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BackgroundIntracranial aneurysm is an abnormal expansion in the intracranial arteries, which is associated with growth and apoptosis of vascular smooth muscle cells. Circular RNAs (circRNAs) have implicated in the progression of intracranial aneurysms. The purpose of this paper is to study the function and mechanism of circRNA dedicator of cytokinesis 1 (circ_DOCK1) in regulating proliferation and apoptosis of human brain vascular smooth muscle cells (HBVSMCs).MethodsHBVSMCs were exposed to hydrogen peroxide (H2O2). Cell proliferation and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry, respectively. Circ_DOCK1, microRNA (miR)-409-3p, and myeloid cell leukemia sequence 1 (MCL1) levels were examined by quantitative reverse transcription polymerase chain reaction or western blotting. The target association was assessed by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation assays.ResultsExposure to H2O2 decreased proliferation and increased apoptosis of HBVSMCs. Circ_DOCK1 expression was reduced in H2O2-treated HBVSMCs. Circ_DOCK1 overexpression rescued H2O2-caused reduction of proliferation and PCNA expression and attenuated H2O2-induced apoptosis and expression of Bcl-2, Bax, and cleaved PARP. MiR-409-3p was targeted by circ_DOCK1 and upregulated in H2O2-treated HBVSMCs. MiR-409-3p upregulation mitigated the role of circ_DOCK1 in proliferation and apoptosis of H2O2-treated HBVSMCs. MCL1 was targeted via miR-409-3p and downregulated via H2O2 treatment. Circ_DOCK1 overexpression enhanced MCL1 expression via modulating miR-409-3p. MiR-409-3p knockdown weakened H2O2-induced proliferation reduction and apoptosis promotion via regulating MCL1.ConclusionCirc_DOCK1 overexpression mitigated H2O2-caused proliferation inhibition and apoptosis promotion in HBVSMCs by modulating miR-409-3p/MCL1 axis.

BackgroundA substantial body of research has demonstrated a notable impact of hot temperatures on mortality from cardiovascular diseases (CVDs). However, a paucity of studies has addressed the influence of sweltering conditions on CVD mortality.ObjectiveTo investigate the effect of sweltering conditions on mortality from CVD among permanent residents of Huizhou City, using the temperature-humidity index (THI) as an indicator.MethodsThis study employs descriptive statistics, distributed lag non-linear model (DLNM) and general algebraic modeling system (GAMs) with the THI as an indicator in order to examine the impact of sweltering conditions on the mortality of CVD among permanent residents of Huizhou City.ResultsSweltering conditions increase the risk of death from CVDs and have a cumulative lag effect. The greater the THI, the more pronounced the increase in mortality, and after a certain range, the mortality rate from CVDs increases significantly, and the effect is gender-specific. The lag effect generally peaks in 2–3 days, and the lag effect of stroke mortality is longer and deeper than that of coronary heart disease (CAD) mortality.ConclusionSweltering increased the mortality of cardiovascular diseases in Huizhou city, so we should pay attention to public health intervention strategies under sweltering.

IntroductionDengue viruses (DENVs), the causative agents of dengue hemorrhagic fever and dengue shock syndrome, undergo genetic mutations that result in new strains and lead to ongoing global re-infections.ObjectivesTo address the growing complexity of identifying and tracking biological samples, this study screened RNA barcode segments for the four DENV serotypes, ensuring high specificity and recall rates for DENV identification using segments.ResultsThrough analyzing complete genome sequences of DENVs, we screened eight barcode segments for DENV, DENV-1, DENV-2, DENV-3, and DENV-4 identification. Comparing the screened barcode segments to sequences of known strains and determining the proportion of correctly or incorrectly identified nucleotides, these segments demonstrated an average recall rate at nucleotide level of 91.34% for four DENV serotypes, a specificity of 99.50% at species level within the Flaviviridae family, and a precision rate of 100% for identifying DENVs. For arboviruses, the nucleotide-level specificity was 63.58%. We designed and used the “Barcoding” software to streamline segment design, integrating automated sequence preprocessing, evaluation of barcode segments, and primer design, significantly reducing manual intervention and enhancing overall efficiency. We also established an online database called “Barcodes” for storing and preparing barcode segments.ConclusionThis work established a standard framework for DENV identification and barcode segment selection, promising significant advancements in the real-time management and control of DENVs, thereby enhancing surveillance capabilities and facilitating targeted interventions in dengue outbreak-prone regions.

BackgroundIntestinal malrotation is a rare condition, and its delayed diagnosis can lead to fatal consequences. This study aimed to investigate the identification and treatment of malrotation in children.MethodsClinical data, imaging, operative findings, and early postoperative outcomes of 75 children with malrotation were retrospectively analyzed.ResultsThe mean age was 6.18 ± 4.93 days and 51.26 ± 70.13 months in the neonatal group (56 patients) and non-neonatal group (19 patients), respectively. Sixty-seven patients were under the age of 1 year at the time of diagnosis. The occurrence of bilious vomiting and jaundice was significantly higher in the neonatal group (89.29%) than that in the non-neonatal group (37.5%), p 10% of neonates and nearly 40% of non-neonates. This highlights the importance for emergency physicians and surgeons to be cautious about ruling out malrotation in patients with non-bilious vomiting. Utilizing US can obviate the need for contrast examinations owing to its higher diagnostic accuracy and rapid diagnosis and can be recommended as a first-line imaging technique. Additionally, open surgery is still an option for neonatal patients.

IntroductionMeteorological and environmental factors can affect people’s lives and health, which is crucial among the older adults. However, it is currently unclear how they specifically affect the physical condition of older adults people.MethodsWe collected and analyzed the basic physical examination indicators of 41 older adults people for two consecutive years (2021 and 2022), and correlated them with meteorological and environmental factors. Partial correlation was also conducted to exclude unrelated factors as well.ResultsWe found that among the physical examination indicators of the older adults for two consecutive years, five indicators (HB, WBC, HbAlc, CB, LDL-C) showed significant differences across the population, and they had significantly different dynamic correlation patterns with six meteorological (air pressure, temperature, humidity, precipitation, wind speed, and sunshine duration) and seven air quality factors (NO2, SO2, PM10, O3-1h, O3-8h, CO, PM2.5).DiscussionOur study has discovered for the first time the dynamic correlation between indicators in normal basic physical examinations and meteorological factors and air quality indicators, which will provide guidance for the future development of policies that care for the healthy life of the older adults.

Necroptosis is a type of proinflammatory programmed necrosis essential for innate immunity. The receptor interacting protein kinases 1/3 (RIPK1/3) and the substrate mixed lineage kinase domain-like protein (MLKL) are core components of the necroptotic axis. The activation and immunological function of necroptosis in fish remain elusive. Herein, we studied the function and activation of RIPK1/3 (PoRIPK1/3) and MLKL (PoMLKL) in teleost Paralichthys olivaceus. Bacterial infection increased the expression of RIPK1/3 and MLKL. The N-terminal four-helix bundle (4HB) domain of PoMLKL exhibited necroptosis-inducing activity, and the C-terminal pseudokinase domain exerted auto-inhibitory effect on the 4HB domain. PoRIPK3 was capable of phosphorylating the T360/S361 residues in the PoMLKL C-terminal domain and initiated necroptosis, and this necroptosis-inducing activity was enhanced by PoRIPK1. PoRIPK1/3 interacted with PoMLKL in a manner that depended on the RIP homotypic interaction motif (RHIM), and deletion of RHIM from PoRIPK1/3 led to the dissociation of PoRIPK1/3 with PoMLKL. Inhibition of PoMLKL-mediated necroptosis increased Edwardsiella tarda infection in fish cells and tissues, and led to significantly enhanced lethality of the host. Taken together, these results revealed the activation mechanism of PoRIPK1/3-PoMLKL signaling pathway and the immunological function of necroptosis in the immune defense of teleost.

Creatine kinase (CK) is an enzyme that regulates adenosine triphosphate (ATP) metabolism to maintain energy homeostasis. Although CK has been reported to be involved in pathogen infection, the immune function of CK remains elusive. In this study, we identified two muscle-type CK from the teleost tongue sole Cynoglossus semilaevis (designated CsCKM-1 and CsCKM-2). Bacterial infection modulated CsCKM-1/2 expression in tongue sole tissues and induced the release of CsCKM-1/2 into serum. Recombinant CsCKM-1/2 (rCsCKM-1/2) exhibited robust kinase activity and bound to bacterial pathogens and pathogen-associated molecular patterns. rCsCKM-1/2 also bound to tongue sole peripheral blood leukocytes (PBLs) and promoted PBLs to uptake bacterial pathogens, inhibit bacterial proliferation, and express proinflammatory cytokines. When co-expressed in HEK293T cells, CsCKM-1/2 were found to interact with the leucine rich domain of toll-like receptor 2 (TLR2). The presence of TLR2 antagonist significantly reduced CsCKM-1/2-induced immune response and antibacterial effect. Taken together, these results indicated that tongue sole creatine kinases function as damage-associated molecular pattern (DAMP) molecules and play an important role in antimicrobial immunity via TLR2.

Cantharellus, one of the main genera of Hydnaceae (Cantharellales), is both ecologically and economically important. Although many studies have focused on this genus in China, the taxonomy should be further updated. In the present study, Cantharellus subgenera Afrocantharellus and Magni were investigated based on morphology and molecular phylogenetic analyses with new collections from China. Five phylogenetic species were recognized among the studied collections, three of which were described as new: C. bellus, C. cineraceus, and C. laevigatus; one was previously described taxon: C. hygrophoroides; and the remaining species was not defined due to the paucity of the materials. Among the four described species, both C. bellus and C. laevigatus are members of subgen. Magni, whereas C. cineraceus and C. hygrophoroides belong to subgen. Afrocantharellus.

ObjectivesTo be the first to apply a novel 450 nm blue diode laser in transurethral resection of bladder tumor (TURBt) to treat patients with non-muscle invasive bladder cancer (NMIBC) and evaluate its efficacy and safety during the preoperative period compared to the conventional plasmakinetic electrocautery.Materials and MethodsRandomized controlled trial (RCT) in five medical centers was designed as a non-inferiority study and conducted from October 2018 to December 2019. Patients with NMIBC were randomized to the blue laser or plasmakinetic electrocautery group for TURBt. As the first study to evaluate this novel blue laser device, the primary outcome was the effective resection rate of bladder tumors, including effective dissection and hemostasis. The secondary outcomes were the perioperative records, including surgical time, postoperative indwelling catheter time, hospital stay length, blood loss, reoperation rate, wound healing and adverse events.ResultsA total of 174 patients were randomized to either the blue laser group (85 patients) or plasmakinetic electrocautery group (89 patients). There was no statistical significance in the clinical features of bladder tumors, including tumor site, number and maximum lesion size. Both the blue laser and plasmakinetic electrocautery could effectively dissect all visible bladder tumors. The surgical time for patients in the blue laser group was longer (p=0.001), but their blood loss was less than that of patients in the control group (p=0.003). There were no differences in the postoperative indwelling catheter time, hospital stay length, reoperation rate or other adverse events. However, the patients undergoing TURBt with the blue laser showed a faster wound healing at 3 months after operation.ConclusionThe novel blue laser could be effectively and safely used for TURBt in patients with NMIBC, and this method was not inferior to plasmakinetic electrocautery during the perioperative period. However, TURBt with the blue laser may provide the benefit to reduce preoperative blood loss and accelerate postoperative wound healing. Moreover, longer follow-up to confirm recurrence-free survival benefit was required.

High-resolution radar rainfall data have great potential for rainfall predictions up to 6 h ahead (nowcasting); however, conventional extrapolation approaches based on in-built physical assumptions yield poor performance at longer lead times (3–6 h), which limits their operational utility. Moreover, atmospheric factors in radar estimate errors are often ignored. This study proposed a radar rainfall nowcasting method that attempts to achieve accurate nowcasting of 6 h using long short-term memory (LSTM) networks. Atmospheric conditions were considered to reduce radar estimate errors. To build radar nowcasting models based on LSTM networks (LSTM-RN), approximately 11 years of radar, gauge rainfall, and atmospheric data from the UK were obtained. Compared with the models built on optical flow (OF-RN) and random forest (RF-RN), LSTM-RN had the lowest root-mean-square errors (RMSE), highest correlation coefficients (COR), and mean bias errors closest to 0. Furthermore, LSTM-RN showed a growing advantage at longer lead times, with the RMSE decreasing by 17.99% and 7.17% compared with that of OF-RN and RF-RN, respectively. The results also revealed a strong relationship between LSTM-RN performance and weather conditions. This study provides an effective solution for nowcasting radar rainfall at long lead times, which enhances the forecast value and supports practical utility.

