Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.

Tracing embryonic hematopoiesis guides induction of pluripotent stem cells to hematopoietic progenitors.

In this issue of Developmental Cell, Fowler et al. applied genetic lineage-tracing mouse models to support the notion that artery endothelial cells are the predominant source of hematopoietic stem cells. They leveraged this and developed a method capable of efficiently differentiating human pluripotent stem cells into HLF+HOXA+ hematopoietic progenitors.

A freeway vehicle early warning method based on risk map: Enhancing traffic safety through global perspective characterization of driving risk.

In the era of rapid advancements in intelligent transportation, utilizing vehicle operating data to evaluate the risk of freeway vehicles and study on vehicle early warning methods not only lays a theoretical foundation for improving the active safety of vehicles, but also provides the technical support for reducing accident rate. This paper proposes a freeway vehicle early warning method based on risk map to enhance vehicle safety. Firstly, Modified Time-to-Collision (MTTC), a two-dimensional indicator that describes the risk of inter-vehicle travel, is used as an indicator of road traffic risk. This paper designs a transformation function to probabilistically transform MTTC into Risk Indicators (RI). The single-vehicle risk map is generated based on the mapping relationship between the risk values and the corresponding roadway segments. These single-vehicle risk maps of all vehicles on the road are superimposed to construct the risk map, which is used to describe the risk distribution in the freeway. Then, a vehicle early warning framework is built based on the risk map. The risk values in the risk map are compared with predefined early warning thresholds to alert the vehicle when it enters a high-risk state. Finally, VISSIM is used to carry out simulation experiments. The experiment simulates a freeway accident stopping situation. This scenario includes sub-scenarios such as unplanned stopping and lane-changing, continuous lane-changing, and adjacent lane overtaking. We analyze the risk map and vehicle warning results in different sub-scenarios, evaluate the risk changes of the vehicles before and after receiving the warning, and compare the warning results of the method in this paper with other alternative methods. The method is applied to 17 vehicles in the simulation to adjust their motion states. The results show that the total warning time is reduced by 29.6% and 73.3% of vehicles change lanes away from the accident vehicle. The overall results validate the effectiveness of the vehicle early warning method based on risk map proposed in this paper.

DCLK1-Mediated Regulation of Invadopodia Dynamics and Matrix Metalloproteinase Trafficking Drives Invasive Progression in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major health concern due to its high mortality from poor treatment responses and locoregional tumor invasion into life sustaining structures in the head and neck. A deeper comprehension of HNSCC invasion mechanisms holds the potential to inform targeted therapies that may enhance patient survival. We previously reported that doublecortin like kinase 1 (DCLK1) regulates invasion of HNSCC cells. Here, we tested the hypothesis that DCLK1 regulates proteins within invadopodia to facilitate HNSCC invasion. Invadopodia are specialized subcellular protrusions secreting matrix metalloproteinases that degrade the extracellular matrix (ECM). Through a comprehensive proteome analysis comparing DCLK1 control and shDCLK1 conditions, our findings reveal that DCLK1 plays a pivotal role in regulating proteins that orchestrate cytoskeletal and ECM remodeling, contributing to cell invasion. Further, we demonstrate in TCGA datasets that DCLK1 levels correlate with increasing histological grade and lymph node metastasis. We identified higher expression of DCLK1 in the leading edge of HNSCC tissue. Knockdown of DCLK1 in HNSCC reduced the number of invadopodia, cell adhesion and colony formation. Using super resolution microscopy, we demonstrate localization of DCLK1 in invadopodia and colocalization with mature invadopodia markers TKS4, TKS5, cortactin and MT1-MMP. We carried out phosphoproteomics and validated using immunofluorescence and proximity ligation assays, the interaction between DCLK1 and motor protein KIF16B. Pharmacological inhibition or knockdown of DCLK1 reduced interaction with KIF16B, secretion of MMPs, and cell invasion. This research unveils a novel function of DCLK1 within invadopodia to regulate the trafficking of matrix degrading cargo. The work highlights the impact of targeting DCLK1 to inhibit locoregional invasion, a life-threatening attribute of HNSCC.

Integrated driving risk surrogate model and car-following behavior for freeway risk assessment.

