Endocannabinoids Are Expressed in Bone Marrow Stromal Niches and Play a Role in Interactions of Hematopoietic Stem and Progenitor Cells with the Bone Marrow Microenvironment

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB₁ and CB₂. The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB₁ receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10⁷ cells), and 2-AG (75.2 ng/10⁷ cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10⁷ cells) and 2-AG (98.8 ng/10⁷ cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB₁ agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1^−/− showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)^−/− mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1^−/−, as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.     

TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis

In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutant^cas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1^cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1^cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1^cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.     

On Leukocytes in Mammary Development and Cancer

During metastasis, tumor cells may be copying a program that is executed by hematopoietic stem cells during development.That cancer is development gone awry is not a new concept. Most of the “hallmarks” ascribed to cancer—proliferation, invasion and induction of blood vessel growth—also occur during organogenesis and development. Therefore, tumors are not necessarily learning new tricks during their development, but how about when they metastasize? In colonizing a new organ, often with some degree of specificity, tumor cells may simply be copying a program that is executed during development by hematopoietic stem cells (HSCs)—the stem cells that ultimately generate all of the cells in our blood and maintain its homeostasis. One family of cells generated by HSCs—leukocytes—is the focus of the work by Coussens and Pollard (2012). These two scientists have woven together several studies that revolutionized the way we think of immune cells. As pointed out by the investigators (whose respective laboratories are responsible for much of the seminal work on this subject), immune cells also have a variety of trophic functions, and it is these functions that are used rationally during development, and recklessly during tumor growth.This leads us back to metastasis. There is so much to learn about why a tumor travels from one organ to another, how it does so, and the manner by which it adapts to and ultimately flourishes (or fails) in a foreign microenvironment. And as stated above, immune cell precursors, HSCs, do the same. In the mouse, HSCs have originated in one tissue (the dorsal aorta), traveled to another (the placenta) via the circulation, and matured somewhere else (the liver)—all before birth. Finally, HSCs make their way to the bone marrow, where they reside postnatally. Specialized niches in the bone marrow are thought to mediate HSC dormancy as a means to preserve the “stemness” of this population, and there are mechanisms in place that allow these cells to rapidly exit these environs and proliferate in response to injury. Therefore, it should not come as a surprise that a common site where micrometastases are found is the bone marrow for many cancers (including that of the breast).Uncovering whether the same niches that control HSC expansion in the bone marrow are also responsible for maintaining quiescence of tumor cell populations is an exciting prospect, as is deciphering the precise components of these niches. Such work could explain the seemingly incongruous observation that despite an absence of clinically detectable disease, circulating tumor cells are present in the blood of post-treatment cancer patients sometimes even decades later! Perhaps the niches that regulate prolonged dormancy of tumors are dynamic and inhibit tumor proliferation while allowing them to mobilize periodically, much like for HSCs. It also stands to reason that loss of the same controls that prevent HSC expansion until systemic damage occurs could awaken dormant tumors.Shiozawa et al. (2011) have demonstrated that prostate cancer cells do in fact compete with HSCs for niches within the bone marrow, and that tumor cells are mobilized from HSC niches by similar mechanisms as for HSCs. Whether this is the case for other cancers and whether these similarities can be exploited therapeutically remain to be seen.So what more is there to be learned about immune cells? By furthering our understanding of how solid cancers mimic and hijack components of our immune system, we may not “cure” cancer, but we very well may uncover a means to suppress some cancers into a state of permanent dormancy.     

Posttranscriptional regulation of colonic epithelial repair by RNA binding protein IMP1/IGF2BP1

Correction to: EMBO Reports (2019) 20: e47074. DOI 10.15252/embr.201847074 | Published online 6 May 2019The authors noticed that the control and disease labels had been inverted in their data analysis resulting in publication of incorrect data in Figure 1C. The corrected figure is displayed below. This change affects the conclusions as detailed below. The authors apologize for this error and any confusion it may have caused.In the legend of 1C, change from, “Differential gene expression analysis of pediatric ileal CD patient samples (n = 180) shows increased (> 4‐fold) IMP1 expression as compared to non‐inflammatory bowel disease (IBD) pediatric samples (n = 43)”.Open in a separate windowFigure 1CCorrected Open in a separate windowFigure 1COriginal To, "Differential gene expression analysis of pediatric ileal CD patient samples (n = 180) shows decreased (> 4‐fold) IMP1 expression as compared to non‐inflammatory bowel disease (IBD) pediatric samples (n = 43)”.In abstract, change from, “Here, we report increased IMP1 expression in patients with Crohn''s disease and ulcerative colitis”.To, “Here, we report increased IMP1 expression in adult patients with Crohn''s disease and ulcerative colitis”.In results, change from, “Consistent with these findings, analysis of published the Pediatric RISK Stratification Study (RISK) cohort of RNA‐sequencing data 38 from pediatric patients with Crohn''s disease (CD) patients revealed that IMP1 is upregulated significantly compared to control patients and that this effect is specific to IMP1 (i.e., other distinct isoforms, IMP2 and IMP3, are not changed; Fig 1C)”.To, “Contrary to our findings in colon tissue from adults, analysis of published RNA‐sequencing data from the Pediatric RISK Stratification Study (RISK) cohort of ileal tissue from children with Crohn’s disease (CD) 38 revealed that IMP1 is downregulated significantly compared to control patients in the RISK cohort and that this effect is specific to IMP1 (i.e., other distinct isoforms, IMP2 and IMP3, are not changed; Fig 1C)”.In discussion, change from, “Indeed, we report that IMP1 is upregulated in patients with Crohn''s disease and ulcerative colitis and that mice with Imp1 loss exhibit enhanced repair following DSS‐mediated damage”.To “Indeed, we report that IMP1 is upregulated in adult patients with Crohn''s disease and ulcerative colitis and that mice with Imp1 loss exhibit enhanced repair following DSS‐mediated damage”.     