Hainan is the second largest island in China with the most extensive and well-preserved tropical forests and is also the largest island of the Indo Burma Biodiversity Hotspot. It provides in situ conservation for the unique ecosystem of the island. Recent studies have shown that there are diverse fungal species in Hainan. In this study, about 40 collections of the genus Amanita have been studied based on the morphology and molecular systematics, including 35 Chinese specimens (24 from Hainan, and eleven from other regions) and three specimens from other countries (Singapore and Malaysia). In total, five new species belonging to Amanita section Validae are described: A. cacaina, A. parvigrisea, A. pseudofritillaria, A. pseudosculpta, and A. yangii. Amanita parvifritillaria is recorded for the first time in Hainan. It is also the first report of this fungus occurring, outside Yunnan Province, China. Among the five new species, two are unique in this section because of the appendiculate pileus margin and the absence of an annulus. Based on these new findings, the diagnosis of the section Validae should be slightly modified to include a few species with appendiculate margin and the lack of annulus.

Purpose: Recent reports suggest retinal microvasculature mirror cerebral microcirculation. Using optical coherence tomography angiography (OCTA), we investigated the retinal microvasculature differences between ischemic stroke patients with large artery atherosclerosis (LAA) and small artery disease (SAD).Methods: All patients underwent MR imaging and were classified as SAD and LAA; LAA was subdivided into anterior LAS and posterior LAS depending on the location. Swept-source OCTA (SS-OCTA) was used to image and segment the retina into the superficial vascular complex (SVC) and deep vascular complex (DVC) in a 6 × 6 mm area around the fovea. A deep learning algorithm was used to assess the vessel area density (VAD, %) in the retinal microvasculature.Results: Fifty-eight (mean age = 60.26 ± 10.88 years; 81.03% males) were LAA while 64 (mean age = 55.58 ± 10.34 years; 85.94% males) were SAD. LAS patients had significantly reduced VAD in the DVC (P = 0.022) compared to SAD patients; the VAD in the SVC did not show any significant difference between the two groups (P = 0.580). Anterior LAA ischemic stroke showed significantly lower VAD (P = 0.002) in the SVC compared with posterior LAS patients. There was no significant difference in the DVC between the two groups (P = 0.376).Conclusions: We found LAA patients had significantly reduced DVC density compared with SAD; we also showed anterior LAA patients had significantly reduced SVC density compared with posterior LAA. These findings suggest retinal imaging has the potential to be used to detect microvasculature changes in subtypes of ischemic stroke.

Study designControlled laboratory study.ObjectiveTo determine the 6 degrees of freedom of lumbar vertebra in vivo during different functional activities in young and middle-aged asymptomatic subjects.MethodsA total of 26 asymptomatic subjects (M/F, 15/11; age, 20–55 years) were recruited in this study. They were divided into two groups: young group (number: 14; age: 20–30 years old) and middle-aged group (number: 12; age: 45–55 years old). The lumbar segment of each subject was scanned by computed tomography for the construction of three-dimensional (3D) models of the vertebra from L1 to S1. The lumbar spine was imaged by using a dual fluoroscopic system when the subjects performed different trunk postures. The 3D models of vertebrae were matched to two fluoroscopic images simultaneously in software. The range of motion (ROM) of vertebrae in the young and middle-aged groups was compared by using multiway analysis of variance, respectively.ResultsDuring the supine to the upright posture, vertebral rotation of the L1–S1 occurred mainly around the mediolateral axis (mean: 3.9 ± 2.9°). Along the mediolateral axis, vertebral translation was significantly lower at L1–2 (7.7 ± 2.4 mm) and L2–3 (8.0 ± 3.5 mm) than at L3–4 (1.6 ± 1.2 mm), L4–5 (3.3 ± 2.6 mm), and L5–S1 (2.6 ± 1.9 mm). At the L4–5 level, the young group had a higher rotational ROM than the middle-aged group around all three axes during left–right bending. Along the anteroposterior axis, the young group had a lower translational ROM at L4–5 than the middle-aged group during left–right bending (4.6 ± 3.3 vs. 7.6 ± 4.8 mm; P

ObjectivesIn this study, the objectives were to investigate the clinical efficacy of orthopedic therapeutic surgery (OTS) in patients with bone metastasis of liver cancer and explore the prognostic factors.MethodsThe electronic medical records of patients with bone metastasis of liver cancer in the Third Affiliated Hospital of Naval Medical University from September 2016 to August 2021 were retrospectively collected. A total of 53 patients were included. Patients were assigned to the OTS (n = 35) or the control group (n = 18) based on receiving orthopedic therapeutic surgery or conservative treatment. The pre/posttreatment Karnofsky Performance Status scale (KPS) and numeric rating scale (NRS) scores were compared. Univariate and multivariate Cox regression analyses were used to explore the prognostic factors affecting survival after bone metastasis. Logistic regression analyses were adopted to discover potential factors that contributed to greater KPS score improvement.ResultsThe axial bone accounted for 69.8% of all bone metastases. The proportion of multiple bone metastases was 52.8%. After surgery, the median KPS score of the OTS group increased from 60 to 80 (p

Ultrasonic guided wave (UGW) technology based on piezoelectric sensors is considered a very promising technology for aircraft structural damage detection. Traditional piezoelectric sensors are made of lead zirconate titanate (PZT) ceramics, but their brittleness and hardness make them difficult to apply to curved structure surfaces. In this study, a novel 0–3 flexible piezoelectric composite was fabricated by dispersing PZT particles in silicone resin, and its performance for potential applications in UGW sensors was studied. The effects of polarization conditions, PZT volume fraction, and PZT powder size on the performance of the composite were investigated. The influence of ambient temperature on composite performance was discussed, and temperature adaptability experiments were conducted. The results show that the optimal poling process of 0–3 PZT/silicone resin piezoelectric composite has a poling time of 25 min, a poling electric field of 4 kV/mm, and a poling temperature of 100°C. When the sensor is required to meet the test strain range of 8,000 με, the composite should be fabricated with a PZT volume fraction of 50% and a powder size of 170∼212 μm to obtain optimal sensing sensitivity. At an ambient temperature range of -55–75°C, the fabricated piezoelectric composite sensor has good flexibility and sensitivity in detecting the guide wave signals. These research results provide a new flexible piezoelectric sensing technology for aircraft structural damage detection.

Tight sandstone has low porosity and permeability, a complex pore structure, and strong heterogeneity due to strong diagenetic modifications. Limited intervals of Lianggaoshan Formation in the Fuling area are cored due to high costs, thus, a model for predicting diagenetic facies based on logging curves was established based on few core, thin section, X-ray diffraction (XRD), scanning electron microscopy (SEM), cathodoluminescence, routine core analysis, and mercury injection capillary pressure tests. The results show that tight sandstone in the Lianggaoshan Formation has primary and secondary intergranular pores, secondary intragranular pores, and intergranular micropores in the clay minerals. The compaction experienced by sandstone is medium to strong, and the main diagenetic minerals are carbonates (calcite, dolomite, and ferric dolomite) and clay minerals (chlorite, illite, and mixed illite/montmorillonite). Four types of diagenetic facies are recognized: carbonate cemented (CCF), tightly compacted (TCF), chlorite coating and clay mineral filling (CCCMFF), and dissolution facies (DF). Primary pores develop in the CCCMFF, and secondary pores develop in the DF; The porosities and permeabilities of CCCMFF and DF are better than that of CCF and TCF. The diagenetic facies were converted to logging data, and a diagenetic facies prediction model using four machine learning methods was established. The prediction results show that the random forest model has the highest prediction accuracy of 97.5%, followed by back propagation neural networks (BPNN), decision trees, and K-Nearest Neighbor (KNN). In addition, the random forest model had the smallest accuracy difference between the different diagenetic facies (2.86%). Compared with the other three machine learning models, the random forest model can balance unbalanced sample data and improve the prediction accuracy for the tight sandstone of the Lianggaoshan Formation in the Fuling area, which has a wide application range. It is worth noting that the BPNN may be more advantageous in diagenetic facies prediction when there are more sample data and diagenetic facies types.

Cancer is a disease with high morbidity and mortality in the world. In the past, the main treatment methods for cancer patients were surgery, radiotherapy and chemotherapy. However, with early treatment, the recurrence rate of cancer is higher, and the drug resistance of cancer cells is faster. In recent years, with the discovery of immune escape mechanism of cancer cells, Immunotherapy, especially Immune Checkpoint Inhibitors (ICIs), has made a breakthrough in the treatment of solid tumors, significantly prolonging the overall survival time and disease-free progression in some solid tumors, and its clinical benefits are more prominent than those of traditional anti-tumor drugs, which has become the hope of cancer patients after the failure of multi-line therapy. More and more studies have shown that there is a correlation between cancer driving genes and the clinical benefits of ICIs treatment, and the therapeutic effects and adverse reactions of ICIs can be predicted by the status of driving genes. Therefore, screening potential biomarkers of people who may benefit from immunotherapy in order to maximize the therapeutic benefits is a top priority. This review systematically summarizes the cancer driving genes that may affect the clinical benefits of immune checkpoint inhibitors, and provides accurate scientific basis for clinical practice.

IntroductionThis paper introduces the treatment and clinical outcome of the dorsal wrist ganglion cyst utilizing the Kiss-in method to establish a midcarpal volar portal.Materials and methodsPatients with dorsal ganglia of the wrist (n = 12, 6 females, 6 males) underwent arthroscopic surgery using the Kiss-in method at our hospital between September 2018 and January 2021. All patients underwent preoperative radiological investigations, such as magnetic resonance imaging (MRI; 12 cases) or ultrasonography (12 cases). The mean age of patients was 30.7 years (range: 19–46 years). The time lost from work, the wrist motion and strength, the presence of scarring, residual symptoms, complications, and recurrence were recorded at a mean follow-up of 24 months.ResultsEleven patients showed a good prognosis with active motion recovery. One patient showed the recurrence of ganglion, and the second arthroscopic resection was performed 5 months after the first surgery for this patient. After the surgery, the patient fully recovered.ConclusionsEstablishing the midcarpal volar portal by the Kiss-in method is safe. The dorsal ganglion cyst resection through the established midcarpal volar portal is a promising approach, allowing better visualization and a broader range motion of the arthroscope.

BackgroundOssification of the posterior longitudinal ligament (OPLL) and that of ligamentum flavum (OLF) are the main types of the ossification of spinal ligaments (OSL) that cause the thoracic myelopathy. Although several studies have investigated the relationship of body mass index (BMI) with the onset or severity of OSL, it remains unverified due to the contradictory results of existing evidence. A systematic review and meta-analysis were performed in this work to determine the relationship of BMI with the onset and severity of OSL.MethodsPubMed, EMBASE, Web of Science, and Cochrane Library were comprehensively searched online for relevant studies focusing on the relationship of BMI with the onset or severity of the OSL. The difference in BMI of OSL (or severe OSL group) and non-OSL (or nonsevere OSL group) groups was evaluated using the mean difference (MD) with a corresponding 95% confidence interval (CI).ResultsFifteen studies were included in this systematic review and meta-analysis. The BMI of the OSL group was significantly higher than that of the non-OSL group (MD = 1.70 kg/m2, 95% CI = 1.02–2.39 kg/m2, and P

Gasdermin (GSDM) is a family of pore-forming proteins that induce pyroptosis. To date, the origin and evolution of GSDM in Metazoa remain elusive. Here, we found that GSDM emerged early in Placozoa but is absent in a large number of invertebrates. In the lower vertebrate, fish, three types of GSDME, i.e., GSDMEa, GSDMEb, and a previously unreported type (designated GSDMEc), were idenitied. Evolutionarily, the three GSDMEs are distinctly separated: GSDMEa is closely related to tetrapod GSDME; GSDMEb exists exclusively in fish; GSDMEc forms the lineage root of tetrapod GSDMA/B/C/D. GSDMEc shares conserved genomic features with and is probably the prototype of GSDMA, which we found existing in all tetrapod classes. GSDMEc displays fast evolutionary dynamics, likely as a result of genomic transposition. A cross-metazoan analysis of GSDME revealed that GSDMEa shares a conserved caspase recognition motif with the GSDME of tetrapods and cnidarians, whereas GSDMEb has a unique caspase recognition motif similar to that of mammalian GSDMD, and GSDMEc exhibits no apparent caspase recognition motif. Through functional test, four highly conserved residues in vertebrate GSDME proved to be essential to auto-inhibition. Together our results provide new insights into the origin, evolution, and function of metazoan GSDMs.