Drivers' risk perception plays a crucial role in understanding vehicle interactions and car-following behavior under complex conditions and physical appearances. Therefore, it is imperative to evaluate the variability of risks involved. With advancements in communication technology and computing power, real-time risk assessment has become feasible for enhancing traffic safety. In this study, a novel approach for evaluating driving interaction risk on freeways is presented. The approach involves the integration of an interaction risk perception model with car-following behavior. The proposed model, named the driving risk surrogate (DRS), is based on the potential field theory and incorporates a virtual energy attribute that considers vehicle size and velocity. Risk factors are quantified through sub-models, including an interactive vehicle risk surrogate, a restrictions risk surrogate, and a speed risk surrogate. The DRS model is applied to assess driving risk in a typical scenario on freeways, and car-following behavior. A sensitivity analysis is conducted on the effect of different parameters in the DRS on the stability of traffic dynamics in car-following behavior. This behavior is then calibrated using a naturalistic driving dataset, and then car-following predictions are made. It was found that the DRS-simulated car-following behavior has a more accurate trajectory prediction and velocity estimation than other car-following methods. The accuracy of the DRS risk assessments was verified by comparing its performance to that of traditional risk models, including TTC, DRAC, MTTC, and DRPFM, and the results show that the DRS model can more accurately estimate risk levels in free-flow and congested traffic states. Thus the proposed risk assessment model provides a better approach for describing vehicle interactions and behavior in the digital world for both researchers and practitioners.

Stress-protecting harbors for hematopoietic stem cells.

Hematopoietic stem cells (HSCs) rely on specialized microenvironments known as niches to maintain their self-renewal and multilineage potential to generate diverse types of blood cells continuously. Over the last two decades, substantial advancements have been made in unraveling the niche cell components and HSC localizations under homeostatic and stressed circumstances. Advances in imaging, combined with the discovery of phenotypic surface markers combinations and single cell sequencing, have greatly facilitated the systematic examination of HSC localizations. This review aims to present a summary of HSC localizations, highlighting potential distinctions between phenotypically and functionally defined HSCs, and explore the functionality of niches in ensuring the integrity and long-term maintenance of HSCs.

Understanding and Overcoming Immunosuppression Shaped by Cancer Stem Cells.

Use of immunotherapy in recent years has revolutionized cancer treatment for certain types of cancers. However, the broad utility of immunotherapy is limited because there are still many types of cancer that do not respond effectively. Failure of a cancer to respond is due, at least in part, to its phenotypic plasticity, a feature that is established by cancer stem cells (CSC) and their associated microenvironments. This article discusses the current understanding of CSC-mediated immune evasion and provides a prospective view on how researchers can better understand and overcome the intrinsic immune privilege of CSCs and the extrinsic immune-suppressive microenvironment shaped by them.

Canonical and non-canonical Wnt signal pathway in classic Hodgkin lymphoma and the prognostic significance of LEF1, ß-catenin, FZD6 and Wnt5a/b.

Aberrant Wnt signaling has been found in many solid organ cancers, as well as hematological malignancies. However, its role in classic Hodgkin lymphoma (CHL) remains unclear. We evaluated the expression of Wnt signaling components LEF1, ß-catenin, FZD6 and Wnt5a/b and their correlation with the prognosis in 50 CHL patients by immunohistochemical stains. The neoplastic Hodgkin/Reed-Sternberg (HRS) cells showed expression of LEF1, FZD6, and Wnt5a/b but absent nuclear ß-catenin. Wnt5a/b expression was seen in a significantly higher percentage of stage IV patients (67%) compared to other stages (p=0.03). However, there was no correlation between the expression of LEF1, FZD6 and Wnt5a/b and patients' stage or survival. In summary, our results confirmed decreased ß-catenin expression in HRS. Non-canonical Wnt pathway may play a role in the microenvironment that facilitates HRS migration, however, it is not sufficient to consider LEF1, ß-catenin, FZD6 and Wnt5a/b as prognostic factors for CHL.