LDL Cholesterol Modulates Human CD34+ HSPCs through Effects on Proliferation and the IL-17 G-CSF Axis

Background

Hypercholesterolemia plays a critical role in atherosclerosis. CD34+ CD45dim Lineage- hematopoietic stem/progenitor cells (HSPCs) give rise to the inflammatory cells linked to atherosclerosis. In mice, high cholesterol levels mobilize HSPCs into the bloodstream, and promote their differentiation to granulocytes and monocytes. The objective of our study was to determine how cholesterol levels affect HSPC quantity in humans.

Methods

We performed a blinded, randomized hypothesis generating study in human subjects (n=12) treated sequentially with statins of differing potencies to vary lipid levels. CD34+ HSPC levels in blood were measured by flow cytometry. Hematopoietic colony forming assays confirmed the CD34+ population studied as HSPCs with multlineage differentiation potential. Mobilizing cytokine levels were measured by ELISA.

Results

The quantity of HSPCs was 0.15 ± 0.1% of buffy coat leukocytes. We found a weak, positive correlation between CD34+ HSPCs and both total and LDL cholesterol levels (r²=0.096, p < 0.025). Additionally, we tested whether cholesterol modulates CD34+ HSPCs through direct effects or on the levels of mobilizing cytokines. LDL cholesterol increased cell surface expression of CXCR4, G-CSFR affecting HSPC migration, and CD47 mediating protection from phagocytosis by immune cells. LDL cholesterol also increased proliferation of CD34+ HSPCs (28 ± 5.7%, n=6, p < 0.03). Finally, the HSPC mobilizing cytokine G-CSF (r²=0.0683, p < 0.05), and its upstream regulator IL-17 (r²=0.0891, p < 0.05) both correlated positively with LDL cholesterol, while SDF-1 levels were not significantly affected.

Conclusions

Our findings support a model where LDL cholesterol levels positively correlate with CD34+ HSPC levels in humans through effects on the levels of G-CSF via IL-17 promoting mobilization of HSPCs, and by direct effects of LDL cholesterol on HSPC proliferation. The findings are provocative of further study to determine if HSPCs, like cholesterol levels, are linked to CVD events.     

Genetic Relationship Between Angustifolia3 and Extra-Small Sisters Highlights Novel Mechanisms Controlling Leaf Size

Coordination between cell proliferation and cell expansion is pivotal in leaf size determination. A group of mutants that are impaired in cell proliferation such as the angustifolia3 (an3) has provided a clue to understanding how these cellular processes are coordinated. In these mutants, impaired cell proliferation is accompanied by enhanced cell enlargement. We propose to call this phenomenon “compensated cell enlargement.” Previously, we isolated ten extra-small sisters (xs) mutants that are specifically impaired in post-mitotic cell expansion and found that several xs mutations are able to suppress compensated cell enlargement in an3. Thus, the enhanced cell expansion observed in an3 results from the hyperactivation of post-mitotic cell expansion involving specific members of the XS gene family. These results suggested that cell proliferation process(es) and post-mitotic cell expansion process(es) are somehow linked in an as yet unknown fashion in leaf primordia. In this addendum, we propose possible models for the linking mechanisms that coordinate AN3-dependent cell proliferation and XS-dependent cell expansion in leaf development.Key Words: Arabidopsis, cell expansion, cell proliferation, extra-small sisters (xs), angustifolia3 (an3), compensated cell enlargement, leaf, organ size controlMature leaf size is determined by the final number and size of cells within a leaf. Thus, the spatial and temporal regulation of cell proliferation and cell expansion plays pivotal roles in establishing developmentally programmed leaf size in a reproducible fashion. During leaf development, cell proliferation is maintained in the basal part of the leaf primordium and terminates basipetally.^1,2 Cells that exit cell cycling undergo differentiation and expand enormously.¹ Although many studies have revealed the molecular mechanisms underlying the above processes, the coordination of cell proliferation and cell expansion in the context of organogenesis is not yet understood.In recent years, an interesting phenomenon has been reported in leaves of several mutants or transgenic plants with impaired cell proliferation. These mutants not only have a defect in cell proliferation, but have larger cells than does the wild type, suggesting that the cell proliferation process interacts with the cell expansion process during leaf organogenesis.^3–8 This phenomenon, called “compensation,” has highlighted the existence of a coordination system between cell proliferation and cell expansion in leaf development.^9–13Conceptually, this compensation can be dissected into two processes: the induction process involves the reduction of cell proliferation and the response process directs the enhancement of cell expansion and “compensated cell enlargement.” Recently, we showed that, in the typical compensation-exhibiting mutant angustifolia3 (an3), the expansion of post-mitotic, but not mitotic, cells is specifically enhanced.⁸ Thus, the induction and the response processes should take place separately in proliferating and differentiating cells, respectively. To dissect compensation genetically, with an emphasis on the response process, we isolated 10 mutants, named extra-small sisters (xs), that are specifically impaired in post-mitotic cell expansion.^14–16 We classified xs mutants into three classes based on the effect of each xs mutation on compensated cell enlargement, using an3 as a representative of compensation-exhibiting mutants.¹⁴ As expected, a group of xs mutants (xs1, xs2, xs4 and xs5) completely suppressed compensated cell enlargement in an3 mutants (named the “small-cell” class), whereas the other two classes had either no suppressive or additive effects on cell enlargement.¹⁴ This finding demonstrated that these XS genes act downstream of cell expansion pathways that are regulated by compensation and triggered in an3. How is this relationship established in the context of leaf organogenesis? When considering the above result, one might speculate that, in addition to a promotive role in cell proliferation, AN3 has a role in cell expansion post-mitotic cells. However, AN3 is hardly expressed in differentiating cells, and the overexpression of AN3 has no effects on post-mitotic cell expansion,⁶ suggesting that this possibility is unlikely. Taken together, these results indicate that the AN3-dependent cell proliferation pathway is somehow linked by an intermediary process to post-mitotic cell expansion pathway(s) involving the small-cell class XS.Based on these data, we propose two possible scenarios for the intermediary process, categorized in terms of cell autonomy (Fig. 1). In the non-cell-autonomous case, proliferating cells located in the basal region of the developing leaf may regulate the expansion of differentiating cells located in the upper region of the leaf via unknown cell-cell communications (Fig. 1A and B). In the cell-autonomous case, the activity involved in cell proliferation may be memorized in each cell, and, depending on this memory, each post-mitotic cell determines its own final size (Fig. 1C). Whatever the mechanism, we can assume that regulatory signal(s) would be affected by cell proliferation. Irrespective of cell autonomy, this putative signal acts either positively or negatively on cell expansion. When cell proliferation is impaired by the an3 mutation, the strength of the negative signal would be reduced and become insufficient to prevent differentiating cells from excessive cell expansion. Conversely, if this signal plays a positive role in cell expansion, the signal may be insufficient to positively control cell expansion in the wild type. However, when the cell number is significantly reduced by the an3 mutation, this positive signal(s) would hyperactivate cell expansion pathways. Further analyses of the factors involved in the intermediary process should provide an important insight into signaling mechanisms that control leaf size.Open in a separate windowFigure 1Proposed models for the leaf size control inferred from the analysis of compensated cell enlargement. (A and B) Non-cell-autonomous model. Cells located in the basal part of a leaf primordium would produce signal(s) that inhibit (A) or promote (B) cell expansion of post-mitotic cells present in the apical part of the leaf primordium. (A) If a significant reduction in cell number occurs in an3, the strength of the inhibitory signal would be reduced and cell expansion would be de-repressed, resulting in the abnormal enlargement of leaf cells. (B) When we assume a cell expansion-promoting signal(s), its strength may be insufficient to enhance cell expansion in the wild type. When a significant reduction in cell number occurs in an3, the promoting signal(s) would increase sufficiently to cause compensation. (C) “Cell memory” model. A specific signal reflecting cell proliferation activity in proliferating cells is retained during cell differentiation and affects the magnitude of cell expansion. Inhibitory and stimulatory signal examples are shown in the upper and lower panels, respectively. The relationship between the strength of the signals and the induction of compensation is the same as that described in the non-cell-autonomous model.Before our reports on the actions of an3, fugu and xs mutants,^8,14 compensated cell enlargement was considered to be caused by the uncoupling of cell division and growth. Now, this possibility is clearly ruled out. Cell proliferation and post-mitotic cell expansion are the most basic cellular processes and are each supported by different regulatory networks. The putative signaling systems discussed here provide a new perspective on how developmental programs integrate these networks into a super-network to control organ size.     

Immunodominant HIV-Specific CD8+ T-Cell Responses Are Common to Blood and Gastrointestinal Mucosa,and Gag-Specific Responses Dominate in Rectal Mucosa of HIV Controllers

Previous studies have suggested that polyfunctional mucosal CD8⁺ T-cell responses may be a correlate of protection in HIV controllers. Mucosal T-cell breadth and/or specificity may also contribute to defining protective responses. In this study, rectal CD8⁺ T-cell responses to HIV Gag, Env, and Nef were mapped at the peptide level in four subject groups: elite controllers (n = 16; viral load [VL], <75 copies/ml), viremic controllers (n = 14; VL, 75 to 2,000 copies/ml), noncontrollers (n = 14; VL, >10,000 copies/ml), and antiretroviral-drug-treated subjects (n = 8; VL, <75 copies/ml). In all subject groups, immunodominant CD8⁺ T-cell responses were generally shared by blood and mucosa, although there were exceptions. In HIV controllers, responses to HLA-B27- and HLA-B57-restricted epitopes were common to both tissues, and their magnitude (in spot-forming cells [SFC] per million) was significantly greater than those of responses restricted by other alleles. Furthermore, peptides recognized by T cells in both blood and rectal mucosa, termed “concordant,” elicited higher median numbers of SFC than discordant responses. In magnitude as well as breadth, HIV Gag-specific responses, particularly those targeting p24 and p7, dominated in controllers. Responses in noncontrollers were more evenly distributed among epitopes in Gag, Env, and Nef. Viremic controllers showed significantly broader mucosal Gag-specific responses than other groups. Taken together, these findings demonstrate that (i) Gag-specific responses dominate in mucosal tissues of HIV controllers; (ii) there is extensive overlap between CD8⁺ T cells in blood and mucosal tissues, with responses to immunodominant epitopes generally shared by both sites; and (iii) mucosal T-cell response breadth alone cannot account for immune control.Despite more than two decades of intensive research, the immunologic correlates of protection from human immunodeficiency virus (HIV) infection and disease progression remain incompletely understood. To date, the majority of studies of HIV-specific T-cell responses have focused on the measurement of such responses in peripheral blood lymphocytes. Nevertheless, the majority of the body''s lymphocytes are housed in mucosal tissues, notably the gastrointestinal (GI) tract (18, 33, 40). The gastrointestinal mucosa also serves as a major target of HIV infection and CD4⁺ T-cell depletion (7, 25, 36), as well as an important site of transmission (18, 33, 40). Antigen-experienced T cells may preferentially traffic to tissue sites of infection (50), where they may also expand in an antigen-driven manner. Because of the unique role of the gastrointestinal mucosa in HIV pathogenesis, detailed studies of HIV-specific immune responses in this compartment may contribute important insights to our understanding of the disease process.An important question is the degree to which T-cell responses in mucosal tissues are “compartmentalized” and distinct in specificity and/or clonality from those found elsewhere in the body, including in peripheral blood. Because of the technical challenges associated with obtaining large numbers of viable lymphocytes from mucosal biopsy specimen tissue, comprehensive mapping of the fine specificity of mucosal HIV-specific T-cell responses has been difficult. Relying on a polyclonal expansion approach, Ibarrondo and colleagues successfully mapped HIV-specific CD8⁺ T-cell responses in blood and rectal mucosa of chronically infected persons to the level of peptide pools but not to individual epitopes (29). Their studies revealed a similar pattern of responses, and nearly identical immunodominance hierarchies, in the two tissue sites.We have focused our recent studies of mucosal immunity on a group of individuals who control HIV infection in the absence of antiretroviral therapy. These are often called “long-term nonprogressors” (LTNP) (14), referring to their ability to maintain normal CD4⁺ T-cell counts for more than 10 years without medication. LTNP are believed to account for 5 to 15% of the HIV-infected population. Several recent studies have used the term “HIV controllers,” defined as those who maintain undetectable plasma HIV RNA levels (“elite controllers”) and those who have persistently detectable but low plasma HIV RNA levels (“viremic controllers”). Elite controllers represent less than 1% of the HIV-infected population (14). In contrast, individuals with viral loads of >10,000 copies/ml in the absence of therapy are termed “noncontrollers.” Recently, we found that “polyfunctional” HIV-specific T cells, producing multiple antiviral factors, were significantly more abundant in gastrointestinal mucosa of HIV controllers than in those of noncontrollers or subjects on highly active antiretroviral therapy (HAART) (20). Furthermore, in many cases these strong, polyfunctional mucosal T-cell responses were not mirrored in peripheral blood, suggesting that HIV-specific T cells either preferentially traffic to or undergo expansion within mucosal tissues.Because of these findings, we undertook a follow-up study to determine the breadth and fine specificity, to the peptide level, of mucosal CD8⁺ T-cell responses to HIV Gag, Env, and Nef among HIV controllers, noncontrollers, and individuals on HAART. We hypothesized that controllers might harbor an unusually broad repertoire of HIV-specific CD8⁺ T cells in mucosal tissues. We found a similar response breadth in mucosal tissues of all three subject groups, arguing against a critical role for mucosal T-cell response breadth in determining the extent of HIV control. In contrast, we found that high-magnitude mucosal responses directed at well-conserved regions in Gag were a strong and consistent correlate of control. Finally, concordant responses, defined as those common to blood and mucosa, were generally stronger than discordant responses, underscoring the observation that T cells responding to immunodominant epitopes are broadly distributed throughout the body in both controllers and noncontrollers.     

