Direct Identification of Complex Glycans via a Highly Sensitive Engineered Nanopore.

The crucial roles that glycans play in biological systems are determined by their structures. However, the analysis of glycan structures still has numerous bottlenecks due to their inherent complexities. The nanopore technology has emerged as a powerful sensor for DNA sequencing and peptide detection. This has a significant impact on the development of a related research area. Currently, nanopores are beginning to be applied for the detection of simple glycans, but the analysis of complex glycans by this technology is still challenging. Here, we designed an engineered α-hemolysin nanopore M113R/T115A to achieve the sensing of complex glycans at micromolar concentrations and under label-free conditions. By extracting characteristic features to depict a three-dimensional (3D) scatter plot, glycans with different numbers of functional groups, various chain lengths ranging from disaccharide to decasaccharide, and distinct glycosidic linkages could be distinguished. Molecular dynamics (MD) simulations show different behaviors of glycans with ß1,3- or ß1,4-glycosidic bonds in nanopores. More importantly, the designed nanopore system permitted the discrimination of each glycan isomer with different lengths in a mixture with a separation ratio of over 0.9. This work represents a proof-of-concept demonstration that complex glycans can be analyzed using nanopore sequencing technology.

SARS-CoV-2 envelope protein-derived extracellular vesicles act as potential media for viral spillover.

Extracellular vesicles (EVs) are shown to be a novel viral transmission model capable of increasing a virus's tropism. According to our earlier research, cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or transfected with envelope protein plasmids generate a novel type of EVs that are micrometer-sized and able to encase virus particles. Here, we showed the capacity of these EVs to invade various animals both in vitro and in vivo independent of the angiotensin-converting enzyme 2 receptor. First, via macropinocytosis, intact EVs produced from Vero E6 (monkey) cells were able to enter cells from a variety of animals, including cats, dogs, bats, hamsters, and minks, and vice versa. Second, when given to zebrafish with cutaneous wounds, the EVs showed favorable stability in aqueous environments and entered the fish. Moreover, infection of wild-type (WT) mice with heterogeneous EVs carrying SARS-CoV-2 particles led to a strong cytokine response and a notable amount of lung damage. Conversely, free viral particles did not infect WT mice. These results highlight the variety of processes behind viral transmission and cross-species evolution by indicating that EVs may be possible vehicles for SARS-CoV-2 spillover and raising risk concerns over EVs' potential for viral gene transfer.

Design, synthesis, and evaluation of novel oxyacanthine derivatives for anti-SARS-CoV-2 activity.

Here, we report the synthesis of a series of oxyacanthine derivatives and evaluation for their anti-SARS-CoV-2 activity in Vero E6 cells. In order to eliminate the potential metabolic activation caused by para-methylene phenol moiety in oxyacanthine, totally 29 derivatives were designed and synthesized, resulting in 23 compounds with antivirus IC50 below 5.00 µM and 9 compounds with antivirus IC50 below 1.00 µM. Among them, amides compound 4a and 4d exhibited potent anti-SARS-CoV-2 activity and the most favorable selectivity index (SI) in vitro with the SI values of 115 and 70, respectively. The pharmacokinetic properties of 4a and 4d were also assessed. Much more improved exposure in mice, longer half-life (T1/2), and increased oral bioavailability were observed for both compounds 4a and 4d compared with oxyacanthine.

Mapping the Acetylamino and Carboxyl Groups on Glycans by Engineered α-Hemolysin Nanopores.

Glycan is a crucial class of biological macromolecules with important biological functions. Functional groups determine the chemical properties of glycans, which further affect their biological activities. However, the structural complexity of glycans has set a technical hurdle for their direct identification. Nanopores have emerged as highly sensitive biosensors that are capable of detecting and characterizing various analytes. Here, we identified the functional groups on glycans with a designed α-hemolysin nanopore containing arginine mutations (M113R), which is specifically sensitive to glycans with acetamido and carboxyl groups. Molecular dynamics simulations indicated that the acetamido and carboxyl groups of the glycans produce unique electrical signatures by forming polar and electrostatic interactions with the M113R nanopores. Using these electrical features as the fingerprints, we mapped the length of the glycans containing acetamido and carboxyl groups at the monosaccharide, disaccharide, and trisaccharide levels. This proof-of-concept study provides a promising foundation for developing single-molecule glycan fingerprinting libraries and demonstrates the capability of biological nanopores in glycan sequencing.

