Impact of Microbubble Degradation and Flow Velocity on Subharmonic-aided Pressure Estimation (SHAPE): An Experimental Investigation.

OBJECTIVE: This study aimed to investigate the impact of microbubble degradation and flow velocity on Sub-Harmonic Aided Pressure Estimation (SHAPE), and to explore the correlation between subharmonic amplitude and pressure as a single factor. METHODS: We develop an open-loop vascular phantom platform system and utilize a commercial ultrasound machine and microbubbles for subharmonic imaging. Subharmonic amplitude was measured continuously at constant pressure and flow velocity to assess the impact of microbubble degradation. Flow velocity was varied within a range of 4-14 cm/s at constant pressure to investigate its relationship to subharmonic amplitude. Furthermore, pressure was varied within a range of 10-110 mm Hg at constant flow velocity to assess its isolated effect on subharmonic amplitude. RESULTS: Under constant pressure and flow velocity, subharmonic amplitude exhibited a continuous decrease at an average rate of 0.221 dB/min, signifying ongoing microbubble degradation during the experimental procedures. Subharmonic amplitude demonstrated a positive correlation with flow velocity, with a variation ratio of 0.423 dB/(cm/s). Under controlled conditions of microbubble degradation and flow velocity, a strong negative linear correlation was observed between pressure and subharmonic amplitude across different Mechanical Index (MI) settings (all R2 > 0.90). The sensitivity of SHAPE was determined to be 0.025 dB/mmHg at an MI of 0.04. CONCLUSION: The assessment of SHAPE sensitivity is affected by microbubble degradation and flow velocity. Excluding the aforementioned influencing factors, a strong linear negative correlation between pressure and subharmonic amplitude was still evident, albeit with a sensitivity coefficient lower than previously reported values.

Salinity decreases the contribution of microbial necromass to soil organic carbon pool in arid regions.

Saline soils are widely distributed in arid areas but there is a lack of mechanistic understanding on the effect of salinity on the formation and biochemical composition of soil organic carbon (SOC). We investigated the effects of salinity on the accumulation of microbial necromass under natural vegetation and in cropland in salt-affected arid areas stretching over a 1200-km transect in northwest China. Under both natural vegetation and cropland, microbial physiological activity (indicated by microbial biomass carbon normalized enzymatic activity) decreased sharply where the electrical conductivity approached 4 ds m-1 (a threshold to distinguish between saline and non-saline soils), but microbial biomass was only slightly affected by salinity. These indicated that a larger proportion of microbes could be inactive or dormant in saline soils. The contribution of fungal necromass C to SOC decreased but the contribution of bacterial necromass C to the SOC increased with increasing soil salinity. Adding fungal and bacterial necromass C together, the contribution of microbial necromass C to SOC in saline soils was 32-39 % smaller compared with non-saline soils. Fungal necromass C took up 85-86 % of microbial necromass C in non-saline soils but this proportion dropped to 60-66 % in saline soils. We suggested that the activity, growth, and turnover rate of microbes slowed by salinity was responsible for the decreased accumulation of fungal necromass in saline compared with non-saline soils, while the increased accumulation of bacterial residue in saline soils could be induced mainly by its slower decomposition. Soil microbial biomass was a poor predictor for the accumulation of microbial necromass in saline soils. We demonstrated a reduced contribution of microbial necromass to SOC and a shift in its composition towards the increase in bacterial origin in saline relative to non-saline soils. We concluded that salinity profoundly changes the biochemistry of SOC in arid regions.

Effect of B. subtilis in simulated acid red soil on the corrosion behavior of X80 pipeline steel.

The eastern section of China's West-east gas transmission project is laid in acidic red soil. NRB are widespread in soils and play an important role in metal corrosion. In this article, the corrosion failure behavior and mechanism of X80 pipeline steel under the action of NRB in simulated acidic soil were studied. It was found that the biofilm of B. subtilis had significant inhibitory on the overall corrosion of X80 steel. Electrochemical results prove that the corrosion rate of the sterile group after 14 days of immersion was about 4.5 times that of the bacterial group. However, the biofilm promotes the formation of local corrosion pits. Confocal laser scanning microscopy images indicate that that the corrosion pit depth of the bacterial group (46.1 µm) was three times that of the bacterial-free group (15.7 µm) after 14 days. The pH of the acidic environment was slightly improved by B. subtilis. XPS results proved that B. subtilis complicates the corrosion products of X80 steel through its nitrate reduction ability and metabolism.

