Activation of M2 macrophage autophagy by rapamycin increases the radiosensitivity of colorectal cancer xenografts.

BACKGROUND: Tumor-associated macrophages (TAMs) are intimately involved in cancer radiochemotherapy resistance. However, the mechanism by which macrophages affect radiosensitivity through autophagy remains unclear. The purpose of our study was to investigate how activating autophagy in type-II macrophages (M2) by using rapamycin (RAP) would affect the radiosensitivity of colorectal cancer (CRC) xenografts. MATERIALS AND METHODS: A nude mouse CRC model was established by injecting LoVo CRC cells. After tumor formation, supernatant from M2 cells (autophagy-unactivated), autophagy-activated M2 cells, or autophagy-downregulated M2 cells was injected peritumorally. All tumor-bearing mice were irradiated with 8-Gy X-rays twice, and the radiosensitivity of CRC xenografts was analyzed in each group. RESULTS: The mass, volume, and microvessel density (MVD) of tumors in the autophagy-unactivated M2 group significantly increased; however, supernatant from M2 cells that were autophagy-activated by rapamycin significantly decreased tumor weight, volume, and MVD compared with negative control. Combining bafilomycin A1 (BAF-A1) with RAP treatment restored the ability of the M2 supernatant to increase tumor mass, volume, and MVD. Immunohistochemical and Western blot results showed that compared with the negative control group, supernatant from M2 cells that were not activated by autophagy downregulated the expression of Livin and Survivin in tumor tissues; activation of M2 autophagy further downregulated the protein levels. CONCLUSIONS: Therefore, autophagy-activated M2 supernatant can downregulate the expression of the antiapoptotic genes Livin and Survivin in CRC xenografts, improving the radiosensitivity of CRC by inducing apoptosis in combination with radiotherapy and inhibiting the growth of transplanted tumors.

Tetrahydrobiopterin metabolism attenuates ROS generation and radiosensitivity through LDHA S-nitrosylation: novel insight into radiogenic lung injury.

Genotoxic therapy triggers reactive oxygen species (ROS) production and oxidative tissue injury. S-nitrosylation is a selective and reversible posttranslational modification of protein thiols by nitric oxide (NO), and 5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for NO synthesis. However, the mechanism by which BH4 affects protein S-nitrosylation and ROS generation has not been determined. Here, we showed that ionizing radiation disrupted the structural integrity of BH4 and downregulated GTP cyclohydrolase I (GCH1), which is the rate-limiting enzyme in BH4 biosynthesis, resulting in deficiency in overall protein S-nitrosylation. GCH1-mediated BH4 synthesis significantly reduced radiation-induced ROS production and fueled the global protein S-nitrosylation that was disrupted by radiation. Likewise, GCH1 overexpression or the administration of exogenous BH4 protected against radiation-induced oxidative injury in vitro and in vivo. Conditional pulmonary Gch1 knockout in mice (Gch1fl/fl; Sftpa1-Cre+/- mice) aggravated lung injury following irradiation, whereas Gch1 knock-in mice (Gch1lsl/lsl; Sftpa1-Cre+/- mice) exhibited attenuated radiation-induced pulmonary toxicity. Mechanistically, lactate dehydrogenase (LDHA) mediated ROS generation downstream of the BH4/NO axis, as determined by iodoacetyl tandem mass tag (iodoTMT)-based protein quantification. Notably, S-nitrosylation of LDHA at Cys163 and Cys293 was regulated by BH4 availability and could restrict ROS generation. The loss of S-nitrosylation in LDHA after irradiation increased radiosensitivity. Overall, the results of the present study showed that GCH1-mediated BH4 biosynthesis played a key role in the ROS cascade and radiosensitivity through LDHA S-nitrosylation, identifying novel therapeutic strategies for the treatment of radiation-induced lung injury.

USP11 Exacerbates Radiation-Induced Pneumonitis by Activating Endothelial Cell Inflammatory Response via OTUD5-STING Signaling.