Blood loses it when nerves go bad

Myeloproliferative neoplasms are diseases that arise in the stem cells of the blood. In a recent paper published in Nature, Arranz et al. demonstrated that abrogation of sympathetic nerve fibers reduced bone marrow Nestin⁺ mesenchymal cells, which in turn led to an expansion of hematopoietic stem cells and progression of myeloproliferative neoplasms.The stromal cell compartment, or non-hematopoietic cells, of the bone marrow has emerged as an important driver of cell state in hematopoietic stem cells (HSCs) and hematopoietic stem/progenitor cells (HSPCs) in a non-autonomous manner. The HSC niche is not well defined and a number of studies suggest that there are specialized niches for the unique regulation of HSCs and HSPCs¹. Several studies suggest that HSCs reside in a perivascular niche in which a heterogenic population of perivascular mesenchymal stromal cells (MSCs) with overlapping expression of Nestin, LepR, Prx1, or Mx1 each synthesize multiple factors (e.g., CXCL12 and SCF) that promote maintenance and/or localization of HSCs^1,2. Endothelial cells (Tie2⁺)³, and MSCs (NG2⁺/LepR⁻) surrounding arterial vessels⁴, are other units of the niche reported to regulate HSC numbers and quiescence, respectively. Furthermore, osteoprogenitors lining the endosteal surface have a more indirect role in the regulation of HSCs⁶. Thus, it appears that hematopoietic regulation and differentiation in the bone marrow microenvironment is governed at multiple levels⁵.Increasing evidence suggests that dysregulation of the bone marrow microenvironment may participate in blood malignancies. For instance, perturbations of the miRNA processing or ribosomal components through Dicer1 deletion in immature osteolineage cells induced myelodysplasia in mice, followed by the rare emergence of acute myeloid leukemia (AML)⁶. Others reported that β-catenin stabilization in mature osteolineage cells resulted in Notch pathway activation, myelodysplastic syndrome, and highly penetrant AML in mice⁷. In humans, ∼5% of post-transplant AML patients relapse with a leukemia of donor cell origin, suggesting that some patients may have a microenvironmental driver of leukemogenesis⁸. Together, these studies are consistent with a role of the bone marrow microenvironment in maintaining the integrity of hematopoiesis and restricting oncogenesis. When the well-orchestrated regulation of hematopoiesis is disrupted, blood malignancies might occur.The study by Arranz et al.⁹ is a continuation of prior work identifying perivascular bone marrow Nestin⁺ MSCs affected by sympathetic nerve fibers to regulate HSCs¹⁰. Previous studies that a perturbed bone marrow microenvironment modulates myeloproliferative neoplasms (MPNs)^6,7 prompted the authors to further investigate the role of Nestin⁺ MSCs in MPN, specifically MPN associated with Janus kinase 2 (JAK2) mutations^11,12.The authors first analyzed Nestin expression in bone marrow samples from MPN patients and discovered that despite elevated blood-vessel density, Nestin⁺ cell numbers and mRNA expression were reduced. Similar findings were observed in genetically engineered mice that recapitulate human MPNs (e.g., Mx1-cre; JAK2^V617F), indicating that Nestin⁺ MSCs might play a role in MPN. Arranz et al. proceeded to investigate whether a selective depletion of Nestin⁺ MSCs mimics the MPN mouse model. Mice depleted of Nestin⁺ MSCs showed an expansion of HSCs, due to a drop in CXCL12 expression, accompanied by increased hematopoietic progenitors in bone marrow, peripheral blood and spleen, indicative of MPN. Extensive genome-wide RNA-sequencing studies revealed enrichment of Schwann cell- and neural-related genes in Nestin⁺ MSCs derived from MPN mice. This result prompted the authors to explore the role of sympathetic nerve fibers and nonmyelinating Schwann cells in MPN patients and the MPN mouse model. Strikingly, both MPN patients and MPN mice had reduced sympathetic nerve fibers and nonmyelinating Schwann cells adjacent to Nestin⁺ cells in the bone marrow. Multiplex ELISA experiments identified that mutant HSCs secrete IL-1β, which induced apoptosis in bone marrow Schwann cells by Caspase-1 activation followed by neuronal damage. Neural-glial damage in turn compromised Nestin⁺ MSCs survival and led to MPN. Finally, the authors rescued the MPN phenotype partially in MPN mice by treating the mutant mice with IL-1R antagonist, a neuroglial protection agent (4-methylcatechol), or a β3-adrenergic agonist (BRL37344) which compensated for deficient sympathetic stimulation. This treatment was selective against mutant hematopoietic progenitors and preserved normal HSCs, and this effect could only be observed in vivo. Therefore, the authors concluded that the effect was niche-dependent (Figure 1).Open in a separate windowFigure 1Bone marrow neuropathy leads to mutant HSC expansion in MPN. (1) IL-1β is released by mutant HSCs, which induces Caspase-1-dependent apoptosis in sympathetic nerve fibers, ensheathed by nonmyelinating Schwann cells. (2) Neural-glial damage leads to a reduced noradrenergic sympathetic stimulation of Nestin⁺ MSCs and loss of MSCs. (3) Aberrant neural regulation sensitizes Nestin⁺ MSCs to IL-1β-induced apoptosis with a subsequent drop in CXCL12 expression. (4) HSC and progenitor cell proliferation is increased, followed by MPN pathogenesis. (5) Nestin⁺ MSCs survival and function can be restored by the neuroglial protective agent 4-methylcatechol, the β3-adrenergic agonist BRL37344, or by blocking IL-1R. MPN, myeloproliferative neoplasm; HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; NA, noradrenaline; AR, adrenergic receptor; IL, Interleukin; IL-1R, Interleukin-1 receptor.While the prevailing understanding of cancer as a disease in which changes in the cell of origin drive oncogenic transformation, these studies point to the potential for the microenvironment as a critical cooperator in the malignant process for at least some neoplasms. This study emphasizes that there may be a two-way perturbation process required for MPN. HSCs acquire a mutation (e.g., JAK2-V617F mutation) that leads to cell expansion and the mutant HSC perturbs the bone marrow niche, which further drive HSCs into neoplasia. Based on these findings, the authors postulated that neural-glial protective agents and β3-adrenergic agonists may subvert the process and be therapeutically useful. Therefore, this model provides insight into how the neural compartment of the bone marrow microenvironment can serve as a modulator of malignancy and offers a novel, testable approach for treating MPNs — by not only targeting the malignant cell, but also by selectively targeting the unhealthy niche.     