Discovery of SARS-CoV-2-E channel inhibitors as antiviral candidates.

Lack of efficiency has been a major problem shared by all currently developed anti-SARS-CoV-2 therapies. Our previous study shows that SARS-CoV-2 structural envelope (2-E) protein forms a type of cation channel, and heterogeneously expression of 2-E channels causes host cell death. In this study we developed a cell-based high throughput screening (HTS) assay and used it to discover inhibitors against 2-E channels. Among 4376 compounds tested, 34 hits with cell protection activity were found. Followed by an anti-viral analysis, 15 compounds which could inhibit SARS-CoV-2 replication were identified. In electrophysiological experiments, three representatives showing inhibitory effect on 2-E channels were chosen for further characterization. Among them, proanthocyanidins directly bound to 2-E channel with binding affinity (KD) of 22.14 µM in surface plasmon resonance assay. Molecular modeling and docking analysis revealed that proanthocyanidins inserted into the pore of 2-E N-terminal vestibule acting as a channel blocker. Consistently, mutations of Glu 8 and Asn 15, two residues lining the proposed binding pocket, abolished the inhibitory effects of proanthocyanidins. The natural product proanthocyanidins are widely used as cosmetic, suggesting a potential of proanthocyanidins as disinfectant for external use. This study further demonstrates that 2-E channel is an effective antiviral drug target and provides a potential antiviral candidate against SARS-CoV-2.

Machine learning algorithms' accuracy in predicting kidney disease progression: a systematic review and meta-analysis.

BACKGROUND: Kidney disease progression rates vary among patients. Rapid and accurate prediction of kidney disease outcomes is crucial for disease management. In recent years, various prediction models using Machine Learning (ML) algorithms have been established in nephrology. However, their accuracy have been inconsistent. Therefore, we conducted a systematic review and meta-analysis to investigate the diagnostic accuracy of ML algorithms for kidney disease progression. METHODS: We searched PubMed, EMBASE, Cochrane Central Register of Controlled Trials, the Chinese Biomedicine Literature Database, Chinese National Knowledge Infrastructure, Wanfang Database, and the VIP Database for diagnostic studies on ML algorithms' accuracy in predicting kidney disease prognosis, from the establishment of these databases until October 2020. Two investigators independently evaluate study quality by QUADAS-2 tool and extracted data from single ML algorithm for data synthesis using the bivariate model and the hierarchical summary receiver operating characteristic (HSROC) model. RESULTS: Fifteen studies were left after screening, only 6 studies were eligible for data synthesis. The sample size of these 6 studies was 12,534, and the kidney disease types could be divided into chronic kidney disease (CKD) and Immunoglobulin A Nephropathy, with 5 articles using end-stage renal diseases occurrence as the primary outcome. The main results indicated that the area under curve (AUC) of the HSROC was 0.87 (0.84-0.90) and ML algorithm exhibited a strong specificity, 95% confidence interval and heterogeneity (I2) of (0.87, 0.84-0.90, [I2 99.0%]) and a weak sensitivity of (0.68, 0.58-0.77, [I2 99.7%]) in predicting kidney disease deterioration. And the the results of subgroup analysis indicated that ML algorithm's AUC for predicting CKD prognosis was 0.82 (0.79-0.85), with the pool sensitivity of (0.64, 0.49-0.77, [I2 99.20%]) and pool specificity of (0.84, 0.74-0.91, [I2 99.84%]). The ML algorithm's AUC for predicting IgA nephropathy prognosis was 0.78 (0.74-0.81), with the pool sensitivity of (0.74, 0.71-0.77, [I2 7.10%]) and pool specificity of (0.93, 0.91-0.95, [I2 83.92%]). CONCLUSION: Taking advantage of big data, ML algorithm-based prediction models have high accuracy in predicting kidney disease progression, we recommend ML algorithms as an auxiliary tool for clinicians to determine proper treatment and disease management strategies.

Kinetics of antimony biogeochemical processes under pre-definite anaerobic and aerobic conditions in a paddy soil.

While the transformation of antimony (Sb) in paddy soil has been previously investigated, the biogeochemical processes of highly chemical active Sb in the soil remain poorly understood. In addition, there is a lack of quantitative understanding of Sb transformation in soil. Therefore, in this study, the kinetics of exogenous Sb in paddy soils were investigated under anaerobic and aerobic incubation conditions. The dissolved Sb(V) and the Sb(V) extracted by diffusive gradient technique decreased under anaerobic conditions and then increased under aerobic conditions. The redox reaction of Sb occurred, and Sb bioavailability significantly decreased after 55 days of incubation. The kinetics of Fe and the scanning transmission electron microscopy analysis revealed that the Fe oxides were reduced and became dispersed under anaerobic conditions, whereas they were oxidized and re-aggregated during the aerobic stage. In addition, the redox processes of sulfur and nitrogen were detected under both anaerobic and aerobic conditions. Based on these observations, a simplified kinetic model was established to distinguish the relative contributions of the transformation processes. The bioavailability of Sb was controlled by immobilization as a result of S reduction and by mobilization as a result of Fe reductive dissolution and S oxidation, rather than the pH. These processes coupled with the redox reaction of Sb jointly resulted in the complex behavior of Sb transformation under anaerobic and aerobic conditions. The model-based method and findings of this study provide a comprehensive understanding of the Sb transformation in a complex soil biogeochemical system under changing redox conditions.

Impacts of Redox Conditions on Arsenic and Antimony Transformation in Paddy Soil: Kinetics and Functional Bacteria.

Arsenic (As) and antimony (Sb) are known carcinogens and are present as contaminants in paddy soils. However, the complicated dynamics of the mobility of these metalloids have not been well understood due to changing redox conditions in paddy soils. Herein, the kinetics of dissolved As and Sb, and functional bacteria/genes were examined in a paddy soil cultured under aerobic and anaerobic conditions. Under aerobic condition, dissolved As(V) and Sb(V) increased constantly due to sulfide oxidation by O2 and bound As and Sb were released. Under anaerobic condition, the reduction of As(V) and Sb(V) occurred, and the mobility of As and Sb were affected by soil redox processes. The bacteria with functional genes aioA and arrA were responsible for the direct As/Sb transformation, while Fe- and N-related bacteria had an indirect effect on the fate of As/Sb via coupling with the redox processes of Fe and N. These findings improve understanding of the mobility of As and Sb in paddy soil systems under different redox conditions.

High-Resolution Breast MRI Reconstruction Using a Deep Convolutional Generative Adversarial Network.

BACKGROUND: A generative adversarial network could be used for high-resolution (HR) medical image synthesis with reduced scan time. PURPOSE: To evaluate the potential of using a deep convolutional generative adversarial network (DCGAN) for generating HRpre and HRpost images based on their corresponding low-resolution (LR) images (LRpre and LRpost ). STUDY TYPE: This was a retrospective analysis of a prospectively acquired cohort. POPULATION: In all, 224 subjects were randomly divided into 200 training subjects and an independent 24 subjects testing set. FIELD STRENGTH/SEQUENCE: Dynamic contrast-enhanced (DCE) MRI with a 1.5T scanner. ASSESSMENT: Three breast radiologists independently ranked the image datasets, using the DCE images as the ground truth, and reviewed the image quality of both the original LR images and the generated HR images. The BI-RADS category and conspicuity of lesions were also ranked. The inter/intracorrelation coefficients (ICCs) of mean image quality scores, lesion conspicuity scores, and Breast Imaging Reporting and Data System (BI-RADS) categories were calculated between the three readers. STATISTICAL TEST: Wilcoxon signed-rank tests evaluated differences among the multireader ranking scores. RESULTS: The mean overall image quality scores of the generated HRpre and HRpost were significantly higher than those of the original LRpre and LRpost (4.77 ± 0.41 vs. 3.27 ± 0.43 and 4.72 ± 0.44 vs. 3.23 ± 0.43, P < 0.0001, respectively, in the multireader study). The mean lesion conspicuity scores of the generated HRpre and HRpost were significantly higher than those of the original LRpre and LRpost (4.18 ± 0.70 vs. 3.49 ± 0.58 and 4.35 ± 0.59 vs. 3.48 ± 0.61, P < 0.001, respectively, in the multireader study). The ICCs of the image quality scores, lesion conspicuity scores, and BI-RADS categories had good agreements among the three readers (all ICCs >0.75). DATA CONCLUSION: DCGAN was capable of generating HR of the breast from fast pre- and postcontrast LR and achieved superior quantitative and qualitative performance in a multireader study. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 2 J. MAGN. RESON. IMAGING 2020;52:1852-1858.

