Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in dystrophin and the subsequent disruption of the dystrophin-associated protein complex (DAPC). Utrophin is a dystrophin homolog expressed at high levels in developing muscle that is an attractive target for DMD therapy. Here we show that the extracellular matrix protein biglycan regulates utrophin expression in immature muscle and that recombinant human biglycan (rhBGN) increases utrophin expression in cultured myotubes. Systemically delivered rhBGN up-regulates utrophin at the sarcolemma and reduces muscle pathology in the mdx mouse model of DMD. RhBGN treatment also improves muscle function as judged by reduced susceptibility to eccentric contraction-induced injury. Utrophin is required for the rhBGN therapeutic effect. Several lines of evidence indicate that biglycan acts by recruiting utrophin protein to the muscle membrane. RhBGN is well tolerated in animals dosed for as long as 3 months. We propose that rhBGN could be a therapy for DMD.

Genome-wide loss-of-function screen using human pluripotent stem cells to study virus-host interactions for SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019, has become a global health concern. Therefore, there is an immense need to understand the network of virus-host interactions by using human disease-relevant cells. We have thus conducted a loss-of-function genome-wide screen using haploid human embryonic stem cells (hESCs) to identify genes involved in SARS-CoV-2 infection. Although the undifferentiated hESCs are resistant to SARS-CoV-2, their differentiated definitive endoderm (DE) progenies, which express high levels of ACE2, are highly sensitive to the virus. Our genetic screening was able to identify the well-established entry receptor ACE2 as a host factor, along with additional potential novel modulators of SARS-CoV-2. Two such novel screen hits, the transcription factor MAFG and the transmembrane protein TMEM86A, were further validated as conferring resistance against SARS-CoV-2 by using CRISPR-mediated mutagenesis in hESCs, followed by differentiation of mutant lines into DE cells and infection by SARS-CoV-2. Our genome-wide genetic screening investigated SARS-CoV-2 host factors in non-cancerous human cells with endogenous ACE2 expression, providing a unique platform to identify novel modulators of SARS-CoV-2 cytopathology in human cells.

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

Fragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

Genome-wide analysis of haploinsufficiency in human embryonic stem cells.

Haploinsufficiency describes a phenomenon where one functioning allele is insufficient for a normal phenotype, underlying several human diseases. The effect of haploinsufficiency on human embryonic stem cells (hESC) has not been thoroughly studied. To establish a genome-wide loss-of-function screening for heterozygous mutations, we fuse normal haploid hESCs with a library of mutant haploid hESCs. We identify over 600 genes with a negative effect on hESC growth in a haploinsufficient manner and characterize them as genes showing less tolerance to mutations, conservation during evolution, and depletion from telomeres and X chromosome. Interestingly, a large fraction of these genes is associated with extracellular matrix and plasma membrane and enriched for genes within WNT and TGF-ß pathways. We thus identify haploinsufficiency-related genes that show growth retardation in early embryonic cells, suggesting dosage-dependent phenotypes in hESCs. Overall, we construct a unique model for studying haploinsufficiency and identified important dosage-dependent pathways involved in hESC growth and survival.

Identifying regulators of parental imprinting by CRISPR/Cas9 screening in haploid human embryonic stem cells.

In mammals, imprinted genes are regulated by differentially methylated regions (DMRs) that are inherited from germ cells, leading to monoallelic expression in accordance with parent-of-origin. Yet, it is largely unknown how imprinted DMRs are maintained in human embryos despite global DNA demethylation following fertilization. Here, we explored the mechanisms involved in imprinting regulation by employing human parthenogenetic embryonic stem cells (hpESCs), which lack paternal alleles. We show that although global loss of DNA methylation in hpESCs affects most imprinted DMRs, many paternally-expressed genes (PEGs) remain repressed. To search for factors regulating PEGs, we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs. This revealed ATF7IP as an essential repressor of a set of PEGs, which we further show is also required for silencing sperm-specific genes. Our study reinforces an important role for histone modifications in regulating imprinted genes and suggests a link between parental imprinting and germ cell identity.

Cardiac structure and function in response to a multi-stage marathon over 4486 km.

AIMS: To investigate whether participation in the Trans Europe Foot Race 2009 (TEFR), an ultramarathon race held over 64 consecutive days and 4486 km, led to changes in cardiac structure and function. METHODS: Cardiac magnetic resonance imaging was performed in 20 of 67 participating runners (two women; mean ± SD age 47.8 ± 10.4 years) at three time points (baseline scan at 294 ± 135 km (B), scan two at 1735 ± 86 km (T1) and scan three at 3370 ± 90 km (T2)) during the TEFR. Imaging included an assessment of left ventricular structure (mass) and function (strain). In parallel, cardiac troponin I, NT-pro-BNP, myostatin and GDF11 were determined in venous blood samples. A subsample of ten runners returned for a follow-up scan eight months after the race. RESULTS: Left ventricular mass increased significantly (B, 158.5 ± 23.8 g; T1, 165.1 ± 23.2 g; T2, 167 ± 24.6 g; p < 0.001) over the course of the race, although no significant change was seen in the remaining structural and functional parameters. Serum concentrations of cardiac troponin I and NT-proBNP significantly increased 1.5 - and 3.5-fold, respectively, during the first measurement interval, with no further increase thereafter (cardiac troponin I, 6.8 ± 3.1 (B), 16.9 ± 10.4 (T1) and 17.1 ± 9.7 (T2); NT-proBNP, 30.3 ± 22.8 (B), 135.9 ± 177.5 (T1) and 111.2 ± 87.3 (T2)), whereas the growth markers myostatin and GDF11 did not change. No association was observed with functional parameters, including the ejection fraction and the volume of both ventricles. The follow-up scans showed a reduction to baseline values (left ventricular mass 157 ± 19.3 g). CONCLUSIONS: High exercise-induced cardiac volume load for >2 months in ultra-endurance runners results in a physiological structural adaptation with no sign of adverse cardiovascular remodelling.

Mapping Gene Circuits Essential for Germ Layer Differentiation via Loss-of-Function Screens in Haploid Human Embryonic Stem Cells.

Pluripotent stem cells can differentiate into all embryonic germ layers, yet the genes essential for these cell fate transitions in human remain elusive. Here, we mapped the essential genes for the differentiation of human pluripotent stem cells (hPSCs) into the three germ layers by using a genome-wide loss-of-function library established in haploid hPSCs. Strikingly, we observed a high fraction of essential genes associated with plasma membrane, highlighting signaling pathways needed for each lineage differentiation. Interestingly, analysis of all hereditary neurological disorders uncovered high essentiality among microcephaly-causing genes. Furthermore, we demonstrated lineage-specific hierarchies among essential transcription factors and a set of Golgi- and endoplasmic reticulum-related genes needed for the differentiation into all germ layers. Our work sheds light on the gene networks regulating early gastrulation events in human by defining essential drivers of specific embryonic germ layer fates and essential genes for the exit from pluripotency.

Defining Human Pluripotency.

Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.

The essentiality landscape of cell cycle related genes in human pluripotent and cancer cells.

BACKGROUND: Cell cycle regulation is a complex system consisting of growth-promoting and growth-restricting mechanisms, whose coordinated activity is vital for proper division and propagation. Alterations in this regulation may lead to uncontrolled proliferation and genomic instability, triggering carcinogenesis. Here, we conducted a comprehensive bioinformatic analysis of cell cycle-related genes using data from CRISPR/Cas9 loss-of-function screens performed in four cancer cell lines and in human embryonic stem cells (hESCs). RESULTS: Cell cycle genes, and in particular S phase and checkpoint genes, are highly essential for the growth of cancer and pluripotent cells. However, checkpoint genes are also found to underlie the differences between the cell cycle features of these cell types. Interestingly, while growth-promoting cell cycle genes overlap considerably between cancer and stem cells, growth-restricting cell cycle genes are completely distinct. Moreover, growth-restricting genes are consistently less frequent in cancer cells than in hESCs. Here we show that most of these genes are regulated by the tumor suppressor gene TP53, which is mutated in most cancer cells. Therefore, the growth-restriction system in cancer cells lacks important factors and does not function properly. Intriguingly, M phase genes are specifically essential for the growth of hESCs and are highly abundant among hESC-enriched genes. CONCLUSIONS: Our results highlight the differences in cell cycle regulation between cell types and emphasize the importance of conducting cell cycle studies in cells with intact genomes, in order to obtain an authentic representation of the genetic features of the cell cycle.

Defining essential genes for human pluripotent stem cells by CRISPR-Cas9 screening in haploid cells.

The maintenance of pluripotency requires coordinated expression of a set of essential genes. Using our recently established haploid human pluripotent stem cells (hPSCs), we generated a genome-wide loss-of-function library targeting 18,166 protein-coding genes to define the essential genes in hPSCs. With this we could allude to an intrinsic bias of essentiality across cellular compartments, uncover two opposing roles for tumour suppressor genes and link autosomal-recessive disorders with growth-retardation phenotypes to early embryogenesis. hPSC-enriched essential genes mainly encode transcription factors and proteins related to cell-cycle and DNA-repair, revealing that a quarter of the nuclear factors are essential for normal growth. Our screen also led to the identification of growth-restricting genes whose loss of function provides a growth advantage to hPSCs, highlighting the role of the P53-mTOR pathway in this context. Overall, we have constructed an atlas of essential and growth-restricting genes in hPSCs, revealing key aspects of cellular essentiality and providing a reference for future studies on human pluripotency.

Cooperative effects of cofilin (ADF) on actin structure suggest allosteric mechanism of cofilin function.

Using site-specific fluorescence probes and cross-linking we demonstrated that cofilin (ADF), a key regulator of actin cellular dynamics, weakens longitudinal contacts in F-actin in a cooperative manner. Differential scanning calorimetry detected a dual nature of cofilin effects on F-actin conformation. At sub-stoichiometric cofilin to actin ratios, cofilin stabilized sterically and non-cooperatively protomers at the points of attachment, and destabilized allosterically and cooperatively protomers in the cofilin-free parts of F-actin. This destabilizing effect had a long range, with one cofilin molecule affecting more than 100 protomers, and concentration-dependent amplitude that reached maximum at about 1:2 molar ratio of cofilin to actin. In contrast to existing models, our results suggest an allosteric mechanism of actin depolymerization by cofilin. We propose that cofilin is less likely to sever actin filaments at the points of attachment as thought previously. Instead, due to its dual structural effect, spontaneous fragmentation occurs most likely in cofilin-free segments of filaments weakened allosterically by nearby cofilin molecules.

Haploid Human Embryonic Stem Cells: Half the Genome, Double the Value.

Recent advances in the generation of haploid embryonic stem cells (ESCs), capable of self-renewal and differentiation, have laid the groundwork for numerous biomedical applications in developmental biology and reproductive medicine. When combined with the power of genetic screening, haploid human ESCs could advance cancer research, regenerative medicine, and disease modeling.

MuSK is a BMP co-receptor that shapes BMP responses and calcium signaling in muscle cells.

Bone morphogenetic proteins (BMPs) function in most tissues but have cell type-specific effects. Given the relatively small number of BMP receptors, this exquisite signaling specificity requires additional molecules to regulate this pathway's output. The receptor tyrosine kinase MuSK (muscle-specific kinase) is critical for neuromuscular junction formation and maintenance. Here, we show that MuSK also promotes BMP signaling in muscle cells. MuSK bound to BMP4 and related BMPs with low nanomolar affinity in vitro and to the type I BMP receptors ALK3 and ALK6 in a ligand-independent manner both in vitro and in cultured myotubes. High-affinity binding to BMPs required the third, alternatively spliced MuSK immunoglobulin-like domain. In myoblasts, endogenous MuSK promoted BMP4-dependent phosphorylation of SMADs and transcription of Id1, which encodes a transcription factor involved in muscle differentiation. Gene expression profiling showed that MuSK was required for the BMP4-induced expression of a subset of genes in myoblasts, including regulator of G protein signaling 4 (Rgs4). In myotubes, MuSK enhanced the BMP4-induced expression of a distinct set of genes, including transcripts characteristic of slow muscle. MuSK-mediated stimulation of BMP signaling required type I BMP receptor activity but was independent of MuSK tyrosine kinase activity. MuSK-dependent expression of Rgs4 resulted in the inhibition of Ca(2+) signaling induced by the muscarinic acetylcholine receptor in myoblasts. These findings establish that MuSK has dual roles in muscle cells, acting both as a tyrosine kinase-dependent synaptic organizing molecule and as a BMP co-receptor that shapes BMP transcriptional output and cholinergic signaling.

Ectopic expression of Msx2 in mammalian myotubes recapitulates aspects of amphibian muscle dedifferentiation.

In contrast to urodele amphibians and teleost fish, mammals lack the regenerative responses to replace large body parts. Amphibian and fish regeneration uses dedifferentiation, i.e., reversal of differentiated state, as a means to produce progenitor cells to eventually replace damaged tissues. Therefore, induced activation of dedifferentiation responses in mammalian tissues holds an immense promise for regenerative medicine. Here we demonstrate that ectopic expression of Msx2 in cultured mouse myotubes recapitulates several aspects of amphibian muscle dedifferentiation. We found that MSX2, but not MSX1, leads to cellularization of myotubes and downregulates the expression of myotube markers, such as MHC, MRF4 and myogenin. RNA sequencing of myotubes ectopically expressing Msx2 showed downregulation of over 500 myotube-enriched transcripts and upregulation of over 300 myoblast-enriched transcripts. MSX2 selectively downregulated expression of Ptgs2 and Ptger4, two members of the prostaglandin pathway with important roles in myoblast fusion during muscle differentiation. Ectopic expression of Msx2, as well as Msx1, induced partial cell cycle re-entry of myotubes by upregulating CyclinD1 expression but failed to initiate S-phase. Finally, MSX2-induced dedifferentiation in mouse myotubes could be recapitulated by a pharmacological treatment with trichostatin A (TSA), bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 1 (FGF1). Together, these observations indicate that MSX2 is a major driver of dedifferentiation in mammalian muscle cells.

Does polycystic ovary syndrome itself have additional effect on apelin levels?

Objective. The present study was designed to compare serum levels of apelin between lean PCOS women and healthy women with regular menses. Study Design. A total of 30 lean patients with PCOS and 30 healthy subjects were included in this study. Serum apelin levels were compared between groups. Results. Serum apelin levels in lean PCOS patients were not significantly different from the control subjects. Conclusion. Our findings indicate that PCOS itself does not seem to change apelin levels. Further investigation on a large number of subjects will need to be conducted to prove the consistent or variable association in PCOS.