Experts in science communication: A shift from neutral encyclopedia to equal participant in dialogue

Even if the predominant model of science communication with the public is now based on dialogue, many experts still adhere to the outdated deficit model of informing the public. Subject Categories: Genetics, Gene Therapy & Genetic Disease, S&S: History & Philosophy of Science, S&S: Ethics

During the past decades, public communication of science has undergone profound changes: from policy‐driven to policy‐informing, from promoting science to interpreting science, and from dissemination to interaction (Burgess, 2014). These shifts in communication paradigms have an impact on what is expected from scientists who engage in public communication: they should be seen as fellow citizens rather than experts whose task is to increase scientific literacy of the lay public. Many scientists engage in science communication, because they see this as their responsibility toward society (Loroño‐Leturiondo & Davies, 2018). Yet, a significant proportion of researchers still “view public engagement as an activity of talking to rather than with the public” (Hamlyn et al, 2015). The highly criticized “deficit model” that sees the role of experts as educating the public to mitigate skepticism still persists (Simis et al, 2016; Suldovsky, 2016).Indeed, a survey we conducted among experts in training seems to corroborate the persistence of the deficit model even among younger scientists. Based on these results and our own experience with organizing public dialogues about human germline gene editing (Box 1), we discuss the implications of this outdated science communication model and an alternative model of public engagement, that aims to align science with the needs and values of the public.Box 1

The DNA‐dialogue project

The Dutch DNA‐dialogue project invited citizens to discuss and form opinions about human germline gene editing. During 2019 and 2020, this project organized twenty‐seven dialogues with professionals, such as embryologists and midwives, and various lay audiences. Different scenarios of a world in 2039 (https://www.rathenau.nl/en/making‐perfect‐lives/discussing‐modification‐heritable‐dna‐embryos) served as the starting point. Participants expressed their initial reactions to these scenarios with emotion‐cards and thereby explored the values they themselves and other participants deemed important as they elaborated further. Starting each dialogue in this way provides a context that enables everyone to participate in dialogue about complex topics such as human germline gene editing and demonstrates that scientific knowledge should not be a prerequisite to participate.An important example of “different” relevant knowledge surfaced during a dialogue with children between 8 and 12 years in the Sophia Children’s Hospital in Rotterdam (Fig 1). Most adults in the DNA‐dialogues accepted human germline gene modification for severe genetic diseases, as they wished the best possible care and outcome for their children. The children at Sophia, however, stated that they would find it terrible if their parents had altered something about them before they had been born; their parents would not even have known them. Some children went so far to say they would no longer be themselves without their genetic condition, and that their condition had also given them experiences they would rather not have missed.Open in a separate windowFigure 1 Children participating in a DNA‐dialogue meeting. Photographed by Levien Willemse.     

