The c-di-GMP signalling component YfiR regulates multiple bacterial phenotypes and virulence in Pseudomonas plecoglossicida.

AIMS: Pseudomonas plecoglossicida (P. plecoglossicida) is the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea) and it causes severe economic loss to its industry. Biofilm formation, related to intracellular cyclic bis (3'-5') diguanylic acid (c-di-GMP) levels, is essential for the lifestyle of P. plecoglossicida. This research aims to investigate the role of YfiR-a key regulator of the diguanylate cyclase YfiN to regulate c-di-GMP levels and reveal its regulatory function of bacterial virulence expression in P. plecoglossicida. METHODS AND RESULTS: A genetic analysis was carried out to identify the yfiBNR operon for c-di-GMP regulation in P. plecoglossicida. Then, we constructed a yfiR mutant and observed increased c-di-GMP levels, enhanced biofilm formation, increased exopolysaccharides, and diminished swimming and swarming motility in this strain. Moreover, through establishing a yolk sac microinjection infection model in zebrafish larvae, an attenuated phenotype of yfiR mutant that manifested as restored survival and lower bacterial colonization was found. CONCLUSIONS: YfiR is the key regulator of virulence in P. plecoglossicida, which contributes to c-di-GMP level, biofilm formation, exopolysaccharides production, swimming, swarming motility, and bacterial colonization in zebrafish model.

Assessment of Hepatocyte Ploidy by Flow Cytometry.

Polyploidy is a common and dynamic feature of mature rodent and human hepatocytes. While polyploidization occurs naturally during growth, alterations in the distribution of diploid and polyploid cells in the liver can be indicative of tissue stress or a pathologic state. Here, we describe a method for flow cytometric quantification of ploidy distribution by staining with propidium iodide. We first outline a hepatocyte isolation procedure from mouse liver through a two-step perfusion system for analysis of cellular ploidy. In an alternative approach, we employ a nuclei isolation protocol to assess nuclear ploidy. Finally, we describe how the use of fluorescent cell markers is compatible with these methods and helps retain information on cellular position within the tissue.

EGFR signaling activates intestinal stem cells by promoting mitochondrial biogenesis and ß-oxidation.

EGFR-RAS-ERK signaling promotes growth and proliferation in many cell types, and genetic hyperactivation of RAS-ERK signaling drives many cancers. Yet, despite intensive study of upstream components in EGFR signal transduction, the identities and functions of downstream effectors in the pathway are poorly understood. In Drosophila intestinal stem cells (ISCs), the transcriptional repressor Capicua (Cic) and its targets, the ETS-type transcriptional activators Pointed (pnt) and Ets21C, are essential downstream effectors of mitogenic EGFR signaling. Here, we show that these factors promote EGFR-dependent metabolic changes that increase ISC mass, mitochondrial growth, and mitochondrial activity. Gene target analysis using RNA and DamID sequencing revealed that Pnt and Ets21C directly upregulate not only DNA replication and cell cycle genes but also genes for oxidative phosphorylation, the TCA cycle, and fatty acid beta-oxidation. Metabolite analysis substantiated these metabolic functions. The mitochondrial transcription factor B2 (mtTFB2), a direct target of Pnt, was required and partially sufficient for EGFR-driven ISC growth, mitochondrial biogenesis, and proliferation. MEK-dependent EGF signaling stimulated mitochondrial biogenesis in human RPE-1 cells, indicating the conservation of these metabolic effects. This work illustrates how EGFR signaling alters metabolism to coordinately activate cell growth and cell division.

Wnt signaling regulates hepatocyte cell division by a transcriptional repressor cascade.

Cell proliferation is tightly controlled by inhibitors that block cell cycle progression until growth signals relieve this inhibition, allowing cells to divide. In several tissues, including the liver, cell proliferation is inhibited at mitosis by the transcriptional repressors E2F7 and E2F8, leading to formation of polyploid cells. Whether growth factors promote mitosis and cell cycle progression by relieving the E2F7/E2F8-mediated inhibition is unknown. We report here on a mechanism of cell division control in the postnatal liver, in which Wnt/ß-catenin signaling maintains active hepatocyte cell division through Tbx3, a Wnt target gene. The TBX3 protein directly represses transcription of E2f7 and E2f8, thereby promoting mitosis. This cascade of sequential transcriptional repressors, initiated by Wnt signals, provides a paradigm for exploring how commonly active developmental signals impact cell cycle completion.

Assembly of Poly(ethylene glycol)ylated Oleanolic Acid on a Linear Polymer as a Pseudomucin for Influenza Virus Inhibition and Adsorption.

Biomimicry of the mucin barrier function is an efficient strategy to counteract influenza. We report the simple aminolyzation of poly(methyl vinyl ether-alt-maleic anhydride) (PM) using amine-terminated poly(ethylene glycol)ylated oleanolic acid (OAPEG) to mimic the mucin structure and its adsorption of the influenza virus. Direct interactions between influenza hemagglutinin (HA) and the prepared macromolecule evaluated by surface plasmon resonance and isothermal titration calorimetry demonstrated that the multivalent presentation of OAPEG on PM enhanced the binding affinity to HA with a decrease in KD of approximately three orders of magnitude compared with monomeric OAPEG. Moreover, hemagglutination inhibition assay, viral growth inhibition assay, and cytopathic effect reduction assay indicated that the nonglycosylated polymer could mimic natural heavily glycosylated mucin and thus promote the attachment of the virus in a subnanomolar range. Further investigation of the antiviral effects via time-of-addition assay, dynamic light scattering experiments, and transmission electron microscopy photographs indicated that the pseudomucin could adsorb the virion particles and synergistically inhibit the early attachment and final release steps of the influenza infection cycle. These findings demonstrate the effectiveness of the macromolecule in the physical sequestration and prevention of viral infection. Notably, due to its structural similarities with mucin, the biomacropolymer also has the potential for the rational design of antiviral drugs, influenza adsorbents, or filtration materials and the construction of model systems to explore protection against other pathogenic viruses.

Isolated Metastasis to Spleen-Stomach Space From Follicular Thyroid Cancer Combined With a Benign Subcutaneous Nodule on 18F-FDG PET/CT and 131I Scintigraphy.

ABSTRACT: A 40-year-old woman with follicular thyroid cancer underwent a systemic PET/CT evaluation before operation and then received total thyroidectomy and radioactive 131I ablation therapy. Posttreatment imaging of 131I whole-body scan with SPECT/CT showed that metastasis on the spleen-stomach space coexists with mimicking subcutaneous metastasis, considering their level of 131I accumulation and morphological changes on PET/CT. However, histopathology confirmed the subcutaneous lesion of fibrous hyperplasia by fine-needle aspiration. This case demonstrates a vital role of SPECT/CT in the diagnosis of metastatic thyroid cancer and mimicking metastasis.

Differential Diagnosis of Benign and Malignant Thyroid Nodules Using Deep Learning Radiomics of Thyroid Ultrasound Images.

PURPOSE: We aimed to propose a highly automatic and objective model named deep learning Radiomics of thyroid (DLRT) for the differential diagnosis of benign and malignant thyroid nodules from ultrasound (US) images. METHODS: We retrospectively enrolled and finally include US images and fine-needle aspiration biopsies from 1734 patients with 1750 thyroid nodules. A basic convolutional neural network (CNN) model, a transfer learning (TL) model, and a newly designed model named deep learning Radiomics of thyroid (DLRT) were used for the investigation. Their diagnostic accuracy was further compared with human observers (one senior and one junior US radiologist). Moreover, the robustness of DLRT over different US instruments was also validated. Analysis of receiver operating characteristic (ROC) curves were performed to calculate optimal area under it (AUC) for benign and malignant nodules. One observer helped to delineate the nodules. RESULTS: AUCs of DLRT were 0.96 (95% confidence interval [CI]: 0.94-0.98), 0.95 (95% confidence interval [CI]: 0.93-0.97) and 0.97 (95% confidence interval [CI]: 0.95-0.99) in the training, internal and external validation cohort, respectively, which were significantly better than other deep learning models (P < 0.01) and human observers (P < 0.001). No significant difference was found when applying DLRT on thyroid US images acquired from different US instruments. CONCLUSIONS: DLRT shows the best overall performance comparing with other deep learning models and human observers. It holds great promise for improving the differential diagnosis of benign and malignant thyroid nodules.

Computed Tomography Radiomic Nomogram for Preoperative Prediction of Extrathyroidal Extension in Papillary Thyroid Carcinoma.

Objectives: Determining the presence of extrathyroidal extension (ETE) is important for patients with papillary thyroid carcinoma (PTC) in selecting the proper surgical approaches. This study aimed to explore a radiomic model for preoperative prediction of ETE in patients with PTC. Methods: The study included 624 PTC patients (without ETE, n = 448; with minimal ETE, n = 52; with gross ETE, n = 124) whom were divided randomly into training (n = 437) and validation (n = 187) cohorts; all data were gathered between January 2016 and November 2017. Radiomic features were extracted from computed tomography (CT) images of PTCs. Key radiomic features were identified and incorporated into a radiomic signature. Combining the radiomic signature with clinical risk factors, a radiomic nomogram was constructed using multivariable logistic regression. Delong test was used to compare different receiver operating characteristic curves. Results: Five key radiomic features were incorporated into the radiomic signature, which were significantly associated with ETE (p < 0.001 for both cohorts) and slightly better than clinical model integrating significant clinical risk factors in the training cohort (area under the receiver operating characteristic curve (AUC), 0.791 vs. 0.778; F1 score, 0.729 vs. 0.714) and validation cohort (AUC, 0.772 vs. 0.756; F1 score, 0.710 vs. 0.692). The radiomic nomogram significantly improved predictive value in the training cohort (AUC, 0.837, p < 0.001; F1 score, 0.766) and validation cohort (AUC, 0.812, p = 0.024; F1 score, 0.732). Conclusions: The radiomic nomogram significantly improved the preoperative prediction of ETE in PTC patients. It indicated that radiomics could be a valuable method in PTC research.

Radiomic analysis for preoperative prediction of cervical lymph node metastasis in patients with papillary thyroid carcinoma.

PURPOSE: Cervical lymph node (LN) metastasis of papillary thyroid carcinoma (PTC) is critical for treatment and prognosis. We explored the feasibility of using radiomics to preoperatively predict cervical LN metastasis in PTC patients. METHOD: Total 221 PTC patients (training cohort: n = 154; validation cohort: n = 67; divided randomly at the ratio of 7:3) were enrolled and divided into 2 groups based on LN pathologic diagnosis (N0: n = 118; N1a and N1b: n = 88 and 15, respectively). We extracted 546 radiomic features from non-contrast and venous contrast-enhanced computed tomography (CT) images. We selected 8 groups of candidate feature sets by minimum redundancy maximum relevance (mRMR), and obtained 8 radiomic sub-signatures by support vector machine (SVM) to construct the radiomic signature. Incorporating the radiomic signature, CT-reported cervical LN status and clinical risk factors, a nomogram was constructed using multivariable logistic regression. The nomogram's calibration, discrimination, and clinical utility were assessed. RESULTS: The radiomic signature was associated significantly with cervical LN status (p < 0.01 for both training and validation cohorts). The radiomic signature showed better predictive performance than any radiomic sub-signatures devised by SVM. Addition of radiomic signature to the nomogram improved the predictive value (area under the curve (AUC), 0.807 to 0.867) in the training cohort; this was confirmed in an independent validation cohort (AUC, 0.795 to 0.822). Good agreement was observed using calibration curves in both cohorts. Decision curve analysis demonstrated the radiomic nomogram was worthy of clinical application. CONCLUSIONS: Our radiomic nomogram improved the preoperative prediction of cervical LN metastasis in PTC patients.

Synergistic triple-combination therapy with hyaluronic acid-shelled PPy/CPT nanoparticles results in tumor regression and prevents tumor recurrence and metastasis in 4T1 breast cancer.

Breast cancer is characterized by high aggression, poor prognosis, and high recurrence rate. Early detection and specific targeted treatment with less toxicity are the ultimate goals for breast cancer therapy. To improve antitumor therapeutic effects, we developed a novel polypyrrole nanoparticle using the near infrared dye IRDye800CW with camptothecin (CPT)-conjugated hyaluronic acid (HA) shell (PPy@CPT-HA-IRDye800CW) and performed a photothermal therapy (PTT), along with chemotherapy, guided by fluorescence and photoacoustic dual-modality imaging, in combination with immunotherapy. Irradiation with near infrared (NIR) light offered a strong PTT effect and promoted CPT drug release in tumors. Moreover, we found that chemo-photothermal therapy with PPy@CPT-HA-IRDye800CW NPs, in combination with immune checkpoint inhibitor anti-PD-L1 immunotherapy, synergistically enhanced the anti-tumor immune response, thereby eliminating primary breast cancer and preventing tumor metastases and recurrences in 4T1 tumor-bearing mice. This approach may provide important clues for the clinical management of breast cancer and other malignant tumors.

Tissue Repair in the Mouse Liver Following Acute Carbon Tetrachloride Depends on Injury-Induced Wnt/ß-Catenin Signaling.

In the liver, Wnt/ß-catenin signaling is involved in regulating zonation and hepatocyte proliferation during homeostasis. We examined Wnt gene expression and signaling after injury, and we show by in situ hybridization that Wnts are activated by acute carbon tetrachloride (CCl4 ) toxicity. Following injury, peri-injury hepatocytes become Wnt-responsive, expressing the Wnt target gene axis inhibition protein 2 (Axin2). Lineage tracing of peri-injury Axin2+ hepatocytes shows that during recovery the injured parenchyma becomes repopulated and repaired by Axin2+ descendants. Using single-cell RNA sequencing, we show that endothelial cells are the major source of Wnts following acute CCl4 toxicity. Induced loss of ß-catenin in peri-injury hepatocytes results in delayed repair and ultimately injury-induced lethality, while loss of Wnt production from endothelial cells leads to a delay in the proliferative response after injury. Conclusion: Our findings highlight the importance of the Wnt/ß-catenin signaling pathway in restoring tissue integrity following acute liver toxicity and establish a role of endothelial cells as an important Wnt-producing regulator of liver tissue repair following localized liver injury.

Advances in molecular imaging of immune checkpoint targets in malignancies: current and future prospect.

OBJECTIVES: This review describes the current status and progress of immune checkpoint targets for imaging of malignancies. Immune checkpoint blockade holds great potential for cancer treatment, and clinical implementation into routine is very rapidly progressing. Therefore, it is an urgent need to become familiar with the vocabulary of immunotherapy and with the evaluation of immune checkpoint and related treatments through noninvasive molecular imaging. Currently, immune target-associated imaging mainly includes PET, SPECT, optical imaging, and MRI. Each imaging method has its own inherent strengths and weaknesses in reflecting tumor morphology and physiology. PD-1, PD-L1, CTLA-4, and LAG-3 are the most commonly considered targets. In this review, the current status and progress of molecular imaging of immune checkpoint targets are discussed. CONCLUSION: Molecular imaging is likely to become a major tool for monitoring immunotherapy. It can help in selecting patients who are suitable for immunotherapy, and also monitor the tumor response. KEY POINTS: • Immune checkpoint blockade holds great promise for the treatment of different malignant tumors. • Molecular imaging can identify the expression of immune checkpoint targets in the tumor microenvironment at the molecular and cellular levels, and therefore helps selecting potential responders, suitable for specific immunotherapy. • Molecular imaging can also monitor immunotherapeutic effects, and therefore participates in the evaluation of tumor response to treatment.

Manganese-Zeolitic Imidazolate Frameworks-90 with High Blood Circulation Stability for MRI-Guided Tumor Therapy.

Zeolitic imidazolate frameworks (ZIFs) as smart drug delivery systems with microenvironment-triggered release have attracted much attention for tumor therapy. However, the exploration of ZIFs in biomedicine still encounters many issues, such as inconvenient surface modification, fast drug release during blood circulation, undesired damage to major organs, and severe in vivo toxicity. To address the above issues, we developed an Mn-ZIF-90 nanosystem functionalized with an originally designed active-targeting and pH-responsive magnetic resonance imaging (MRI) Y1 receptor ligand [Asn28, Pro30, Trp32]-NPY (25-36) for imaging-guided tumor therapy. After Y1 receptor ligand modification, the Mn-ZIF-90 nanosystem exhibited high drug loading, better blood circulation stability, and dual breast cancer cell membrane and mitochondria targetability, further favoring specific microenvironment-triggered tumor therapy. Meanwhile, this nanosystem showed promising T1-weighted magnetic resonance imaging contrast in vivo in the tumor sites. Especially, this nanosystem with fast clean-up had almost no obvious toxicity and no damage occurred to the major organs in mice. Therefore, this nanosystem shows potential for use in imaging-guided tumor therapy.

Inflammatory Cytokine TNFα Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture.

In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.

Diagnostic Value of ER, PR, FR and HER-2-Targeted Molecular Probes for Magnetic Resonance Imaging in Patients with Breast Cancer.

BACKGROUND/AIMS: Smart molecular probes are required in the application of Magnetic resonance imaging (MRI) for biochemical and clinical research. This study aims to investigate the diagnostic values of estrogen receptor (ER), progesterone receptor (PR), folate receptor (FR) and human epidermal growth factor receptor 2 (HER-2)-targeted molecular probes in the MRI diagnosis of breast cancer. METHODS: Initially, a total of 508 female breast cancer patients were selected for breast cancer subtype classification by immunohistochemistry. Subsequently, the tumor size, lymph node metastasis, and histological grade of different breast cancer subtypes were compared. Molecular probes of Ab-ER-USPIO, Ab-PR-USPIO, Ab-FR-USPIO and Ab-HER-2-USPIO were constructed and screened. The specific binding of molecular probes to breast cancer cells was detected both in vitro and in vivo by Prussian blue staining and MRI using T1 and T2 weighted images. Finally, in vivo toxicity of Ab-HER-2-USPIO was analyzed using hematoxylin and eosin staining. RESULTS: We identified the following subtypes of breast cancer: Luminal A (ER-positive, FR-positive, HER-2-negative), Luminal B (ER-positive, FR-positive, HER-2-positive), HER-2 overexpression (ER-negative, FR-negative, HER-2-positive), and triple-negative breast cancer (ER-negative, FR-negative, HER-2-negative). Featuring favorable in vitro biocompatibility and low in vivo toxicity, Ab-HER-2-USPIO can specifically bind to breast cancer cells BT47 and SKBR3, thus enhancing the quality of T1 weighted MRI images. CONCLUSION: The results indicate that HER-2-targeted MRI molecular probes may be used in the clinical diagnosis of breast cancer and facilitate the development of promising strategies for breast cancer treatments.

Y1-receptor-ligand-functionalized ultrasmall upconversion nanoparticles for tumor-targeted trimodality imaging and photodynamic therapy with low toxicity.

Achieving efficient photodynamic therapy (PDT) in deeper biological tissue is still the biggest bottleneck that limits its widespread application in clinic. Although deeper biological tissue PDT could be realized through a combination of upconversion nanoparticles with a photosensitizer, issues with particle-size-induced upconversion fluorescence (UF) reduction and the related in vivo toxicity still cannot be solved properly. In this study, we synthesized Y1Rs-ligand [Pro30, Nle31, Bpa32, Leu34]NPY(28-36) (NPY)-modified and photosensitizer MC540-loaded LiLuF4:Yb,Er@nLiGdF4@mSiO2 multifunctional nanocomposites (MNPs) with a core-multishell structure and ultrasmall size. Their in vitro and in vivo breast tumor targeting, trimodality imaging performance, PDT therapeutic efficacy, and acute toxicity were evaluated. Our results demonstrated that the core-multishell MNPs(MC540) could achieve excellent UF imaging, and that doping with Gd3+ and Lu3+ rare earth ions could enhance the MR and CT imaging performance. In addition, the mSiO2 shell provided a higher loading rate for the photosensitizer MC540, and the DSPE-PEG thin layer coating outside the MNPs(MC540) further improved the water solubility and biocompatibility, reducing the acute toxicity of the nanocomposites. Finally, the NPY modification enhanced the targetability of MNPs(MC540)/DSPE-PEG-NPY to breast tumors, improving the trimodality UF, CT, and MR imaging performance and PDT efficacy for Y1-receptor-overexpressed breast cancer. In general, our developed multifunctional nanocomposites can serve as a theranostic agent with low toxicity, providing great potential for their use in clinical breast cancer diagnosis and therapy.

Recent Advances in Superparamagnetic Iron Oxide Based Nanoprobes as Multifunctional Theranostic Agents for Breast Cancer Imaging and Therapy.

BACKGROUND: Breast cancer accounts for nearly one in three cancers, and it is the most common cancer diagnosed among women. The death rate of breast cancer is estimated to be 14%. Hence, accurate diagnosis in early stage and effective treatment in any stage are critical for the survival of breast cancer. Mammogram has been the most common technique administered to detect breast cancer. However, the radiation dose from mammogram is harmful to patients. Fortunately, magnetic resonance imaging (MRI) can diagnose breast cancer without any radiation dose, and enhanced MRI can make earlier and differential diagnosis. Therefore, as contrast materials, superparamagnetic iron oxide based nanoprobes (SPIONs) have generated a great deal of attention. OBJECTIVE: This review covers recent advances in SPIONs as multifunctional theranostic agents. METHODS: Besides synthesis and surface modification of SPIONs, passive and active targeted imaging is also discussed. Moreover, a serial of potential therapy for breast cancer is further described, such as photodynamic therapy, photothermal therapy, chemotherapy and magnetic hyperthermia therapy. CONCLUSION: Preparation and surface modification of SPIONs is critical for imaging diagnosis of breast cancer and further potential treatment.

Plasma cell-free DNA level and its integrity as biomarkers to distinguish non-small cell lung cancer from tuberculosis.

AIMS: The aim of this study was to evaluate the potential clinical value of the plasma cell-free DNA (cfDNA) concentrations and strand integrity as an auxiliary tool for Non-small cell lung cancer (NSCLC) differential diagnosis from tuberculosis in patients with solitary pulmonary nodules detected by computed tomography (CT). METHODS: This research was divided into 3 groups: NSCLC (n=106), tuberculosis (n=105) and healthy controls (n=107). The quantization of plasma DNA fragments was performed by quantitative real-time PCR. Amplifying and quantifying shorter (115bp) and longer (247bp) fragments from abundant genomic ALU repeats. RESULTS: The level of cfDNA (ALU115) in patients with NSCLC [95.67 (51.28, 238.85) ng/µl] was significantly higher than that in patients with tuberculosis [59.60 (34.25, 102.53) ng/µl, P=0.001] and that in healthy controls [44.66 (24.56, 66.54) ng/µl, P=0.001]. The integrity of cfDNA in patients with NSCLC [5.91(4.14, 7.45)] was also significantly higher than that in patients with tuberculosis [3.85 (2.91, 5.06), P=0.000] and that in healthy controls [2.78(2.18, 4.82), P=0.000]. Receiver-operating characteristic (ROC) curve analysis showed that cfDNA(ALU115) and its integrity could be used as biomarkers to distinguish NSCLC from tuberculosis (AUC=0.628, P=0.001, cut-off values=91.48, sensitivity=57.50%, specificity=64.80%; AUC=0.722, P=0.000, cut-off values=5.54, sensitivity=55.70%, specificity=82.90%, respectively). In addition, the effect of integrity of cfDNA(AUC=0.722) to distinguish NSCLC from tuberculosis was higher than traditional tumor marker Carbohydrate antigen 125(CA125) (AUC=0.626), Neuron-specific enolase(NSE) (AUC=0.716) and Carcino-embryonic antigen(CEA) (AUC=0.589). CONCLUSIONS: cfDNA and its integrity could be used as indicators for identification of NSCLC from tuberculosis. Moreover, the effect of integrity of cfDNA to distinguish NSCLC from tuberculosis was higher than traditional tumor marker CA125, NSE and CEA.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration.

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair.

Intestinal Stem Cell Pool Regulation in Drosophila.

Intestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, we performed partial ISC depletion. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. We discovered, however, that the loss of differentiated midgut enterocytes (ECs) slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. Our study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis.