+), originally developed as a cancer therapeutic, caused lethality of adult worms in vitro, suggesting that it may be a potential therapeutic that can be repurposed for treating lymphatic filariasis.IMPORTANCE The current treatment regimen for lymphatic filariasis is mostly microfilaricidal. In an effort to identify new drug candidates for lymphatic filariasis, we conducted a three-way transcriptomics/systems biology study of one of the causative agents of lymphatic filariasis, Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host Aedes aegypti at 16 distinct B. malayi life stages. B. malayi upregulates the expression of bromodomain-containing proteins in the adult female, embryo, and microfilaria stages. In vitro, we find that the existing cancer therapeutic JQ1(+), which is a bromodomain and extraterminal protein inhibitor, has adulticidal activity in B. malayi.

Cold acclimation increases levels of some heat shock protein and sirtuin isoforms in threespine stickleback.

Molecular chaperones [heat shock proteins (HSPs)] increase in response to rapid changes in temperatures, but long-term acclimation to cold temperature may also warrant elevations in HSPs. In fishes, cold acclimation increases mitochondrial density and oxidative stress in some tissues, which may increase demand for HSPs. We hypothesized that levels of HSPs, as well as sirtuins (SIRTs), NAD-dependent deacetylases that mediate changes in metabolism and responses to oxidative stress (including increases in HSPs), would increase during cold acclimation of threespine stickleback (Gasterosteus aculeatus). Transcript levels of hsp70, hsc70, hsp60 and hsp90-α, sirts1-4, as well as protein levels of HSP60, HSP90 and HSC70 were quantified in liver and pectoral adductor muscle of stickleback during cold acclimation from 20 °C to 8 °C. In liver, cold acclimation stimulated a transient increase in mRNA levels of hsp60 and hsc70. Transcript levels of sirt1 and sirt2 also increased in response to cold acclimation and remained elevated. In pectoral muscle, mRNA levels of hsp60, hsp90-α, hsc70 and sirt1 all transiently increased in response to cold acclimation, while levels of sirts2-4 remained constant or declined. Similar to transcript levels, protein levels of HSC70 increased in both liver and pectoral muscle. Levels of HSP90 also increased in liver after 4 weeks at 8 °C. HSP60 remained unchanged in both tissues, as did HSP90 in pectoral muscle. Our results indicate that while both HSPs and SIRTs increase in response to cold acclimation in stickleback, the response is tissue and isoform specific, likely reflecting differences in metabolism and oxidative stress.