An Atlas of Grass Carp IgM+ B Cells in Homeostasis and Bacterial Infection Helps to Reveal the Unique Heterogeneity of B Cells in Early Vertebrates.

Teleost B cells are primitive lymphocytes with both innate and adaptive immune functions. However, the heterogeneity and differentiation trajectory of teleost B cells remain largely unknown. In this study, the landscape of grass carp IgM+ (gcIgM+) B cells was revealed by single-cell RNA sequencing. The results showed that gcIgM+ B cells mainly comprise six populations: (im)mature B cells, innate B cells, proliferating B cells, plasma cells, CD22+ cells, and CD34+ cells, among which innate B cells and proliferating B cells were uncommon B cell subsets with, to our knowledge, new characteristics. Remarkably, three functional IgMs were discovered in grass carp, and a significant percentage of gcIgM+ B cells, especially plasma cells, expressed multiple Igµ genes (Igµ1, Igµ2, and/or Igµ3). More importantly, through single-cell sorting combined with Sanger sequencing, we found that distinct VHDJH recombination patterns of Igµ genes were present in single IgM+ B cells, indicating that individual teleost B cells might produce multiple Abs by coexpressing rearranged IgM subclass genes. Moreover, the percentage of IgM1highIgM2highIgM3high plasma cells increased significantly after bacterial infection, suggesting that individual plasma cells might tend to produce multiple IgMs to resist the infection in teleost fish. In summary, to our knowledge, this study not only helps to uncover the unique heterogeneity of B cells in early vertebrates but also provided significant new evidence supporting the recently proposed "one cell-multiple Abs" paradigm, challenging the classical rule of "one cell-one Ab."

Identification and functional analyses of CD4-1+ cells in grass carp (Ctenopharyngodon idella).

In mammals, CD4 is found to be expressed on T cells and innate immune cells, however, teleost cells bearing CD4 have not been well identified and characterized. In this study, we identified two different CD4-1+ cell subsets in grass carp (Ctenopharyngodon idella): CD4-1+ lymphocytes (Lym) and CD4-1+ myeloid cells (Mye), both of which had the highest proportions in the head kidney. The mRNA expression analysis showed that CD4-1, CD4-2, TCRß, CD3γ/δ, and LCK1 are highly expressed in CD4-1+ Lym and also expressed in CD4-1+ Mye. Furthermore, we found that CD4-1+ Lym have a Lym morphology and highly express T-cell cytokines, suggesting that they are CD4+ T cells equivalent to mammalian Th cells. On the other hand, CD4-1+ Mye were found to have a morphology of macrophage and highly express macrophage marker gene MCSFR, indicating that they are macrophages. In addition, functional analysis revealed that CD4-1+ Mye possess phagocytic ability and great antigen-processing ability. Taken together, our study sheds further light on the composition and function of CD4+ cells in teleost fish.

Athermal panoramic annular lens design with a thermal analysis method.

An athermal 360∘×(30∘-100∘) and F/3.5 panoramic annular lens (PAL) system is designed. Through the optical mechanical thermal analysis results based on finite element analysis (FEA), it is expected that the system will have excellent performance under extreme temperature fluctuations. Simulation shows that the system is thermally insensitive in the temperature range from -40∘ C to 60°C, consistently providing great imaging capability with the modulation transfer function (MTF) value at 133 lp/mm greater than 0.3. The proposed design and analysis workflow contains an evaluation of thermal optical performance with a higher accuracy, thus having significance in future athermal optical design. We expect the designed PAL system to have a broad application prospect in the field of outdoor applications, including automatic navigation of vehicles and all-weather surveillance systems.

Universality and scaling in complex networks from periods of Chinese history.

Critical physical systems with large numbers of molecules can show universal and scaling behaviors. It is of interest to know whether human societies with large numbers of people can show the same behaviors. Here, we use network theory to analyze Chinese history in periods 209 BCE-23 CE and 515-618 CE) related to the Western Han-Xin Dynasty and the late Northern Wei-Sui Dynasty, respectively. Two persons are connected when they appear in the same historical event. We find that the historical networks from two periods separated about 500 years have interesting universal and scaling behaviors, and they are small-world networks; their average cluster coefficients as a function of degree are similar to the network of movie stars. In the historical networks, the persons with larger degrees prefer to connect with persons with a small degree; however, in the network of movie stars, the persons with larger degrees prefer to connect with persons with large degrees. We also find an interesting similar mechanism for the decline or collapse of historical Chinese dynasties. The collapses of the Xin dynasty (9-23 CE) and the Sui dynasty (581-618 CE) were initiated from their arrogant attitude toward neighboring states.

Elaiophylin Elicits Robust Anti-Tumor Responses via Apoptosis Induction and Attenuation of Proliferation, Migration, Invasion, and Angiogenesis in Pancreatic Cancer Cells.

Pancreatic cancer remains a formidable challenge in oncology due to its aggressive nature and limited treatment options. In this study, we investigate the potential therapeutic efficacy of elaiophylin, a novel compound, in targeting BxPC-3 and PANC-1 pancreatic cancer cells. We comprehensively explore elaiophylin's impact on apoptosis induction, proliferation inhibition, migration suppression, invasion attenuation, and angiogenesis inhibition, key processes contributing to cancer progression and metastasis. The results demonstrate that elaiophylin exerts potent pro-apoptotic effects, inducing a substantial increase in apoptotic cells. Additionally, elaiophylin significantly inhibits proliferation, migration, and invasion of BxPC-3 and PANC-1 cells. Furthermore, elaiophylin exhibits remarkable anti-angiogenic activity, effectively disrupting tube formation in HUVECs. Moreover, elaiophylin significantly inhibits the Wnt/ß-Catenin signaling pathway. Our findings collectively demonstrate the multifaceted potential of elaiophylin as a promising therapeutic agent against pancreatic cancer via inhibition of the Wnt/ß-Catenin signaling pathway. By targeting diverse cellular processes crucial for cancer progression, elaiophylin emerges as a prospective candidate for future targeted therapies. Further investigation of the in vivo efficacy of elaiophylin is warranted, potentially paving the way for novel and effective treatment approaches in pancreatic cancer management.

Targeting autophagy regulation in NLRP3 inflammasome-mediated lung inflammation in COVID-19.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Emerging evidence indicates that the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome is activated, which results in a cytokine storm at the late stage of COVID-19. Autophagy regulation is involved in the infection and replication of SARS-CoV-2 at the early stage and the inhibition of NLRP3 inflammasome-mediated lung inflammation at the late stage of COVID-19. Here, we discuss the autophagy regulation at different stages of COVID-19. Specifically, we highlight the therapeutic potential of autophagy activators in COVID-19 by inhibiting the NLRP3 inflammasome, thereby avoiding the cytokine storm. We hope this review provides enlightenment for the use of autophagy activators targeting the inhibition of the NLRP3 inflammasome, specifically the combinational therapy of autophagy modulators with the inhibitors of the NLRP3 inflammasome, antiviral drugs, or anti-inflammatory drugs in the fight against COVID-19.

Response of serum LDL cholesterol to oatmeal consumption depends on CYP7A1_rs3808607 genotype in Chinese.

BACKGROUND AND OBJECTIVES: Notable inter-individual differences in cholesterol-lowering effects following oatmeal consumption have been previously reported. Genetic variations may among the reasons for the heterogeneous response to lipid modulations. And to determine whether SNP of cytochrome P450 family 7 subfamily A member 1 gene rs3808607 and isoforms of apolipoprotein E are associated with the inter-individual variations in cholesterol-lowering effects of oatmeal consumption, we did this study. METHODS AND STUDY DESIGN: Data in this study were extracted from a parallel, controlled trial, in which 62 medication-naive hypercholesterolemic patients provided with staple food substitute of either 80 g/d oatmeal (n=31) or 80 g/d refined white rice (n=31) for 45 days. Fasting blood samples were collected at baseline and endpoint of the study for lipid profiling, glycemic testing, and genotyping. RESULTS: Totally, 56 of 62 participants completed the study and were thus included. Genotype- diet interactions were observed between oatmeal consumption and SNP in the cytochrome P450 family 7 subfamily A member 1 gene rs3808607 in regulating LDL cholesterol (p=0.04); rs3808607-TT homozygotes exhibited significantly higher responsiveness to oatmeal (reduction in LDL cholesterol) than G allele carriers (GG/GT) (p=0.02). However, obvious genotype-diet interactions were not observed between oatmeal consumption and apolipoprotein E isoforms in cholesterol and glycemic modulation (p>0.05). CONCLUSIONS: SNP in cytochrome P450 family 7 subfamily A member 1 gene rs3808607 was associated with the extent of LDL cholesterol reduction following oatmeal consumption. Trials with larger sample sizes are required to confirm the findings.

Phosphorylation of moesin by Jun N-terminal kinase is important for podosome rosette formation in Src-transformed fibroblasts.

Podosomes are actin-based membrane protrusions that facilitate extracellular matrix degradation and motility of invasive cells. Podosomes can self-organize into large rosette-like structures in Src-transformed fibroblasts, osteoclasts and some highly invasive cancer cells. However, the mechanism of this assembly remains obscure. In this study, we show that the suppression of Jun N-terminal kinase (JNK) by the JNK inhibitor SP600125 or short-hairpin RNA inhibited podosome rosette formation in SrcY527F-transformed NIH3T3 fibroblasts. In addition, SrcY527F was less able to induce podosome rosettes in JNK1-null or JNK2-null mouse embryo fibroblasts than in wild-type counterparts. The kinase activity of JNK was essential for promoting podosome rosette formation but not for its localization to podosome rosettes. Moesin, a member of the ERM (ezrin, radixin and moesin) protein family, was identified as a substrate of JNK. We show that the phosphorylation of moesin at Thr558 by JNK was important for podosome rosette formation in SrcY527F-transformed NIH3T3 fibroblasts. Taken together, our results unveil a novel role of JNK in podosome rosette formation through the phosphorylation of moesin.

Protein tyrosine phosphatase SHP2 suppresses podosome rosette formation in Src-transformed fibroblasts.

Podosomes are actin-enriched membrane protrusions that play important roles in extracellular matrix degradation and invasive cell motility. Podosomes undergo self-assembly into large rosette-like structures in Src-transformed fibroblasts, osteoclasts and certain highly invasive cancer cells. Several protein tyrosine kinases have been shown to be important for the formation of podosome rosettes, but little is known regarding the role of protein tyrosine phosphatases in this process. We found that knockdown of the Src homolog domain-containing phosphatase 2 (SHP2) significantly increased podosome rosette formation in Src-transformed fibroblasts. By contrast, SHP2 overexpression suppressed podosome rosette formation in these cells. The phosphatase activity of SHP2 was essential for the suppression of podosome rosette formation. SHP2 selectively suppressed the tyrosine phosphorylation of Tks5, a scaffolding protein required for podosome formation. The inhibitory effect of SHP2 on podosome rosette formation was associated with the increased activation of Rho-associated kinase (ROCK) and the enhanced polymerization of vimentin filaments. A higher content of polymerized vimentin filaments was correlated with a lower content of podosome rosettes. Taken together, our findings indicate that SHP2 serves as a negative regulator of podosome rosette formation through the dephosphorylation of Tks5 and the activation of ROCK-mediated polymerization of vimentin in Src-transformed fibroblasts.

[Low fat milk powder containing esterified plant sterols improves the blood lipid profile of adults with hypercholesterolemia].

OBJECTIVE: To observe the impact of plant sterol esters (PSE) mixed in low fat milk powder (2.5 g of PSE/day) on plasma cholesterol levels in hypercholesterolemic subjects during a 6-week intervention period. METHODS: In this double-blind, randomized, placebo-controlled study, 59 subjects (19 males, mean age (60.28 ± 6.98) years) with primary hypercholesterolemia (fasting LDL cholesterol between 3.4-6.0 mmol/L) were randomly divided into two groups (treatment group, 2.5 g of plant sterol esters a day, n = 30) and placebo group (n = 29). Blood samples were collected at week 0, 3 and 6. The primary outcome was change in plasma LDL-cholesterol (LDL-C). Secondary outcomes were changes in total cholesterol (TC), HDL cholesterol (HDL-C), triglycerides (TG), anthropometry and blood biochemistry. RESULTS: LDL-C significantly reduction from baseline (4.18 ± 0.54) mmol/L to (3.44 ± 0.61) mmol/L (-17.7%, P < 0.05) at week 3 and (3.35 ± 0.39) mmol/L (-19.9%, P < 0.05) at week 6 in the treatment group, whereas in placebo group from (4.11 ± 0.54) mmol/L at baseline to (3.47 ± 0.60) mmol/L (-15.57%, P < 0.05) and (3.61 ± 0.39) mmol/L (-12.17%, P < 0.05) at week 3 and week 6, respectively. TC was reduced from (6.30 ± 0.86) mmol/L at baseline to (5.92 ± 0.75) mmol/L (-6.03%, P > 0.05) at week 3 and (5.43 ± 0.77) mmol/L (-13.8%, P < 0.05) at week 6 in treatment group, from (6.20 ± 0.76) mmol/L at week 0 to (5.70 ± 0.76) mmol/L (-8.06%, P < 0.05) at week 3 and (5.84 ± 0.75) mmol/L (-5.81%, P < 0.05) at week 6 in placebo group. PSE-enriched milk did not affect plasma HDL-C level and TG level at both week 3 and week 6. After normalization to the placebo group, the treatment group showed significant reduction in LDL-C and total cholesteron after 6 weeks. The observed difference of reduction was 7.69% (-0.33 mmol/L, P < 0.05) for LDL-C and 8.00% (-0.51 mmol/L, P < 0.05) for TC between the two groups. There were no significant changes in safety parameters, including blood biochemistry tests during the study period. CONCLUSION: Plant sterol ester enriched milk powder is effective in reducing LDL-C among Chinese hypercholesterolemic subjects at a dosage recommended by EFSA.

Trametinib boosts palbociclib's efficacy in breast cancer via autophagy inhibition.

Breast cancer, a predominant global health issue, requires ongoing exploration of new therapeutic strategies. Palbociclib (PAL), a well-known cyclin-dependent kinase (CDK) inhibitor, plays a critical role in breast cancer treatment. While its efficacy is recognized, the interplay between PAL and cellular autophagy, particularly in the context of the RAF/MEK/ERK signaling pathway, remains insufficiently explored. This study investigates PAL's inhibitory effects on breast cancer using both in vitro (MCF7 and MDA-MB-468 cells) and in vivo (tumor-bearing nude mice) models. Aimed at elucidating the impact of PAL on autophagic processes and exploring the potential of combining it with trametinib (TRA), an MEK inhibitor, our research seeks to address the challenge of PAL-induced drug resistance. Our findings reveal that PAL significantly decreases the viability of MCF7 and MDA-MB-468 cells and reduces tumor size in mice while showing minimal cytotoxicity in MCF10A cells. However, PAL also induces protective autophagy, potentially leading to drug resistance via the RAF/MEK/ERK pathway activation. Introducing TRA effectively neutralized this autophagy, enhancing PAL's anti-tumor efficacy. A combination of PAL and TRA synergistically reduced cell viability and proliferation, and in vivo studies showed notable tumor size reduction. In conclusion, the PAL and TRA combination emerges as a promising strategy for overcoming PAL-induced resistance, offering a new horizon in breast cancer treatment.

Synergistic effects of the combination of trametinib and alpelisib in anaplastic thyroid cancer with BRAF and PI3KCA co-mutations.

Background: Anaplastic thyroid cancer (ATC), a rare and aggressive malignancy with a poor prognosis, has shown promise with the approved dabrafenib/trametinib combination for BRAFV600E mutation. Co-occurring PI3KCA mutations, identified as negative prognostic factors in lung cancer with BRAFV600E mutation, emphasize the need to target both pathways. Exploring trametinib and alpelisib combination becomes crucial for ATC. Methods: A patient-derived xenograft (PDX) and primary cell line were obtained from an ATC patient with BRAF and PI3KCA co-mutation. Individual testing of targeted therapies against BRAF, MEK, and PI3KCA was followed by a combination treatment. Synergistic effects were evaluated using the combination index. Immunoblotting assessed the efficacy, with validation performed using a PDX model. Results: In this study, the ATC0802 cell line and PDX were established from a refractory ATC patient. NGS revealed BRAF and PI3KCA co-mutations pre- and post-dabrafenib/trametinib treatment. Trametinib/alpelisib combination showed synergy, suppressing both pERK and pAKT levels, unlike monotherapies or BRAF knockdown. The combination induced apoptosis and, in the PDX model, demonstrated superior tumor growth inhibition compared to monotherapies. Conclusions: The combination of trametinib and alpelisib showed promise as a strategy for treating ATC with co-mutations in BRAF and PI3KCA, both in vitro and in vivo. This combination offers insights into overcoming resistance to BRAF-targeted treatments in ATC with mutations in BRAF and PI3KCA.

PLK1 and its substrate MISP facilitate intrahepatic cholangiocarcinoma progression by promoting lymphatic invasion and impairing E-cadherin adherens junctions.

Intrahepatic cholangiocarcinoma (iCCA) is a subtype of CCA and has a high mortality rate and a relatively poor prognosis. However, studies focusing on increased cell motility and loss of epithelial integrity during iCCA progression remain relatively scarce. We collected seven fresh tumor samples from four patients to perform RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) to determine the transcriptome profile and chromatin accessibility of iCCA. The increased expression of cell cycle regulators, including PLK1 and its substrate MISP, was identified. Ninety-one iCCA patients were used to validate the clinical significance of PLK1 and MISP. The upregulation of PLK1 and MISP was determined in iCCA tissues. Increased expression of PLK1 and MISP was significantly correlated with tumor number, N stage, and lymphatic invasion in an iCCA cohort. Knockdown of PLK1 or MISP reduced trans-lymphatic endothelial migration and wound healing and affected focal adhesions in vitro. In cell‒cell junctions, MISP localized to adherens junctions and suppressed E-cadherin dimerization. PLK1 disrupted adherens junctions in a myosin-dependent manner. Furthermore, PLK1 and MISP promoted cell proliferation in vitro and tumorigenesis in vivo. In iCCA, PLK1 and MISP promote aggressiveness by increasing lymphatic invasion, tumor growth, and motility through the repression of E-cadherin adherens junctions.

Intestinal flora in the constipation patients before versus after lactulose intervention.

This study aimed to investigate the characteristics of intestinal flora in patients with chronic functional constipation before and after lactulose intervention. Twenty-nine patients with constipation in the treatment group received oral lactulose (15 mL/d) for a month. Twenty healthy subjects served as controls. Stool specimens were collected before and after lactulose treatment. Fecal bacteria were examined by 16SrRNA gene sequencing and bioinformatics analysis. After lactulose treatment, most bacteria in the constipation group, including Bifidobacteria, Bacillus cereus, Prevotella, Bacillus, Anaerostipes, Oribacterium, and Mogibacterium increased as compared to those in the healthy control group. Anaerotruncus declined in the healthy control group after lactulose treatment. Our study shows lactulose can increase the abundance of probiotics, optimize the intestinal microenvironment, and alleviate constipation.

Comparative study on antibacterial characteristics of the multiple liver expressed antimicrobial peptides (LEAPs) in teleost fish.

Antimicrobial peptides are important components of the host innate immune system, forming the first line of defense against infectious microorganisms. Among them, liver-expressed antimicrobial peptides (LEAPs) are a family of antimicrobial peptides that widely exist in vertebrates. LEAPs include two types, named LEAP-1 and LEAP-2, and many teleost fish have two or more LEAP-2s. In this study, LEAP-2C from rainbow trout and grass carp were discovered, both of which are composed of 3 exons and 2 introns. The antibacterial functions of the multiple LEAPs were systematically compared in rainbow trout and grass carp. The gene expression pattern revealed that rainbow trout and grass carp LEAP-1, LEAP-2A, LEAP-2B and/or LEAP-2C were differentially expressed in various tissues/organs, mainly in liver. After bacterial infection, the expression levels of LEAP-1, LEAP-2A, LEAP-2B and/or LEAP-2C in the liver and gut of rainbow trout and grass carp increased to varying degrees. Moreover, the antibacterial assay and bacterial membrane permeability assay showed that rainbow trout and grass carp LEAP-1, LEAP-2A, LEAP-2B and LEAP-2C all have antibacterial activities against a variety of Gram-positive and Gram-negative bacteria with varying levels through membrane rupture. Furthermore, cell transfection assay showed that only rainbow trout LEAP-1, but not LEAP-2, can lead to the internalization of ferroportin, the only iron exporter on cell surface, indicating that only LEAP-1 possess iron metabolism regulation activity in teleost fish. Taken together, this study systematically compared the antibacterial function of LEAPs in teleost fish and the results suggest that multiple LEAPs can enhance the immunity of teleost fish through different expression patterns and different antibacterial activities to various bacteria.

A Potential Association of Zinc Deficiency and Tyrosine Kinase Inhibitor-Induced Hand-Foot Skin Reaction.

Hand-foot skin reaction (HFSR) is a common skin-related adverse event induced by multikinase inhibitors targeting both platelet-derived growth factor receptor and vascular endothelial growth factor receptor, possibly due to inadequate repair following frictional trauma. Zinc is a trace element and essential nutrient in humans that plays critical roles in the development and differentiation of skin cells. Zinc transporters (Zrt- and Irt-like proteins and Zn transporters) and metallothioneins are involved in zinc efflux, uptake, and homeostasis and have been reported to be involved in skin differentiation. The underlying mechanism of HFSR remains unclear, and the association between HFSR and zinc has not been previously studied. However, some case reports and case series provide potential evidence to suggest that zinc deficiency may be involved in HFSR development and zinc supplementation may relieve HFSR symptoms. However, no large-scale clinical studies have been conducted to examine this role. Therefore, this review summarizes the evidence supporting a possible link between HFSR development and zinc and proposes potential mechanisms underlying this association based on current evidence.

Wee1 inhibition by MK1775 potentiates gemcitabine through accumulated replication stress leading to apoptosis in biliary tract cancer.

Patients with advanced biliary tract cancer (BTC) have a poor prognosis, and novel treatments are needed. Gemcitabine, the standard of care for BTC, induces DNA damage; however, the ability of cancer cells to repair DNA dampens its effects. To improve the efficacy of gemcitabine, we combined it with MK1775, a Wee1 inhibitor that prevents activation of the G2/M checkpoint. BTC cell lines were treated with gemcitabine only or in combination with MK1775 to determine the therapeutic potential of BTC. Gemcitabine inhibited the growth and induced the apoptosis of four BTC cell lines to a greater extent when added with MK1775 than when added alone. The effects of the combination treatment were observed in both p53 wild-type and p53 mutant cell lines and were unaffected by knockdown of wild-type p53. The combination treatment increased the percentage of apoptotic cells and decreased the percentage of cells synthesizing DNA, suggesting that it caused DNA-damaged cells to accumulate and possibly die in S phase. It did not induce apoptosis when cells were arrested in mitosis using nocodazole. In a xenograft mouse model, gemcitabine plus MK1775 (but not either alone) inhibited the growth of tumors generated from inoculated BTC cells. Our results show that MK1775 highly enhances gemcitabine cytotoxicity in BTC regardless of p53 status. We suggest that the combination treatment elicits a DNA damage response and consequent apoptosis. Our preclinical study provides a basis for future clinical trials of gemcitabine plus MK1775 in patients with BTC.

Mucin 4 Confers Gemcitabine Resistance and an Unfavorable Prognosis in Patients with Cholangiocarcinoma via AKT Activation.

Cholangiocarcinoma (CCA) exhibits aggressive biological behavior and a poor prognosis. Gemcitabine (GEM)-based chemotherapy is the first-line chemotherapy for advanced CCA but has a response rate of only 20-30%. Therefore, investigating treatments to overcome GEM resistance in advanced CCA is crucial. Among mucin (MUC) family members, MUC4 showed the greatest increase in the resistant versus parental sublines. MUC4 was upregulated in whole-cell lysates and conditioned media from gemcitabine-resistant (GR) CCA sublines. MUC4 mediated GEM resistance by activating AKT signaling in GR CCA cells. The MUC4-AKT axis induced BAX S184 phosphorylation to inhibit apoptosis and downregulated GEM transporter human equilibrative nucleoside transporter 1 (hENT1) expression. The combination of AKT inhibitors and GEM or afatinib overcame GEM resistance in CCA. In vivo, capivasertib (an AKT inhibitor) increased GEM sensitivity in GR cells. MUC4 promoted EGFR and HER2 activation to mediate GEM resistance. Finally, MUC4 expression in patient plasma correlated with MUC4 expression. Paraffin-embedded specimens from non-responders expressed significantly more MUC4 than did those from responders, and this upregulation was associated with poor progression-free survival and overall survival. In GR CCA, high MUC4 expression promotes sustained EGFR/HER2 signaling and AKT activation. The combination of AKT inhibitors with GEM or afatinib might overcome GEM resistance.