Multiparameter analysis of timelapse imaging reveals kinetics of megakaryocytic erythroid progenitor clonal expansion and differentiation.

Single-cell assays have enriched our understanding of hematopoiesis and, more generally, stem and progenitor cell biology. However, these single-end-point approaches provide only a static snapshot of the state of a cell. To observe and measure dynamic changes that may instruct cell fate, we developed an approach for examining hematopoietic progenitor fate specification using long-term (> 7-day) single-cell time-lapse imaging for up to 13 generations with in situ fluorescence staining of primary human hematopoietic progenitors followed by algorithm-assisted lineage tracing. We analyzed progenitor cell dynamics, including the division rate, velocity, viability, and probability of lineage commitment at the single-cell level over time. We applied a Markov probabilistic model to predict progenitor division outcome over each generation in culture. We demonstrated the utility of this methodological pipeline by evaluating the effects of the cytokines thrombopoietin and erythropoietin on the dynamics of self-renewal and lineage specification in primary human bipotent megakaryocytic-erythroid progenitors (MEPs). Our data support the hypothesis that thrombopoietin and erythropoietin support the viability and self-renewal of MEPs, but do not affect fate specification. Thus, single-cell tracking of time-lapse imaged colony-forming unit assays provides a robust method for assessing the dynamics of progenitor self-renewal and lineage commitment.

Correlation between hand grip strength and regional muscle mass in older Asian adults: an observational study.

BACKGROUND: Previous research has demonstrated a correlation between hand grip strength (HGS) and muscle strength. This study aims to determine the relationship between HGS and muscle mass in older Asian adults. METHODS: We retrospectively reviewed the dual-energy X-ray absorptiometry (DXA) records of 907 older adults (239 (26.4%) men and 668 (73.6%) women) at one medical institution in Taipei, Taiwan, from January 2019, to December 2020. Average age was 74.80 ± 9.43 and 72.93 ± 9.09 for the males and females respectively. The inclusion criteria were: 1) aged 60 and older, 2) underwent a full-body DXA scan, and 3) performed hand grip measurements. Patients with duplicate results, incomplete records, stroke history, and other neurological diseases were excluded. Regional skeletal muscle mass was measured using DXA. HGS was measured using a Jamar handheld dynamometer. RESULTS: Total lean muscle mass (kg) averaged 43.63 ± 5.81 and 33.16 ± 4.32 for the males and females respectively. Average HGS (kg) was 28.81 ± 9.87 and 19.19 ± 6.17 for the males and females respectively. In both sexes, HGS and regional muscle mass consistently declined after 60 years of age. The rates of decline per decade in upper and lower extremity muscle mass and HGS were 7.06, 4.95, and 12.30%, respectively, for the males, and 3.36, 4.44, and 12.48%, respectively, for the females. In men, HGS significantly correlated with upper (r = 0.576, p < 0.001) and lower extremity muscle mass (r = 0.532, p < 0.001). In women, the correlations between HGS and upper extremity muscle mass (r = 0.262, p < 0.001) and lower extremity muscle mass (r = 0.364, p < 0.001) were less strong, though also statistically significant. CONCLUSION: Muscle mass and HGS decline with advancing age in both sexes, though the correlation is stronger in men. HGS measurements are an accurate proxy for muscle mass in older Asian adults, particularly in males.

BatchBMD as an Efficient and Accurate Dual-Energy X-ray Absorptiometry Report Generator.

Dual-energy X-ray absorptiometry is the gold standard for evaluating Bone Mineral Density (BMD); however, a typical BMD report is generated in a time-inefficient manner and is prone to error. We developed a rule-based automated reporting system, BatchBMD, that accelerates DXA reporting while improving its accuracy over current systems. BatchBMD generates a structured report, customized to the specific clinical purpose. To compare BatchBMD to a Web-based Reporting (WBR) system for efficiency and accuracy, 500 examinations were randomly chosen from those performed at the Taipei Municipal Wanfang Hospital from January to March 2021. The final assessment included all 2326 examinations conducted from September 2020 to March 2021. The average reporting times were 6.7 and 10.8 min for BatchBMD and the WBR system, respectively, while accuracy was 99.4% and 98.2%, respectively. Most of the errors made by BatchBMD were digit errors in the appendicular skeletal muscle index. After correcting this, 100% accuracy across all 2326 examinations was validated. This automated and accurate BMD reporting system significantly reduces report production workload for radiologists and technicians while increasing productivity and quality. Additionally, the portable software, which employs a simple framework, can reduce deployment costs in clinical practice.

Muscle Mass and Gait Characteristics in Older Women Fallers vs. Non-Fallers.

BACKGROUND: Falling is a major public health concern of elderly people. We aimed to determine if lean mass and spatiotemporal gait parameters could predict the risk of falling in elderly women and also study the relationships between lean mass and gait characteristics. METHODS: Twenty-four community women were prospectively recruited (mean age, 72.30 ± 5.31 years). Lean mass was measured using dual-energy fan-beam X-ray absorptiometry. Gait characteristics were assessed using spatiotemporal analysis. Fall risks were assessed using the Berg Balance Scale (BBS) and the Falls Efficacy Scale-International. Fall histories were recorded. Appropriate statistical analyses were applied to determine lean mass and gait characteristics in predicting the risk of fall and the associations between lean mass and gait characteristics. RESULTS: There were 14 participants (58.33%) with fall histories. Patients with fall histories had a significantly narrower base of support and lower BBS score. However, only the base of support was significantly associated with fall risk (odds ratio, 0.415; p = 0.022). Lean mass was significantly negatively associated with proportion of swing phase and positively associated with proportions of stance and double-support phases. CONCLUSION: Fall risk among elderly women can be predicted using base of support, where a narrower base predicts a greater fall risk. Although the lean mass was not related to risk of fall, lean mass is still related to some gait characteristics.

Current understanding of human megakaryocytic-erythroid progenitors and their fate determinants.

PURPOSE OF REVIEW: This review focuses on our current understanding of fate decisions in bipotent megakaryocyte-erythroid progenitors (MEPs). Although extensive research has been carried out over decades, our understanding of how MEP commit to the erythroid versus megakaryocyte fate remains unclear. RECENT FINDINGS: We discuss the isolation of primary human MEP, and focus on gene expression patterns, epigenetics, transcription factors and extrinsic factors that have been implicated in MEP fate determination. We conclude with an overview of the open debates in the field of MEP biology. SUMMARY: Understanding MEP fate is important because defects in megakaryocyte and erythrocyte development lead to disease states such as anaemia, thrombocytopenia and leukaemia. MEP also represent a model system for studying fundamental principles underlying cell fate decisions of bipotent and pluripotent progenitors, such that discoveries in MEP are broadly applicable to stem/progenitor cell biology.

Selecting Appropriate Sarcopenia Screening Methods for Asian Populations.

We aimed to determine the most appropriate sarcopenia screening method for Asian populations. We retrospectively studied the physiological differences between the sexes in healthy individuals and prospectively compared using skeletal muscle mass versus handgrip strength (HS) to screen for sarcopenia in a community-based population. Skeletal muscle mass was determined using dual-energy X-ray absorptiometry. Of 5881 healthy individuals recruited, 101 were from urban populations and 349 from a community-based population. The sexes were comparable in total lean muscle mass declines after peaking around 20 years of age. An age-dependent decline in total fat mass was found only among men;a persistent increase in total fat mass was observed only among women. The prevalence of low skeletal muscle mass significantly increased with age in both sexes only when applying the weight-adjusted skeletal muscle index (wSMI); it was significant only among men when applying the height-adjusted skeletal muscle index (hSMI). Using HS resulted in a much higher prevalence of sarcopenia in both sexes. A significant age-dependent increase in fat mass in women showed that the most appropriate adjustment method is wSMI for women and hSMI for men. Nevertheless, a primary HS survey is recommended for both sexes in Asian populations.

Dual-energy X-ray absorptiometry-assessed adipose tissues in metabolically unhealthy normal weight Asians.

