SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.

WTAP-induced N6-methyladenosine of PD-L1 blocked T-cell-mediated antitumor activity under hypoxia in colorectal cancer.

N6-Methyladenosine (m6A) is a important process regulating gene expression post-transcriptionally. Programmed death ligand 1 (PD-L1) is a major immune inhibitive checkpoint that facilitates immune evasion and is expressed in tumor cells. In this research we discovered that Wilms' tumor 1-associated protein (WTAP) degradation caused by ubiquitin-mediated cleavage in cancer cells (colorectal cancer, CRC) under hypoxia was inhibited by Pumilio homolog 1 (PUM1) directly bound to WTAP. WTAP enhanced PD-L1 expression in a way that was m6A-dependent. m6A "reader," Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) identified methylated PD-L1 transcripts and subsequently fixed its mRNA. Additionally, we found that T-cell proliferation and its cancer cell-killing effects were prevented by overexpression of WTAP in vitro and in vivo. Overexpression prevented T cells from proliferating and killing CRC by maintaining the expression of PD-L1. Further evidence supporting the WTAP-PD-L1 regulatory axis was found in human CRC and organoid tissues. Tumors with high WTAP levels appeared more responsive to anti-PD1 immunotherapy, when analyzing samples from patients undergoing treatment. Overall, our findings demonstrated a novel PD-L1 regulatory mechanism by WTAP-induced mRNA epigenetic regulation and the possible application of targeting WTAP as immunotherapy for tumor hypoxia.

The importance of preoperative T3 stage substaging by 3D endorectal ultrasonography for the prognosis of middle and low rectal cancer.

OBJECTIVE: The study aimed to validate the role of 3D-endorectal ultrasonography in prognosis and recurrence for patients with T3-stage rectal cancer by evaluating the preoperative extramural depth of tumor invasion. METHODS: In this study, we investigated the medical records of rectal cancer patients who were admitted to Changhai Hospital's Colorectal Surgery Division. The sample group was categorized into three subgroups (T3a, T3b, and T3c) based on the extent of tumor progression (<5 mm, 5-10 mm, and >10 mm) to assess the endorectal ultrasonography diagnostic performance. The 5-year disease-free survival and overall survival were assessed using the Kaplan-Meier method and a log rank test. Cox regression analysis verified the tumor invasion depth's significance as a prognostic predictor, and it was also utilized to evaluate other independent risk variables for recurrence after surgery. RESULTS: The study included 72 individuals with low and middle rectal cancer from January 2014 to November 2019. Twenty-two individuals had stage T3a, 22 had stage T3b, and 28 had stage T3c based on preoperative endorectal ultrasonography. Endorectal ultrasonography had 88.0%, 86.8%, and 76.2% overall accuracy for stratifying subgroups, respectively. According to the Kaplan-Meier curve, 5-year OS was 100%, 83.5%, and 92.9% for T3a, T3b, and T3c (p = 0.172), and 5-year disease-free survival was 100%, 80.8%, and 72.9% for T3a, T3b, and T3c, respectively (p = 0.014). A distinct risk factor for 5-year disease-free survival was the degree of tumor infiltration (p = 0.039). CONCLUSION: Preoperative T3 stage subdivision allows for categorization of prognosis and survival. Endorectal ultrasonography reports should make explicit declarations of T3a, T3b, and T3c scales.

Pyruvate enhances oral tolerance via GPR31.

CX3CR1high myeloid cells in the small intestine mediate the induction of oral tolerance by driving regulatory T (Treg) cells. Bacterial metabolites, e.g. pyruvate and lactate, induce a dendrite extension of CX3CR1high myeloid cells into the intestinal lumen via GPR31. However, it remains unclear whether the pyruvate-GPR31 axis is involved in the induction of oral tolerance. Here, we show that pyruvate enhances oral tolerance in a GPR31-dependent manner. In ovalbumin (OVA)-fed Gpr31-deficient mice, an OVA-induced delayed-type hypersensitivity response was substantially induced, demonstrating the defective induction of oral tolerance in Gpr31-deficient mice. The percentage of RORγt+ Treg cells in the small intestine was reduced in Gpr31-deficient mice. In pyruvate-treated wild-type mice, a low dose of OVA efficiently induced oral tolerance. IL-10 production from intestinal CX3CR1high myeloid cells was increased by OVA ingestion in wild-type mice, but not in Gpr31-deficient mice. CX3CR1high myeloid cell-specific IL-10-deficient mice showed a defective induction of oral tolerance to OVA and a decreased accumulation of OVA-specific Treg cells in the small intestine. These findings demonstrate that pyruvate enhances oral tolerance through a GPR31-dependent effect on intestinal CX3CR1high myeloid cells.

Transcriptomic analysis provides insight into defensive strategies in response to continuous cropping in strawberry (Fragaria × ananassa Duch.) plants.

BACKGROUND: Strawberries are an important economic fruit crop world-wide. In strawberry cultivation, continuous cropping (CC) can seriously threaten yield and quality. However, our understanding of the gene expression changes in response to CC and during subsequent defense processes is limited. In this study, we analyzed the impact of CC on the transcriptome of strawberry roots using RNA-Seq technology to elucidate the effect of CC and the subsequent molecular changes. RESULTS: We found that CC significantly affects the growth of strawberry plants. The transcriptome analysis identified 136 differentially expressed genes (DEGs), including 49 up-regulated and 87 down-regulated DEGs. A Gene Ontology (GO) analysis indicated that the up-regulated DEGs were mainly assigned to defense-related GO terms, and most down-regulated DEGs were assigned to nutrient-related GO terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the responsive DEGs were classified in a large number of important biological pathways, such as phenylalanine metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, glutathione metabolism and plant-pathogen interaction. We also found that four WRKY transcription factors and three peroxidase genes involved in plant defense pathways were up-regulated in the roots of strawberry plants subjected to CC. CONCLUSION: Several unigenes involved in plant defense processes, such as CNGCs, WRKY transcription factors, PR1, and peroxidase genes with highly variable expression levels between non-CC and CC treatments may be involved in the regulation of CC in strawberry. These results indicate that strawberry roots reallocate development resources to defense mechanisms in response to CC. This study will further deepen our understanding of the fundamental regulatory mechanisms of strawberry resource reallocation in response to CC.

Transcriptomic analysis reveals recovery strategies in strawberry roots after using a soil amendment in continuous cropping soil.

BACKGROUND: In strawberry cultivation, continuous cropping (CC) obstacles seriously threaten production. A patented soil amendment (SA) can effectively relieve the CC obstacles to strawberry cultivation, but knowledge of the recovery mechanisms underlying this phenomenon is limited. RESULTS: In this study, transcriptomic profiling of strawberry roots in soil with and without the SA was conducted using RNA-Seq technology to reveal gene expression changes in response to SA treatment. In total, 188 differentially expressed genes (DEGs), including 144 upregulated and 44 downregulated DEGs, were identified. SA treatment resulted in genotype-dependent responses, and the response pattern, including an overall increase in the expression of nutrient transport genes and a decrease in the expression of defense response genes, may be a possible mechanism underlying recovery strategies in strawberry roots after the application of the SA to CC soil. We also found that 9 Hsp genes involved in plant defense pathways were all downregulated in the SA-treated roots. CONCLUSIONS: This research indicated that strawberry plants reallocated defense resources to development when SA treatment alleviated the stress caused by a CC soil environment. The present study provides an opportunity to reveal the fundamental mechanisms of the tradeoff between growth and defense in strawberry.

Blockage of macrophage migration inhibitory factor (MIF) suppressed uric acid-induced vascular inflammation, smooth muscle cell de-differentiation, and remodeling.

Hyperuricemia contributes to vascular injury and dysfunction, yet the potential mechanisms are not well understood. Uric acid (UA) has been found to stimulate macrophage migration inhibitory factor (MIF) up-regulation in renal tubules from rats subjected to UA-induced nephropathy. Given that MIF is able to induce vascular smooth muscle cell (VSMC) de-differentiation (from contractile state to a secretory state), we thus hypothesized that UA-induced vascular injury is via up-regulating of MIF in VSMCs, which enhancing vascular inflammation and VSMC transition. Within a mouse model of UA injection (500 mg/kg, twice/day, 14 days), we measured circulating and vascular MIF levels under UA stimulation at 6 h, day 1, and 14. We tested the efficacy of MIF inhibitor (10 mg/kg, twice/day, 14 days) on UA-induced vascular inflammation and remodeling. High plasma level of UA induced vascular MIF release into the plasma at acute phase. In the chronic phase, the protein level of MIF is up-regulated in the vessels. MIF inhibitor suppressed vascular inflammatory responses, repressed VSMC de-differentiation, and attenuated vascular remodeling and dysfunction following UA stimulation. Knockdown of MIF in cultured VSMCs repressed UA-induced de-differentiation. Our results provided a novel mechanism for MIF-mediated vascular injury in response to UA stimulation, and suggested that anti-MIF interventions may be of therapeutic value in hyperuricemic patients.

Persistence and Transcription of Paternal mtDNA Dependent on the Delivery Strategy Rather than Mitochondria Source in Fish Embryos.

BACKGROUND/AIMS: Mitochondria (MT) and mitochondrial DNA (mtDNA) show maternal inheritance in most eukaryotic organisms; the sperm mtDNA is usually delivered to the egg during fertilization and then rapidly eliminated to avoid heteroplasmy, which can affect embryogenesis. In our previous study, fertilization-delivered sperm mtDNA exhibited late elimination and transcriptional quiescence in cyprinid fish embryos. However, the mechanisms underlying elimination and transcriptional quiescence of paternal mtDNA are unclear. METHODS: Goldfish and zebrafish were used to investigate the fate of mtDNAs with different parental origins delivered by fertilization or microinjection in embryos. Goldfish MT from heart, liver and spermatozoa were microinjected into zebrafish zygotes, respectively. Specific PCR primers were designed so that the amplicons have different sizes to characterize goldfish and zebrafish cytb genes or their cDNAs. RESULTS: The MT injection-delivered paternal mtDNA from sperm, as well as those from the heart and liver, was capable of persistence and transcription until birth, in contrast to the disappearance and transcriptional quiescence at the heartbeat stage of fertilization-delivered sperm mtDNA. In addition, the exogenous MT-injected zebrafish embryos have normal morphology during embryonic development. CONCLUSIONS: The fate of paternal mtDNA in fishes is dependent on the delivery strategy rather than the MT source, suggesting that the presence of sperm factor(s) is responsible for elimination and transcriptional quiescence of fertilization-delivered sperm mtDNA. These findings provide insights into the mechanisms underlying paternal mtDNA fate and heteroplasmy in cyprinid fishes.

Identification and Characterization of C-type Lectins in Ostrinia furnacalis (Lepidoptera: Pyralidae).

C-type lectins (CTLs) are a large family of calcium-dependent carbohydrate-binding proteins. They function primarily in cell adhesion and immunity by recognizing various glycoconjugates. We identified 14 transcripts encoding proteins with one or two CTL domains from the transcriptome from Asian corn borer, Ostrinia furnacalis (Guenée; Lepidoptera: Pyralidae). Among them, five (OfCTL-S1 through S5) only contain one CTL domain, the remaining nine (OfIML-1 through 9) have two tandem CTL domains. Five CTL-Ss and six OfIMLs have a signal peptide are likely extracellular while another two OfIMLs might be cytoplasmic. Phylogenetic analysis indicated that OfCTL-Ss had 1:1 orthologs in Lepidoptera, Diptera, Coleoptera and Hymenoptera species, but OfIMLs only clustered with immulectins (IMLs) from Lepidopteran. Structural modeling revealed that the 22 CTL domains adopt a similar double-loop fold consisting of ß-sheets and α-helices. The key residues for calcium-dependent or independent binding of specific carbohydrates by CTL domains were predicted with homology modeling. Expression profiles assay showed distinct expression pattern of 14 CTLs: the expression and induction were related to the developmental stages and infected microorganisms. Overall, our work including the gene identification, sequence alignment, phylogenetic analysis, structural modeling, and expression profile assay would provide a valuable basis for the further functional studies of O. furnacalis CTLs.

Multicenter analysis of risk factors for anastomotic leakage after middle and low rectal cancer resection without diverting stoma: a retrospective study of 319 consecutive patients.

PURPOSE: The purpose of this study was to evaluate the risk factors for anastomotic leakage (AL) after anterior resection for middle and low rectal cancer in order to help surgeons to decide which patients could benefit from a diverting stoma. METHODS: Data on 319 patients having a middle and low rectal cancer resection with anastomosis between May 2011 and October 2015 from two hospitals were included in the study. The analysis included the following variables: patient-related variables (gender, age, diabetes mellitus, ASA score, preoperative radiochemotherapy, body mass index, blood hemoglobin, and serum albumin level), tumor-related variables (K-ras status, distance of tumor from the anal verge, histopathologic grade, pathological T stage, pathological N stage, pathological M stage, TNM stage, and tumor size), and surgery-related variables (laparoscopic or open surgery, blood loss, and operative time). Univariate and multivariate regression analysis were carried out to identify risk factors for AL. RESULTS: The AL rate was 11.91% (38/319). Male (OR 2.898, 95% CI 1.265-6.637, p = 0.012), diabetes mellitus (OR 2.482, 95% CI 1.004-6.134, p = 0.049), K-ras mutation (OR 2.544, 95% CI 1.210-5.348, p = 0.014), distance of tumor from the anal verge (OR 3.445, 95% CI 1.631-7.279, p = 0.001), and preoperative radiochemotherapy (OR 2.790, 95% CI 1.056-7.372, p = 0.039) were independent risk factors of AL. One (2.63%) in 38 patients with AL presented with no risk factor of AL, 6 (15.8%) in 38 patients with 1 risk factor, 16 (42.1%) in 38 patients with 2 risk factors, 9 (23.7%) in 38 patients with 3 risk factors, and 6 (15.7%) in 38 patients with 4 risk factors. No patient with 5 risk factors in our study. AL rate increased with the elevated number of risk factors clustering in individuals. CONCLUSIONS: K-ras mutation is first reported to be an independent risk factor for AL after sphincter-preserving surgery without diverting stoma. A diverting stoma should be performed in sphincter-preserving surgery for middle and low rectal cancer patients with 2 or more risk factors identified in this analysis.

Activity changes of antioxidant and detoxifying enzymes in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae infected by the entomopathogenic nematode Heterorhabditis beicherriana (Rhabditida: Heterorhabditidae).

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis are lethal parasites of many insect species. To investigate defensive mechanisms towards EPNs in relation to antioxidative and detoxifying enzymes, we chose Tenebrio molitor (Coleoptera: Tenebrionidae) as experimental insect. We studied the activity changes of superoxide dismutases (SODs), peroxidases (PODs), and catalases (CATs), as well as tyrosinase (TYR), acetylcholinesterase (AChE), carboxylesterase (CarE), and glutathione S-transferase (GSTs) for 40 h in T. molitor larvae infected with Heterorhabditis beicherriana infective juveniles (IJs) at 5 rates (0, 20, 40, 80, and 160 IJs/larva). We found that when T. molitor larvae infected with H. beicherriana at higher rates (80 and 160 IJs/larva), SOD activity quickly increased to more than 70 % higher than that control levels. The activities of POD and CAT increased after 24 h. TYR activity increased slowly at lower rates of infection for 16 h, followed by a slight decrease, and then increasing from 32 to 40 h. The other detoxifying enzymes (GST, CarE, and AChE) were enhanced at lower infection rates, but were inhibited at higher rates. Our results suggested that host antioxidative response and detoxification reactions played a central role in the defensive reaction to EPNs, and that this stress which was reflected by the higher level enzymes activity contributed to the death of hosts. Further study should explore the exact function of these enzymes using different species of EPNs and investigate the links between enzyme activity and host susceptibility to EPNs.

Insecticidal Potential of Clove Essential Oil and Its Constituents on Cacopsylla chinensis (Hemiptera: Psyllidae) in Laboratory and Field.

Cacopsylla chinensis (Yang and Li) (Hemiptera: Psyllidae) is an important pest of pear in China. As an alternative to conventional chemical pesticides, botanicals including essential oils and their constituents could provide an eco-friendly and nonhazardous control method. In this study, the essential oil of clove buds (Syzygium aromaticum) was obtained by hydrodistillation. Five constituents, accounting for 99.89% of the oil, were identified by gas chromatography-mass spectrometry, and the major constituents were eugenol (88.61%) and eugenol acetate (8.89%), followed by ß-caryophyllene (1.89%). In a laboratory bioassay, clove essential oil, commercial eugenol (99.00%) and ß-caryophyllene (98.00%) exhibited strong contact toxicity against the summerform adults of C. chinensis with LD50 values of 0.730, 0.673, and 0.708 µg/adult, and against the nymphs with LD50 values of 1.795, 1.668, and 1.770 µg/nymph, respectively. In contrast, commercial eugenol acetate (98%) had LD50 values of 9.266 µg/adult and 9.942 µg/nymph. In a field trial, clove essential oil caused significant population reductions of 73.01% (4.80 mg/ml), 66.18% (2.40 mg/ml) and 46.56% (1.20 mg/ml), respectively. Our results demonstrated that clove essential oil and its constituents have potential as a source of natural insecticides.

Gene expression profiling-derived immunohistochemistry signature with high prognostic value in colorectal carcinoma.

OBJECTIVE: Gene expression profiling provides an opportunity to develop robust prognostic markers of colorectal carcinoma (CRC). However, the markers have not been applied for clinical decision making. We aimed to develop an immunohistochemistry signature using microarray data for predicting CRC prognosis. DESIGN: We evaluated 25 CRC gene signatures in independent microarray datasets with prognosis information and constructed a subnetwork using signatures with high concordance and repeatable prognostic values. Tumours were examined immunohistochemically for the expression of network-centric and the top overlapping molecules. Prognostic values were assessed in 682 patients from Shanghai, China (training cohort) and validated in 343 patients from Guangzhou, China (validation cohort). Median follow-up duration was 58 months. All p values are two-sided. RESULTS: Five signatures were selected to construct a subnetwork. The expression of GRB2, PTPN11, ITGB1 and POSTN in cancer cells, each significantly associated with disease-free survival, were selected to construct an immunohistochemistry signature. Patients were dichotomised into high-risk and low-risk subgroups with an optimal risk score (1.55). Compared with low-risk patients, high-risk patients had shorter disease-specific survival (DSS) in the training (HR=6.62; 95% CI 3.70 to 11.85) and validation cohorts (HR=3.53; 95% CI 2.13 to 5.84) in multivariate Cox analyses. The signature better predicted DSS than did tumour-node-metastasis staging in both cohorts. In those who received postoperative chemotherapy, high-risk score predicted shorter DSS in the training (HR=6.35; 95% CI 3.55 to 11.36) and validation cohorts (HR=5.56; 95% CI 2.25 to 13.71). CONCLUSIONS: Our immunohistochemistry signature may be clinically practical for personalised prediction of CRC prognosis.

Effects of male hepatitis B virus infection and serostatus on sperm quality, pregnancy outcomes, and neonatal outcomes following intrauterine insemination.

OBJECTIVE: To evaluate the impact of male hepatitis B virus (HBV) infection and serostatus on sperm quality, pregnancy outcomes, and neonatal outcomes following intrauterine insemination for infertility. DESIGN AND METHODS: We retrospectively analyzed data from 962 infertile couples undergoing intrauterine insemination treatment at a single center. The case group comprised 212 infertile couples with male HBV infection, and the control group comprised 750 noninfected infertile couples. The couples were further divided into subgroups according to their hepatitis B e antigen (HBeAg)/anti-HBe status: hepatitis B surface antigen (HBsAg)+HBeAg- (group A), HBsAg+HBeAg+ (group B), and HBsAg-HBeAg- (control group). The main outcome parameters, including the seminal parameters, clinical pregnancy rate, miscarriage rate, live birth rate, preterm delivery rate, multiple pregnancy rate, delivery type, birth weight, and sex ratio, were compared. RESULTS: A lower sperm acrosin activity, higher cesarean rate, and newborn sex ratio were observed in the HBV-infected group and group A in comparison with the control group (P < 0.05). However, the standard sperm parameters, clinical pregnancy rate, miscarriage rate, live birth rate, preterm delivery, and birth weight showed no statistically significant differences among the groups. CONCLUSION: Male HBV infection does not adversely impact standard sperm parameters or pregnancy outcomes but can influence sperm acrosin activity and some neonatal outcomes. Moreover, the effect may vary among different HBV serostatuses.

Hijacking of nucleotide biosynthesis and deamidation-mediated glycolysis by an oncogenic herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS) and multiple types of B cell malignancies. Emerging evidence demonstrates that KSHV reprograms host-cell central carbon metabolic pathways, which contributes to viral persistence and tumorigenesis. However, the mechanisms underlying KSHV-mediated metabolic reprogramming remain poorly understood. Carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) is a key enzyme of the de novo pyrimidine synthesis, and was recently identified to deamidate the NF-κB subunit RelA to promote aerobic glycolysis and cell proliferation. Here we report that KSHV infection exploits CAD for nucleotide synthesis and glycolysis. Mechanistically, KSHV vCyclin binds to and hijacks cyclin-dependent kinase CDK6 to phosphorylate Ser-1900 on CAD, thereby activating CAD-mediated pyrimidine synthesis and RelA-deamidation-mediated glycolytic reprogramming. Correspondingly, genetic depletion or pharmacological inhibition of CDK6 and CAD potently impeded KSHV lytic replication and thwarted tumorigenesis of primary effusion lymphoma (PEL) cells in vitro and in vivo. Altogether, our work defines a viral metabolic reprogramming mechanism underpinning KSHV oncogenesis, which may spur the development of new strategies to treat KSHV-associated malignancies and other diseases.

Expression of ZNF148 in different developing stages of colorectal cancer and its prognostic value: a large Chinese study based on tissue microarray.

BACKGROUND: It has been speculated that zinc finger protein 148 (ZNF148) is a tumor suppressor. However, to the authors' knowledge, little is known about the clinical significance of ZNF148 expression in patients with colorectal cancer (CRC). The objective of the current study was to clarify the association between ZNF148 expression and the postoperative prognosis of patients with CRC. METHODS: Tissue microarrays containing 56 normal mucosa, 51 adenoma, 742 CRC (TNM stage I-IV), 16 familial adenomatous polyposis, and 21 metastatic CRC specimens were examined immunohistochemically for ZNF148 expression. RESULTS: Expression of ZNF148 was found to increase consecutively from normal mucosa to stage I CRC, and then decreased consecutively from stage I to stage IV CRC. Lower expression of ZNF148 in tumors was found to be significantly associated with lymph node metastases, advanced TNM disease stage, poor differentiation, higher rate of disease recurrence, worse overall survival (OS), and shorter disease-free survival. High expression of ZNF148 was also associated with improved OS (P = .025) and disease-free survival (P = .042) in patients with stages II to III CRC. On multivariate Cox analysis, lower ZNF148 expression in tumors, advanced TNM stage, colon cancer, and elevated serum carbohydrate antigen 19-9 (CA19-9) were found to be significant factors for a worse OS. In 16 patients with familial adenomatous polyposis, ZNF148 expression was upregulated at steps toward carcinogenesis. In 21 patients with metastatic CRC, although ZNF148 expression was higher in primary tumors compared with adjacent mucosa, its expression in metastatic tumors was significantly lower than that in primary tumors. CONCLUSIONS: Although ZNF148 expression is related to colorectal carcinogenesis, high ZNF148 expression in patients with CRC appears to be inversely associated with malignant phenotypes and may serve as a significant prognostic factor after surgery for patients with CRC.

Erratum: miR-365a-3p regulates ADAM10-JAK-STAT signaling to suppress the growth and metastasis of colorectal cancer cells: Erratum.

[This corrects the article DOI: 10.7150/jca.42731.].

Helicase-independent function of RIG-I against murine gammaherpesvirus 68 via blocking the nuclear translocation of viral proteins.

Innate immunity is the first line of defense against viral pathogens. Retinoic Acid-Inducible Gene 1 (RIG-I) is a pattern recognition receptor that recognizes virus-associated double-stranded RNA and initiates the interferon responses. Besides signal transduction, RIG-I exerts direct antiviral functions to displace viral proteins on dsRNA via its Helicase activity. Nevertheless, this effector-like activity of RIG-I against herpesviruses remains largely unexplored. It has been previously reported that herpesviruses deamidate RIG-I, resulting in the abolishment of its Helicase activity and signal transduction. In this study, we discovered that RIG-I possessed signaling-independent antiviral activities against murine gamma herpesviruses 68 (γHV68, murid herpesvirus 4). Importantly, a Helicase-dead mutant of RIG-I (K270A) demonstrated comparable inhibition on herpesviruses lytic replication, indicating that this antiviral activity is Helicase-independent. Mechanistically, RIG-I bound the Replication and Transcription Activator (RTA) and diminished its nuclear localization to repress viral transcription. We further demonstrated that RIG-I blocked the nuclear translocation of ORF21 (Thymidine Kinase), ORF75c (vGAT), both of which form a nuclear complex with RTA and RNA polymerase II (Pol II) to facilitate viral transcription. Moreover, RIG-I retained ORF59 (DNA processivity factor) in the cytoplasm to repress viral DNA replication. Altogether, we illuminated a previously unidentified, Helicase-independent effector-like function of RIG-I against γHV68, representing an exquisite host strategy to counteract viral manipulations on innate immune signaling. IMPORTANCE: Retinoic acid-inducible gene I (RIG-I), a member of DExD/H box RNA helicase family, functions as a key pattern recognition receptor (PRR) responsible for the detection of intracellular double-stranded RNA (dsRNA) from virus-infected cells and induction of type I interferon (IFN) responses. Nevertheless, our understanding of the helicase-independent effector-like activity of RIG-I against virus infection, especially herpesvirus infection, remains largely unknown. Herein, by deploying murine gamma herpesviruses 68 (γHV68) as a model system, we demonstrated that RIG-I possessed an interferon and helicase-independent antiviral activity against γHV68 via blocking the nuclear trafficking of viral proteins, which concomitantly repressed the viral early transcription and genome replication thereof. Our work illuminates a previously unidentified antiviral strategy of RIG-I against herpesvirus infection.

Protein-encapsulated doxorubicin reduces cardiotoxicity in hiPSC-cardiomyocytes and cardiac spheroids while maintaining anticancer efficacy.

The chemotherapeutic doxorubicin (DOX) detrimentally impacts the heart during cancer treatment. This necessitates development of non-cardiotoxic delivery systems that retain DOX anticancer efficacy. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), cardiac fibroblasts (hiPSC-CFs), multi-lineage cardiac spheroids (hiPSC-CSs), patient-specific hiPSCs, and multiple human cancer cell lines to compare the anticancer efficacy and reduced cardiotoxicity of single protein encapsulated DOX (SPEDOX-6), to standard unformulated (UF) DOX. Cell viability assays and immunostaining in human cancer cells, hiPSC-ECs, and hiPSC-CFs revealed robust uptake of SPEDOX-6 and efficacy in killing these proliferative cell types. In contrast, hiPSC-CMs and hiPSC-CSs exhibited substantially lower cytotoxicity during SPEDOX-6 treatment compared with UF DOX. SPEDOX-6-treated hiPSC-CMs and hiPSC-CSs maintained their functionality, as indicated by sarcomere contractility assessment, calcium imaging, multielectrode arrays, and RNA sequencing. This study demonstrates the potential of SPEDOX-6 to alleviate cardiotoxic side effects associated with UF DOX, while maintaining its anticancer potency.

Up-regulation of PUM1 by miR-218-5p promotes colorectal tumor-initiating cell properties and tumorigenesis by regulating the PI3K/AKT axis.

Background: Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer-related death worldwide. Advanced stage CRC, during the recent past, had a dismal prognosis and only a few available treatments. Pumilio homologous protein 1 (PUM1) is reportedly aberrant in human malignancies, including CRC. However, the role of PUM1 in the regulation of tumor-initiating cells (T-ICs) remains unknown. Methods: The levels of messenger RNAs (mRNAs) were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunoblot analyses. Statistical analyses were performed to determine the associations between the levels of PUM1 and tumor features and patient outcomes. Whether PUM1 is a downstream target of miR-218-5p was verified by bioinformatics target gene prediction and qRT-PCR. Results: Herein, it was found that T-ICs, chemoresistance, and recurrent CRC samples all manifest increased PUM1 expression. Functional investigations have shown that PUM1 increased the self-renewal, tumorigenicity, malignant proliferation, and chemoresistance of colorectal cells. PUM1 activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway biochemically. Furthermore, it was discovered that miR-218-5p specifically targets T-ICs' PUM1 3'-untranslated region (3'-UTR). More importantly, the PUM1/PI3K/AKT axis regulates CRC cells' responses to treatment with cetuximab, and PUM1 overexpression increased cetuximab resistance. More evidence points to the possibility that low PUM1 may predict cetuximab benefits in CRC patients after analysis of the patient cohort, patient-derived tumor organoids, and patient-derived xenografts (PDXs). Conclusions: Taken together, the result of this work points to the critical function of the miR-218-5p/PUM1/PI3K/AKT regulatory circuit in regulating T-ICs characteristics and thus suggests possible therapeutic targets for CRC.