Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of the cell cycle.

ABSTRACT: Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.

Enhanced recovery in patients with gestational diabetes mellitus and MTHFR 677 TT genotype after taking high-dose folic acid supplements during mid-late pregnancy: an open-label interventional study.

Objective: To explore the relationship between folic acid supplementation and the recovery rate of gestational diabetes mellitus (GDM) in women with methylenetetrahydrofolate (MTHFR) 677 TT genotypes in mid-late pregnancy. Methods: 9, 096 pregnant women were recruited with their MTHFR gene genotyped. 5,111 women underwent a 75-g oral glucose tolerance test (OGTT) and 2,097 were confirmed with GDM. The association between MTHFR genotypes and GDM risk was estimated using logistic and log-binomial regression, with age and parity set as the covariates to control their confounding effects. Further assessment of GDM risk on glucose levels was done using the ANCOVA model. As an open-label intervention study, 53 GDM patients with TT genotype were prescribed 800µg/day of folic acid as the high-dose group, while 201 GDM patients were given 400µg/day as the standard-dose group at their 24-28 weeks of pregnancy. A rate ratio (RR) of GDM recovery was estimated at each available time point for both groups. The time-to-GDM persistence events were analyzed with the Kaplan-Meier method and Cox-regression model. The trend of glucose levels over time was estimated using the linear model. Results: MTHFR 677 TT genotype has no significant association with the glucose levels and GDM risk, with an adjusted OR of 1.105 (95% CI 0.853, 1.431; p=0.452) and an adjusted PR of 1.050 (95% CI 0.906, 1.216; p=0.518) compared to the wildtype CC group. Patients in the high-dose group (n=38; 15 drop-outs; 40.69 days (95% CI 33.22, 48.15)) recovered from GDM approximately 27 days faster than those in the standard-dose group (n=133; 68 drop-outs; 68.09 days (95% CI 63.08, 73.11)). Concomitantly, the RR of GDM recovery rose and reached 1.247 (95% CI 1.026, 1.515) at 100 days of treatment with the standard-dose group as reference. Conclusion: High-dose folic acid supplement intake in mid-late pregnancy is associated with faster GDM relief in patients with MTHFR 677 TT genotype compared to the standard dose, which would be served as a novel and low-cost alternative therapy for the treatment of GDM.

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization.

In the plant meristem, tissue-wide maturation gradients are coordinated with specialized cell networks to establish various developmental phases required for indeterminate growth. Here, we used single-cell transcriptomics to reconstruct the protophloem developmental trajectory from the birth of cell progenitors to terminal differentiation in the Arabidopsis thaliana root. PHLOEM EARLY DNA-BINDING-WITH-ONE-FINGER (PEAR) transcription factors mediate lineage bifurcation by activating guanosine triphosphatase signaling and prime a transcriptional differentiation program. This program is initially repressed by a meristem-wide gradient of PLETHORA transcription factors. Only the dissipation of PLETHORA gradient permits activation of the differentiation program that involves mutual inhibition of early versus late meristem regulators. Thus, for phloem development, broad maturation gradients interface with cell-type-specific transcriptional regulators to stage cellular differentiation.

Single-cell molecular profiling provides a high-resolution map of basophil and mast cell development.

BACKGROUND: Basophils and mast cells contribute to the development of allergic reactions. Whereas these mature effector cells are extensively studied, the differentiation trajectories from hematopoietic progenitors to basophils and mast cells are largely uncharted at the single-cell level. METHODS: We performed multicolor flow cytometry, high-coverage single-cell RNA sequencing analyses, and cell fate assays to chart basophil and mast cell differentiation at single-cell resolution in mouse. RESULTS: Analysis of flow cytometry data reconstructed a detailed map of basophil and mast cell differentiation, including a bifurcation of progenitors into two specific trajectories. Molecular profiling and pseudotime ordering of the single cells revealed gene expression changes during differentiation. Cell fate assays showed that multicolor flow cytometry and transcriptional profiling successfully predict the bipotent phenotype of a previously uncharacterized population of peritoneal basophil-mast cell progenitors. CONCLUSIONS: A combination of molecular and functional profiling of bone marrow and peritoneal cells provided a detailed road map of basophil and mast cell development. An interactive web resource was created to enable the wider research community to explore the expression dynamics for any gene of interest.

Evaluating scenarios toward zero plastic pollution.