PURPOSE: Radiation-induced pneumonitis (RIP) seriously limits the application of radiation therapy in the treatment of thoracic tumors, and its etiology and pathogenesis remain elusive. This study aimed to elucidate the role of ubiquitin-specific peptidase 11 (USP11) in the progression of RIP and the associated underlying mechanisms. METHODS AND MATERIALS: Changes in cytokines and infiltrated immune cells were detected by enzyme-linked immunosorbent assays and immunohistochemistry after exposure to 20 Gy x-ray with whole-thorax irradiation. The effects of USP11 expression on endothelial cell proliferation and apoptosis were analyzed by costaining of CD31/Ki67 and CD31/caspase-3 in vivo, and the production of cytokines and reactive oxygen species was confirmed by reverse-transcription polymerase chain reaction and flow cytometry in vitro. Comprehensive proteome and ubiquitinome analyses were used for USP11 substrate screening after radiation. Results were verified by Western blotting and coimmunoprecipitation experiments. Recombinant adeno-associated virus lung vectors expressing OTUD5 were used for localized overexpression of OTUD5 in mouse pulmonary tissue, and immunohistochemistry was conducted to analyze cytokine expression. RESULTS: The progression of RIP was significantly alleviated by reduced expression of proinflammatory cytokines in both Usp11-knockout (Usp11-/-) mice and in mice treated with the USP11 inhibitor mitoxantrone. Likewise, the absence of USP11 resulted in decreased permeability of pulmonary vessels and neutrophils and macrophage infiltration. The proliferation rates of endothelial cells were prominently increased in the Usp11-/- lung, whereas apoptosis in Usp11-/- lungs decreased after irradiation compared with that observed in Usp11+/+ lungs. Conversely, USP11 overexpression increased proinflammatory cytokine expression and reactive oxygen species production in endothelial cells after radiation. Comprehensive proteome and ubiquitinome analyses indicated that USP11 overexpression upregulates the expression of several deubiquitinating enzymes, including USP22, USP33, and OTUD5. We demonstrate that USP11 deubiquitinates OTUD5 and implicates the OTUD5-STING signaling pathway in the progression of the inflammatory response in endothelial cells. CONCLUSIONS: USP11 exacerbates RIP by triggering an inflammatory response in endothelial cells both in vitro and in vivo, and the OTUD5-STING pathway is involved in the USP11-dependent promotion of RIP. This study provides experimental support for the development of precision intervention strategies targeting USP11 to mitigate RIP.

Molecular discrimination of G1 and G3 genotypes of Echinococcus granulosus sensu stricto obtained from human, cattle, and sheep using the mitochondrial NADH dehydrogenase subunit 5 marker.

Cystic echinococcosis (CE) is a common zoonotic disease caused by the larval form of Echinococcus granulosus sensu lato. This study determined the genotype and haplotype differences using the NADH dehydrogenase subunit 5 gene in hydatid cyst samples. Human (n = 12), cattle (n = 28), and sheep (n = 31) hydatid cyst isolates were included. Seventy-one genomic DNA samples were successfully extracted, and a 759 bp mitochondrial NADH dehydrogenase subunit 5 gene fragment was amplified by PCR. Following the sequence analysis, E. granulosus sensu stricto isolates were identified as G1 (n = 61) and G3 (n = 10). A total of 23 haplotypes were obtained from the 71 E. granulosus s.s. G1 and G3 samples. The main haplotype was Hap01 (60.56 %), which consisted of the G1 genotype. The second largest haplotype was Hap04, which consisted entirely of the G3 genotype. Hap14 acted as a bridge between the G1 and G3 genotypes. This study identifies G1 as the dominant genotype in humans and farm animals in Turkey. High haplotype and nucleotide diversity in genotypes were observed. Additionally, this is the first report on the phylogeography and gene flow models of the E. granulosus s.s. population in Turkey using the NADH dehydrogenase subunit 5 gene, the best marker distinguishing between G1 and G3 genotypes.

The mir-199b-5p encapsulated in adipocyte-derived exosomes mediates radioresistance of colorectal cancer cells by targeting JAG1.