A novel partitivirus conferring hypovirulence by affecting vesicle transport in the fungus Colletotrichum.

Colletotrichum spp. are economically important phytopathogenic fungi that cause anthracnose in a variety of plant species worldwide. Hypovirulence-associated mycoviruses provide new options for the biological control of plant fungal diseases. Here, we found a novel partitivirus from Colletotrichum alienum and named it Colletotrichum alienum partitivirus 1 (CaPV1). CaPV1 contained two dsRNA segments encoding an RNA-dependent RNA polymerase and a capsid protein and was classified under the genus Gammapartitivirus of the family Partitiviridae. CaPV1 significantly decreased host virulence, mycelial growth, appressorial development, and appressorium turgor but increased conidial production with abnormal morphology. In addition, CaPV1 could be successfully transfected into other Colletotrichum species, including C. fructicola, C. spaethianum, and C. gloeosporioides, and caused hypovirulence, indicating the broad application potential of this virus. CaPV1 caused significant transcriptional rewiring of the host fungus C. alienum. Notably, some genes related to vesicle transport in the CaPV1-infected strain were downregulated, consistent with the impaired endocytosis pathway in this fungus. When the Rab gene CaRab7, which is associated with endocytosis in vesicle transport, was knocked out, the virulence of the mutants was reduced. Overall, our findings demonstrated that CaPV1 has the potential to control anthracnose caused by Colletotrichum, and the mechanism by which Colletotrichum induces hypovirulence is caused by affecting vesicle transport.IMPORTANCEColletotrichum is a kind of economically important phytopathogenic fungi that cause anthracnose disease in a variety of plant species worldwide. We found a novel mycovirus of the Gammapartitivirus genus and Partitiviridae family from the phytopathogenic fungus Colletotrichum alienum and named it CaPV1. This study revealed that CaPV1 infection significantly decreased host virulence and fitness by affecting mycelial growth, appressorial development, and appressorium turgor. In addition, CaPV1 could also infect other Colletotrichum species, including C. fructicola, C. spaethianum, and C. gloeosporioides, by viral particle transfection and resulting in hypovirulence of these Colletotrichum species. Transcriptomic analysis showed that CaPV1 caused significant transcriptional rewiring of the host fungus C. alienum, especially the genes involved in vesicle transport. Moreover, endocytosis and gene knockout assays demonstrated that the mechanism underlying CaPV1-induced hypovirulence is, at least in part, caused by affecting the vesicle transport of the host fungus. This study provided insights into the mechanisms underlying the pathogenesis of Colletotrichum species and mycovirus-fungus interactions, linking the role of mycovirus and fungus vesicle transport systems in shaping fungal pathogenicity.

Effect of the Water Content on the Adsorption of CO2 and CH4 in Calcite Slit Nanopores: Insights from GCMC, MD, and DFT.

It is significant to understand the adsorption mechanisms of shale gas (CH4) and CO2 in shale formations to enhance CH4 recovery rates and enable geological CO2 storage. This study provides a comprehensive investigation into the adsorption behaviors of CO2 and CH4 within dry and hydrous calcite nanopores, utilizing a combination of grand canonical Monte Carlo simulations, molecular dynamics simulations, and density functional theory calculations. In dry calcite slits, the calculated results for the adsorption capacity, density profile, and isosteric heat of CO2 and CH4 reveal that CO2 possesses a stronger adsorption affinity, making it preferentially adsorb on the pore surface compared to CH4. In hydrous calcite slits, calculating the adsorption capacity and density profile of CO2 and CH4, the results show that the gas adsorption sites become progressively occupied by H2O molecules, leading to a substantial decrease in the adsorption capacity of CO2 and CH4. Furthermore, by analysis of the adsorption energy and electronic structure, the reason for the reduction of gas adsorption capacity caused by H2O is further revealed. This work has a deep understanding of the adsorption mechanisms of shale gas and CO2 in calcite and can offer valuable theoretical insights for the development of a CO2-enhanced shale gas recovery technology.

Characterization and biocontrol mechanism of Streptomyces olivoreticuli as a potential biocontrol agent against Rhizoctonia solani.