Plastic pollution is a pervasive and growing problem. To estimate the effectiveness of interventions to reduce plastic pollution, we modeled stocks and flows of municipal solid waste and four sources of microplastics through the global plastic system for five scenarios between 2016 and 2040. Implementing all feasible interventions reduced plastic pollution by 40% from 2016 rates and 78% relative to "business as usual" in 2040. Even with immediate and concerted action, 710 million metric tons of plastic waste cumulatively entered aquatic and terrestrial ecosystems. To avoid a massive build-up of plastic in the environment, coordinated global action is urgently needed to reduce plastic consumption; increase rates of reuse, waste collection, and recycling; expand safe disposal systems; and accelerate innovation in the plastic value chain.

Lgr5+ stem and progenitor cells reside at the apex of a heterogeneous embryonic hepatoblast pool.

During mouse embryogenesis, progenitors within the liver known as hepatoblasts give rise to adult hepatocytes and cholangiocytes. Hepatoblasts, which are specified at E8.5-E9.0, have been regarded as a homogeneous progenitor population that initiate differentiation from E13.5. Recently, scRNA-seq analysis has identified sub-populations of transcriptionally distinct hepatoblasts at E11.5. Here, we show that hepatoblasts are not only transcriptionally but also functionally heterogeneous, and that a subpopulation of E9.5-E10.0 hepatoblasts exhibit a previously unidentified early commitment to cholangiocyte fate. Importantly, we also identify a subpopulation constituting 2% of E9.5-E10.0 hepatoblasts that express the adult stem cell marker Lgr5, and generate both hepatocyte and cholangiocyte progeny that persist for the lifespan of the mouse. Combining lineage tracing and scRNA-seq, we show that Lgr5 marks E9.5-E10.0 bipotent liver progenitors residing at the apex of a hepatoblast hierarchy. Furthermore, isolated Lgr5+ hepatoblasts can be clonally expanded in vitro into embryonic liver organoids, which can commit to either hepatocyte or cholangiocyte fates. Our study demonstrates functional heterogeneity within E9.5 hepatoblasts and identifies Lgr5 as a marker for a subpopulation of bipotent liver progenitors.

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19-CD34+CD38-CD45RA-CD49f+CD90+ (49f+) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f+ compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9AhiCD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9AloCD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment.

T cell cytolytic capacity is independent of initial stimulation strength.

How cells respond to myriad stimuli with finite signaling machinery is central to immunology. In naive T cells, the inherent effect of ligand strength on activation pathways and endpoints has remained controversial, confounded by environmental fluctuations and intercellular variability within populations. Here we studied how ligand potency affected the activation of CD8+ T cells in vitro, through the use of genome-wide RNA, multi-dimensional protein and functional measurements in single cells. Our data revealed that strong ligands drove more efficient and uniform activation than did weak ligands, but all activated cells were fully cytolytic. Notably, activation followed the same transcriptional pathways regardless of ligand potency. Thus, stimulation strength did not intrinsically dictate the T cell-activation route or phenotype; instead, it controlled how rapidly and simultaneously the cells initiated activation, allowing limited machinery to elicit wide-ranging responses.

A single-cell hematopoietic landscape resolves 8 lineage trajectories and defects in Kit mutant mice.

Hematopoietic stem and progenitor cells (HSPCs) maintain the adult blood system, and their dysregulation causes a multitude of diseases. However, the differentiation journeys toward specific hematopoietic lineages remain ill defined, and system-wide disease interpretation remains challenging. Here, we have profiled 44 802 mouse bone marrow HSPCs using single-cell RNA sequencing to provide a comprehensive transcriptional landscape with entry points to 8 different blood lineages (lymphoid, megakaryocyte, erythroid, neutrophil, monocyte, eosinophil, mast cell, and basophil progenitors). We identified a common basophil/mast cell bone marrow progenitor and characterized its molecular profile at the single-cell level. Transcriptional profiling of 13 815 HSPCs from the c-Kit mutant (W41/W41) mouse model revealed the absence of a distinct mast cell lineage entry point, together with global shifts in cell type abundance. Proliferative defects were accompanied by reduced Myc expression. Potential compensatory processes included upregulation of the integrated stress response pathway and downregulation of proapoptotic gene expression in erythroid progenitors, thus providing a template of how large-scale single-cell transcriptomic studies can bridge between molecular phenotypes and quantitative population changes.

Refractive Errors and Concomitant Strabismus: A Systematic Review and Meta-analysis.