Radiotherapy is a key treatment option for colorectal cancer, but its efficacy varies among patients. Our previous studies suggested that adipose tissue may confer the radioresistance of several abdominal tumors, such as pancreatic cancer, biliary cancer, and others. In the present work, the effects of adipocytes in regulating the radiosensitivity of colorectal cancer are explored for the first time. It was found that colony formation was increased and radiation-induced apoptosis decreased in colorectal cancer cells HCT8 and HCT116 co-cultured with adipocytes, which verified the mediation of adipocyte-driven radioresistance in colorectal cancer in vitro. Next, the colorectal cancer cells were incubated with adipocyte-derived exosomes, and a perceptible reduction in radiosensitivity was detected. Furthermore, to investigate the possible mechanisms involved, the exosomes were isolated, the encapsulated microRNAs were extracted and analyzed by small RNA sequencing. Based on bioinformatics analysis and qRT-PCR verification, miR-199b-5p was chosen for functional annotation. It was shown that miR-199b-5p expression was significantly upregulated after 6 Gy irradiation, and overexpressed miR-199b-5p significantly suppressed the radiosensitivity of HCT8 and HCT116 cells. In addition, jagged canonical Notch ligand 1(JAG1) was identified as the target gene of miR-199b-5p by using bioinformatics prediction and dual luciferase reporter gene assay. It was demonstrated that JAG1 conferred the radioresistance of colorectal cancer cells both in vivo and in vitro. Taken together, the present study demonstrates that adipocytes trigger the radioresistance of colorectal cancer cells, probably by targeting JAG1 through an adipocyte-derived exosomal miR-199b-5p.

Metabolomic Signatures Associated with Radiation-Induced Lung Injury by Correlating Lung Tissue to Plasma in a Rat Model.

The lung has raised significant concerns because of its radiosensitivity. Radiation-induced lung injury (RILI) has a serious impact on the quality of patients' lives and limits the effect of radiotherapy on chest tumors. In clinical practice, effective drug intervention for RILI remains to be fully elucidated. Therefore, an in-depth understanding of the biological characteristics is essential to reveal the mechanisms underlying the complex biological processes and discover novel therapeutic targets in RILI. In this study, Wistar rats received 0, 10, 20 or 35 Gy whole-thorax irradiation (WTI). Lung and plasma samples were collected within 5 days post-irradiation. Then, these samples were processed using liquid chromatography-mass spectrometry (LC-MS). A panel of potential plasma metabolic markers was selected by correlation analysis between the lung tissue and plasma metabolic features, followed by the evaluation of radiation injury levels within 5 days following whole-thorax irradiation (WTI). In addition, the multiple metabolic dysregulations primarily involved amino acids, bile acids and lipid and fatty acid ß-oxidation-related metabolites, implying disturbances in the urea cycle, intestinal flora metabolism and mitochondrial dysfunction. In particular, the accumulation of long-chain acylcarnitines (ACs) was observed as early as 2 d post-WTI by dynamic plasma metabolic data analysis. Our findings indicate that plasma metabolic markers have the potential for RILI assessment. These results reveal metabolic characteristics following WTI and provide new insights into therapeutic interventions for RILI.

Acrolein increases the concentration of intracellular Zn2⁺ by producing mitochondrial reactive oxygen species in A549 cells.

Smoking or occupational exposure leads to low concentrations of acrolein on the surface of the airways. Acrolein is involved in the pathophysiological processes of various respiratory diseases. Reports showed that acrolein induced an increase in mitochondrial reactive oxygen species (mROS). Furthermore, exogenous H2O2 was found to increase intracellular Zn2⁺ concentration ([Zn2⁺]ᵢ). However, the specific impact of acrolein on changes in intracellular Zn2⁺ levels has not been fully investigated. Therefore, this study aimed to investigate the effects of acrolein on mROS and [Zn2⁺]ᵢ in A549 cells. We used Mito Tracker Red CM-H2Xros (MitoROS) and Fluozin-3 fluorescent probes to observe changes in mROS and intracellular Zn2⁺. The results revealed that acrolein increased [Zn2⁺]ᵢ in a time- and dose-dependent manner. Additionally, the production of mROS was observed in response to acrolein treatment. Subsequent experiments showed that the intracellular Zn2⁺ chelator TPEN could inhibit the acrolein-induced elevation of [Zn2⁺]ᵢ but did not affect the acrolein-induced mROS production. Conversely, the acrolein-induced elevation of mROS and [Zn2⁺]ᵢ were significantly decreased by the inhibitors of ROS formation (NaHSO3, NAC). Furthermore, external oxygen free radicals increased both [Zn2⁺]ᵢ levels and mROS production. These results demonstrated that acrolein-induced elevation of [Zn2⁺]ᵢ in A549 cells was mediated by mROS generation, rather than through a pathway where [Zn2⁺]ᵢ elevation leads to mROS production.

