Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

DNMT1 Deficiency Impacts on Plasmacytoid Dendritic Cells in Homeostasis and Autoimmune Disease.

Dendritic cells (DCs) are heterogeneous immune regulators involved in autoimmune diseases. Epigenomic mechanisms orchestrating DC development and DC subset diversification remain insufficiently understood but could be important to modulate DC fate for clinical purposes. By combining whole-genome methylation assessment with the analysis of mice expressing reduced DNA methyltransferase 1 levels, we show that distinct DNA methylation levels and patterns are required for the development of plasmacytoid DC and conventional DC subsets. We provide clonal in vivo evidence for DC lineage establishment at the stem cell level, and we show that a high DNA methylation threshold level is essential for Flt3-dependent survival of DC precursors. Importantly, reducing methylation predominantly depletes plasmacytoid DC and alleviates systemic lupus erythematosus in an autoimmunity mouse model. This study shows how DNA methylation regulates the production of DC subsets and provides a potential rationale for targeting autoimmune disease using hypomethylating agents.

Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.

Rare multipotent haematopoietic stem cells (HSCs) in adult bone marrow with extensive self-renewal potential can efficiently replenish all myeloid and lymphoid blood cells, securing long-term multilineage reconstitution after physiological and clinical challenges such as chemotherapy and haematopoietic transplantations. HSC transplantation remains the only curative treatment for many haematological malignancies, but inefficient blood-lineage replenishment remains a major cause of morbidity and mortality. Single-cell transplantation has uncovered considerable heterogeneity among reconstituting HSCs, a finding that is supported by studies of unperturbed haematopoiesis and may reflect different propensities for lineage-fate decisions by distinct myeloid-, lymphoid- and platelet-biased HSCs. Other studies suggested that such lineage bias might reflect generation of unipotent or oligopotent self-renewing progenitors within the phenotypic HSC compartment, and implicated uncoupling of the defining HSC properties of self-renewal and multipotency. Here we use highly sensitive tracking of progenitors and mature cells of the megakaryocyte/platelet, erythroid, myeloid and B and T cell lineages, produced from singly transplanted HSCs, to reveal a highly organized, predictable and stable framework for lineage-restricted fates of long-term self-renewing HSCs. Most notably, a distinct class of HSCs adopts a fate towards effective and stable replenishment of a megakaryocyte/platelet-lineage tree but not of other blood cell lineages, despite sustained multipotency. No HSCs contribute exclusively to any other single blood-cell lineage. Single multipotent HSCs can also fully restrict towards simultaneous replenishment of megakaryocyte, erythroid and myeloid lineages without executing their sustained lymphoid lineage potential. Genetic lineage-tracing analysis also provides evidence for an important role of platelet-biased HSCs in unperturbed adult haematopoiesis. These findings uncover a limited repertoire of distinct HSC subsets, defined by a predictable and hierarchical propensity to adopt a fate towards replenishment of a restricted set of blood lineages, before loss of self-renewal and multipotency.

Multiple Levels of Control Determine How E4bp4/Nfil3 Regulates NK Cell Development.

The transcription factor E4bp4/Nfil3 has been shown to have a critical role in the development of all innate lymphoid cell types including NK cells. In this study, we show that posttranslational modifications of E4bp4 by either SUMOylation or phosphorylation have profound effects on both E4bp4 function and NK cell development. We examined the activity of E4bp4 mutants lacking posttranslational modifications and found that Notch1 was a novel E4bp4 target gene. We observed that abrogation of Notch signaling impeded NK cell production and the total lack of NK cell development from E4bp4-/- progenitors was completely rescued by short exposure to Notch peptide ligands. This work reveals both novel mechanisms in NK cell development by a transcriptional network including E4bp4 with Notch, and that E4bp4 is a central hub to process extrinsic stimuli.

Disrupted brain network dynamics and cognitive functions in methamphetamine use disorder: insights from EEG microstates.

BACKGROUND: Dysfunction in brain network dynamics has been found to correlate with many psychiatric disorders. However, there is limited research regarding resting electroencephalogram (EEG) brain network and its association with cognitive process for patients with methamphetamine use disorder (MUD). This study aimed at using EEG microstate analysis to determine whether brain network dynamics in patients with MUD differ from those of healthy controls (HC). METHODS: A total of 55 MUD patients and 27 matched healthy controls were included for analysis. The resting brain activity was recorded by 64-channel electroencephalography. EEG microstate parameters and intracerebral current sources of each EEG microstate were compared between the two groups. Generalized linear regression model was used to explore the correlation between significant microstates with drug history and cognitive functions. RESULTS: MUD patients showed lower mean durations of the microstate classes A and B, and a higher global explained variance of the microstate class C. Besides, MUD patients presented with different current density power in microstates A, B, and C relative to the HC. The generalized linear model showed that MA use frequency is negatively correlated with the MMD of class A. Further, the generalized linear model showed that MA use frequency, scores of Two-back task, and the error rate of MA word are correlated with the MMD and GEV of class B, respectively. CONCLUSIONS: Intracranial current source densities of resting EEG microstates are disrupted in MUD patients, hence causing temporal changes in microstate topographies, which are correlated with attention bias and history of drug use.

Improvement of wheat seed vitality by dielectric barrier discharge plasma treatment.

Influences of discharge voltage on wheat seed vitality were investigated in a dielectric barrier discharge (DBD) plasma system at atmospheric pressure and temperature. Six different treatments were designed, and their discharge voltages were 0.0, 9.0, 11.0, 13.0, 15.0, and 17.0 kV, respectively. Fifty seeds were exposed to the DBD plasma atmosphere with an air flow rate of 1.5 L min-1 for 4 min in each treatment, and then the DBD plasma-treated seeds were prepared for germination in several Petri dishes. Each treatment was repeated three times. Germination indexes, growth indexes, surface topography, water uptake, permeability, and α-amylase activity were measured. DBD plasma treatment at appropriate energy levels had positive effects on wheat seed germination and seedling growth. The germination potential, germination index, and vigor index significantly increased by 31.4%, 13.9%, and 54.6% after DBD treatment at 11.0 kV, respectively, in comparison to the control. Shoot length, root length, dry weight, and fresh weight also significantly increased after the DBD plasma treatment. The seed coat was softened and cracks were observed, systematization of the protein was strengthened, and amount of free starch grain increased after the DBD plasma treatment. Water uptake, relative electroconductivity, soluble protein, and α-amylase activity of the wheat seed were also significantly improved after the DBD plasma treatment. Roles of active species and ultraviolet radiation generated in the DBD plasma process in wheat seed germination and seedling growth are proposed. Bioelectromagnetics. 39:120-131, 2018. © 2017 Wiley Periodicals, Inc.

Identification of functionally important residues in TFPI Kunitz domain 3 required for the enhancement of its activity by protein S.

Protein S is a cofactor for tissue factor pathway inhibitor (TFPI) that critically reduces the inhibition constant for FXa to below the plasma concentration of TFPI. TFPI Kunitz domain 3 is required for this enhancement to occur. To delineate the molecular mechanism underlying enhancement of TFPI function, in the present study, we produced a panel of Kunitz domain 3 variants of TFPI encompassing all 12 surface-exposed charged residues. Thrombin-generation assays in TFPI-depleted plasma identified a novel variant, TFPI E226Q, which exhibited minimal enhancement by protein S. This was confirmed in purified FXa inhibition assays in which no protein S enhancement of TFPI E226Q was detected. Surface plasmon resonance demonstrated concentration-dependent binding of protein S to wild-type TFPI, but almost no binding to TFPI E226Q. We conclude that the TFPI Kunitz domain 3 residue Glu226 is essential for TFPI enhancement by protein S.

Application value of peripheral blood IgG and IgM combined with ultrasonic echo parameters of substantia nigra in the diagnosis of Parkinson's disease.

We study the clinical value of peripheral blood immunoglobulin G (IgG) and immunoglobulin M (IgM) combined with ultrasonic echo parameters of substantia nigra (SN) in the diagnosis of Parkinson's disease (PD). The clinical data of 121 patients with PD (case group) in our hospital from November 2020 to November 2022 were selected for retrospective analysis, and 9 patients with poor sound transmission of temporal window were excluded. Finally, this study included 112 patients with PD and selected 108 health examination population in the same period (control group). The levels of IgG and IgM in both groups were detected, and ultrasound examination was carried out to observe the structure of SN and obtain strong echo area of SN, midbrain area and strong echo area of SN/midbrain area. The receiver operator characteristic curve of serum IgG and IgM combined with ultrasonic echo parameters of SN in the diagnosis of PD was drawn to evaluate the clinical efficacy of single diagnosis and combined diagnosis. Compared with the control group, the serum levels of IgG and IgM, strong echo area of SN, midbrain area and strong echo area of SN/midbrain area in the case group were obviously higher (P < 0.001), while the folic acid level was notably lower (P < 0.05). The AUC value, Youden index and sensitivity of combined diagnosis were higher than those of single detection. Peripheral blood IgG and IgM combined with ultrasonic echo parameters of SN have high clinical value in the diagnosis of PD, which can provide a new direction for the subsequent diagnosis of PD.

Epigenetic programming defines haematopoietic stem cell fate restriction.

Haematopoietic stem cells (HSCs) are multipotent, but individual HSCs can show restricted lineage output in vivo. Currently, the molecular mechanisms and physiological role of HSC fate restriction remain unknown. Here we show that lymphoid fate is epigenetically but not transcriptionally primed in HSCs. In multi-lineage HSCs that produce lymphocytes, lymphoid-specific upstream regulatory elements (LymUREs) but not promoters are preferentially accessible compared with platelet-biased HSCs that do not produce lymphoid cell types, providing transcriptionally silent lymphoid lineage priming. Runx3 is preferentially expressed in multi-lineage HSCs, and reinstating Runx3 expression increases LymURE accessibility and lymphoid-primed multipotent progenitor 4 (MPP4) output in old, platelet-biased HSCs. In contrast, platelet-biased HSCs show elevated levels of epigenetic platelet-lineage priming and give rise to MPP2 progenitors with molecular platelet bias. These MPP2 progenitors generate platelets with faster kinetics and through a more direct cellular pathway compared with MPP2s derived from multi-lineage HSCs. Epigenetic programming therefore predicts both fate restriction and differentiation kinetics in HSCs.

Deubiquitinase USP29 correlates RORγt expression and its association with thymoma myasthenia gravis.

OBJECTIVE: The objective of this study was to examine the expression of deubiquitylases USP29 in thymomas with myasthenia gravis (MG) and research associated immunological processes. METHODS: 69 MG patients with thymomas, 21 thymoma patients without MG, and 31 healthy controls were classified into three groups (categories): group with MG-associated thymoma (MG-T), group with non-MG-associated thymoma (NMG-T), and group with healthy controls (HC). In thymomas, the mRNA and protein levels of RORγt and USP29 were examined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and western blotting. Th17 cell counts in MG patients with thymomas were investigated by flow cytometry. RESULTS: In MG-related thymomas, the mRNA and protein levels of deubiquitylases USP29 were substantially elevated. USP29 post-transcriptionally regulated RORγt. In MG patients with thymomas, the expression of USP29 was positively linked to the RORγt expression and Th17 cell frequency. CONCLUSION: This work exhibited that the elevated USP29 enhanced RORγt expression, which in turn affected the Th17 cell growth in thymomatous MG. Our data suggest that USP29 might take part in the regulation of RORγt expression and Th17 cell generation and constitute an innovative regulatory function for USP29 in autoimmune disease.

Modulation of Methamphetamine-Related Attention Bias by Intermittent Theta-Burst Stimulation on Left Dorsolateral Prefrontal Cortex.

BACKGROUND: Previous studies have identified the treatment effect of repetitive transcranial magnetic stimulation (rTMS) on cravings of patients with methamphetamine use disorder (MUD). However, the mechanism underlying the treatment effect remains largely unknown. A potential candidate mechanism could be that rTMS over the dorsolateral prefrontal cortex (DLPFC) modulates the attention bias to methamphetamine-related cues. The purpose of this study is therefore to determine the modulation of rTMS on methamphetamine-related attention bias and the corresponding electrophysiological changes. METHODS: Forty-nine patients with severe MUD were included for analysis. The subjects were randomized to receive the active intermittent theta-burst stimulation (iTBS) or sham iTBS targeting DLPFC for 20 sessions. Participants performed the Addiction Stroop Task before and after the treatment while being recorded by a 64-channel electroencephalogram. Baseline characteristics were collected through the Addiction Severity Index. RESULTS: Post-treatment evaluations showed a reduced error rate in discriminating the color of methamphetamine words in the active iTBS group compared with the sham iTBS group. Following rTMS treatment, we found the significant time-by-group effect for the N1 amplitude (methamphetamine words > neutral words) and P3 latency (methamphetamine words > neutral words). The change of N1 amplitude was positively correlated with cravings in the active group. Moreover, reduced power of neural oscillation in the beta band, manifesting at frontal central areas, was also found in the active group. CONCLUSION: This study suggests that attention bias and the beta oscillation during the attentional processing of methamphetamine words in patients with MUD could be modulated by iTBS applied to left DLPFC.

Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors.

Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta-gonad-mesonephros (AGM) blood development. Loss of EZH2 activity in hemogenic endothelium (HE) leads to the generation of phenotypically intact but functionally deficient erythro-myeloid progenitors (EMPs), while the generation of primitive erythroid cells is not affected. EZH2 activity is critical for the generation of functional EMPs at the onset of the endothelial-to-hematopoietic transition but subsequently dispensable. We identify a lack of Wnt signaling downregulation as the primary reason for the production of non-functional EMPs. Together, our findings demonstrate a critical and stage-specific role of Ezh2 in modulating Wnt signaling during the generation of EMPs from YS HE.

Micro-environmental sensing by bone marrow stroma identifies IL-6 and TGFß1 as regulators of hematopoietic ageing.

Hematopoietic ageing involves declining erythropoiesis and lymphopoiesis, leading to frequent anaemia and decreased adaptive immunity. How intrinsic changes to the hematopoietic stem cells (HSCs), an altered microenvironment and systemic factors contribute to this process is not fully understood. Here we use bone marrow stromal cells as sensors of age-associated changes to the bone marrow microenvironment, and observe up-regulation of IL-6 and TGFß signalling-induced gene expression in aged bone marrow stroma. Inhibition of TGFß signalling leads to reversal of age-associated HSC platelet lineage bias, increased generation of lymphoid progenitors and rebalanced HSC lineage output in transplantation assays. In contrast, decreased erythropoiesis is not an intrinsic property of aged HSCs, but associated with decreased levels and functionality of erythroid progenitor populations, defects ameliorated by TGFß-receptor and IL-6 inhibition, respectively. These results show that both HSC-intrinsic and -extrinsic mechanisms are involved in age-associated hematopoietic decline, and identify therapeutic targets that promote their reversal.

Cognitive and emotional predictors of real versus sham repetitive transcranial magnetic stimulation treatment response in methamphetamine use disorder.

BACKGROUND AND AIMS: Repetitive transcranial magnetic stimulation (rTMS) of the left dorsolateral prefrontal cortex (DLPFC) can effectively reduce cravings in methamphetamine use disorder (MUD). However, a considerable group still fails to respond. Cognitive and emotional disturbance, as well as impulsive features, are widespread in patients with MUD and might mediate the treatment response of rTMS. The purpose of this study is to figure out whether these variables can help predicting patients' responses to rTMS treatment. METHODS: Ninety-seven patients with severe MUD and thirty-one gender- and age-matched healthy subjects were included. Patients were randomized to receive 20 sessions of real or sham rTMS. Intermittent theta burst protocols (iTBS) or sham iTBS were applied every weekday over the DLPFC for 20 daily sessions. Both groups received regular treatment. Craving induced by drug-related cue was measured before and after stimulation. Cognition was evaluated by using the CogState Battery. Baseline characteristics were collected through the Addiction Severity Index, Patient Health Questionnaire-9, General Anxiety Disorder Scale-7, and Barrett Impulsivity Scale-11. RESULTS: Results showed that patients with MUD have worse spatial working memory, problem-solving ability, as well as depression and anxiety symptoms compared with healthy controls. Cognition and emotion differed between responders (craving decrease ≥60%) and non-responders in real rTMS group but not in the sham group. Better cognitive and emotional functions means that patients have higher possibility for better response to real rTMS treatment. CONCLUSIONS: This study suggests that cognitive, emotional and impulsive features could be used to predict the prospective treatment responses of rTMS in patients with MUD.

C/EBPα and GATA-2 Mutations Induce Bilineage Acute Erythroid Leukemia through Transformation of a Neomorphic Neutrophil-Erythroid Progenitor.

Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia.