Convergent somatic evolution commences in utero in a germline ribosomopathy.

Clonal tracking of cells using somatic mutations permits exploration of clonal dynamics in human disease. Here, we perform whole genome sequencing of 323 haematopoietic colonies from 10 individuals with the inherited ribosomopathy Shwachman-Diamond syndrome to reconstruct haematopoietic phylogenies. In ~30% of colonies, we identify mutually exclusive mutations in TP53, EIF6, RPL5, RPL22, PRPF8, plus chromosome 7 and 15 aberrations that increase SBDS and EFL1 gene dosage, respectively. Target gene mutations commence in utero, resulting in a profusion of clonal expansions, with only a few haematopoietic stem cell lineages (mean 8, range 1-24) contributing ~50% of haematopoietic colonies across 8 individuals (range 4-100% clonality) by young adulthood. Rapid clonal expansion during disease transformation is associated with biallelic TP53 mutations and increased mutation burden. Our study highlights how convergent somatic mutation of the p53-dependent nucleolar surveillance pathway offsets the deleterious effects of germline ribosomopathy but increases opportunity for TP53-mutated cancer evolution.

eIF6 rebinding dynamically couples ribosome maturation and translation.

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome - license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.

Somatic genetic rescue of a germline ribosome assembly defect.

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.

ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia.

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double-cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

Novel RPL13 Variants and Variable Clinical Expressivity in a Human Ribosomopathy With Spondyloepimetaphyseal Dysplasia.

Spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of disorders with variable growth failure and skeletal impairments affecting the spine and long bone epiphyses and metaphyses. Here we report on four unrelated families with SEMD in which we identified two monoallelic missense variants and one monoallelic splice site variant in RPL13, encoding the ribosomal protein eL13. In two out of four families, we observed autosomal dominant inheritance with incomplete penetrance and variable clinical expressivity; the phenotypes of the mutation-positive subjects ranged from normal height with or without hip dysplasia to severe SEMD with severe short stature and marked skeletal dysplasia. In vitro studies on patient-derived dermal fibroblasts harboring RPL13 missense mutations demonstrated normal eL13 expression, with proper subcellular localization but reduced colocalization with eL28 (p < 0.001). Cellular functional defects in fibroblasts from mutation-positive subjects indicated a significant increase in the ratio of 60S subunits to 80S ribosomes (p = 0.007) and attenuated global translation (p = 0.017). In line with the human phenotype, our rpl13 mutant zebrafish model, generated by CRISPR-Cas9 editing, showed cartilage deformities at embryonic and juvenile stages. These findings extend the genetic spectrum of RPL13 mutations causing this novel human ribosomopathy with variable skeletal features. Our study underscores for the first time incomplete penetrance and broad phenotypic variability in SEMD-RPL13 type and confirms impaired ribosomal function. Furthermore, the newly generated rpl13 mutant zebrafish model corroborates the role of eL13 in skeletogenesis. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Loss of Kat2a enhances transcriptional noise and depletes acute myeloid leukemia stem-like cells.

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy with abnormal progenitor self-renewal and defective white blood cell differentiation. Its pathogenesis comprises subversion of transcriptional regulation, through mutation and by hijacking normal chromatin regulation. Kat2a is a histone acetyltransferase central to promoter activity, that we recently associated with stability of pluripotency networks, and identified as a genetic vulnerability in AML. Through combined chromatin profiling and single-cell transcriptomics of a conditional knockout mouse, we demonstrate that Kat2a contributes to leukemia propagation through preservation of leukemia stem-like cells. Kat2a loss impacts transcription factor binding and reduces transcriptional burst frequency in a subset of gene promoters, generating enhanced variability of transcript levels. Destabilization of target programs shifts leukemia cell fate out of self-renewal into differentiation. We propose that control of transcriptional variability is central to leukemia stem-like cell propagation, and establish a paradigm exploitable in different tumors and distinct stages of cancer evolution.