Normal body mass index (BMI) is associated with lower risk for cardiometabolic diseases. However, there is a subset of individuals with BMI in this range who present with this metabolic abnormality (called metabolically unhealthy normal weight, MUHNW). Here we aimed to assess the adipose characteristics of people with MUHNW using dual-energy X-ray absorptiometry (DXA). This study included 3259 people with normal BMI who underwent health examinations from January 1, 2007 through December 31, 2016. Body fat percentage (%BF), android-gynoid percent fat ratio (AG ratio), and visceral adipose tissue (VAT) were measured simultaneously using DXA CoreScan software. Those with MUHNW comprised 12.67% of the sample. Among those with MUHNW, 71.6% of the women and 56.5% of the men showed high VAT amounts, but less than 40% of either showed high %BFs. Furthermore, considering the combined effects of fat amount and distribution, a normal BMI accompanied by high AG ratio and/or high VAT mass but low %BF presents a much higher risk for metabolic syndrome than when %BF is high, most predominantly in women. In conclusion, using DXA-measured abdominal fat, particularly VAT accumulation, is clinically more important than using %BF when assessing metabolic syndrome in those with normal BMI.

Single-cell microRNA-mRNA co-sequencing reveals non-genetic heterogeneity and mechanisms of microRNA regulation.

Measuring multiple omics profiles from the same single cell opens up the opportunity to decode molecular regulation that underlies intercellular heterogeneity in development and disease. Here, we present co-sequencing of microRNAs and mRNAs in the same single cell using a half-cell genomics approach. This method demonstrates good robustness (~95% success rate) and reproducibility (R2 = 0.93 for both microRNAs and mRNAs), yielding paired half-cell microRNA and mRNA profiles, which we can independently validate. By linking the level of microRNAs to the expression of predicted target mRNAs across 19 single cells that are phenotypically identical, we observe that the predicted targets are significantly anti-correlated with the variation of abundantly expressed microRNAs. This suggests that microRNA expression variability alone may lead to non-genetic cell-to-cell heterogeneity. Genome-scale analysis of paired microRNA-mRNA co-profiles further allows us to derive and validate regulatory relationships of cellular pathways controlling microRNA expression and intercellular variability.

The Molecular Signature of Megakaryocyte-Erythroid Progenitors Reveals a Role for the Cell Cycle in Fate Specification.

Megakaryocytic-erythroid progenitors (MEPs) give rise to the cells that produce red blood cells and platelets. Although the mechanisms underlying megakaryocytic (MK) and erythroid (E) maturation have been described, those controlling their specification from MEPs are unknown. Single-cell RNA sequencing of primary human MEPs, common myeloid progenitors (CMPs), megakaryocyte progenitors, and E progenitors revealed a distinct transitional MEP signature. Inferred regulatory transcription factors (TFs) were associated with differential expression of cell cycle regulators. Genetic manipulation of selected TFs validated their role in lineage specification and demonstrated coincident modulation of the cell cycle. Genetic and pharmacologic modulation demonstrated that cell cycle activation is sufficient to promote E versus MK specification. These findings, obtained from healthy human cells, lay a foundation to study the mechanisms underlying benign and malignant disease states of the megakaryocytic and E lineages.

Surfactant protein C dampens inflammation by decreasing JAK/STAT activation during lung repair.

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

Functional evaluation of therapeutic response of HCC827 lung cancer to bevacizumab and erlotinib targeted therapy using dynamic contrast-enhanced and diffusion-weighted MRI.

This study aimed to investigate the therapeutic responses of lung cancer mice models with adenocarcinoma HCC827 (gefitinib sensitive) and HCC827R (gefitinib resistant) to the epidermal growth factor receptor-tyrosine kinase inhibitor erlotinib alone and in combination with the anti-angiogenesis agent bevacizumab using dynamic contrast enhanced (DCE) and diffusion-weighted MRI. In the HCC827 model, temporal changes in DCE-MRI derived parameters (Ktrans, kep, and iAUC90) and apparent diffusion coefficient (ADC) were significantly correlated with tumor size. Ktrans and iAUC90 significantly decreased at week 2 in the groups receiving erlotinib alone and in combination with bevacizumab, whereas kep decreased at week 1 and 2 in both treatment groups. In addition, there was a significant difference in iAUC90 between the treatment groups at week 1. Compared to the control group of HCC827, there was a significant reduction in microvessel density and increased tumor apoptosis in the two treatment group. ADC value increased in the erlotinib alone group at week 1 and week 2, and in the erlotinib combined with bevacizumab group at week 2. Enlarged areas of central tumor necrosis were associated with a higher ADC value. However, progressive enlargement of the tumors but no significant differences in DCE parameters or ADC were noted in the HCC827R model. These results showed that both erlotinib alone and in combination with bevacizumab could effectively inhibit tumor growth in the gefitinib-sensitive lung cancer mice model, and that this was associated with decreased vascular perfusion, increased ADC percentage, decreased microvessel density, and increased tumor apoptosis with a two-week treatment cycle.