This systematic review and meta-analysis is to evaluate the risk of development of concomitant strabismus due to refractive errors. Eligible studies published from 1946 to April 1, 2016 were identified from MEDLINE and EMBASE that evaluated any kinds of refractive errors (myopia, hyperopia, astigmatism and anisometropia) as an independent factor for concomitant exotropia and concomitant esotropia. Totally 5065 published records were retrieved for screening, 157 of them eligible for detailed evaluation. Finally 7 population-based studies involving 23,541 study subjects met our criteria for meta-analysis. The combined OR showed that myopia was a risk factor for exotropia (OR: 5.23, P = 0.0001). We found hyperopia had a dose-related effect for esotropia (OR for a spherical equivalent [SE] of 2-3 diopters [D]: 10.16, P = 0.01; OR for an SE of 3-4D: 17.83, P < 0.0001; OR for an SE of 4-5D: 41.01, P < 0.0001; OR for an SE of ≥5D: 162.68, P < 0.0001). Sensitivity analysis indicated our results were robust. Results of this study confirmed myopia as a risk for concomitant exotropia and identified a dose-related effect for hyperopia as a risk of concomitant esotropia.

Risk of recurrence of retinopathy of prematurity after initial intravitreal ranibizumab therapy.

We report our experience with the use of intravitreal ranibizumab for the treatment of retinopathy of prematurity (ROP). A retrospective review was performed on 138 consecutive infants screened at a single centre over 18 months. Intravitreal ranibizumab was offered in selected cases requiring treatment, such as aggressive posterior ROP or poor mydriasis. 2 eyes of 1 infant received intravitreal ranibizumab alone and 8 eyes of 5 infants received combined intravitreal ranibizumab and laser therapy. 3 out of 8 eyes treated initially with intravitreal ranibizumab monotherapy had persistent disease requiring laser therapy, and 3 out of 5 eyes with initial regression suffered disease recurrence at a mean of 7.6 weeks post-injection. 2 eyes treated first with laser followed by intravitreal ranibizumab had disease regression without recurrence. Our cohort demonstrate a significant rate of persistent disease and recurrence in ROP eyes treated initially with intravitreal ranibizumab monotherapy, which is greater and earlier than that reported for intravitreal bevacizumab in the BEAT-ROP study. Intravitreal ranibizumab may be useful as an initial treatment in selected cases of ROP when laser therapy as first line is suboptimal. However, close monitoring is important and adjunctive laser therapy may subsequently be needed in a majority of cases.

SR and LR Union Suture for the Treatment of Myopic Strabismus Fixus: Is Scleral Fixation Necessary?

PURPOSE: To evaluate and compare the effectiveness of scleral fixation SR and LR union suture and nonscleral fixation union suture for the treatment of myopic strabismus fixus. METHODS: Retrospective review of 32 eyes of 22 patients with myopic strabismus fixus who had undergone union suture of superior rectus (SR) and lateral rectus (LR) with or without scleral fixation, and follow-up longer than 6 months at Hong Kong Eye Hospital from 2006 to 2013. Surgical techniques and outcomes in terms of ocular alignment are analyzed. RESULTS: There is significant overall improvement both in postoperative angle of esodeviation (P < 0.01) and postoperative range of movement (P = 0.042). Comparing between the sclera fixation group (11 eyes) versus nonscleral fixation group (21 eyes), the postoperative horizontal deviation, the postoperative vertical deviation, successful outcome, and the change in horizontal deviation were not significantly different (P > 0.05). CONCLUSIONS: Union suture of SR and LR is an effective procedure in correcting myopic strabismus fixus. Fixation of the union suture to the sclera does not improve surgical outcome.

Risk association of HbA1c variability with chronic kidney disease and cardiovascular disease in type 2 diabetes: prospective analysis of the Hong Kong Diabetes Registry.

