The effect of surgery and metastatic risk prediction in patients with gastrointestinal tract signet-ring cell carcinoma.

PURPOSE: This study aimed to assess the efficacy of surgery as a treatment option for patients with signet-ring cell carcinoma in the gastrointestinal tract (GI-SRCC). METHODS: Using the Surveillance Epidemiology and End Results (SEER) database, GI-SRCC patients who underwent surgery or received non-surgical treatment were included. Propensity score matching (PSM) analysis was used to balance baseline characteristics and reduce bias. Overall survival (OS) was calculated in matching cohorts to estimate prognosis for GI-SRCC patients. Nomogram was established to predict metastasis for GI-SRCC patients. RESULTS: The study enrolled a total of 9428 GI-SRCC patients, with 1689 patients in the non-surgery group and 7739 patients in the surgery group. Following 1:1 PSM, we analyzed 743 patients from each group. Our survival analyses revealed that surgery independently correlated with improved OS for GI-SRCC patients (HR = 0.37, 95% CI: 0.33-0.42, P < 0.001). Subgroup analysis further confirmed the positive impact of surgery on the prognosis of patients with non-metastatic GI-SRCC. Notably, distinct subsets of metastatic patients, particularly those originating from the upper GI (esophagus, proximal stomach, distal stomach) and left colon, demonstrated a significant improvement in OS post-surgery. However, no significant survival difference was observed for patients with metastatic right colon-SRCC and rectum-SRCC. Utilizing nomogram, we quantitatively assessed the risk of metastasis in patients with right colon and rectum SRCC, which exhibited robust predictive accuracy, with area under the curve (AUC) values of 0.829. CONCLUSIONS: Our study highlights surgery's positive impact on prognosis for both non-metastatic and metastatic upper GI-SRCC and left colon-SRCC patients. Hence, we recommend surgery as a treatment option for these groups. Additionally, for metastatic right colon and rectum SRCC patients ineligible for surgery, our predictive nomogram can offer a convenient tool to aid early intervention and improve prognosis.

FEN1-assisted DNA logic amplifier circuit for fast and compact DNA computing.

This work developed DNA amplifier logic gates (AND-OR, OR-AND, FAN-IN, FAN-OUT, and 4-bit square-root circuits) using a flap endonuclease 1 (FEN1)-catalyzed signal amplification reaction, for the fastest and compact DNA computing. Moreover, the logic circuit can use input strands with concentrations of less than 1 nM, which is more than 100 times lower than the input concentration of other DNA logic circuits, providing a promising methodology for constructing fast and compact DNA computations.

Combined Study of Gene Expression and Chromosome Three-Dimensional Structure in Escherichia coli During Growth Process.

The chromosome structure of different bacteria has its unique organization pattern, which plays an important role in maintaining the spatial location relationship between genes and regulating gene expression. Conversely, transcription also plays a global role in regulating the three-dimensional structure of bacterial chromosomes. Therefore, we combine RNA-Seq and Hi-C technology to explore the relationship between chromosome structure changes and transcriptional regulation in E. coli at different growth stages. Transcriptome analysis indicates that E. coli synthesizes many ribosomes and peptidoglycan in the exponential phase. In contrast, E. coli undergoes more transcriptional regulation and catabolism during the stationary phase, reflecting its adaptability to changes in environmental conditions during growth. Analyzing the Hi-C data shows that E. coli has a higher frequency of global chromosomal interaction in the exponential phase and more defined chromosomal interaction domains (CIDs). Still, the long-distance interactions at the replication termination region are lower than in the stationary phase. Combining transcriptome and Hi-C data analysis, we conclude that highly expressed genes are more likely to be distributed in CID boundary regions during the exponential phase. At the same time, most high-expression genes distributed in the CID boundary regions are ribosomal gene clusters, forming clearer CID boundaries during the exponential phase. The three-dimensional structure of chromosome and expression pattern is altered during the growth of E. coli from the exponential phase to the stationary phase, clarifying the synergy between the two regulatory aspects.