Sex chromosome evolution: life,death and repetitive DNA

Dimorphic sex chromosomes create problems. Males of many species, including Drosophila, are heterogametic, with dissimilar X and Y chromosomes. The essential process of dosage compensation modulates the expression of X-linked genes in one sex to maintain a constant ratio of X to autosomal expression. This involves the regulation of hundreds of dissimilar genes whose only shared property is chromosomal address. Drosophila males dosage compensate by up regulating X-linked genes 2 fold. This is achieved by the Male Specific Lethal (MSL) complex, which is recruited to genes on the X chromosome and modifies chromatin to increase expression. How the MSL complex is restricted to X-linked genes remains unknown. Recent studies of sex chromosome evolution have identified a central role for 2 types of repetitive elements in X recognition. Helitrons carrying sites that recruit the MSL complex have expanded across the X chromosome in at least one Drosophila species.¹ Our laboratory found that siRNA from an X-linked satellite repeat promotes X recognition by a yet unknown mechanism.² The recurring adoption of repetitive elements as X-identify elements suggests that the large and mysterious fraction of the genome called “junk” DNA is actually instrumental in the evolution of sex chromosomes.     

Pharmacological Modulation of Hemodynamics in Adult Zebrafish In Vivo

Introduction

Hemodynamic parameters in zebrafish receive increasing attention because of their important role in cardiovascular processes such as atherosclerosis, hematopoiesis, sprouting and intussusceptive angiogenesis. To study underlying mechanisms, the precise modulation of parameters like blood flow velocity or shear stress is centrally important. Questions related to blood flow have been addressed in the past in either embryonic or ex vivo-zebrafish models but little information is available for adult animals. Here we describe a pharmacological approach to modulate cardiac and hemodynamic parameters in adult zebrafish in vivo.

Materials and Methods

Adult zebrafish were paralyzed and orally perfused with salt water. The drugs isoprenaline and sodium nitroprusside were directly applied with the perfusate, thus closely resembling the preferred method for drug delivery in zebrafish, namely within the water. Drug effects on the heart and on blood flow in the submental vein were studied using electrocardiograms, in vivo-microscopy and mathematical flow simulations.

Results

Under control conditions, heart rate, blood flow velocity and shear stress varied less than ± 5%. Maximal chronotropic effects of isoprenaline were achieved at a concentration of 50 μmol/L, where it increased the heart rate by 22.6 ± 1.3% (n = 4; p < 0.0001). Blood flow velocity and shear stress in the submental vein were not significantly increased. Sodium nitroprusside at 1 mmol/L did not alter the heart rate but increased blood flow velocity by 110.46 ± 19.64% (p = 0.01) and shear stress by 117.96 ± 23.65% (n = 9; p = 0.03).

Discussion

In this study, we demonstrate that cardiac and hemodynamic parameters in adult zebrafish can be efficiently modulated by isoprenaline and sodium nitroprusside. Together with the suitability of the zebrafish for in vivo-microscopy and genetic modifications, the methodology described permits studying biological processes that are dependent on hemodynamic alterations.     

Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Background

Cord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34⁺ cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion.

Methodology/Principal Findings

CB CD34⁺ cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34⁺ cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone.

Conclusion/Significance

Our data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34⁺ cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts.     

Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT

In “Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT” by Lyu et al (2021), the authors depict the inhibitor MMV007839 in its hemiketal form in Fig 3A and F, Fig 4C, and Appendix Figs S10A, B and S13. We note that Golldack et al (2017) reported that the linear vinylogous acid tautomer of MMV007839 constitutes the binding and inhibitory entity of PfFNT. The authors are currently obtaining higher resolution cryo‐EM structural data of MMV007839‐bound PfFNT to ascertain which of the interconvertible isoforms is bound and the paper will be updated accordingly.     

Dan Salah Tawfik (1955‐2021)—A giant of protein evolution

It was with great sorrow that we have learned of the untimely death of our friend, mentor, collaborator, and hero, Dan Tawfik. Danny was a true legend in the field of protein function and evolution. He had an incredibly creative mind and a breadth of knowledge—his interests spanned chemistry and engineering to genetics and evolution—that allowed him to see connections that the rest of us could not. More importantly, he made solving biochemical mysteries fun: He was passionate about his work, and his face lit up with joy whenever he talked about scientific topics that excited him (of which there were a lot). Conversations with Danny made us all smarter by osmosis.Danny’s own evolution in science began with physical organic chemistry and biochemistry. His PhD at the Weizmann Institute of Science, awarded in 1995, was on catalytic antibodies under the supervision of Zelig Eshhar and Michael Sela. It was followed by a highly productive period at the University of Cambridge’s Centre for Protein Engineering, first as a postdoctoral fellow with Alan Fersht and Tony Kirby, and then as a senior researcher. Among his many achievements during his time in Cambridge was the demonstration that off‐the‐shelf proteins—the serum albumins—could rival the best catalytic antibodies in accelerating the Kemp elimination reaction due to non‐specific medium effects. This work was an early example of unexpected catalytic promiscuity, and it sowed the seed for Danny’s later fascination with “esoteric, niche enzymology” that went far beyond convenient model systems.It was also in Cambridge where Danny first realized the power of the then new field of directed evolution, both for biotechnology and for elucidating evolutionary processes. He and Andrew Griffiths pioneered emulsion‐based in vitro compartmentalization. The idea of controlling biochemical reactions in separate aqueous droplets inspired emulsion PCR and next‐generation sequencing technologies, whereas Danny used it to solve a long‐standing problem in directed evolution; in vitro selection techniques had always been good at identifying ligand‐binding proteins, but compartmentalization finally enabled the directed evolution of ultra‐fast catalysts.Danny returned to Israel in 2001 to join the faculty of the Weizmann Institute of Science where his scientific trajectory further evolved, diverged, and even “drifted”. He developed new methods for enzyme engineering and applied his evolutionary insights into de novo protein design efforts. In this context, Danny’s interest was always focused on how proteins evolve, particularly the connection between promiscuity, conformational diversity, and evolvability. His depth of understanding underpinned both applied research, such as engineering enzymes to detoxify nerve agents, and fundamental research, such as the evolution of enzymes from non‐catalytic scaffolds.Through it all, Danny retained his sense of joy and wonder at the “beautiful aspects of Nature’s chemistry”. This includes his discovery of an exquisite molecular specificity mechanism mediated by a single, short H‐bond that enables microbes to scavenge phosphate in arsenate‐rich environments. In recent years, he deciphered the biosynthetic mechanism of dimethyl sulfide, “the smell of the sea”, and homed in on the interplay between the evolution of an enzyme, its host organism, and environmental complexity. His insights into how the first proteins emerged caused tremendous excitement in the field. He established the roots of two common enzyme lineages, the Rossmann and P‐loop NTPases, as simple polypeptides, and suggested ornithine as the first cationic amino acid. Prior to his death, he published the results of another tour de force: evidence that the first organisms to utilize oxygen may have appeared much earlier than thought.His work impacted many research fields, and he won many significant awards. Most recently, Danny was awarded the EMET Prize for Art, Science and Culture (2020), informally dubbed “Israel’s Nobel Prize”. He was an active and valued member of the EMBO community, having been elected in 2009, and, until his passing, served on the Editorial Advisory Board of EMBO Reports.Danny was also a superb science communicator. Both his research articles and reviews are a joy to read. What stood out just as much as his brilliance was his personality, as he embodied the Yiddish concept of being a true “mensch”. Danny was humble, was down‐to‐earth, and treated all his colleagues—including the most junior members of our research teams—as equals. He championed the careers of others, both those who worked directly for him and those who were lucky enough to be “just” his friends and collaborators. He believed in us even when we did not believe in ourselves, and he was always there to answer questions both scientific and professional. While he loved to share his own ideas, he would be just as excited about ours. Despite his own busy schedule, he always found the time to help others. He was also excellent company, with a great, very dry, sense of humor, and endless interesting stories, including from his own colorful life. In the days after his untimely death, an often‐repeated phrase was “he was my best friend”. Danny’s former group members have gone on to be highly successful in both industry and academia, including more than 15 former doctoral and postdoctoral researchers who are now faculty. The network of researchers Danny has trained, mentored, or influenced is broad, and this legacy is testament to his qualities as both a scientist and a person.Danny was born in Jerusalem to an Iraqi Jewish family, and his Arabic Jewish identity was important to him. He believed strongly in coexistence and peace, and very much valued the Arabic part of his heritage. In his own words: “I am an Israeli, a Jew, an Arab, but first and foremost a human being”. He would often speak of the achievements of his children with immense pride. Danny also had a passion for being outdoors, especially climbing and hiking—when the best discussions were often to be had (Fig ​(Fig1).1). One of the easiest ways to persuade him to come for a seminar, a collaborative visit, or a conference was to have access to high‐quality climbing in the area. He passed away in a tragic rock‐climbing accident, doing what he loved most outside of science. Our thoughts are with his partner Ita and his children, and we join the much broader community of friends, collaborators, and colleagues whose hearts are broken by his sudden loss.Open in a separate windowFigure 1Dan Salah Tawfik (1955–2021)Photo courtesy of Prof. Joel Mackay, The University of Sydney.