High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30–50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1407-3) contains supplementary material, which is available to authorized users.
The newly developed ATT measurement for evaluation of liver steatosis was closely correlated with steatosis grade and automated quantification of fat area, and it provides clinically relevant information.
Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells. STEM CELLS 2014;32:3099-3111
BackgroundThe present study aimed to investigate the impact of pre-sarcopenia on the prognosis of patients with advanced hepatocellular carcinoma (HCC) treated with sorafenib.MethodsWe enrolled 214 patients (71 ± 10 years old; 166 men and 48 women; 90% Child–Pugh grade A and 10% Child–Pugh grade B) treated with sorafenib in our hospital from July 2009 to August 2016. The muscle volume was measured from CT images just before sorafenib administration using software (SliceOmatic). Skeletal muscle mass index was calculated, and the presence of pre-sarcopenia was judged according to the standard (42 cm2/m2 for men and 38 cm2/m2 for women) proposed by the Japan Society of Hepatology.ResultsPre-sarcopenia was found in 123 patients (57%). The overall survival (OS) in patients with pre-sarcopenia tended to be worse than in patients without pre-sarcopenia (median 252 vs. 284 days, respectively; p = 0.16). Multivariate Cox hazard analysis revealed a baseline serum albumin level of ≤3.5 g/dl [hazard ratio (HR) 1.9; p = 0.0006], a baseline alpha-fetoprotein(AFP) level of ≥100 ng/ml (HR 2.1; p = 0.002), presence of lesions in bilateral hepatic lobes (HR 1.7; p = 0.03), and presence of major portal vein invasion (HR 1.8; p = 0.01) to be independent prognostic factors. In the 68 patients who had three or more negative prognostic factors, the presence of pre-sarcopenia did not correlate with prognosis. Of the 146 patients who had two or less prognostic factors, OS was significantly worse in 84 patients (58%) with pre-sarcopenia than in 62 patients without pre-sarcopenia (median 417 vs. 562 days, respectively; p = 0.047), and Cox hazard analysis revealed pre-sarcopenia to be an important prognostic factor (HR 1.6; p = 0.047).ConclusionIn sorafenib treatment for advanced HCC, pre-sarcopenia is a significant prognostic factor in patients with two or less negative prognostic factors, and could be the target of intervention to improve prognosis.
Human adipose‐derived stem/stromal cell spheroids enriched in undifferentiated cells were efficiently prepared by using three‐dimensional floating culture in non‐cross‐linked hyaluronic acid gel. The results suggest the superior plasticity and growth factor‐secreting potential of the spheroids and clearly indicate their therapeutic power for promoting tissue regeneration. As an easy local injectable, spheroids are expected to be a powerful tool to treat various stem cell‐depleted conditions/organs through nonintravenous administration.
Based on these data, we developed a simple scoring system to predict HCC development using these three factors. Using these scores, patients were classified into four groups; cumulative incidence of HCC development significantly increased with increasing scores (p < 0.001).
Conclusions
Aim:The presence of resistance-associated variants (RAV) may attenuate the efficacy of direct-acting antivirals (DAA) in combination therapy for hepatitis C. The aim of this study was to characterize the NS3 and NS5A regions of hepatitis C virus (HCV) in naturally occurring RAV.
Methods:The NS3 and NS5A regions of HCV were amplified by nested polymerase chain reaction and their nucleotide sequences were determined by direct sequencing in 493 genotype 1b patients naive to DAA-based therapies. The effect of baseline RAV on response to pegylated interferon and ribavirin therapy was analyzed in 65 patients after stratification by interleukin (IL)-28B genotype.Results: The incidence of RAV was 7.9% in NS3 (V36I/L, 1.2%; T54S, 2.8%; Q80K/R, 3.0%; A156S, 0.2%; and D168E/T, 2.4%) and 20.2% in NS5A (L31I/M, 2.2%; and Y93H, 19.0%). The incidence in interferon experienced and naive patients was similar. The incidence of Y93H in NS5A was significantly higher in the IL-28B TT genotype (rs8099917) than non-TT (27.1% vs 9.5%, P < 0.001). The virological response to pegylated interferon plus ribavirin therapy was not affected by the presence of RAV in IL-28B TT genotype.Conclusion: RAV, especially Y93H in the NS5A region, were highly prevalent in DAA naive patients with genotype 1b HCV in Japan and were linked to IL-28B TT genotype. Interferonbased therapy could be an alternative for patients with RAV because these variants did not attenuate the response to that therapy. The analysis of RAV may impact the selection of the optimal treatment strategy.
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