Less than 30% of patients with acute myeloid leukaemia ­ an aggressive cancer of the white blood cells ­ survive five years post-diagnosis. This disease disrupts the maturation of white blood cells, resulting in the accumulation of immature cells that multiply and survive but are incapable of completing their maturation process. Amongst these, a group of cancer cells known as leukemic stem cells is responsible for continually replenishing the leukaemia, thus perpetuating its growth. Cancers develop when cells in the body acquire changes or mutations to their genetic makeup. The mutations that lead to acute myeloid leukaemia often affect the activity of genes known as epigenetic regulators. These genes regulate which proteins and other molecules cells make by controlling the way in which cells 'read' their genetic instructions. The epigenetic regulator Kat2a is thought to 'tune' the frequency at which cells read their genetic instructions. This tuning mechanism decreases random fluctuations in the execution of the instructions cells receive to make proteins and other molecules. In turn, this helps to ensure that individual cells of the same type behave in a similar way, for example by keeping leukaemia cells in an immature state. Here, Domingues, Kulkarni et al. investigated whether interfering with Kat2a can make acute myeloid leukaemia less aggressive by allowing the immature white blood cells to mature. Domingues, Kulkarni et al. genetically engineered mice to remove Kat2a from blood cells on demand and then inserted a mutation that causes acute myeloid leukaemia. The experiments showed that the loss of Kat2a delayed the development of leukaemia in the mice and progressively depleted leukaemia stem cells, causing the disease to become less aggressive. The results also showed that loss of Kat2a caused more fluctuations in how the white blood cells read their genetic code, which resulted in more variability in the molecules they produced and increased the tendency of the cells to mature. These findings establish that loss of Kat2a causes leukaemia stem cells to mature and stop multiplying by untuning the frequency at which the cells read their genetic instructions. In the future, it may be possible to develop drugs that target human KAT2A to treat acute myeloid leukaemia.

EFL1 mutations impair eIF6 release to cause Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.

Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells.

Serotonin or 5-hydroxytryptamine (5-HT) has been shown to be essential in lots of physiological and pathological processes. It is well known that 5-HT and 5-HT transporter (5-HTT) play important roles in the pulmonary artery in pulmonary hypertension. However, little is known about the function of 5-HTT in other arteries. In this study we found that the expression of 5-HTT was elevated in injured carotid arteries and over-expression of 5-HTT induced proliferation of smooth muscle cells (SMCs); however, this phenotype could be reversed by knocking-down of 5-HTT or endothelial cells conditional medium (EC-CM). A 5-HTT inhibitor, fluoxetine, treated animals also exhibited reduced restenosis after injury. We identified that miR-195 was packaged in the extracellular vesicles from EC-CM. We further confirmed that extracellular vesicles could transfer miR-195 from ECs to SMCs to inhibit the expression of 5-HTT in SMCs and the proliferation of SMCs. These results provide the first evidence that ECs communicate with SMCs via micro-RNA195 in the regulation of the proliferation of SMCs through 5-HTT, which will contribute to a better understanding of communications between ECs and SMCs via micro-RNA. Our findings suggest a potential target for the control of vessel restenosis.

The role of monoxide hemoglobin in color improvement of chicken sausage.

The role of monoxide hemoglobin (COHb) in improvement of chicken sausage color was investigated. COHb and NaNO2 synergistically increased a* values. Addition of 0.1% COHb decreased the residual nitrite content in the presence of 0.001% NaNO2. Compared with controls, the combined treatment resulted in significantly higher nitroso pigment contents while the single treatment resulted in slightly higher nitroso pigment contents. Visible spectrometry indicated that both nitrosohemochrome (NH) and hematin were the main ingredients of pigments extracted from chicken sausage treated with a combination of 0.006% NaNO2 and 0.6% COHb. Formation of NH and hematin caused an increase in a* values and a decrease in L* values, respectively. COHb showed potential for use in meat product formulations.

Nifedipine promotes the proliferation and migration of breast cancer cells.