Rhizoctonia solani is a widespread and devastating plant pathogenic fungus that infects many important crops. This pathogen causes tobacco target spot, a disease that is widespread in many tobacco-growing countries and is destructive to tobacco. To identify antagonistic microorganisms with biocontrol potential against this disease, we isolated Streptomyces strains from forest inter-root soil and screened a promising biocontrol strain, ZZ-21. Based on in vitro antagonism assays, ZZ-21 showed a significant inhibitory effect on R. solani and various other phytopathogens. ZZ-21 was identified as Streptomyces olivoreticuli by its phenotypic, genetic, physiological and biochemical properties. Complete genome sequencing revealed that ZZ-21 harbored numerous antimicrobial biosynthesis gene clusters. ZZ-21 significantly reduced the lesion length in detached inoculated leaf assays and reduced the disease index under greenhouse and field conditions. Based on an in vitro antagonistic assay of ZZ-21 culture, the strain exhibited an antifungal activity against R. solani in a dose-dependent manner. The culture filtrate could impair membrane integrity, possibly through membrane lipid peroxidation. ZZ-21 could secrete multiple extracellular enzymes and siderophores. According to a series of antifungal assays, the extracellular metabolites of ZZ-21 contained antimicrobial bioactive compounds composed of proteins/peptides extracted using ammonium sulfate precipitation, which were stable under stress caused by high temperature and protease K. The EC50 value for ammonium sulfate precipitation was determined to be 21.11 µg/mL in this study. Moreover, the proteins/peptides also exhibited biocontrol ability and were observed to alter the plasma membrane integrity of R. solani which were evaluated by biocontrol efficacy assays on detached tobacco leaves and PI staining. Overall, strain ZZ-21 shows the potential to be developed into a biopesticide against tobacco target spot disease.

Physiological response mechanism of heavy metal-resistant endophytic fungi isolated from the roots of Polygonatum kingianum.

This study aims to evaluate the tolerance of endophytic fungi isolated from the fibrous roots of Polygonatum kingianum to arsenic (As) and cadmium (Cd) and their physiological response mechanisms. Five isolated strains were obtained with EC50 values for As(V) ranging from 421 to 1281 mg/L, while the other three strains tolerated Cd(II) with an EC50 range of 407-1112 mg/L. Morphological and molecular identification indicated that these eight strains were Cladosporium spp. belonging to dark septate endophytes (DSEs). The contents of metal ions in mycelium sharply increased, reaching 38.87 mg/kg for strain MZ-11 under As(V) stress and 0.33 mg/kg for fungus PR-2 under Cd(II). The physiological response revealed that the biomass decreased with increasing concentrations of As(V) or Cd(II), and the activity of superoxide dismutase significantly improved under the corresponding EC50 -concentration As/Cd of the strains, as well as the contents of antioxidant substances, including metallothionein, glutathione, malondialdehyde, melanin, and proline. Taken together, the filamentous fungi of Cladosporium spp. accounted for a high proportion of fungi isolated from the fibrous roots of P. kingianum and had a strong capacity to tolerate As(V) or Cd(II) stress by improving antioxidase activities and the content of antioxidant substances, and immobilization of metal ions in hyphae.

Autophagy suppression facilitates macrophage M2 polarization via increased instability of NF-κB pathway in hepatocellular carcinoma.

The tumor microenvironment is a highly heterogeneous circumstance composed of multiple components, while tumor-associated macrophages (TAMs) are major innate immune cells with highly plastic and are always educated by tumor cells to structure an advantageous pro-tumor immune microenvironment. Despite emerging evidence focalizing the role of autophagy in other immune cells, the regulatory mechanism of autophagy in macrophage polarization remains poorly understood. Herein, we demonstrated that hepatocellular carcinoma (HCC) cells educated macrophages toward M2-like phenotype polarization under the condition of coculture. Moreover, we observed that inhibition of macrophage autophagy promoted M2-like macrophage polarization, while the tendency was impeded when autophagy was motivated. Mechanistically, macrophage autophagy inhibition inactivates the NF-κB pathway by increasing the instability of TAB3 via ubiquitination degradation, which leads to the M2-like phenotype polarization of macrophages. Both immunohistochemistry staining using human HCC tissues and experiment in vivo verified autophagy inhibition is correlated with M2 macrophage polarization. Altogether, we illustrated that macrophage autophagy was involved in the process of HCC cells domesticating M2 macrophage polarization via the NF-κB pathway. These results provide a new target to interfere with the polarization of macrophages to M2-like phenotype during HCC progression.

A-to-I RNA Editing in Klebsiella pneumoniae Regulates Quorum Sensing and Affects Cell Growth and Virulence.

Millions of adenosine (A) to inosine (I) RNA editing events are reported and well-studied in eukaryotes; however, many features and functions remain unclear in prokaryotes. By combining PacBio Sequel, Illumina whole-genome sequencing, and RNA Sequencing data of two Klebsiella pneumoniae strains with different virulence, a total of 13 RNA editing events are identified. The RNA editing event of badR is focused, which shows a significant difference in editing levels in the two K. pneumoniae strains and is predicted to be a transcription factor. A hard-coded Cys is mutated on DNA to simulate the effect of complete editing of badR. Transcriptome analysis identifies the cellular quorum sensing (QS) pathway as the most dramatic change, demonstrating the dynamic regulation of RNA editing on badR related to coordinated collective behavior. Indeed, a significant difference in autoinducer 2 activity and cell growth is detected when the cells reach the stationary phase. Additionally, the mutant strain shows significantly lower virulence than the WT strain in the Galleria mellonella infection model. Furthermore, RNA editing regulation of badR is highly conserved across K. pneumoniae strains. Overall, this work provides new insights into posttranscriptional regulation in bacteria.

Inhibition of autophagy in macrophage promotes IL-1ß-mediated hepatocellular carcinoma progression via inflammasome accumulation and self-recruitment.

Hepatocellular carcinoma (HCC) has a complex and changeable tumor microenvironment. Despite emerging evidence focusing on autophagy process within immune cells, the function and regulatory mechanism of macrophage autophagy in tumor progression remains unclear. Our results of multiplex-immunohistochemistry and RNA-sequencing identified the reduced levels of autophagy in tumor macrophages in the HCC microenvironment, associated with a poor prognosis and increased microvascular metastasis in HCC patients. Specifically, HCC suppressed the macrophage autophagy initiation through the up-regulation of mTOR and ULK1 phosphorylation at Ser757. Knockdown of autophagy-related proteins to further inhibit autophagy significantly boosted the metastatic potential of HCC. Mechanistically, the accumulation of NLRP3 inflammasome mediated by autophagy inhibition promoted the cleavage, maturation, and release of IL-1ß, which facilitated the HCC progression, eventually accelerating HCC metastasis via the epithelial-mesenchymal transition. Autophagy inhibition provoked macrophage self-recruitment through the CCL20-CCR6 signaling was also a crucial account of HCC progression. Recruited macrophages mediated the cascade amplification of IL-1ß and CCL20 to form a novel pro-metastatic positive feedback loop through promoting HCC metastasis and increased macrophage recruitment, respectively. Notably, targeting IL-1ß/IL-1 receptor signaling impaired lung metastasis induced by macrophage autophagy inhibition in a mice HCC lung metastasis model. In summary, this study highlighted that inhibition of tumor macrophage autophagy facilitated HCC progression by increasing IL-1ß secretion via NLRP3 inflammasome accumulation and by macrophage self-recruitment through the CCL20 signaling pathway. Interruption of this metastasis-promoting loop by IL-1ß blockade may provide a promising therapeutic strategy for HCC patients.

Screening and identification of hub-gene associated with brain metastasis in breast cancer.

BACKGROUND: The presence of breast cancer in the brain, also known as brain metastasis (BMS), is the primary reason for a bad prognosis in cases of breast cancer. Breast cancer is the most prevalent malignant tumor seen in women in developing nations. At present, there is no effective method to inhibit brain metastasis of breast cancer. Therefore, it is necessary to conduct a systematic study on BMS of breast cancer, which will not provide ideas and sites for follow-up studies on the treatment and inhibition of BMS. METHODS: In this study, data set GSE43837 was screened from gene expression omnibus database, and then R language tool was used for differential analysis of its expression spectrum, The gene ontology functional enrichment and Kyoto encyclopedia of genes and genomes signal pathway enrichment analyses, as well as the interactive gene retrieval tool for hub-gene analysis, were performed. RESULTS: According to the findings, the primary genes linked to breast cancer brain metastases are those that involve interactions between cytokines and their respective receptors and between neuroactive ligands and their respective receptors. The majority of the gene ontology enrichment took place in the extracellular structural tissues, the extracellular matrix tissues, and the second message-mediated signaling. We were able to identify 8 genes that are linked to breast cancer spreading to the brain. The gene score for matrix metallopeptidase1 (MMP-1) was the highest among them, and the genes MMP10, tumor necrosis factor alpha-inducible protein 8, collagen type I alpha 2 chain, vascular cell adhesion molecule 1, and TNF superfamily member 11 were all connected to 1 another in an interaction way. CONCLUSIONS: There is a possibility that the 8 key genes that were identified in this research are connected to the progression of BMS in breast cancer. Among them, MMP1 is 1 that has the potential to have a role in the diagnosis and treatment of BMS in breast cancer.

Evaluation of left atrial reservoir function and sphericity index in patients with mitral valve disease: a cardiac magnetic resonance imaging study.

To assess the performance of biplane area-length method in measuring left atrial (LA) volume and sphericity index and to investigate the correlation of LA reservoir function and sphericity index with atrial fibrosis on cardiac magnetic resonance imaging (MRI) in patients with mitral valve disease (MVD). Forty-eight patients with MVD undergoing cardiac MRI scan were enrolled in this retrospective study. LA reservoir function, measured as maximum volume (LAVmax), minimum volume (LAVmin) and ejection fraction (LAEF), and sphericity index were quantified using the biplane area-length method and standard short-axis approach, respectively. Comparisons of LA reservoir function and sphericity index between the two different methods were performed, as well as between positive (n = 17, 35%) and negative atrial wall fibrosis groups (n = 31, 65%). There was no difference in the values of LAVmax index and sphericity index between the two different methods. The biplane area-length method had poor performance in assessing LAVmin index and LAEF compared to standard short-axis approach, with an underestimation of 13.5% for LAVmin index and an overestimation of 27% for LAEF. Patients with positive atrial fibrosis had larger LAVmax index, LAVmin index and sphericity index, and lower LAEF levels in comparison to the negative atrial fibrosis group. The biplane area-length method has good performance in assessing LA sphericity index for patients with MVD, not in LA reservoir function. Patients with positive atrial fibrosis tend to suffer from more adverse LA remodelling.

Novel and diverse mycoviruses co-infecting a single strain of the phytopathogenic fungus Alternaria dianthicola.

Alternaria dianthicola is a pathogenic fungus that causes serious leaf or flower blight on some medicinal plants worldwide. In this study, multiple dsRNA bands in the range of 1.2-10 kbp were found in a Alternaria dianthus strain HNSZ-1, and eleven full-length cDNA sequences of these dsRNA were obtained by high-throughput sequencing, RT-PCR detection and conventional Sanger sequencing. Homology search and phylogenetic analyses indicated that the strain HNSZ-1 was infected by at least nine mycoviruses. Among the nine, five viruses were confirmed to represent novel viruses in the families Hypoviridae, Totiviridae, Mymonaviridae and a provisional family Ambiguiviridae. Virus elimination and horizontal transmission indicated that the (-) ssRNA virus, AdNSRV1, might be associated with the slow growth and irregular colony phenotype of the host fungus. As far as we know, this is the first report for virome characterization of A. dianthus, which might provide important insights for screening of mycovirus for biological control and for studying of the interactions between viruses or viruses and their host.

[Effects of Low-dose Antibiotic Exposure on Fetal Growth and Development].

Objective To analyze maternal blood exposure to non-therapeutic antibiotics in late pregnancy and explore the effects of low-dose antibiotics on fetal growth and development.Methods A total of 104 pregnant women in late pregnancy (28-32 weeks) without serious pregnancy complications were enrolled,who had regular antenatal examination and delivery in Peking Union Medical College Hospital and did not use therapeutic antibiotics 2 months before pregnancy and in the whole pregnant process.The levels of antibiotics in the maternal blood were detected by liquid chromatography-tandem mass spectrometry,and the pregnant women were assigned into an antibiotic exposure group (antibiotic positive) and a non-exposure group (antibiotic negative).The length,weight,placental weight,and placental volume of the newborns in the two groups were measured,and the data were statistically analyzed by t test or χ2 test.Results The maternal blood antibiotic test showed 7 positive cases (6.73%,antibiotic exposure group) and 97 negative cases (93.27%,non-exposure group). The average length of newborns in the antibiotic exposure group and the non-exposure group was (49.57±1.40) cm and (48.85±1.77) cm,respectively,with no significant difference (t=1.060,P=0.363).The average weight of newborns in the antibiotic exposure group and the non-exposure group was (3558.57±382.95) g and (3275.36±356.41) g,respectively,with significant difference (t=2.021,P=0.046).The mean placental weight in the antibiotic exposure group and the non-exposure group was (676.43±124.59) g and (631.96±129.25) g,respectively,with no significant difference (t=0.881,P=0.380).The mean placental volume in the antibiotic exposure group and the non-exposure group was (724.67±174.91) cm3 and (676.82±220.86) cm3,respectively,with no significant difference (t=0.560,P=0.388).Compared with those in the non-exposure group,the neonatal length,neonatal weight,placental weight,and placental volume in the antibiotic exposure group increased by 1.47%,8.65%,7.04%,and 7.07%,respectively.Conclusion There are antibiotics in the environment,and maternal blood exposure to non-therapeutic antibiotics can promote the growth and development of the fetus and placenta,especially increasing the fetal weight.

Identification and Characterization of Colletotrichum fructicola and Colletotrichum siamense Causing Anthracnose on Luffa Sponge Gourd in China.

Luffa sponge gourd (Luffa cylindrica) is an important cucurbitaceous vegetable and is known as the source of loofah. From 2020 to 2021, a leaf disease occurred on luffa leaves in the Hunan Province of China. Symptoms were displayed as oval to irregular chlorotic lesions surrounded by yellow halos. The pathogens were isolated from the affected leaves. According to morphological characterization and molecular identification using multi-locus phylogenetic analysis of the internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), and partial mating type (Mat1-2) gene (ApMAT) regions, the pathogens were identified as two Colletotrichum species: Colletotrichum fructicola and C. siamense. Koch's postulates were identified by a pathogenicity test and re-confirmation. To the best of our knowledge, C. fructicola and C. siamense are two new species associated with luffa sponge gourd anthracnose.

The glacial-terrestrial-fluvial pathway: A multiparametrical analysis of spatiotemporal dissolved organic matter variation in three catchments of Lake Nam Co, Tibetan Plateau.

The Tibetan Plateau (TP) is a sensitive alpine environment of global importance, being Asia's water tower, featuring vast ice masses and comprising the world's largest alpine grasslands. Intensified land-use and pronounced global climate change have put pressure on the environment of the TP. We studied the tempo-spatial variability of dissolved organic matter (DOM) to better understand the fluxes of nutrients and energy from terrestrial to aquatic ecosystems in the TP. We used a multiparametrical approach, based on inorganic water chemistry, dissolved organic carbon (DOC) concentration, dissolved organic matter (DOM) characteristics (chromophoric DOM, fluorescence DOM and δ13C of DOM) in stream samples of three catchments of the Nam Co watershed and the lake itself. Satellite based plant cover estimates were used to link biogeochemical data to the structure and degradation of vegetation zones in the catchments. Catchment streams showed site-specific DOM signatures inherited from glaciers, wetlands, groundwater, and Kobresia pygmaea pastures. By comparing stream and lake samples, we found DOM processing and unification by loss of chromophoric DOM signatures and a change towards an autochthonous source of lake DOM. DOM diversity was largest in the headwaters of the catchments and heavily modified in terminal aquatic systems. Seasonality was characterized by a minor influence of freshet and by a very strong impact of the Indian summer monsoon on DOM composition, with more microbial DOM sources. The DOM of Lake Nam Co differed chemically from stream water samples, indicating the lake to be a quasi-marine environment in regards to the degree of chemical modification and sources of DOM. DOM proved to be a powerful marker to elucidate consequences of land use and climatic change on biogeochemical processes in High Asian alpine ecosystems.

KRAS acting through ERK signaling stabilizes PD-L1 via inhibiting autophagy pathway in intrahepatic cholangiocarcinoma.

BACKGROUND: While the correlation between PD-L1 expression and KRAS mutation has been previously reported in other solid tumors such as non-small cell lung cancer (NSCLC), whether PD-L1 can be modulated by ERK signaling downstream of KRAS in intrahepatic cholangiocarcinoma (iCCA) and the underlying molecular regulatory mechanism remain unclear. METHODS: The expression of ERK, p-ERK, PD-L1 and autophagy markers following KRAS knockdown or Ras/Raf/MEK/ERK signaling inhibitors treatment was examined in two human iCCA cell lines (HuCCT1 and RBE) using western blotting and immunofluorescence. Both pharmacological autophagy inhibitors and short-interfering RNA against ATG7 were applied to inhibit autophagy. The apoptosis rates of iCCA cell lines were detected by flow cytometry and CCK-8 after co-culturing with CD3/CD28-activated human CD8+ T lymphocytes. Immunohistochemistry was applied to detect the correlation of ERK, p-ERK and PD-L1 in 92 iCCA tissues. RESULTS: The present study demonstrated that the PD-L1 expression level was distinctly reduced in KRAS-mutated iCCA cell lines when ERK signaling was inhibited and ERK phosphorylation levels were lowered. The positive association between p-ERK and PD-L1 was also verified in 92 iCCA tissue samples. Moreover, ERK inhibition induced autophagy activation. Both inhibiting autophagy via autophagy inhibitors and genetically silencing the ATG7 expression partially reversed the reduced PD-L1 expression caused by ERK inhibition. In addition, ERK-mediated down-regulation of PD-L1 via autophagy pathways induced the apoptosis of iCCA cells when co-cultured with CD3/CD28-activated human CD8+ T lymphocytes in vitro. CONCLUSIONS: Our results suggest that ERK signaling inhibition contributes to the reduction of PD-L1 expression through the autophagy pathway in iCCA. As a supplement to anti-PD-1/PD-L1 immunotherapy, ERK-targeted therapy may serve as a potentially novel treatment strategy for human KRAS-mutated iCCA.

Relationship between epicardial fat volume on cardiac CT and atherosclerosis severity in three-vessel coronary artery disease: a single-center cross-sectional study.

BACKGROUND: The ideal treatment strategy for stable three-vessel coronary artery disease (CAD) patients are difficult to determine and for patients undergoing conservative treatment, imaging evidence of coronary atherosclerotic severity progression remains limited. Epicardial fat volume (EFV) on coronary CT angiography (CCTA) has been considered to be associated with coronary atherosclerosis. Therefore, this study aims to evaluate the relationship between EFV level and coronary atherosclerosis severity in three-vessel CAD. METHODS: This retrospective study enrolled 252 consecutive patients with three-vessel CAD and 252 normal control group participants who underwent CCTA between January 2018 and December 2019. A semi-automatic method was developed for EFV quantification on CCTA images, standardized by body surface area. Coronary atherosclerosis severity was evaluated and scored by the number of coronary arteries with ≥ 50% stenosis on coronary angiography. Patients were subdivided into groups on the basis of lesion severity: mild (score = 3 vessels, n = 85), moderate (3.5 vessels ≤ score < 4 vessels, n = 82), and severe (4 vessels ≤ score ≤ 7 vessels, n = 85). The independent sample t-test, analysis of variance, and logistic regression analysis were used to evaluate the associations between EFV level and severity of coronary atherosclerosis. RESULTS: Compared with normal controls, three-vessel CAD patients had significantly higher EFV level (65 ± 22 mL/m2 vs. 48 ± 19 mL/m2; P < 0.001). In patients with three-vessel CAD, there was a progressive decline in EFV level as the score of coronary atherosclerosis severity increased, especially in those patients with a body mass index (BMI) ≥ 25 kg/m2 (75 ± 21 mL/m2 vs. 72 ± 22 mL/m2 vs. 62 ± 17 mL/m2; P < 0.05). Multivariable regression analysis showed that both BMI (OR 3.40, 95% CI 2.00-5.78, P < 0.001) and the score of coronary atherosclerosis severity (OR 0.49, 95% CI 0.26-0.93, P < 0.05) were independently related to the change of EFV level. CONCLUSION: Three-vessel CAD patients do have higher EFV level than the normal controls. While, there may be an inverse relationship between EFV level and the severity of coronary atherosclerosis in patients with three-vessel CAD.

A Panax notoginseng phosphate transporter, PnPht1;3, greatly contributes to phosphate and arsenate uptake.

The crisis of arsenic (As) accumulation in rhizomes threatens the quality and safety of Panax notoginseng (Burk.) F.H. Chen, which is a well-known traditional Chinese herb with a long clinical history. The uptake of arsenate (AsV) could be suppressed by supplying phosphate (Pi), in which Pi transporters play important roles in the uptake of Pi and AsV. Herein, the P . notoginseng Pi transporter-encoding gene PnPht1;3 was identified and characterised under Pi deficiency and AsV exposure. In this study, the open reading frame (ORF) of PnPht1;3 was cloned according to RNA-seq and encoded 545 amino acids. The relative expression levels revealed that PnPht1;3 was significantly upregulated under phosphate deficiency and AsV exposure. Heterologous expression in Saccharomyces cerevisiae MB192 demonstrated that PnPht1;3 performed optimally in complementing the yeast Pi-transport defect and accumulated more As in the cells. Combined with the subcellular localisation prediction, it was concluded that PnPht1;3 encodes a functional plasma membrane-localised transporter protein that mediates putative high-affinity Pi/H+ symport activity and enhances the uptake of Pi and AsV. Therefore, a better understanding of the roles of the P . notoginseng Pi transporter could provide new insight for solving As accumulation in medicinal plants.

First Report of Southern Blight on Aloe vera Caused by Athelia rolfsii in China.

Aloe vera (L.) Burm f., which belongs to the family Aloaceae, is a perennial succulent plant and cultivated for its medicinal, cosmetic, vegetable and ornamental uses. In summer of 2021, about 15% (60 infected among 400 surveyed plants) of A. vera (A. barbadensis) plants in two gardens in Lishui, Zhejiang Province of China showed symptoms of southern blight disease. Symptomatic plants primarily exhibited slightly sunken water-soaked, dark brown lesions on taproot and basal part of the stems. As the disease progressed, leaves in the basal part of stems and subsequently the whole plant rotted and withered, with white mycelial mats occurring on infected stems and leaves. Numerous brown, spherical sclerotia were observed on the colonized tissues and soil surfaces around the infected plants. Mycelial fragments and sclerotia from symptomatic leaves were plated directly to potato dextrose agar (PDA) amended with 100 µg/ml streptomycin and incubated at 26°C in the dark. By hyphal-tip method, a total of five pure isolates were obtained from five diseased leaf samples. When cultured on PDA at 26°C for three days, colonies showed white and thick aerial mycelium, with a radial growth rate of 23.7 mm/day. Typical clamp connection structures were observed microscopically after three days and numerous globoid, rapeseed shape sclerotia, measuring 1 to 2 mm in diameter (n=50) formed after six days. These sclerotia were initially white and gradually turned dark brown with age. On the basis of morphological characteristics, the fungal isolates were identified as Athelia rolfsii (Curzi) C.C. Tu & Kimbr (anamorph Sclerotium rolfsii Sacc) (Mordue 1974). The internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1) regions of a representative isolate LHBJ2-4 were amplified and sequenced using the primers ITS4/ITS5 (White et al. 1990) and EF1/EF2, respectively (accession no. MZ956758 and OL365370). BLASTn search showed that the amplified ITS and TEF1 sequences had 99.71% (680/682 bp) and 99.80% (498/499 bp) identity with the A. rolfsii isolates CBS 115.22 (MH854711.1) and Sr_286 (JF267815), respectively. Neighbor-joining phylogenetic tree based on the ITS sequences revealed that LHBJ2-4 clustered with A. rolfsii isolates. For pathogenicity test, three potted A. vera plants (~30 cm tall) were inoculated by placing a 0.5 cm mycelial plug of isolate LHBJ2-4 (three-day old) at the base of each A. vera plant. Three A. vera plants inoculated with sterile PDA plugs served as controls. All the inoculated plants were placed in a growth chamber at 27°C under a 12/12 h light/dark cycle. The inoculation assays were carried out twice. After 5 to 7 days, stem bases of the inoculated plants showed brown lesions that were similar to those observed in the field. However, control plants remained symptomless. Athelia rolfsii was re-isolated from all the inoculated plants and identified using morphological and molecular method described above, thus confirming Koch's postulates. Although A. rolfsii has been reported to cause disease on A. vera in India (Dubey and Pandey 2009), to the best of our knowledge, this is the first report of A. rolfsii causing southern blight on A. vera in China. Because A. rolfsii has a wide host range and is difficult to control (Punja 1985), occurrence of southern blight in China might be a serious threat for A. vera production and appropriate management strategies should be developed to control this disease.