Establishment of image-guided radiotherapy of orthotopic hepatocellular carcinoma mouse model.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Recently, developments in radiotherapy technology have led to radiotherapy becoming one of the main therapeutics of HCC. Therefore, a suitable animal model for radiotherapy of the orthotopic HCC mouse model is urgently needed. METHODS: In the present study, Hepa1-6 cells were injected into the liver of C57BL/6 mice in situ to mimic the pathological characteristics of the original HCC. Tumor formation was monitored by applying magnetic resonance imaging techniques and verified by H&E histopathological staining, AFP staining, and Ki67 staining. A single dose of 10 Gy X-ray was applied to simulate clinical radiotherapy plans using image-guided radiotherapy (IGRT) equipment. The efficiency of radiotherapy was then assessed by examining tumor size and weight one week after radiation. Cleaved-caspase3 staining and TUNEL were used to assess apoptosis in tumor tissues. RESULTS: Intrahepatic tumor development was detected in the liver according using MRI. A high-density shadow could be seen 10 days after cell injection, which indicated the formation of HCC in vivo. The tumors grew steadily bigger, and underwent precision radiotherapy 20 days after injection. The typical pathological characteristics of HCC, such as large, deeply stained nuclei and irregular cell size, were visible with H&E staining. After radiotherapy, significantly higher expression of the immunohistochemical markers Ki67 and AFP were detected in tumor tissue than in the nearby normal tissue. Compared with the control group, the tumor volume (p = 0.05) and weight (p < 0.05) of the irradiated group were significantly reduced. In addition, a higher frequency of apoptosis was identified in irradiated HCC tumor tissue using the TUNEL and cleaved-caspase3 staining assay. CONCLUSIONS: In a well-established orthotopic HCC model, MRI was utilized to monitor the formation of tumors, and IGRT was used to simulate clinical radiotherapy. The present study could provide a suitable preclinical system for HCC radiotherapy-related studies.

Extracellular vesicles secreted by Echinococcus multilocularis: important players in angiogenesis promotion.

The involvement of Echinococcus multilocularis, and other parasitic helminths, in regulating host physiology is well recognized, but molecular mechanisms remain unclear. Extracellular vesicles (EVs) released by helminths play important roles in regulating parasite-host interactions by transferring materials to the host. Analysis of protein cargo of EVs from E. multilocularis protoscoleces in the present study revealed a unique composition exclusively associated with vesicle biogenesis. Common proteins in various Echinococcus species were identified, including the classical EVs markers tetraspanins, TSG101 and Alix. Further, unique tegumental antigens were identified which could be exploited as Echinococcus EV markers. Parasite- and host-derived proteins within these EVs are predicted to support important roles in parasite-parasite and parasite-host communication. In addition, the enriched host-derived protein payloads identified in parasite EVs in the present study suggested that they can be involved in focal adhesion and potentially promote angiogenesis. Further, increased angiogenesis was observed in livers of mice infected with E. multilocularis and the expression of several angiogenesis-regulated molecules, including VEGF, MMP9, MCP-1, SDF-1 and serpin E1 were increased. Significantly, EVs released by the E. multilocularis protoscolex promoted proliferation and tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. Taken together, we present the first evidence that tapeworm-secreted EVs may promote angiogenesis in Echinococcus-infections, identifying central mechanisms of Echinococcus-host interactions.

METTL3 Mediates Epithelial-Mesenchymal Transition by Modulating FOXO1 mRNA N6 -Methyladenosine-Dependent YTHDF2 Binding: A Novel Mechanism of Radiation-Induced Lung Injury.

The biological roles of epithelial-mesenchymal transition (EMT) in the pathogenesis of radiation-induced lung injury (RILI) have been widely demonstrated, but the mechanisms involved have been incompletely elucidated. N6 -methyladenosine (m6 A) modification, the most abundant reversible methylation modification in eukaryotic mRNAs, plays vital roles in multiple biological processes. Whether and how m6 A modification participates in ionizing radiation (IR)-induced EMT and RILI remain unclear. Here, significantly increased m6 A levels upon IR-induced EMT are detected both in vivo and in vitro. Furthermore, upregulated methyltransferase-like 3 (METTL3) expression and downregulated α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) expression are detected. In addition, blocking METTL3-mediated m6 A modification suppresses IR-induced EMT both in vivo and in vitro. Mechanistically, forkhead box O1 (FOXO1) is identified as a key target of METTL3 by a methylated RNA immunoprecipitation (MeRIP) assay. FOXO1 expression is downregulated by METTL3-mediated mRNA m6 A modification in a YTH-domain family 2 (YTHDF2)-dependent manner, which subsequently activates the AKT and ERK signaling pathways. Overall, the present study shows that IR-responsive METTL3 is involved in IR-induced EMT, probably by activating the AKT and ERK signaling pathways via YTHDF2-dependent FOXO1 m6 A modification, which may be a novel mechanism involved in the occurrence and development of RILI.

Surgical Implications and Radiologic Classification for the Upward Bulging of the Planum Sphenoidale in Patients With Anterior Skull Base Meningiomas Involving the Tuberculum Sellae Area.

OBJECTIVE: To investigate the surgical implications and morphologic type of upward bulging of the planum sphenoidale (PS) in anterior skull base meningiomas involving the tuberculum sellae area. METHODS: Between January 2014 and June 2021, 96 patients with anterior skull base meningiomas underwent surgery at the Sanbo Brain Hospital of Capital Medical University. A total of 96 patients with nonintracranial space-occupying lesions were selected as the control group. The height of upward bulging of the PS was measured and classified. The authors performed univariate and multivariate analyses to evaluate the rate and effects of upward bulging of the PS. RESULTS: The PS upward bulging rate was 23.00% versus 66.70% (P<0.001) between the control and meningioma groups. Multiple linear regression showed that it was correlated with the tumor midsagittal anteroposterior length (P=0.025) and the midsagittal height diameter (P=0.012). According to the height of PS upward bulging, it was divided into types 1, 2, and 3. The tumor gross-total resection rates were 96.9%, 92.3%, and 76.0%, respectively (P=0.042). CONCLUSIONS: Anterior skull base meningiomas involving the tuberculum sellae area can cause PS upward bulging, which lowers the tumor resection rate and should be considered while determining the treatment approach.

Environmental Factors Associated with Cryptosporidium and Giardia.

Environmental factors significantly influence the transmission of intestinal protozoan diseases. Cryptosporidiosis and giardiasis are important zoonotic diseases characterized by diarrhea, and are mainly water or foodborne diseases caused by fecal-borne oocysts. The One Health approach effectively addresses environmentally influenced zoonotic diseases. However, the impact of environmental factors on the survival of Cryptosporidium/Giardia (oo)cysts or disease transmission is mostly uncharacterized. Associations between cryptosporidiosis and giardiasis incidence and environmental variables (e.g., climatic conditions, soil characteristics, and water characteristics) have been reported; however, the identified relationships are not consistently reported. Whether these are country-specific or global observations is unclear. Herein, we review the evidence for the influence of environmental factors on Cryptosporidium/Giardia and corresponding diseases from three perspectives: climatic, soil, and water characteristics. The (oo)cyst concentration or survival of Cryptosporidium/Giardia and the incidence of corresponding diseases are related to environmental variables. The associations identified varied among studies and have different levels of importance and lag times in different locations. This review summarizes the influence of relevant environmental factors on Cryptosporidium/Giardia from the One Health perspective and provides recommendations for future research, monitoring, and response.

Inhibition of hepatic natural killer cell function via the TIGIT receptor in schistosomiasis-induced liver fibrosis.

Schistosomiasis is a zoonotic parasitic disease. Schistosoma japonicum eggs deposited in the liver tissue induce egg granuloma formation and liver fibrosis, seriously threatening human health. Natural killer (NK) cells kill activated hepatic stellate cells (HSCs) or induce HSC apoptosis and inhibit the progression of liver fibrosis. However, the function of NK cells in liver fibrosis caused by S. japonicum infection is significantly inhibited. The mechanism of this inhibition remains unclear. Twenty mice were percutaneously infected with S. japonicum cercariae. Before infection and 2, 4, 6, and 8 weeks after infection, five mice were euthanized and dissected at each time point. Hepatic NK cells were isolated and transcriptome sequenced. The sequencing results showed that Tigit expression was high at 4-6 weeks post infection. This phenomenon was verified by reverse transcription quantitative PCR (RT-qPCR) and flow cytometry. NK cells derived from Tigit-/- and wild-type (WT) mice were co-cultured with HSCs. It was found that Tigit-/- NK cells induced apoptosis in a higher proportion of HSCs than WT NK cells. Schistosomiasis infection models of Tigit-/- and WT mice were established. The proportion and killing activity of hepatic NK cells were significantly higher in Tigit-/- mice than in WT mice. The degree of liver fibrosis in Tigit-/- mice was significantly lower than that in WT mice. NK cells were isolated from Tigit-/- and WT mice and injected via the tail vein into WT mice infected with S. japonicum. The degree of liver fibrosis in mice that received NK cell infusion reduced significantly, but there was no significant difference between mice that received NK cells from Tigit-/- and WT mice, respectively. Our findings indicate that Tigit knockout enhanced the function of NK cells and reduced the degree of liver fibrosis in schistosomiasis, thus providing a novel strategy for treating hepatic fibrosis induced by schistosomiasis.

Molecular identification and genetic characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in human immunodeficiency virus/acquired immunodeficiency syndrome patients in Shanghai, China.

BACKGROUND: Opportunistic infections are a ubiquitous complication in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients. Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans. In China, despite the number of HIV/AIDS patients being extremely large, only a few studies have investigated opportunistic infections caused by intestinal pathogens in this patient population. The aims of this study were to elucidate the occurrence and genetic characteristics of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients. METHODS: We collected fecal specimens from 155 HIV/AIDS patients (one from each patient). All of the specimens were examined for the presence of the pathogens by genotyping using polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene for Cryptosporidium spp.; the triosephosphate isomerase, ß-giardin and glutamate dehydrogenase genes for G. duodenalis; and the internal transcribed spacer region of the rRNA gene for E. bieneusi. The Cryptosporidium-positive specimens were further subtyped by polymerase chain reacion and sequencing of the 60-kDa glycoprotein gene. RESULTS: Six (3.9%), three (1.9%), and eight (5.2%) HIV/AIDS patients were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. No statistical differences were observed in occurrence rate between the groups by gender, clinical symptom (diarrhea), and CD4+ cell count. Four Cryptosporidium species were identified: Cryptosporidium hominis (n = 2), Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). Furthermore, two C. hominis subtypes (IeA12G3T3 and IaA28R4) were detected. Three G. duodenalis-positive specimens were successfully amplified and sequenced at the triosephosphate isomerase and ß-giardin loci, which led to the identification of assemblages C and B, respectively. Seven genotypes (D, Type IV, EbpC, Peru11, EbpD, A, and I) were identified in E. bieneusi-positive specimens. CONCLUSIONS: Our findings should increase awareness of AIDS-related opportunistic intestinal pathogens, and indicate the need for routine examination in clinical practice for the detection of Cryptosporidium spp., G. duodenalis, and E. bieneusi. Homology analyses of the three intestinal pathogens at the nucleotide and/or amino acid levels indicated their zoonotic potential.

An Epidemiological Survey to Investigate the Prevalence of Cystic Echinococcosis in Slaughtered Bovine Hosts in Punjab, Pakistan.

Cystic echinococcosis (CE) is a neglected zoonotic disease of worldwide geographical distribution. CE is most common in underdeveloped and herding communities where people survive on animal husbandry and agricultural activities. The prevalence of CE in livestock and its risk factors are widely underreported, because of inefficient surveillance systems. The aim of this study was to evaluate the epidemiological characteristics and prevalence of CE in cattle in Punjab, Pakistan. Data were collected from slaughterhouses from September 2021 to February 2022. Ante- and postmortem examination and cyst characterization were performed. Epidemiological, demographic, and one health data were collected. A total of 8877 animals (8096 buffalo, 781 cattle) were examined, and the prevalence of CE was 6.22% (n = 552) in all animals, with a higher prevalence in cattle (15.20% vs. buffalo 5.83%). Prevalence was not significantly different in males and females. Of the 23 districts studied, the highest prevalence was in the Haripur district of KP (20.85%). The majority of animals studied were older than 3 years. Most cysts were found in animals older than 5 years. Lungs and liver were the predominant sites for the presence of cysts (65.58% and 31.34%, respectively. Of the collected cysts, 29.71% were fertile. The findings may be useful in estimating the eco-epidemiology of CE and improving surveillance and prevention programs in Pakistan.

The polymorphic analysis of cox1 and cob genes of Echinococcus granulosus in the Ngari region of Tibet in China.

Cystic echinococcosis (CE) is an important zoonotic parasitic disease caused by Echinococcus granulosus (E. granulosus). CE seriously threatens human health and the development of animal husbandry. The Ngari region is one of the world's highest endemic regions for CE, while genetic polymorphisms of E. granulosus were unclear. Paraffin slices of liver Cyst were collected from seventy-nine surgical patients with echinococcosis in the Ngari region. DNA was extracted from samples. The cox1 and cob genes of mitochondrial DNA of E. granulosus were simultaneously amplified and sequenced. The sequencing results were compared with the standard sequence (KU925397.1and HF947574.1). Phylogenetic trees and the haplotype network of cob and cox1 genes were constructed and analyzed genotypes of E. granulosus isolated from humans in the Ngari Region of Tibet. Out of 79 hydatid cyst samples collected from surgery patients, 60 isolates were identified as G1/ G3, and two isolates were identified as G6/ G7. Analysis of the cob/ cox1 genes revealed 9/7 mutations resulting in 8/6 haplotypes, respectively. The cob and cox1 neutrality indices computed by Tajima's D and Fu's Fs tests showed high negative values in Echinococcus granulosus sensu stricto (E. granulosus s. s.). The result suggested that E. granulosus in the Ngari region experienced population expansion or a negative selection. We found that G1/ G3 was still the main genotype, and G6/ G7 was found occasionally in humans of the Ngari region. Therefore, we recommend future surveys and control efforts to investigate G1/ G3 and G6/ G7 transmission in the Ngari region.

The single-cell landscape of cystic echinococcosis in different stages provided insights into endothelial and immune cell heterogeneity.

Introduction: Hydatid cysts and angiogenesis are the key characteristics of cystic echinococcosis, with immune cells and endothelial cells mediating essential roles in disease progression. Recent single-cell analysis studies demonstrated immune cell infiltration after Echinococcus granulosus infection, highlighting the diagnostic and therapeutic potential of targeting certain cell types in the lesion microenvironment. However, more detailed immune mechanisms during different periods of E. granulosus infection were not elucidated. Methods: Herein, we characterized immune and endothelial cells from the liver samples of mice in different stages by single-cell RNA sequencing. Results: We profiled the transcriptomes of 45,199 cells from the liver samples of mice at 1, 3, and 6 months after infection (two replicates) and uninfected wild-type mice. The cells were categorized into 26 clusters with four distinct cell types: natural killer (NK)/T cells, B cells, myeloid cells, and endothelial cells. An SPP1+ macrophage subset with immunosuppressive and pro-angiogenic functions was identified in the late infection stage. Single-cell regulatory network inference and clustering (SCENIC) analysis suggested that Cebpe, Runx3, and Rora were the key regulators of the SPP1+ macrophages. Cell communication analysis revealed that the SPP1+ macrophages interacted with endothelial cells and had pro-angiogenic functions. There was an obvious communicative relationship between SPP1+ macrophages and endothelial cells via Vegfa-Vegfr1/Vegfr2, and SPP1+ macrophages interacted with other immune cells via specific ligand-receptor pairs, which might have contributed to their immunosuppressive function. Discussion: Our comprehensive exploration of the cystic echinococcosis ecosystem and the first discovery of SPP1+ macrophages with infection period specificity provide deeper insights into angiogenesis and the immune evasion mechanisms associated with later stages of infection.

In Silico Evaluation of the Haplotype Diversity, Phylogenetic Variation and Population Structure of Human E. granulosus sensu stricto (G1 Genotype) Sequences.

Echinococcus granulosus sensu lato is the causative agent of cystic echinococcosis (CE), which is a neglected zoonotic disease with an important role in human morbidity. In this study, we aimed to investigate the haplotype diversity, genetic variation, population structure and phylogeny of human E. granulosus sensu stricto (s.s.) (G1 genotype) isolates submitted to GenBank from different parts of the world by sequencing the mitochondrial CO1 and ND1 genes. The sequences of the mt-CO1 (401 bp; n = 133) and mt-ND1 (407 bp; n = 140) genes were used to analyze the haplotype, polymorphism and phylogenetic of 273 E. granulosus s.s. (G1 genotype) isolates. Mutations were observed at 31 different points in the mt-CO1 gene sequences and at 100 different points in the mt-ND1 gene sequences. Furthermore, 34 haplotypes of the mt-CO1 sequences and 37 haplotypes of the mt-ND1 sequences were identified. Tajima's D, Fu's Fs, and Fu's LD values showed high negative values in both mt-CO1 and mt-ND1 gene fragments. The haplotype diversities in the sequences retrieved from GenBank in this study indicate that the genetic variation in human isolates of E. granulosus s.s. in western countries is higher than in eastern countries. This may be due to demographic expansions due to animal trades and natural selections.

Differentially Expressed mRNAs and Potential Mechanisms of Radiation-Induced TUT4-/- Esophageal Cell Injury.

Radiation-induced esophageal injury remains a limitation for the process of radiotherapy for lung and esophageal cancer patients. Esophageal epithelial cells are extremely sensitive to irradiation, nevertheless, factors involved in the radiosensitivity of esophageal epithelial cells are still unknown. Terminal uridyl transferase 4 (TUT4) could modify the sequence of miRNAs, which affect their regulation on miRNA targets and function. In this study, we used transcriptome sequencing technology to identify mRNAs that were differentially expressed before and after radiotherapy in esophageal epithelial cells. We further explored the mRNA expression profiles between wild-type and TUT4 knockout esophageal epithelial cells. Volcano and heatmap plots unsupervised hierarchical clustering analysis were performed to classify the samples. Enrichment analysis on Gene Ontology functional annotations and Kyoto Encyclopedia of Genes and Genomes pathways was performed. We annotated differential genes from metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems human diseases. The aberrantly expressed genes are significantly enriched in irradiation-related biological processes, such as DNA replication, ferroptosis, and cell cycle. Moreover, we explored the distribution of transcription factor family and its target genes in differential genes. These mRNAs might serve as therapeutic targets in TUT4-related radiation-induced esophageal injury.

Echinococcus granulosus Protoscoleces-Derived Exosome-like Vesicles and Egr-miR-277a-3p Promote Dendritic Cell Maturation and Differentiation.

Cystic echinococcosis, a major parasitic disease caused by Echinococcus granulosus, seriously threatens human health. The excretory-secretory (ES) products of E. granulosus can induce immune tolerance in dendritic cells (DCs) to downregulate the host's immune response; however, the effect of exosomes in the ES products on the DCs has remained unclear. This study showed that E. granulosus protoscoleces-derived exosome-like vesicles (PSC-ELVs) could be internalized by bone marrow-derived dendritic cells (BMDCs), allowing for the delivery of the parasite microRNAs to the BMDCs. Moreover, PSC-ELVs induced BMDCs to produce the proinflammatory cytokinesinterleukin (IL)-6, IL-12, IL-ß, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ). PSC-ELVs also upregulated the BMDCs surface marker major histocompatibility complex class II (MHC II), as well as costimulatory molecules CD40, CD80, and CD86. PSC-ELV-derived egr-miR-277a-3p upregulated the IL-6, IL-12, and TNF-α mRNA levels in BMDCs. Moreover, egr-miR-277a-3p directly targeted Nfkb1 (encoding nuclear factor kappa B 1) to significantly suppress the mRNA and protein levels of NF-κB1 in BMDCs, while the expression of NF-κB p65 significantly increased, suggesting that egr-miR-277a-3p induces the production of proinflammatory cytokines by the modification of the NF-kB p65/p50 ratio in BMDCs. These results demonstrated that PSC-ELVs and egr-miR-277a-3p might enhance DCs maturation and differentiation in a cross-species manner, which in turn may modulate the host immune responses and offer a new approach to echinococcosis prevention and treatment.