Lack of VMP1 impairs hepatic lipoprotein secretion and promotes non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that regulates the formation of autophagosomes and lipid droplets. Recent evidence suggests that VMP1 plays a critical role in lipoprotein secretion in zebra fish and cultured cells. However, the pathophysiological roles and mechanisms by which VMP1 regulates lipoprotein secretion and lipid accumulation in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are unknown. METHODS: Liver-specific and hepatocyte-specific Vmp1 knockout mice as well as Vmp1 knock-in mice were generated by crossing Vmp1flox or Vmp1KI mice with albumin-Cre mice or by injecting AAV8-TBG-cre, respectively. Lipid and energy metabolism in these mice were characterized by metabolomic and transcriptome analyses. Mice with hepatic overexpression of VMP1 who were fed a NASH diet were also characterized. RESULTS: Hepatocyte-specific deletion of Vmp1 severely impaired VLDL secretion resulting in massive hepatic steatosis, hepatocyte death, inflammation and fibrosis, which are hallmarks of NASH. Mechanistically, loss of Vmp1 led to decreased hepatic levels of phosphatidylcholine and phosphatidylethanolamine as well as to changes in phospholipid composition. Deletion of Vmp1 in mouse liver also led to the accumulation of neutral lipids in the ER bilayer and impaired mitochondrial beta-oxidation. Overexpression of VMP1 ameliorated steatosis in diet-induced NASH by improving VLDL secretion. Importantly, we also showed that decreased liver VMP1 is associated with NAFLD/NASH in humans. CONCLUSIONS: Our results provide novel insights on the role of VMP1 in regulating hepatic phospholipid synthesis and lipoprotein secretion in the pathogenesis of NAFLD/NASH. LAY SUMMARY: Non-alcoholic fatty liver disease and its more severe form, non-alcoholic steatohepatitis, are associated with a build-up of fat in the liver (steatosis). However, the exact mechanisms that underly steatosis in patients are not completely understood. Herein, the authors identified that the lack of a protein called VMP1 impairs the secretion and metabolism of fats in the liver and could therefore contribute to the development and progression of non-alcoholic fatty liver disease.

An optimized chromatin immunoprecipitation protocol using Staph-seq for analyzing genome-wide protein-DNA interactions.

Genome-wide mapping of transcription factors (TFs) is critical to understand their functions. In chromatin immunoprecipitation (ChIP)-seq assay, it's challenging to study recruitment of low-abundant TFs transiently boud to the genome. Here, we present an optimized protocol using ChIP Next-Gen Seq Sepharose (Staph-seq) to efficiently pull down chromatin complexes. The double size selection promotes sensitive capture of genome-wide protein-DNA associations while eliminating potential Staph A contamination, which is a common problem in protocols using Staph A cells. For complete details on the use and execution of this protocol, please refer to Tao et al. (2020).1.

Controlling the Connected Vehicle with Bi-Directional Information: Improved Car-Following Models and Stability Analysis.

Connected vehicle (CV) technologies are changing the form of traditional traffic models. In the CV driving environment, abundant and accurate information is available to vehicles, promoting the development of control strategies and models. Under these circumstances, this paper proposes a bidirectional vehicles information structure (BDVIS) by making use of the acceleration information of one preceding vehicle and one following vehicle to improve the car-following models. Then, we deduced the derived multiple vehicles information structure (DMVIS), including historical movement information of multiple vehicles, without the acceleration information. Next, the paper embeds the four kinds of basic car-following models into the framework to investigate the stability condition of two structures under the small perturbation of traffic flow and explored traffic response properties with different proportions of forward-looking or backward-looking terms. Under the open boundary condition, simulations on a single lane are conducted to validate the theoretical analysis. The results indicated that BDVIS and the DMVIS perform better than the original car-following model in improving the traffic flow stability, but that they have their own advantages for differently positioned vehicles in the platoon. Moreover, increasing the proportions of the preceding and following vehicles presents a benefit to stability, but if traffic is stable, an increase in any of the parameters would extend the influence time, which reveals that neither ß1 or ß2 is the biggest the best for the traffic.

Tumor-initiating stem cell shapes its microenvironment into an immunosuppressive barrier and pro-tumorigenic niche.

Tumor-initiating stem cells (TSCs) are critical for drug resistance and immune escape. However, the mutual regulations between TSC and tumor microenvironment (TME) remain unclear. Using DNA-label retaining, single-cell RNA sequencing (scRNA-seq), and other approaches, we investigated intestinal adenoma in response to chemoradiotherapy (CRT), thus identifying therapy-resistant TSCs (TrTSCs). We find bidirectional crosstalk between TSCs and TME using CellPhoneDB analysis. An intriguing finding is that TSCs shape TME into a landscape that favors TSCs for immunosuppression and propagation. Using adenoma-organoid co-cultures, niche-cell depletion, and lineaging tracing, we characterize a functional role of cyclooxygenase-2 (Cox-2)-dependent signaling, predominantly occurring between tumor-associated monocytes and macrophages (TAMMs) and TrTSCs. We show that TAMMs promote TrTSC proliferation through prostaglandin E2 (PGE2)-PTGER4(EP4) signaling, which enhances ß-catenin activity via AKT phosphorylation. Thus, our study shows that the bidirectional crosstalk between TrTSC and TME results in a pro-tumorigenic and immunosuppressive contexture.

In Situ Hematopoietic Stem Cell Imaging.

Although immunohistochemistry of tissue sections has been the gold standard for analyzing tissue structure and cellular localization, this approach has significant shortcomings when it comes to analyzing complex and heterogeneous tissues such as the bone marrow with rare cells like hematopoietic stem cells (HSCs). Hence, studying rare cells and their relationship with the surrounding heterogenous microenvironment requires visualization of specifically labeled cells within large intact tissues in three dimensions. Here, we describe a whole mount sternal bone marrow imaging method which has enabled detailed quantitative and qualitative analysis of rare HSCs within the sternal tissue. The methodology is broadly applicable for examining the 3D architecture of niche cells in relation to HSCs.

Risk perception and the warning strategy based on safety potential field theory.

Benefiting from the rapid development of communication and intelligent vehicle technology in recent years, most traffic information is capable of being collected, processed, and transmitted to each vehicle through a connected and automated vehicles (CAVs) system. To meet the higher requirements of driving safety in CAVs environment, it is necessary to develop more effective safety evaluation indicators that combine all the traffic information received by the vehicle. To this end, this study proposes a novel methodology for risk perception and warning strategy based on safety potential field model to minimize driving risk in the CAVs environment. A dynamic safety potential field model was constructed to describe the spatial distribution of driving risk encountered by vehicles. This safety potential field model can comprehensively consider the impact of various types of traffic information on driving risk. And then, a novel driving risk indicator, named potential field indicator (PFI), was established to evaluate the level of driving risk. Finally, an early warning strategy was proposed to prevent accidents, whose performance was evaluated by several simulations carried out through SUMO simulator. The comparison with some classic risk indicators indicate that our proposed PFI can more accurately reflect the actual driving risk faced by vehicles under different vehicle motion states and thus is more suitable for driving risk assessment in the CAVs environment. It is expected that the findings in this study could be valuable in improving the performance of strategic decision-making in driver assistance systems in the CAVs environment.

Overcoming resistance to immunotherapy by teaching old drugs new tricks.

Cancer stem cells (CSCs) underlie resistance to therapy. Cancer develops only in the context of failing immunosurveillance, and stem cells occupy immune privileged microenvironments. Recent evidence demonstrates that CSCs borrow immune privilege from their normal counterparts. However, low doses of doxorubicin can target CSCs by restoring anticancer immunity.

ß-Catenin and Associated Proteins Regulate Lineage Differentiation in Ground State Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. ß-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of ß-catenin in restraining germline and somatic lineage differentiation genes. We show that ß-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help ß-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of ß-catenin in preserving lineage differentiation integrity in ground state ESCs.

Overcoming Wnt-ß-catenin dependent anticancer therapy resistance in leukaemia stem cells.

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-ß-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate ß-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-ß-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated ß-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, ß-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated ß-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.

Intestinal epithelial regeneration: active versus reserve stem cells and plasticity mechanisms.

The gastrointestinal system is arguably one of the most complicated developmental systems in a multicellular organism, as it carries out at least four major functions: digestion of food, absorption of nutrients, excretion of hormones, and defense against pathogens. Anatomically, the fetal gut has a tubular structure with an outer layer of smooth muscle derived from lateral splanchnic mesoderm and an inner lining of epithelium derived from the definitive endoderm. During morphogenesis of the gut tube, the definitive endoderm transforms into a primitive gut tube with a foregut, midgut, and hindgut. During the course of further development, the midgut gives rise to the small and proximal large intestine and the hindgut gives rise to the distal large intestine and rectum. The small intestine is subdivided into three parts: duodenum, jejunum, and ileum, whereas the large intestine is subdivided into the cecum, colon, and rectum.