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"Xi, Lei"
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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
Water as elemental medium and heritage: The case of Sangyuanwei Polder embankment system
This paper argues that elemental media studies, which emphasize the entanglements between humans and non-humans, can offer new avenues for addressing the challenges faced by post-humanist heritage studies. Due to the importance of tourism for heritage revitalization, this paper examines the limitations of the local tourism industry’s understanding of the water element in the context of the tourism plan of the Sangyuanwei Polder Embankment System, particularly the neglect of the destructiveness of water. It also investigates human-water interactions in the history of SPES through elemental analysis, examining how water as a medium of life has inspired human affects, feelings, actions, as well as facilitated the transformation of and communication with water through the development of water-related engineering and social institutions. By focusing on the affective aspects of the elements, as well as revisiting the histories and local knowledge, elemental aesthetics derived from elemental analysis aims to reconnect humans to the elements as media of life, thus allowing for the initiation of dialogs with heritage management and tourism. The elemental aesthetics of water for the life of heritage sites aiming at flood control has often included the destructive characteristics of water, as well as the complex feelings of fear, awe, reverence, and dedication that it stirs. Based on this, this paper also points out a possible new orientation for the future development of water-related heritage sites.
Journal Article
Systemic immune-inflammation index predicting chemoradiation resistance and poor outcome in patients with stage III non-small cell lung cancer
Background
There is increasing evidence that the existence of systemic inflammation response is correlated with poor prognosis in several solid tumors. The aim of this retrospective study was to investigate the association between systemic immune-inflammation index (SII) and therapy response and overall survival in patients with stage III non-small cell lung cancer (NSCLC). The prognostic values of neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), and prognostic nutritional index (PNI) were also evaluated.
Methods
In total, 332 patients with new diagnosis of stage III NSCLC were included in this retrospective analysis. SII was defined as platelet counts × neutrophil counts/lymphocyte counts. Receiver operating characteristic (ROC) curve was used to evaluate the optimal cut-off value for SII, NLR, PLR and PNI. Univariate and multivariate survival analysis were performed to identify the factors correlated with overall survival.
Results
Applying cut-offs of ≥ 660 (SII), ≥ 3.57 (NLR), ≥ 147 (PLR), ≤ 52.95 (PNI), SII ≥ 660 was significantly correlated with worse ECOG PS (< 0.001), higher T stage (< 0.001), advanced clinical stage (
p
= 0.019), and lower response rate (
p
= 0.018). In univariate analysis, SII ≥ 660, NLR ≥ 3.57, PLR ≥ 147, and PNI ≤ 52.95 were significantly associated with worse overall survival (
p
all
< 0.001). Patients with SII ≥ 660 had a median overall survival of 10 months, and patients with SII < 660 showed a median overall survival of 30 months. In multivariate analysis only ECOG PS (HR, 1.744; 95% CI 1.158–2.626;
p
= 0.008), T stage (HR, 1.332; 95% CI 1.032–1.718;
p
= 0.028), N stage (HR, 1.848; 95% CI 1.113–3.068;
p
= 0.018), SII (HR, 2.105; 95% CI 1.481–2.741;
p
< 0.001) and NLR ≥ 3.57 (HR, 1.934; 95% CI 1.448–2.585;
p
< 0.001) were independently correlated with overall survival.
Conclusions
This study demonstrates that the SII is an independent prognostic indicator of poor outcomes for patients with stage III NSCLC and is superior to other inflammation-based factors in terms of prognostic ability.
Journal Article
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
Sfcnn: a novel scoring function based on 3D convolutional neural network for accurate and stable protein–ligand affinity prediction
Background
Computer-aided drug design provides an effective method of identifying lead compounds. However, success rates are significantly bottlenecked by the lack of accurate and reliable scoring functions needed to evaluate binding affinities of protein–ligand complexes. Therefore, many scoring functions based on machine learning or deep learning have been developed to improve prediction accuracies in recent years. In this work, we proposed a novel featurization method, generating a new scoring function model based on 3D convolutional neural network.
Results
This work showed the results from testing four architectures and three featurization methods, and outlined the development of a novel deep 3D convolutional neural network scoring function model. This model simplified feature engineering, and in combination with Grad-CAM made the intermediate layers of the neural network more interpretable. This model was evaluated and compared with other scoring functions on multiple independent datasets. The Pearson correlation coefficients between the predicted binding affinities by our model and the experimental data achieved 0.7928, 0.7946, 0.6758, and 0.6474 on CASF-2016 dataset, CASF-2013 dataset, CSAR_HiQ_NRC_set, and Astex_diverse_set, respectively. Overall, our model performed accurately and stably enough in the scoring power to predict the binding affinity of a protein–ligand complex.
Conclusions
These results indicate our model is an excellent scoring function, and performs well in scoring power for accurately and stably predicting the protein–ligand affinity. Our model will contribute towards improving the success rate of virtual screening, thus will accelerate the development of potential drugs or novel biologically active lead compounds.
Journal Article
Hypoxia, HIF-1α, and COVID-19: from pathogenic factors to potential therapeutic targets
The pandemic of coronavirus disease 2019 (COVID-19) and its pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the greatest current threat to global public health. The highly infectious SARS-CoV-2 virus primarily attacks pulmonary tissues and impairs gas exchange leading to acute respiratory distress syndrome (ARDS) and systemic hypoxia. The current pharmacotherapies for COVID-19 largely rely on supportive and anti-thrombi treatment and the repurposing of antimalarial and antiviral drugs such as hydroxychloroquine and remdesivir. For a better mechanistic understanding of COVID-19, our present review focuses on its primary pathophysiologic features: hypoxia and cytokine storm, which are a prelude to multiple organ failure and lethality. We discussed a possible link between the activation of hypoxia inducible factor 1α (HIF-1α) and cell entry of SARS-CoV-2, since HIF-1α is shown to suppress the angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease serine 2 (TMPRSS2) and upregulate disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). In addition, the protein targets of HIF-1α are involved with the activation of pro-inflammatory cytokine expression and the subsequent inflammatory process. Furthermore, we hypothesized a potential utility of so-called “hypoxic conditioning” to activate HIF-1α-induced cytoprotective signaling for reduction of illness severity and improvement of vital organ function in patients with COVID-19. Taken together, we would propose further investigations into the hypoxia-related molecular mechanisms, from which novel targeted therapies can be developed for the improved management of COVID-19.
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
Human norovirus exhibits strain-specific sensitivity to host interferon pathways in human intestinal enteroids
Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2. An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more wide-spread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.
Journal Article