Nifedipine is widely used as a calcium channel blocker (CCB) to treat angina and hypertension,but it is controversial with respect the risk of stimulation of cancers. In this study, we demonstrated that nifedipine promoted the proliferation and migration of breast cancer cells both invivo and invitro. However, verapamil, another calcium channel blocker, didn't exert the similar effects. Nifedipine and high concentration KCl failed to alter the [Ca2+]i in MDA-MB-231 cells, suggesting that such nifedipine effect was not related with calcium channel. Moreover, nifedipine decreased miRNA-524-5p, resulting in the up-regulation of brain protein I3 (BRI3). Erk pathway was consequently activated and led to the proliferation and migration of breast cancer cells. Silencing BRI3 reversed the promoting effect of nifedipine on the breast cancer. In a summary, nifedipine stimulated the proliferation and migration of breast cancer cells via the axis of miRNA-524-5p-BRI3-Erk pathway independently of its calcium channel-blocking activity. Our findings highlight that nifedipine but not verapamil is conducive for breast cancer growth and metastasis, urging that the caution should be taken in clinic to prescribe nifedipine to women who suffering both hypertension and breast cancer, and hypertension with a tendency in breast cancers.

Carbon monoxide inhibits inward rectifier potassium channels in cardiomyocytes.

Reperfusion-induced ventricular fibrillation (VF) severely threatens the lives of post-myocardial infarction patients. Carbon monoxide (CO)--produced by haem oxygenase in cardiomyocytes--has been reported to prevent VF through an unknown mechanism of action. Here, we report that CO prolongs action potential duration (APD) by inhibiting a subset of inward-rectifying potassium (Kir) channels. We show that CO blocks Kir2.2 and Kir2.3 but not Kir2.1 channels in both cardiomyocytes and HEK-293 cells transfected with Kir. CO directly inhibits Kir2.3 by interfering with its interaction with the second messenger phosphatidylinositol (4,5)-bisphosphate (PIP2). As the inhibition of Kir2.2 and Kir2.3 by CO prolongs APD in myocytes, cardiac Kir2.2 and Kir2.3 are promising targets for the prevention of reperfusion-induced VF.

A novel method to stabilize meat colour: ligand coordinating with hemin.

Carbon monoxide (CO), L-cysteine and L-histidine were tested to coordinate with hemin chloride (pigment containing haem iron). In the presence of sodium dithionite, both CO and L-cysteine could react with hemin to afford respectively the corresponding complexes: CO-hemin and L-cysteine hemin; while L-histidine could not react with hemin. Both CO-hemin and L-cysteine hemin could decompose and release the corresponding ligand to generate hemin. Both light and temperature had an obvious effect on stabilization of these complexes. By sensory evaluation, both CO-hemin and L-cysteine hemin have bright red colour and show a potential as cured cooked-meat pigments (CCMP) in the manufacture of meat product.

Selective extraction of berberine and palmatine from Huangbai powder.

Berberine and palmatine are two of the main bioactive components in Huangbai, a major Chinese medicinal herb. The current methods to extract these compounds usually involving the usage of inorganic acid and base, are not only complex and time-consuming, but have a low selectivity. In this paper, it was reported that hexane, ethyl acetate and dichloromethane were tested to extract berberine and palmatine from Huangbai powder. The results showed that dichloromethane extracted selectively and effectively berberine and palmatine from Huangbai powder among the examined solvents. In addition, dichloromethane can be recycled and reused, making it a potential candidate for large scale extraction of berberine and palmatine from Huangbai.

Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation.

MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. We report here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. We show that pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. We propose that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics.

Mars promotes dTACC dephosphorylation on mitotic spindles to ensure spindle stability.

Microtubule-associated proteins (MAPs) ensure the fidelity of chromosome segregation by controlling microtubule (MT) dynamics and mitotic spindle stability. However, many aspects of MAP function and regulation are poorly understood in a developmental context. We show that mars, which encodes a Drosophila melanogaster member of the hepatoma up-regulated protein family of MAPs, is essential for MT stabilization during early embryogenesis. As well as associating with spindle MTs in vivo, Mars binds directly to protein phosphatase 1 (PP1) and coimmunoprecipitates from embryo extracts with minispindles and Drosophila transforming acidic coiled-coil (dTACC), two MAPs that function as spindle assembly factors. Disruption of binding to PP1 or loss of mars function results in elevated levels of phosphorylated dTACC on spindles. A nonphosphorylatable form of dTACC is capable of rescuing the lethality of mars mutants. We propose that Mars mediates spatially controlled dephosphorylation of dTACC, which is critical for spindle stabilization.