Folded or Degraded in Endoplasmic Reticulum.

In consistent with other membrane-bound and secretory proteins, immune checkpoint proteins go through a set of modifications in the endoplasmic reticulum (ER) to acquire their native functional structures before they function at their destinations. There are various ER-resident chaperones and enzymes synergistically regulate and catalyze the glycosylation, folding and transporting of proteins. The whole processing is under the surveillance of ER quality control system which allows the correctly folded proteins to exit from the ER with the help of coat proteinII(COPII) coated vesicles, while retains the rest of terminally misfolded ones in the ER and then eliminates them via ER-associated degradation (ERAD) or ER-to-lysosomes-associated degradation (ERLAD). The dysfunction of the ER causes ER stress which triggers unfolded protein response (UPR) to restore ER proteostasis. Unsolvable prolonged ER stress ultimately results in cell death. This chapter reviews the process that proteins undergo in the ER, and the glycosylation, folding and degradation of immune checkpoint proteins as well as the associated potential immunotherapies to date.

A Combination of Stable Isotope Probing, Illumina Sequencing, and Co-occurrence Network to Investigate Thermophilic Acetate- and Lactate-Utilizing Bacteria.

Anaerobic digestion is a complicated microbiological process that involves a wide diversity of microorganisms. Acetate is one of the most important intermediates, and interactions between acetate-oxidizing bacteria and archaea could play an important role in the formation of methane in anoxic environments. Anaerobic digestion at thermophilic temperatures is known to increase methane production, but the effects on the microbial community are largely unknown. In the current study, stable isotope probing was used to characterize acetate- and lactate-oxidizing bacteria in thermophilic anaerobic digestion. In microcosms fed 13C-acetate, bacteria related to members of Clostridium, Hydrogenophaga, Fervidobacterium, Spirochaeta, Limnohabitans, and Rhodococcus demonstrated elevated abundances of 13C-DNA fractions, suggesting their activities in acetate oxidation. In the treatments fed 13C-lactate, Anaeromyxobacter, Desulfobulbus, Syntrophus, Cystobacterineae, and Azospira were found to be the potential thermophilic lactate utilizers. PICRUSt predicted that enzymes related to nitrate and nitrite reduction would be enriched in 13C-DNA fractions, suggesting that the acetate and lactate oxidation may be coupled with nitrate and/or nitrite reduction. Co-occurrence network analysis indicated bacterial taxa not enriched in 13C-DNA fractions that may also play a critical role in thermophilic anaerobic digestion.

Response of Soil Microbial Communities to Elevated Antimony and Arsenic Contamination Indicates the Relationship between the Innate Microbiota and Contaminant Fractions.

Mining of sulfide ore deposits containing metalloids, such as antimony and arsenic, has introduced serious soil contamination around the world, posing severe threats to food safety and human health. Hence, it is important to understand the behavior and composition of the microbial communities that control the mobilization or sequestration of these metal(loid)s. Here, we selected two sites in Southwest China with different levels of Sb and As contamination to study interactions among various Sb and As fractions and the soil microbiota, with a focus on the microbial response to metalloid contamination. Comprehensive geochemical analyses and 16S rRNA gene amplicon sequencing demonstrated distinct soil taxonomic inventories depending on Sb and As contamination levels. Stochastic gradient boosting indicated that citric acid extractable Sb(V) and As(V) contributed 5% and 15%, respectively, to influencing the community diversity. Random forest predicted that low concentrations of Sb(V) and As(V) could enhance the community diversity but generally, the Sb and As contamination impairs microbial diversity. Co-occurrence network analysis indicated a strong correlation between the indigenous microbial communities and various Sb and As fractions. A number of taxa were identified as core genera due to their elevated abundances and positive correlation with contaminant fractions (total Sb and As concentrations, bioavailable Sb and As extractable fractions, and Sb and As redox species). Shotgun metagenomics indicated that Sb and As biogeochemical redox reactions may exist in contaminated soils. All these observations suggest the potential for bioremediation of Sb- and As-contaminated soils.

Clinical Outcomes in Pediatric Patients Hospitalized with Fulminant Myocarditis Requiring Extracorporeal Membrane Oxygenation: A Meta-analysis.

We conducted a meta-analysis to provide the survival rates for pediatric patients hospitalized with fulminant myocarditis requiring ECMO. The literature search was conducted using Embase, PubMed, MEDLINE and Elsevier for studies published before April 1, 2016. We focus on survival rates for pediatric patients hospitalized with fulminant myocarditis requiring ECMO, and studies that reported only on adult patients were excluded. Summary of the survival rates was obtained using fixed-effect or random-effect meta-analysis which determined by I 2. Six studies were included in the analysis, encompassing 172 patients. The minimum and maximum reported rates of survival to hospital discharge were 53.8 and 83.3%, respectively. The cumulative rate was 107/172. The calculated Cochran Q value was 3.73, which was not significant for heterogeneity (P = 0.588). The I 2 value was 0%. The pooled estimate rate was 62.9% with a 95% confidence interval of 55.3-69.8%. In pediatric patients with cardiac failure who have failed conventional therapies in FM, venoarterial ECMO should be considered. In total, 62.9% of patients with FM and either cardiogenic shock and/or cardiac arrest survived to hospital discharge with ECMO.

Bimodal voltage dependence of TRPA1: mutations of a key pore helix residue reveal strong intrinsic voltage-dependent inactivation.

Transient receptor potential A1 (TRPA1) is implicated in somatosensory processing and pathological pain sensation. Although not strictly voltage-gated, ionic currents of TRPA1 typically rectify outwardly, indicating channel activation at depolarized membrane potentials. However, some reports also showed TRPA1 inactivation at high positive potentials, implicating voltage-dependent inactivation. Here we report a conserved leucine residue, L906, in the putative pore helix, which strongly impacts the voltage dependency of TRPA1. Mutation of the leucine to cysteine (L906C) converted the channel from outward to inward rectification independent of divalent cations and irrespective to stimulation by allyl isothiocyanate. The mutant, but not the wild-type channel, displayed exclusively voltage-dependent inactivation at positive potentials. The L906C mutation also exhibited reduced sensitivity to inhibition by TRPA1 blockers, HC030031 and ruthenium red. Further mutagenesis of the leucine to all natural amino acids individually revealed that most substitutions at L906 (15/19) resulted in inward rectification, with exceptions of three amino acids that dramatically reduced channel activity and one, methionine, which mimicked the wild-type channel. Our data are plausibly explained by a bimodal gating model involving both voltage-dependent activation and inactivation of TRPA1. We propose that the key pore helix residue, L906, plays an essential role in responding to the voltage-dependent gating.

Nanopore-based glycan sequencing: state of the art and future prospects.

Sequencing of biomacromolecules is a crucial cornerstone in life sciences. Glycans, one of the fundamental biomolecules, derive their physiological and pathological functions from their structures. Glycan sequencing faces challenges due to its structural complexity and current detection technology limitations. As a highly sensitive sensor, nanopores can directly convert nucleic acid sequence information into electrical signals, spearheading the revolution of third-generation nucleic acid sequencing technologies. However, their potential for deciphering complex glycans remains untapped. Initial attempts demonstrated the significant sensitivity of nanopores in glycan sensing, which provided the theoretical basis and insights for the realization of nanopore-based glycan sequencing. Here, we present three potential technical routes to employ nanopore technology in glycan sequencing for the first time. The three novel technical routes include: strand sequencing, capturing glycan chains as they translocate through nanopores; sequential hydrolysis sequencing, capturing released monosaccharides one by one; splicing sequencing, mapping signals from hydrolyzed glycan fragments to an oligosaccharide database/library. Designing suitable nanopores, enzymes, and motors, and extracting characteristic signals pose major challenges, potentially aided by artificial intelligence. It would be highly desirable to design an all-in-one high-throughput glycan sequencer instrument by integrating a sample processing unit, nanopore array, and signal acquisition system into a microfluidic device. The nanopore sequencer invention calls for intensive multidisciplinary cooperation including electrochemistry, glycochemistry, engineering, materials, enzymology, etc. Advancing glycan sequencing will promote the development of basic research and facilitate the discovery of glycan-based drugs and disease markers, fostering progress in glycoscience and even life sciences.