BACKGROUND: In type 2 diabetes, tight glycaemic control lowers the risk of diabetic complications, but it remains uncertain whether variability of glycaemia influences outcomes. We examined the association of glycated haemoglobin (HbA1c ) variability with incident chronic kidney disease and cardiovascular disease in a prospective cohort of 8439 Chinese patients with type 2 diabetes recruited from 1994 to 2007. METHODS: Intrapersonal mean and SD of serially measured HbA1c were calculated. Chronic kidney disease was defined as estimated glomerular filtration rate <60 ml/min per 1.73 m². Cardiovascular disease was defined as events of ischemic heart disease, heart failure, ischemic stroke or peripheral vascular disease. RESULTS: Over a median follow-up period of 7.2 years, 19.7 and 10.0% of patients developed chronic kidney disease and cardiovascular disease, respectively. Patients who progressed to chronic kidney disease had higher mean HbA1c (7.8 ± 1.3% vs 7.4 ± 1.2%, p < 0.001) and SD (1.0 ± 0.8% vs 0.8 ± 0.6%, p < 0.001) than nonprogressors. Similarly, patients who developed cardiovascular disease had higher mean HbA1c (7.7 ± 1.3% vs 7.4 ± 1.2%, p < 0.001) and SD (1.4 ± 1.1% vs 1.1 ± 0.8%, p < 0.001) than patients who did not develop cardiovascular disease. By using multivariate-adjusted Cox regression analysis, adjusted SD was associated with incident chronic kidney disease and cardiovascular disease with corresponding hazard ratios of 1.16 (95% CI 1.11-1.22), p < 0.001) and 1.27 (95% CI 1.15-1.40, p < 0.001), independent of mean HbA1c and other confounding variables. CONCLUSIONS: Long-term glycaemic variability expressed by SD of HbA1c predicted development of renal and cardiovascular complications.

High risk for cardiovascular disease in Chinese type 2 diabetic patients with major depression--a 7-year prospective analysis of the Hong Kong Diabetes Registry.

BACKGROUND: Depression is known to be associated with premature mortality and cardiovascular disease (CVD) in type 2 diabetes, although there is a paucity of similar data in Chinese population. In this study, we examined the risk association of major depression with premature mortality and CVD in a hospital clinic-based cohort. METHODS: In a prospective cohort of 7835 Hong Kong Chinese with type 2 diabetes but without CVD at baseline, 153 patients were diagnosed with major depression by psychiatrists in public hospitals. After a median follow-up period of 7.4 years, 827 patients died and 829 patients developed CVD mainly due to stroke (n=384). We used Cox proportional hazard regression to obtain the hazard ratio (HR, 95% confidence interval, CI) of depression for the risk of mortality and CVD. RESULTS: Depressed patients were younger (51.6 versus 56.6 years, p<0.001), more likely to be female (78.4% versus 53.0%, p<0.001), had higher LDL-cholesterol (3.2 versus 3.0 mmol/L, p=0.038) at baseline and longer hospitalization stays per year (median:0.8 nights per 100-person-years versus 0.1 nights per 100-person-years, p<0.001). After adjusting for conventional risk factors, depression independently predicted CVD [HR=2.18(95% CI=1.45-3.27)], mainly due to stroke [HR=3.55(95% CI=2.15-5.84)]. LIMITATIONS: The young age and small sample size of patients with depression did not give sufficient power to confirm risk association of depression with premature mortality and myocardial infarction. CONCLUSIONS: In Chinese type 2 diabetic patients, depression was associated with a 2-3 fold increase in the risk of incident CVD, especially stroke.

Metabolic syndrome predicts new onset of chronic kidney disease in 5,829 patients with type 2 diabetes: a 5-year prospective analysis of the Hong Kong Diabetes Registry.

OBJECTIVE: Type 2 diabetes is the leading cause of end-stage renal disease worldwide. Aside from hyperglycemia and hypertension, other metabolic factors may determine renal outcome. We examined risk associations of metabolic syndrome with new onset of chronic kidney disease (CKD) in 5,829 Chinese patients with type 2 diabetes enrolled between 1995 and 2005. RESEARCH DESIGN AND METHODS: Metabolic syndrome was defined by National Cholesterol Education Program Adult Treatment Panel III criteria with the Asian definition of obesity. Estimated glomerular filtration rate (eGFR) was calculated using the abbreviated Modification of Diet in Renal Disease formula modified for the Chinese population. New onset of CKD was defined as eGFR <60 ml/min per 1.73 m(2) at the time of censor. Subjects with CKD at baseline were excluded from the analysis. RESULTS: After a median follow-up duration of 4.6 years (interquartile range: 1.9-7.3 years), 741 patients developed CKD. The multivariable-adjusted hazard ratio (HR) of CKD was 1.31 (95% CI 1.12-1.54, P = 0.001) for subjects with metabolic syndrome compared with those without metabolic syndrome. Relative to subjects with no other components of metabolic syndrome except for diabetes, those with two, three, four, and five metabolic syndrome components had HRs of an increased risk of CKD of 1.15 (0.83-1.60, P = 0.407) 1.32 (0.94-1.86, P = 0.112), 1.64 (1.17-2.32, P = 0.004), and 2.34 (1.54-3.54, P < 0.001), respectively. The metabolic syndrome traits of central obesity, hypertriglyceridemia, hypertension, and low BMI were independent predictors for CKD. CONCLUSIONS: The presence of metabolic syndrome independently predicts the development of CKD in subjects with type 2 diabetes.

Clinical features and outcomes of Fusarium keratitis associated with contact lens wear.

PURPOSE: To describe the clinical features and outcomes among contact lens wearers with Fusarium keratitis. METHODS: A retrospective observational review of all cases of culture-proven Fusarium keratitis among contact lens wearers from three hospitals in Hong Kong Island were included. The clinical features, hygiene habits, and clinical outcomes were reviewed. RESULTS: Sixteen patients (17 eyes) were diagnosed with Fusarium keratitis associated with contact lens wear. One patient had bilateral involvement. Six patients had a central lesion; four had paraxial lesions; one had paraxial and peripheral lesions; and the rest had peripheral lesions. Ten (62.5%) patients reported using ReNu multipurpose cleaning solution. Most patients had poor contact lens hygiene habits. One patient required systemic antifungal treatment. No surgical intervention was required in any of the patients. CONCLUSIONS: The clinical features of Fusarium keratitis in contact lens wearers can be variable. Although fungal infection is reported rarely, clinicians should maintain a high index of suspicion for it when examining patients with contact lens-associated microbial keratitis. Education on proper contact lens care should be reinforced. Early and appropriate treatment may lead to satisfactory visual outcomes.

Orbital involvement in Erdheim-Chester disease.

Erdheim-Chester disease is a rare, idiopathic, non-Langerhans' cell, histiocytic disorder. To our knowledge this is only the second case of Erdheim-Chester disease reported in the Chinese population. We describe a 45-year-old woman presenting with unilateral proptosis and periorbital xanthelasma. Histopathological examination revealed a xanthogranulomatous lesion expressing CD68, but negative for S100 protein, CD1a, CD3, or CD20. Systemic involvement was evident on bone scanning, and involvement of the thorax and abdominal aorta was seen on computed tomography. Despite treatment with systemic steroids, immunosuppressants, chemotherapy and interferon, progressive deterioration occurred. Our patient's clinical course was consistent with reports in the literature. Unfortunately, our patient developed neutropenic fever and died from septicaemic shock. Although Erdheim-Chester disease is a rare entity, especially in the Chinese population, an unusual presentation with orbital masses and bilateral xanthelasma, associated with systemic features, should raise the suspicion of this serious and potentially fatal disease.

Succession and diel transcriptional response of the glycolate-utilizing component of the bacterial community during a spring phytoplankton bloom.

The influence of the phytoplankton-specific organic compound glycolate on bacterial community structure was examined during the 2004 spring phytoplankton bloom (February to April) in Dabob Bay in Washington. The diversity of the bacteria able to utilize glycolate during the phytoplankton bloom was determined using previously developed PCR primers to amplify the gene for the D subunit of glycolate oxidase (glcD). Many of the glcD sequences obtained represented novel sequences that appeared to be specific to marine environments. Overall, the glcD sequence diversity decreased as the phytoplankton bloom progressed. Phylotype-specific glcD quantitative PCR primers were designed for the six most commonly detected glcD phylotypes that represented distinct phylogenetic groups of heterotrophic bacteria. Three patterns of phylotype abundance were detected: four phylotypes were most abundant during the onset of the bloom; the abundance of one phylotype increased as the bloom progressed; and one phylotype was abundant throughout the bloom. Quantitative reverse transcriptase PCR with the same phylotype-specific primers was used to determine the levels of day and night glcD RNA transcription over the course of the bloom. glcD transcripts, when detectable, were always more abundant in the day than at night for each phylotype, suggesting that the bacteria responded to the glycolate produced by phytoplankton during the day. The nearly constant low in situ glycolate concentrations suggested that bacteria rapidly utilized the available glycolate. This study provided evidence for direct phytoplankton-bacterium interactions and the resulting succession in a single functional group of marine bacteria.

The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism.

Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for approximately 20% of global carbon fixation. We report the 34 million-base pair draft nuclear genome of the marine diatom Thalassiosira pseudonana and its 129 thousand-base pair plastid and 44 thousand-base pair mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle, all attributes that allow diatoms to prosper in aquatic environments.