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"Zeng, Xi"
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Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning
Human intestinal tissue–derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR–Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR–Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2–small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2–3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.The authors present a protocol for using the CRISPR–Cas9 genome editing system to knock out a gene of interest in human intestinal tissue–derived enteroids by lentiviral transduction and single-cell cloning.
Journal Article
التخفيف من حدة الفقر في الصين المعاصرة
استنادا إلى نظرة عامة على أوضاع الفقر، يقدم هذا الكتاب مسار التخفيف من حدة الفقر والتنمية في الصين، ويشرح نموذج التنمية والتخفي من حدة الفقر بخصائص صينية والتمسك بمباديء (سيطرة الحكومة ومشاركة المجتمع والاعتماد على الذات والتنمية الموجهة والتنمية الشاملة) كما يقدم الكتاب تلخيصا شاملا لإنجازات الصين العظيمة وخبراتها الهامة وإسهاماتها الرئيسية في قضية التخفيف من حدة الفقر في العالم، ويعرض بإيجاز نظرات وممارسات التخفيف المستهدف من الفقر في العصر الجديد من أجل توفير مراجع لكسب المعركة ضد الفقر في الصين وقضية التخفيف من حدة الفقر في العالم.
Book
Replication of human noroviruses in stem cell-derived human enteroids
The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for straindependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.
Journal Article
Highly selective cesium(I) capture under acidic conditions by a layered sulfide
Radiocesium remediation is desirable for ecological protection, human health and sustainable development of nuclear energy. Effective capture of Cs
+
from acidic solutions is still challenging, mainly due to the low stability of the adsorbing materials and the competitive adsorption of protons. Herein, the rapid and highly selective capture of Cs
+
from strongly acidic solutions is achieved by a robust K
+
-directed layered metal sulfide KInSnS
4
(InSnS-1) that exhibits excellent acid and radiation resistance. InSnS-1 possesses high adsorption capacity for Cs
+
and can serve as the stationary phase in ion exchange columns to effectively remove Cs
+
from neutral and acidic solutions. The adsorption of Cs
+
and H
3
O
+
is monitored by single-crystal structure analysis, and thus the underlying mechanism of selective Cs
+
capture from acidic solutions is elucidated at the molecular level.
The removal of radiocesium from acidic solutions is challenging. Here, the authors report the rapid and highly selective capture of cesium(I) from strongly acidic solutions by a robust layered metal sulfide.
Journal Article
Heterologous sequential immunization using chimeric mRNA and protein vaccines with HA-stem and S-RBD enhanced protective mucosal immunity against influenza and COVID-19
Influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as two highly contagious respiratory pathogens, continue to pose critical challenges to global public health. In our previous research, we successfully developed mRNA and protein vaccines by integrating the HA stem of H1N1 influenza virus with the RBD from SARS-CoV-2, which demonstrated broad protective efficacy against multiple strains of both viruses in mouse models. Here, we compared the immunogenicity and protective efficacy of heterologous sequential immunization regimens based on this chimeric antigen design, employing mRNA vaccine priming followed by intranasal protein vaccine boosting. Our results show that an mRNA vaccine prime followed by an intranasally administered protein vaccine with PICKCa adjuvant boost not only induces robust systemic humoral immunity but also significantly enhances respiratory mucosal immunity. Compared to boosting with an adjuvant-free intranasal protein vaccine, the inclusion of PICKCa adjuvant markedly elevated mucosal IgA levels in both nasal washes and bronchoalveolar lavage fluid (BALF). Furthermore, the adjuvanted intranasal protein vaccine boost regimen provided optimal protection against high-dose lethal influenza virus challenge. These findings provide valuable insights for refining immunization strategies to enhance the efficacy of combined influenza-COVID-19 vaccines.
Journal Article
A novel angiogenesis-related gene signature predicting prognosis in clear cell renal cell carcinoma
Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer and is characterized by high recurrence and metastasis rates. A key feature of ccRCC is its rich vascular network, which supports tumor growth and metastasis through angiogenesis, the process of forming new blood vessels. Anti-angiogenic therapy targeting the vascular endothelial growth factor (VEGF) pathway has shown efficacy in treating advanced ccRCC, but resistance often develops, highlighting the need for new therapeutic targets. We systematically screened differentially expressed genes (DEGs) between ccRCC and normal tissues using TCGA-KIRC data, and intersected them with angiogenesis-related genes from GeneCards and MSigDB. Survival analysis identified prognostic genes, and a multigene risk signature was established and validated using an independent GEO cohort (GSE29609). Functional analyses and immunohistochemistry were performed to explore the biological roles of the key gene RUNX1. A total of 27 ccRCC-related angiogenesis genes were identified, of which four formed a prognostic signature with strong predictive value. Among them, RUNX1 carried the greatest prognostic weight. Functional validation revealed that RUNX1 promotes ccRCC progression by regulating immune infiltration and lipid metabolism. In particular, RUNX1 expression was associated with enhanced CD8⁺ T-cell infiltration and upregulation of lipid metabolism genes such as FASN and SCD1. Our study identifies a novel angiogenesis-related prognostic signature for ccRCC and highlights RUNX1 as a key regulator linking tumor angiogenesis, immune infiltration, and lipid metabolism. Compared with conventional anti-VEGF therapies, targeting RUNX1 may provide broader therapeutic benefits and represents a promising translational strategy for improving ccRCC management.
Journal Article
Serum AKR1B10 as an indicator of unfavorable survival of hepatocellular carcinoma
Background and AimsA large-scale multicenter study validated aldo–keto reductase 1B10 (AKR1B10) as a new serum marker of hepatocellular carcinoma (HCC). This study aimed to evaluate the prognostic value of serum AKR1B10 in HCC.Methods273 naïve HCC patients enrolled for serum AKR1B10 tests were followed up for 2 years. Survival and clinical data were collected. Kaplan–Meier survival analysis and log-rank tests were used to estimate correlation of patient survival with serum AKR1B10. Univariate and multivariate COX regression analyses were used to evaluate the prognostic value of serum AKR1B10 level independently or in combination with other clinicopathological factors. α-fetoprotein (AFP) was analyzed in parallel for comparison.ResultsSerum AKR1B10 associated with tumor stage (p = 0.012), size (p = 0.004), primary tumor number (p = 0.019), and Child–Pugh classification (p = 0.003). HCC patients with a high level of serum AKR1B10 (≥ 267.9 pg/ml) had median survival (MS) of 25 months (95% confidence interval [CI] 20.788–29.212) vs. MS of 34 months (CI 28.911–39.089) in patients with normal serum AKR1B10 (p < 0.001). Univariate and multivariate COX regression analyses showed that serum AKR1B10 level was an unfavorable prognostic marker of HCC independently (HR 1.830, 95% CI 1.312–2.552; p < 0.001) or in combination with other clinical factors (HR 1.883, 95% CI 1.264–2.806; p = 0.002), such as TNM stage, tumor size and portal invasion. In the same cohort of HCC patients, AFP exhibited prognostic value at a cut-off of 400 ng/ml, but not at 20 ng/ml and 200 ng/ml.ConclusionsSerum AKR1B10 is a new prognostic marker of HCC, better than AFP.
Journal Article
Human intestinal organoids from Cronkhite-Canada syndrome patients reveal link between serotonin and proliferation
Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.
Journal Article
Investigating function roles of hypothetical proteins encoded by the Mycobacterium tuberculosis H37Rv genome
Background
Mycobacterium tuberculosis
(MTB) is a common bacterium causing tuberculosis and remains a major pathogen for mortality. Although the MTB genome has been extensively explored for two decades, the functions of 27% (1051/3906) of encoded proteins have yet to be determined and these proteins are annotated as hypothetical proteins.
Methods
We assigned functions to these hypothetical proteins using SSEalign, a newly designed algorithm utilizing structural information. A set of rigorous criteria was applied to these annotations in order to examine whether they were supported by each parameter. Virulence factors and potential drug targets were also screened among the annotated proteins.
Results
For 78% (823/1051) of the hypothetical proteins, we could identify homologs in
Escherichia coli
and
Salmonella typhimurium
by using SSEalign. Functional classification analysis indicated that 62.2% (512/823) of these annotated proteins were enzymes with catalytic activities and most of these annotations were supported by at least two other independent parameters. A relatively high proportion of transporter was identified in MTB genome, indicating the potential frequent transportation of frequent absorbing essential metabolites and excreting toxic materials in MTB. Twelve virulence factors and ten vaccine candidates were identified within these MTB hypothetical proteins, including two genes (rpoS and pspA) related to stress response to the host immune system. Furthermore, we have identified six novel drug target candidates among our annotated proteins, including Rv0817 and Rv2927c, which could be used for treating MTB infection.
Conclusions
Our annotation of the MTB hypothetical proteins will probably serve as a useful dataset for future MTB studies.
Journal Article