In vivo correction of anaemia in ß-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery.

The blood disorder, ß-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human ß-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% ß-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.

Adult human megakaryocyte-erythroid progenitors are in the CD34+CD38mid fraction.

Bipotent megakaryocyte/erythroid progenitors (MEPs) give rise to progeny limited to the megakaryocyte (Mk) and erythroid (E) lineages. We developed a novel dual-detection functional in vitro colony-forming unit (CFU) assay for single cells that differentiates down both the Mk and E lineages (CFU-Mk/E), which allowed development and validation of a novel purification strategy for the identification and quantitation of primary functional human MEPs from granulocyte colony-stimulating factor-mobilized peripheral blood and bone marrow. Applying this assay to fluorescence-activated cell sorter-sorted cell populations, we found that the Lin(-)CD34(+)CD38(mid)CD45RA(-)FLT3(-)MPL(+)CD36(-)CD41(-) population is much more highly enriched for bipotent MEPs than any previously reported subpopulations. We also developed purification strategies for primary human lineage-committed Mk and E progenitors identified as CFU-Mk and burst forming unit-E. Comparative expression analyses in MEP, MkP, and ErP populations revealed differential expression of MYB We tested whether alterations in MYB concentration affect the Mk-E fate decision at the single cell level in MEPs and found that short hairpin RNA-mediated MYB knockdown promoted commitment of MEPs to the Mk lineage, further defining its role in MEP lineage fate. There are numerous applications for these novel enrichment strategies, including facilitating mechanistic studies of MEP lineage commitment, improving approaches for in vitro expansion of Mk and E cells, and developing improved therapies for benign and malignant hematologic disease.

Prevalence of Osteoporosis and Low Bone Mass in Older Chinese Population Based on Bone Mineral Density at Multiple Skeletal Sites.

Diagnosis of osteoporosis is based on bone mineral density (BMD) measurement, which is site dependent and commonly discordant between measurement sites. We aimed to determine the prevalence of osteoporosis diagnosed based on BMD T-scores measured by dual-energy x-ray absorptiometry (DXA) at different sites: the lumbar spine (LS) alone, femoral neck (FN) alone, or both. A total of 1712 women and 2028 men with LS and FN BMD measurements were enrolled. Over 50% discordance was found between osteoporosis classifications based on T-scores measured at the LS and FN. Use of the lowest T-scores measured at both the LS and right and left FN (rather than one site) significantly increased the prevalence of osteoporosis from 4.03 to 10.75% in postmenopausal women and 1.82 to 4.29% in men aged ≧50 years (p < 0.001). The trends of overall and age-adjusted prevalence of osteoporosis were similar in women and men. Osteoporosis was diagnosed at a higher rate if the USA reference rather than the Asia reference was used to calculate the T-score (26.64% vs. 10.75%). In conclusion, diagnosis based on the lowest T-score from multiple site BMD measurement can increase the prevalence of osteoporosis, demonstrating the higher sensitivity of the multiple site measurement strategy.

Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis.

Genome-wide association studies (GWASs) have linked genes to various pathological traits. However, the potential contribution of regulatory noncoding RNAs, such as microRNAs (miRNAs), to a genetic predisposition to pathological conditions has remained unclear. We leveraged GWAS meta-analysis data from >188,000 individuals to identify 69 miRNAs in physical proximity to single-nucleotide polymorphisms (SNPs) associated with abnormal levels of circulating lipids. Several of these miRNAs (miR-128-1, miR-148a, miR-130b, and miR-301b) control the expression of key proteins involved in cholesterol-lipoprotein trafficking, such as the low-density lipoprotein (LDL) receptor (LDLR) and the ATP-binding cassette A1 (ABCA1) cholesterol transporter. Consistent with human liver expression data and genetic links to abnormal blood lipid levels, overexpression and antisense targeting of miR-128-1 or miR-148a in high-fat diet-fed C57BL/6J and Apoe-null mice resulted in altered hepatic expression of proteins involved in lipid trafficking and metabolism, and in modulated levels of circulating lipoprotein-cholesterol and triglycerides. Taken together, these findings support the notion that altered expression of miRNAs may contribute to abnormal blood lipid levels, predisposing individuals to human cardiometabolic disorders.

Functional roles of calreticulin in cancer biology.

Calreticulin is a highly conserved endoplasmic reticulum chaperone protein which participates in various cellular processes. It was first identified as a Ca(2+)-binding protein in 1974. Accumulated evidences indicate that calreticulin has great impacts for the development of different cancers and the effect of calreticulin on tumor formation and progression may depend on cell types and clinical stages. Cell surface calreticulin is considered as an "eat-me" signal and promotes phagocytic uptake of cancer cells by immune system. Moreover, several reports reveal that manipulation of calreticulin levels profoundly affects cancer cell proliferation and angiogenesis as well as differentiation. In addition to immunogenicity and tumorigenesis, interactions between calreticulin and integrins have been described during cell adhesion, which is an essential process for cancer metastasis. Integrins are heterodimeric transmembrane receptors which connect extracellular matrix and intracellular cytoskeleton and trigger inside-out or outside-in signaling transduction. More and more evidences reveal that proteins binding to integrins might affect integrin-cytoskeleton interaction and therefore influence ability of cell adhesion. Here, we reviewed the biological roles of calreticulin and summarized the potential mechanisms of calreticulin in regulating mRNA stability and therefore contributed to cancer metastasis.

Calreticulin Regulates VEGF-A in Neuroblastoma Cells.

Calreticulin (CRT) has been previously correlated with the differentiation of neuroblastoma (NB), implying a favorable prognostic factor. Vascular endothelial growth factor (VEGF) has been reported to participate in the behavior of NB. This study investigated the association of CRT and VEGF-A in NB cells. The expressions of VEGF-A and HIF-1α, with overexpression or knockdown of CRT, were measured in three NB cells (SH-SY5Y, SK-N-DZ, and stNB-V1). An inducible CRT NB cell line and knockdown CRT stable cell lines were also established. The impacts of CRT overexpression on NB cell apoptosis, proliferation, and differentiation were also evaluated. We further examined the role of VEGF-A in the NB cell differentiation via VEGF receptor blockade. Constitutive overexpression of CRT led to NB cell differentiation without proliferation. Thus, an inducible CRT stNB-V1 cell line was generated by a tetracycline-regulated gene system. CRT overexpression increased VEGF-A and HIF-1α messenger RNA (mRNA) expressions in SH-SY5Y, SK-N-DZ, and stNB-V1 cells. CRT overexpression also enhanced VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. Knockdown of CRT decreased VEGF-A and HIF-1α mRNA expressions and lowered VEGF-A protein expression and secretion level in conditioned media in different NB cell lines. We further demonstrated that NB cell apoptosis was not affected by CRT overexpression in stNB-V1 cells. Nevertheless, overexpression of CRT suppressed cell proliferation and enhanced cell differentiation in stNB-V1 cells, whereas blockage of VEGFR-1 markedly suppressed the expression of neuron-specific markers including GAP43, NSE2, and NFH, as well as TrkA, a molecular marker indicative of NB cell differentiation. Our findings suggest that VEGF-A is involved in CRT-related neuronal differentiation in NB. Our work may provide important information for developing a new therapeutic strategy to improve the outcome of NB patients.

A Heuristic Framework for Image Filtering and Segmentation: Application to Blood Vessel Immunohistochemistry.

The blood vessel density in a cancerous tissue sample may represent increased levels of tumor growth. However, identifying blood vessels in the histological (tissue) image is difficult and time-consuming and depends heavily on the observer's experience. To overcome this drawback, computer-aided image analysis frameworks have been investigated in order to boost object identification in histological images. We present a novel algorithm to automatically abstract the salient regions in blood vessel images. Experimental results show that the proposed framework is capable of deriving vessel boundaries that are comparable to those demarcated manually, even for vessel regions with weak contrast between the object boundaries and background clutter.