Species of Cantharellus subgenus Cantharellus are interesting and important for their mycorrhizal properties, medicinal values, and edibility. In China, there are many undescribed species of the subgenus. In this study, four new species of subg. Cantharellus, viz. Cantharellus albopileatus, Cantharellus chuiweifanii, Cantharellus pinetorus, and Cantharellus ravus from Hainan and Hunan Provinces, respectively, were described based on morphological and phylogenetic evidence as a contribution to the knowledge of the species diversity in China. Detailed descriptions, color photographs of fresh basidiomata, and line drawings of microstructures of these four new species are presented as well as comparisons with related species.

ObjectiveImmune checkpoint inhibitors (ICIs) have changed the outcomes of a variety of cancers in an unprecedented manner. Gut microbiome plays a crucial regulatory role in the antineoplastic therapy of ICIs, which can be influenced by antibiotic (ABX) administration. In this efficacy evaluation, we aimed to clarify the correlations of ABX administration with the survival of cancer patients receiving ICIs treatment.MethodThe eligible literatures were searched using PubMed, Cochrane Library, Web of Science, and Clinical trials.gov databases before Nov 2021. The correlations of ABX administration with progression-free survival (PFS) and overall survival (OS) were determined using Hazard ratios (HRs) coupled with 95% confidence intervals (CIs).ResultsA total of 12 studies enrolling 6010 cancer patients receiving ICIs treatment were included in this efficacy evaluation. ABX administration was significantly correlated worse PFS (HR=1.60, 95%CI=1.33-1.92, P

PurposeTo assess the retinal microvasculature, choriocapillaris, and choroidal thickness in recent single subcortical infarction (RSSI) patients compared with healthy controls. We also assessed the correlation between the macular microvascular changes and choroidal changes with their clinical implications in RSSI patients.MethodsForty-six RSSI patients and 39 healthy controls (HC) were enrolled in our study. Magnetic resonance imaging (MRI) was done for all RSSI patients, and a total cerebral small vessel disease (CSVD) score was assessed for all patients. Swept-source optical coherence tomography (SS-OCT) was used to image and assess the choroidal thickness and SS-OCT angiography (SS-OCTA) was used to image and assess the macular microvasculature and choriocapillaris in all participants. Clinical information was collected for all participants.ResultsRSSI patients showed significantly sparser inner retinal microvasculature (P = 0.003) when compared with healthy controls. RSSI patients showed significantly thinner choroidal thickness (P < 0.001) when compared with HC. No significant difference (P = 0.247) was seen when the choriocapillaris was compared between the two groups. CSVD burden (P = 0.014) and NIHSS score (P = 0.010) showed significant correlation with the inner retinal microvasculature of RSSI patients. The inner retinal microvasculature (P = 0.016) and choroidal thickness (P = 0.018) showed a significant correlation with the MoCA scores in RSSI patients.ConclusionsOur report suggests that retinal and choroidal imaging may serve as useful indicators to expand our understanding of RSSI and its clinical validity.

With the development of anti-tumor drugs, tyrosine kinase inhibitors (TKIs) are an indispensable part of targeted therapy. They can be superior to traditional chemotherapeutic drugs in selectivity, safety, and efficacy. However, they have been found to be associated with serious adverse effects in use, such as myocardial infarction, fluid retention, hypertension, and rash. Although TKIs induced arrhythmia with a lower incidence than other cardiovascular diseases, much clinical evidence indicated that adequate attention and management should be provided to patients. This review focuses on QT interval prolongation and atrial fibrillation (AF) which are conveniently monitored in clinical practice. We collected data about TKIs, and analyzed the molecule mechanism, discussed the actual clinical evidence and drug-drug interaction, and provided countermeasures to QT interval prolongation and AF. We also pooled data to show that both QT prolongation and AF are related to their multi-target effects. Furthermore, more than 30 TKIs were approved by the FDA, but most of the novel drugs had a small sample size in the preclinical trial and risk/benefit assessments were not perfect, which led to a suspension after listing, like nilotinib. Similarly, vandetanib exhibits the most significant QT prolongation and ibrutinib exhibits the highest incidence in AF, but does not receive enough attention during treatment.

High-grade glioma is one of the most lethal human cancers characterized by extensive tumor heterogeneity. In order to identify cellular and molecular mechanisms that drive tumor heterogeneity of this lethal disease, we performed single-cell RNA sequencing analysis of one high-grade glioma. Accordingly, we analyzed the individual cellular components in the ecosystem of this tumor. We found that tumor-associated macrophages are predominant in the immune microenvironment. Furthermore, we identified five distinct subpopulations of tumor cells, including one cycling, two OPC/NPC-like and two MES-like cell subpopulations. Moreover, we revealed the evolutionary transition from the cycling to OPC/NPC-like and MES-like cells by trajectory analysis. Importantly, we found that SPP1/CD44 interaction plays a critical role in macrophage-mediated activation of MES-like cells by exploring the cell-cell communication among all cellular components in the tumor ecosystem. Finally, we showed that high expression levels of both SPP1 and CD44 correlate with an increased infiltration of macrophages and poor prognosis of glioma patients. Taken together, this study provided a single-cell atlas of one high-grade glioma and revealed a critical role of macrophage-mediated SPP1/CD44 signaling in glioma progression, indicating that the SPP1/CD44 axis is a potential target for glioma treatment.

Background: Colorectal cancer (CRC) is a leading cause of cancer death, and early diagnosis of CRC could significantly reduce its mortality rate. Previous studies suggest that the DNA methylation status of zinc finger genes (ZFGs) could be of potential in CRC early diagnosis. However, the comprehensive evaluation of ZFGs in CRC is still lacking.Methods: We first collected 1,426 public samples on genome-wide DNA methylation, including 1,104 cases of CRC tumors, 54 adenomas, and 268 para-tumors. Next, the most differentially methylated ZFGs were identified and validated in two replication cohorts comprising 218 CRC patients. Finally, we compared the prediction capabilities between the ZFGs and the SEPT9 in all CRC patients and the KRAS + and KRAS- subgroup.Results: Five candidate ZFGs were selected: ESR1, ZNF132, ZNF229, ZNF542, and ZNF677. In particular, ESR1 [area under the curve (AUC) = 0.91] and ZNF132 (AUC = 0.93) showed equivalent or better diagnostic capability for CRC than SEPT9 (AUC = 0.91) in the validation dataset, suggesting that these two ZFGs might be of potential for CRC diagnosis in the future. Furthermore, we performed subgroup analysis and found a significantly higher diagnostic capability in KRAS + (AUC ranged from 0.97 to 1) than that in KRAS- patients (AUC ranged from 0.74 to 0.86) for all these five ZFGs, suggesting that these ZFGs could be ideal diagnostic markers for KRAS mutated CRC patients.Conclusion: The methylation profiles of the candidate ZFGs could be potential biomarkers for the early diagnosis of CRC, especially for patients carrying KRAS mutations.

We report the experimental and numerical realization of a pseudospin-dependent acoustic topological insulator based on two sonic crystals constructed by the same regular hexagonal rods. Based on the zone folding mechanism, we obtain double Dirac cones with a four-fold deterministic degeneracy in the sonic crystal, and realize a band inversion and topological phase transition by rotating the rods. We observe the topologically protected one-way sound propagation of pseudospin-dependent edge states in a designed topological insulator composed of two selected sonic crystals with different rotation angles of the rods. Furthermore, we experimentally demonstrate the robustness of topological sound propagation against two types of defects, in which the edge states are almost immune to backscattering, and remain pseudospin-dependent characteristics. Our work provides a diverse route for designing tunable topological functional sound devices.

BackgroundHyperhomocysteinemia (HHcy) causes cardiovascular diseases via regulating inflammatory responses. We investigated whether and how the epithelial sodium channel (ENaC), a recently identified ion channel in endothelial cells, plays a role in HHcy-induced endothelial dysfunction.MethodsCell-attached patch-clamp recording in acute split-open aortic endothelial cells, western blot, confocal imaging, and wire myograph combined with pharmacological approaches were used to determine whether HHcy-mediated inflammatory signaling leads to endothelial dysfunction via stimulating ENaC.ResultsThe data showed that 4 weeks after L-methionine diet the levels of plasma Hcy were significantly increased and the ENaC was dramatically activated in mouse aortic endothelial cells. Administration of benzamil, a specific ENaC blocker, ameliorated L-methionine diet-induced impairment of endothelium-dependent relaxation (EDR) and reversed Hcy-induced increase in ENaC activity. Pharmacological inhibition of NADPH oxidase, reactive oxygen species (ROS), cyclooxygenase-2 (COX-2)/thromboxane B2 (TXB2), or serum/glucocorticoid regulated kinase 1 (SGK1) effectively attenuated both the Hcy-induced activation of endothelial ENaC and impairment of EDR. Our in vitro data showed that both NADPH oxidase inhibitor and an ROS scavenger reversed Hcy-induced increase in COX-2 expression in human umbilical vein endothelial cells (HUVECs). Moreover, Hcy-induced increase in expression levels of SGK-1, phosphorylated-SGK-1, and phosphorylated neural precursor cell-expressed developmentally downregulated protein 4-2 (p-Nedd4-2) in HUVECs were significantly blunted by a COX-2 inhibitor.ConclusionWe show that Hcy activates endothelial ENaC and subsequently impairs EDR of mouse aorta, via ROS/COX-2-dependent activation of SGK-1/Nedd4-2 signaling. Our study provides a rational that blockade of the endothelial ENaC could be potential method to prevent and/or to treat Hcy-induced cardiovascular disease.

Diabetic nephropathy (DN), a common diabetic microvascular complication, is characterized by its complex pathogenesis, higher risk of mortality, and the lack of effective diagnosis and treatment methods. Many studies focus on the diagnosis and treatment of diabetes mellitus (DM) and have reported that the pathophysiology of DN is very complex, involving many molecules and abnormal cellular activities. Given the respective pivotal roles of NF-κB, Nrf2, and TGF-β in inflammation, oxidative stress, and fibrosis during DN, we first review the effect of posttranslational modifications on these vital molecules in DN. Then, we describe the relationship between these molecules and related abnormal cellular activities in DN. Finally, we discuss some potential directions for DN treatment and diagnosis. The information reviewed here may be significant in the design of further studies to identify valuable therapeutic targets for DN.

The use of cyclosporine A (CsA) in transplant recipients is limited due to its side effects of causing severe hypertension. We have previously shown that CsA increases the activity of the epithelial sodium channel (ENaC) in cultured distal nephron cells. However, it remains unknown whether ENaC mediates CsA-induced hypertension and how we could prevent hypertension. Our data show that the open probability of ENaC in principal cells of split-open cortical collecting ducts was significantly increased after treatment of rats with CsA; the increase was attenuated by lovastatin. Moreover, CsA also elevated the levels of intracellular cholesterol (Cho), intracellular reactive oxygen species (ROS) via activation of NADPH oxidase p47phox, serum- and glucocorticoid-induced kinase isoform 1 (Sgk1), and phosphorylated neural precursor cell–expressed developmentally downregulated protein 4–2 (p-Nedd4-2) in the kidney cortex. Lovastatin also abolished CsA-induced elevation of α-, ß-, and γ-ENaC expressions. CsA elevated systolic blood pressure in rats; the elevation was completely reversed by lovastatin (an inhibitor of cholesterol synthesis), NaHS (a donor of H2S which ameliorated CsA-induced elevation of reactive oxygen species), or amiloride (a potent ENaC blocker). These results suggest that CsA elevates blood pressure by increasing ENaC activity via a signaling cascade associated with elevation of intracellular ROS, activation of Sgk1, and inactivation of Nedd4-2 in an intracellular cholesterol-dependent manner. Our data also show that NaHS ameliorates CsA-induced hypertension by inhibition of oxidative stress.

DM9 refers to an uncharacterized protein domain that is originally discovered in Drosophila melanogaster. Two proteins with DM9 repeats have been recently identified from Pacific oyster Crassostrea gigas as mannose-specific binding pattern-recognition receptors (PRRs). In the present study, a novel member of DM9 domain containing protein (designated as CgDM9CP-4) was identified from C. gigas. CgDM9CP-4, about 16 kDa with only two tandem DM9 domains, was highly enriched in hemocytes and gill. The transcripts level of CgDM9CP-4 in circulating hemocytes were decreased after LPS, PGN and Vibrio splendidus stimulations. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) displayed a broad binding spectrum towards various pathogen-associated molecular patterns (PAMPs) (LPS, PGN, β-glucan and Mannose) and microorganisms (Staphylococcus aureus, Micrococcus luteus, V. splendidus, V. anguillarum, Escherichia coli, Pichia pastoris and Yarrowia lipolytica). CgDM9CP-4 was mostly expressed in gill and some of the hemocytes. Flow cytometry analysis demonstrated that the CgDM9CP-4-positive hemocytes accounted for 7.3% of the total hemocytes, and they were small in size and less in granularity. CgDM9CP-4 was highly expressed in non-phagocytes (~82% of total hemocytes). The reactive oxygen species (ROS) and the expression levels of cytokines in CgDM9CP-4-positive hemocytes were much lower than that in CgDM9CP-4-negative hemocytes. The mRNA expression level of CgDM9CP-4 in hemocytes was decreased after RNAi of hematopoietic-related factors (CgGATA, CgRunt, CgSCL, and CgNotch). In addition, CgDM9CP-4-positive cells were found to be much more abundant in hemocytes from gill than that from hemolymph, with most of them located in the gill filament. All these results suggested that CgDM9CP-4 was a novel member of PRR that expressed in undifferentiated pro-hemocytes to mediate immune recognition of pathogens.

Sex-determining region Y-related high mobility group-box 10 (SOX10), a member of the SOX family, has recently been highlighted as an essential transcriptional factor involved in developmental biology. Recently, the functionality of SOX 10 has been increasingly revealed by researchers worldwide. It has been reported that SOX10 significantly regulates the proliferation, migration, and apoptosis of tumors and is closely associated with the progression of cancer. In this review, we first introduce the basic background of the SOX family and SOX10 and then discuss the pathophysiological roles of SOX10 in cancer. Besides, we enumerate the application of SOX10 in the pathological diagnosis and therapeutic potential of cancer. Eventually, we summarize the potential directions and perspectives of SOX10 in neoplastic theranostics. The information compiled herein may assist in additional studies and increase the potential of SOX10 as a therapeutic target for cancer.

Overexpression of Golgi phosphoprotein 3 (GOLPH3) predicts poor prognosis and is a potential therapeutic target in pancreatic ductal adenocarcinoma (PDAC). However, its role and underlying molecular mechanisms in the progression of PDAC remain unknown. In the present study, using high-throughput bimolecular fluorescence complementation (BiFC) analysis, we identified that stress-inducible protein-1 (STIP1) interacts with GOLPH3 and confirmed the interaction using co-localization and co-immunoprecipitation. The levels of GOLPH3 and STIP1 in PDAC tissues and adjacent non-cancerous pancreatic tissues were determined using immunohistochemistry (IHC) and quantitative real-time reverse transcription PCR. Real-time Quantitative-telomere repeat amplification (Q-TRAP) was applied to detect relative telomerase activity, and cell proliferation was measured when small interfering RNAs targeting GOLPH3 or STIP1 were transfected into PDAC cell lines. BALB/c nude mice were used to assess tumor growth inhibition of BXPC3 cells stably transfected with GOLPH3 short hairpin RNA. In summary, GOLPH3 was found to interact with STIP1 and both proteins were overexpressed and co-localized in PDAC tissues and cell lines. Moreover, suppression of GOLPH3 expression using shRNAs in PANC1 and BXPC3 cells inhibited tumor cell proliferation both in vitro and in vivo. Mechanistically, GOLPH3 interacts with STIP1 to activate telomerase reverse transcriptase (hTERT) and telomerase activity by c-Myc, and then upregulates cell cycle-related signaling proteins, including cyclin D1, to promote tumor cell growth, suggesting that disrupting the interaction between STIP1 and GOLPH3 would be a promising new strategy to treat PDAC.

Cluster of differentiation 63 (CD63), a four-transmembrane glycoprotein in the subfamily of tetraspanin, has been widely recognized as a gateway from the infection of foreign invaders to the immune defense of hosts. Its role in Pacific oyster Crassostrea gigas is, however, yet to be discovered. This work makes contributions by identifying CgCD63H, a CD63 homolog with four transmembrane domains and one conservative CCG motif, and establishing its role as a receptor that participates in immune recognition and hemocyte phagocytosis. The presence of CgCD63H messenger RNA (mRNA) in hepatopancreas, labial palps, gill, and hemocytes is confirmed. The expression level of mRNA in hemocytes is found significantly (p < 0.01) upregulated after the injection of Vibrio splendidus. CgCD63H protein, typically distributed over the plasma membrane of oyster hemocytes, is recruited to the Yarrowia lipolytica-containing phagosomes after the stimulation of Y. lipolytica. The recombinant CgCD63H protein expresses binding capacity to glucan (GLU), peptidoglycan (PGN), and lipopolysaccharide (LPS) in the presence of lyophilized hemolymph. The phagocytic rate of hemocytes toward V. splendidus and Y. lipolytica is significantly inhibited (p < 0.01) after incubation with anti-CgCD63H antibody. Our work further suggests that CgCD63H functions as a receptor involved in the immune recognition and hemocyte phagocytosis against invading pathogen, which can be a marker candidate for the hemocyte typing in C. gigas.

As a gene therapy strategy, RNA interference (RNAi) offers tremendous tumor therapy potential. However, its therapeutic efficacy is restricted by its inferior ability for targeted delivery and cellular uptake of small interfering RNA (siRNA). This study sought to develop a dual-ligand nanoparticle (NP) system loaded with siRNA to promote targeted delivery and therapeutic efficacy. We synthesized a dual receptor-targeted chitosan nanosystem (GCGA), whose target function was controlled by the ligands of galactose of lactobionic acid (LA) and glycyrrhetinic acid (GA). By loading siPAK1, an siRNA targeting P21-activated kinase 1 (PAK1), a molecular-targeted therapeutic dual-ligand NP (GCGA–siPAK1) was established. We investigated the synergistic effect of these two targeting units in hepatocellular carcinoma (HCC). In particular, GCGA–siPAK1 enhanced the NP targeting ability and promoted siPAK1 cell uptake. Subsequently, dramatic decreases in cell proliferation, invasion, and migration, with an apparent increase in cell apoptosis, were observed in treated cells. Furthermore, this dual-ligand NP gene delivery system demonstrated significant anti-tumor effects in tumor-bearing mice. Finally, we illuminated the molecular mechanism, whereby GCGA–siPAK1 promotes endogenous cell apoptosis through the PAK1/MEK/ERK pathway. Thus, the dual-target property effectively promotes the HCC therapeutic effect and provides a promising gene therapy strategy for clinical applications.

Non-coding RNAs (ncRNAs) are functional RNA molecules that are transcribed from DNA but not translated into proteins. ncRNAs function as key regulators of gene expression and chromatin modification. Recently, the functional role of ncRNAs in teleost fish has been extensively studied. Teleost fish are a highly diverse group among the vertebrate lineage. Fish are also important in terms of aquatic ecosystem, food production and human life, being the source of animal proteins worldwide and models of biomedical research. However, teleost fish always suffer from the invasion of infectious pathogens including viruses and bacteria, which has resulted in a tremendous economic loss to the fishing industry worldwide. Emerging evidence suggests that ncRNAs, especially miRNAs and lncRNAs, may serve as important regulators in cytokine and chemokine signaling, antigen presentation, complement and coagulation cascades, and T cell response in teleost fish. In this review, we summarize current knowledge and understanding of the roles of both miRNAs and lncRNAs in immune regulation in teleost fish. Molecular mechanism insights into the function of ncRNAs in fish immune response may contribute to the development of potential biomarkers and therapeutic targets for the prevention and treatment of fish diseases.

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation–reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p

Genome rearrangements are large-scale evolutionary events that shuffle genomic architectures. The minimal number of such events between two genomes is often used in phylogenomic studies to measure the evolutionary distance between the genomes. Double-Cut-and-Join (DCJ) operations represent a convenient model of most common genome rearrangements (reversals, translocations, fissions, and fusions), while other genome rearrangements, such as transpositions, can be modeled by pairs of DCJs. Since the DCJ model does not directly account for transpositions, their impact on DCJ scenarios is unclear. In the present work, we study implicit appearance of transpositions (as pairs of DCJs) in DCJ scenarios. We consider shortest DCJ scenarios satisfying the maximum parsimony assumption, as well as more general DCJ scenarios based on some realistic but less restrictive assumptions. In both cases, we derive a uniform lower bound for the rate of implicit transpositions, which depends only on the genomes but not a particular DCJ scenario between them. Our results imply that implicit appearance of transpositions in DCJ scenarios may be unavoidable or even abundant for some pairs of genomes. We estimate that for mammalian genomes implicit transpositions constitute at least 6% of genome rearrangements.

DM9 domain was first identified in Drosophila melanogaster, and it was subsequently found to integrate with or without other protein domains across a wide range of invertebrates and vertebrates. In the present study, a member of DM9 domain containing protein (DM9CP) family from marine invertebrate Crassostrea gigas (designated CgDM9CP-1), which was only composed of two DM9 domains, was taken as a protein model to study the biological functions of DM9 domain and its molecular determinants. CgDM9CP-1 was found to exhibit high binding specificity and avidity toward d-mannose residue. It served as a pattern recognition receptor (PRR) with a broad range of recognition spectrum to various pathogen-associated molecular patterns, including lipopolysaccharide, peptidylglycan, mannan, and β-1, 3-glucan in a d-mannose-dependent manner, as well as bacteria and fungi. In order to reveal the molecular mechanism underlying its pattern recognition activity, the crystal structures of wild-type and loss-of-function mutants were solved, and Asp22 and Lys43 were found to be the critical residues for ligand recognition. Moreover, CgDM9CP-1 protein was found to mainly distribute on the surface of C. gigas hemocytes, and it could be translocated into cytoplasm and colocalized with the engulfed microbes during hemocyte phagocytosis. The present result clearly indicated that CgDM9CP-1 was a PRR, and it provided an important clue for the better understanding of DM9CP function.

Edwardsiella tarda is a Gram-negative bacterium that can infect a broad range of hosts including humans and fish. Accumulating evidences have indicated that E. tarda is able to survive and replicate in host phagocytes. However, the pathways involved in the intracellular infection of E. tarda are unclear. In this study, we examined the entry and endocytic trafficking of E. tarda in the mouse macrophage cell line RAW264.7. We found that E. tarda entered RAW264.7 and multiplied intracellularly in a robust manner. Cellular invasion of E. tarda was significantly impaired by inhibition of clathrin- and caveolin-mediated endocytic pathways and by inhibition of endosome acidification, but not by inhibition of macropinocytosis. Consistently, RAW264.7-infecting E. tarda was co-localized with clathrin, caveolin, and hallmarks of early and late endosomes, and intracellular E. tarda was found to exist in acid organelles. In addition, E. tarda in RAW264.7 was associated with actin and microtubule, and blocking of the functions of these cytoskeletons by inhibitors significantly decreased E. tarda infection. Furthermore, formaldehyde-killed E. tarda exhibited routes of cellular uptake and intracellular trafficking similar to that of live E. tarda. Together these results provide the first evidence that entry of live E. tarda into macrophages is probably a passive, virulence-independent process of phagocytosis effected by clathrin- and caveolin-mediated endocytosis and cytoskeletons, and that the intracellular traffic of E. tarda involves endosomes and endolysosomes.