The human microbiota is a beneficial reservoir for SARS-CoV-2 mutations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations are rapidly emerging. In particular, beneficial mutations in the spike (S) protein, which can either make a person more infectious or enable immunological escape, are providing a significant obstacle to the prevention and treatment of pandemics. However, how the virus acquires a high number of beneficial mutations in a short time remains a mystery. We demonstrate here that variations of concern may be mutated due in part to the influence of the human microbiome. We searched the National Center for Biotechnology Information database for homologous fragments (HFs) after finding a mutation and the six neighboring amino acids in a viral mutation fragment. Among the approximate 8,000 HFs obtained, 61 mutations in S and other outer membrane proteins were found in bacteria, accounting for 62% of all mutation sources, which is 12-fold higher than the natural variable proportion. A significant proportion of these bacterial species-roughly 70%-come from the human microbiota, are mainly found in the lung or gut, and share a composition pattern with COVID-19 patients. Importantly, SARS-CoV-2 RNA-dependent RNA polymerase replicates corresponding bacterial mRNAs harboring mutations, producing chimeric RNAs. SARS-CoV-2 may collectively pick up mutations from the human microbiota that change the original virus's binding sites or antigenic determinants. Our study clarifies the evolving mutational mechanisms of SARS-CoV-2. IMPORTANCE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations are rapidly emerging, in particular advantageous mutations in the spike (S) protein, which either increase transmissibility or lead to immune escape and are posing a major challenge to pandemic prevention and treatment. However, how the virus acquires a high number of advantageous mutations in a short time remains a mystery. Here, we provide evidence that the human microbiota is a reservoir of advantageous mutations and aids mutational evolution and host adaptation of SARS-CoV-2. Our findings demonstrate a conceptual breakthrough on the mutational evolution mechanisms of SARS-CoV-2 for human adaptation. SARS-CoV-2 may grab advantageous mutations from the widely existing microorganisms in the host, which is undoubtedly an "efficient" manner. Our study might open a new perspective to understand the evolution of virus mutation, which has enormous implications for comprehending the trajectory of the COVID-19 pandemic.

Identification of potential shared gene signatures between gastric cancer and type 2 diabetes: a data-driven analysis.

Background: Gastric cancer (GC) and type 2 diabetes (T2D) contribute to each other, but the interaction mechanisms remain undiscovered. The goal of this research was to explore shared genes as well as crosstalk mechanisms between GC and T2D. Methods: The Gene Expression Omnibus (GEO) database served as the source of the GC and T2D datasets. The differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) were utilized to identify representative genes. In addition, overlapping genes between the representative genes of the two diseases were used for functional enrichment analysis and protein-protein interaction (PPI) network. Next, hub genes were filtered through two machine learning algorithms. Finally, external validation was undertaken with data from the Cancer Genome Atlas (TCGA) database. Results: A total of 292 and 541 DEGs were obtained from the GC (GSE29272) and T2D (GSE164416) datasets, respectively. In addition, 2,704 and 336 module genes were identified in GC and T2D. Following their intersection, 104 crosstalk genes were identified. Enrichment analysis indicated that "ECM-receptor interaction," "AGE-RAGE signaling pathway in diabetic complications," "aging," and "cellular response to copper ion" were mutual pathways. Through the PPI network, 10 genes were identified as candidate hub genes. Machine learning further selected BGN, VCAN, FN1, FBLN1, COL4A5, COL1A1, and COL6A3 as hub genes. Conclusion: "ECM-receptor interaction," "AGE-RAGE signaling pathway in diabetic complications," "aging," and "cellular response to copper ion" were revealed as possible crosstalk mechanisms. BGN, VCAN, FN1, FBLN1, COL4A5, COL1A1, and COL6A3 were identified as shared genes and potential therapeutic targets for people suffering from GC and T2D.

Aberrant methylation of the PTCH1 gene promoter region in aberrant crypt foci.

Patched homolog 1 (PTCH1) is a known tumor suppressor that regulates the Hedgehog (Hh) pathway and has been implicated in tumorigenesis. The role of PTCH1 in colon carcinogenesis, however, is controversial. The aim of the present study was to investigate epigenetic modifications of PTCH1 in aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancer (CRC). Using laser-capture microdissection (LCM), a pure population of ACF epithelial cells was isolated and studied. The inherent protein expression levels of SHH, PTCH1, SMO and GLI1 were assessed by immunohistochemistry for 405 ACF, including 54 dysplastic ACF (d-ACF) and 351 non-dysplastic ACF (n-ACF). The mRNA levels and methylation status of PTCH1 were also determined in 54 d-ACF and 96 n-ACF. Our data showed that the expression of SHH, SMO and GLI1 was significantly up-regulated in d-ACF, compared to n-ACF. Also, the mRNA and protein levels of PTCH1 were lower in d-ACF than n-ACF. Using MSP or MS-HRM, PTCH1 methylation was present in 64.8% (35/54) or 63.3% (34/54), respectively, of d-ACF and 19.8% (19/96) or 22.9% (11/48), respectively, of n-ACF. PTCH1 methylation was more frequent in d-ACF than n-ACF (p < 0.001) and was associated with PTCH1 mRNA levels (r = 0.358, p < 0.01). There was a statistically significant correlation between PTCH1 methylation status and the prevalence of colorectal neoplasms. In conclusion, this study suggests that aberrant methylation of the PTCH1 promoter may be an early, initiating event of colon carcinogenesis.

A small extent of seawater intrusion significantly enhanced Cd uptake by rice in coastal paddy fields.

Paddy fields located around estuaries suffer from seawater intrusion, and how and to what extent salinity levels influence Cd accumulation in rice grains is still unclear. Pot experiments were carried out by cultivating rice under alternating flooding and drainage conditions with different salinity levels (0.2‰, 0.6‰ and 1.8‰). The Cd availability was greatly enhanced at 1.8‰ salinity due to the competition for binding sites by cations and the formation of Cd complexation with anions, which also contributed to Cd uptake by rice roots. The soil Cd fractions were investigated and found that the Cd availability significantly decreased during flooding stage, while it rapidly increased after soil drainage. During drainage stage, Cd availability was greatly enhanced at 1.8‰ salinity mainly attributed to the formation of CdCln2-n. The kinetic model was established to quantitatively evaluate Cd transformation, and it found that the release of Cd from organic matter and Fe-Mn oxides was greatly enhanced at 1.8‰ salinity. The results of pot experiments showed that there was a significant increase in Cd content in rice roots and grains in the treatment of 1.8‰ salinity, because the increasing salinity induced an increase in Cd availability and upregulation of key genes regulating Cd uptake in rice roots. Our findings elucidated the key mechanisms by which high salinity enhanced Cd accumulation in rice grains, and more attention should be given to the food safety of rice cultivated around estuaries.

Extracellular vesicles mediate antibody-resistant transmission of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Antibody resistance dampens neutralizing antibody therapy and threatens current global Coronavirus (COVID-19) vaccine campaigns. In addition to the emergence of resistant SARS-CoV-2 variants, little is known about how SARS-CoV-2 evades antibodies. Here, we report a novel mechanism of extracellular vesicle (EV)-mediated cell-to-cell transmission of SARS-CoV-2, which facilitates SARS-CoV-2 to escape from neutralizing antibodies. These EVs, initially observed in SARS-CoV-2 envelope protein-expressing cells, are secreted by various SARS-CoV-2-infected cells, including Vero E6, Calu-3, and HPAEpiC cells, undergoing infection-induced pyroptosis. Various SARS-CoV-2-infected cells produce similar EVs characterized by extra-large sizes (1.6-9.5 µm in diameter, average diameter > 4.2 µm) much larger than previously reported virus-generated vesicles. Transmission electron microscopy analysis and plaque assay reveal that these SARS-CoV-2-induced EVs contain large amounts of live virus particles. In particular, the vesicle-cloaked SARS-CoV-2 virus is resistant to neutralizing antibodies and able to reinfect naïve cells independent of the reported receptors and cofactors. Consistently, the constructed 3D images show that intact EVs could be taken up by recipient cells directly, supporting vesicle-mediated cell-to-cell transmission of SARS-CoV-2. Our findings reveal a novel mechanism of receptor-independent SARS-CoV-2 infection via cell-to-cell transmission, provide new insights into antibody resistance of SARS-CoV-2 and suggest potential targets for future antiviral therapeutics.