Ultrafast and Highly Specific Detection of One-Base Mutated Cell-Free DNA at a Very Low Abundance.

Liquid biopsy as well as genotyping plays important roles in guiding the use of tumor-targeted drugs and monitoring the generation of drug resistance. However, current methods, such as next-generation sequencing (NGS) and pyrosequencing, require long analysis time and complicated steps. To achieve ultrafast and highly specific detection of cell-free DNA (cfDNA) from blood, we improved our recently developed FEN1-aided RPA (FARPA), which combined flap endonuclease 1 (FEN1)-catalyzed invasive reactions with recombinase polymerase amplification (RPA) by inactivating the RPA enzymes before invasive reactions, designing short RPA primers, and changing invasive reaction conditions. Using the L858R and T790M mutations as examples, FARPA was sensitive to detect 5 copies of targeted mutants, specific to sense the mutants with an abundance as low as 0.01% from blood, and ultrafast to get results within 40 min. The method was readily expended to genotyping, and 15 min was enough to report the allele species directly from oral swab samples by coupling quick DNA extraction reagents. Validation was carried out by detecting clinical samples, including 20 cfDNA from patients with non-small cell lung cancer (NSCLC) for liquid biopsy and 43 human genomic DNA (gDNA) purified from blood (33) or lysed from oral swabs (10) for genotyping, giving 100% agreement with NGS and pyrosequencing, respectively. Furthermore, a portable battery-driven device with dual-channel fluorescence detection was successfully constructed to facilitate point-of-care testing (POCT) of liquid biopsy and genotyping, providing doctors with a potential tool to achieve genotyping- or mutant-guided personalized medicine at emergency or source-limited regions.

TRPV2 inhibitor tranilast prevents atrial fibrillation in rat models of pulmonary hypertension.

Atrial fibrillation (AF) is common in pulmonary hypertension (PH), whereas the mechanisms and treatments remain to be explored. TRPV2 regulates the structure and function of the cardiovascular system; however, little attention has been given to its role in AF. This study was to determine whether TRPV2 was involved in PH-induced AF and the effects of TRPV2 inhibitor tranilast on AF in rat models of PH. Monocrotaline (MCT) and SU5416/hypoxia (SuHx)-induced PH models were performed to detect atrial electrophysiological parameters. Daily tranilast (a TRPV2 inhibitor) or saline was given starting 1 day before PH establishment. PH increased the susceptibility to AF, with TRPV2 up-regulated in the right atria. Compared to PH rats, tranilast reduced AF inducibility and the prolongations of ERP and APD; mitigated cardiopulmonary remodeling and the increases in P-wave duration and P-R interval; partially reversed the down-regulation of ion channels such as Cav1.2, Nav1.5, Kv4.3, Kv4.2, Kv1.5, Kir2.1, Kir3.1, Kir3.4 as well as connexin (Cx) 40 and Cx43; improved right atrial (RA) fibrosis, enlargement, and myocardial hypertrophy; decreased the accumulation of inflammatory cells; down-regulated inflammatory indicators such as TNF-α, IL-1ß, CXCL1, and CXCL2; and inhibited the activation of the PI3K-AKT-NF-κB signaling pathway. Our results reveal that TRPV2 participates in PH-induced AF, and TRPV2 inhibitor tranilast prevents PH-induced RA remodeling. TRPV2 might be a promising target for PH-induced AF.

T2T-YAO: A Telomere-to-telomere Assembled Diploid Reference Genome for Han Chinese.

Since its initial release in 2001, the human reference genome has undergone continuous improvement in quality, and the recently released telomere-to-telomere (T2T) version - T2T-CHM13 - reaches its highest level of continuity and accuracy after 20 years of effort by working on a simplified, nearly homozygous genome of a hydatidiform mole cell line. Here, to provide an authentic complete diploid human genome reference for the Han Chinese, the largest population in the world, we assembled the genome of a male Han Chinese individual, T2T-YAO, which includes T2T assemblies of all the 22 + X + M and 22 + Y chromosomes in both haploid. The quality of T2T-YAO is much better than all currently available diploid assemblies, and its haploid version, T2T-YAO-hp, generated by selecting the better assembly for each autosome, reaches the top quality of fewer than one error per 29.5 Mb, even higher than that of T2T-CHM13. Derived from an individual living in the aboriginal region of the Han population, T2T-YAO shows clear ancestry and potential genetic continuity from the ancient ancestors. Each haplotype of T2T-YAO possesses ∼ 330-Mb exclusive sequences, ∼ 3100 unique genes, and tens of thousands of nucleotide and structural variations as compared with CHM13, highlighting the necessity of a population-stratified reference genome. The construction of T2T-YAO, a truly accurate and authentic representative of the Chinese population, would enable precise delineation of genomic variations and advance our understandings in the hereditability of diseases and phenotypes, especially within the context of the unique variations of the Chinese population.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity.

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

Optical Penetration of Shape-Controlled Metallic Nanosensors across Membrane Barriers.

Precise nanostructure geometry that enables the optical biomolecular delivery of nanosensors to the living intracellular environment is highly desirable for precision biological and clinical therapies. However, the optical delivery through membrane barriers utilizing nanosensors remains difficult due to a lack of design guidelines to avoid inherent conflict between optical force and photothermal heat generation in metallic nanosensors during the process. Here, we present a numerical study reporting significantly enhanced optical penetration of nanosensors by engineering nanostructure geometry with minimized photothermal heating generation for penetrating across membrane barriers. We show that by varying the nanosensor geometry, penetration depths can be maximized while heat generated during the penetration process can be minimized. We demonstrate the effect of lateral stress induced by an angularly rotating nanosensor on a membrane barrier by theoretical analysis. Furthermore, we show that by varying the nanosensor geometry, maximized local stress fields at the nanoparticle-membrane interface enhanced the optical penetration process by four-fold. Owing to the high efficiency and stability, we anticipate that precise optical penetration of nanosensors to specific intracellular locations will be beneficial for biological and therapeutic applications.

Visualised genotyping assay with oral swabs in a closed tube by nested invasive reaction assisted with gold nanoparticle probes.

Single nucleotide polymorphism (SNP) typing is crucial for drug dosage and disease progression. Therefore, a simple and convenient genotyping assay is essential for personalised medicine. Herein, we developed a non-invasive, closed-tube, and visualised method for genotyping. In this method, oral swabs were lysed to directly perform PCR coupled with nested invasive reaction and visualisation based on gold nanoparticle probes in a closed tube. The strategy for genotyping assay depends on the single base recognition property of invasive reaction. This assay allowed quick and simple sample preparation and the detection of 25 copies/µL of CYP2C19*2 and 100 copies/µL of CYP2C19*3 within 90 min. Further, 20 oral swab samples for CYP2C19*2 and CYP2C19*3 were correctly typed, which agreed with pyrosequencing, indicating that this method has great potential for SNP typing in source-limited regions to guide personalised medicine.

Strain-Level Dynamics Reveal Regulatory Roles in Atopic Eczema by Gut Bacterial Phages.

The vast population of bacterial phages or viruses (virome) plays pivotal roles in the ecology of human microbial flora and health conditions. Obstacles, including poor viral sequence inference, strain-sensitive virus-host relationship, and the high diversity among individuals, hinder the in-depth understanding of the human virome. We conducted longitudinal studies of the virome based on constructing a high-quality personal reference metagenome (PRM). By applying long-read sequencing for representative samples, we could build a PRM of high continuity that allows accurate annotation and abundance estimation of viruses and bacterial species in all samples of the same individual by aligning short sequencing reads to the PRM. We applied this approach to a series of fecal samples collected for 6 months from a 2-year-old boy who had experienced a 2-month flare-up of atopic eczema (dermatitis) in this period. We identified 31 viral strains in the patient's gut microbiota and deciphered their strain-level relationship to their bacterial hosts. Among them, a lytic crAssphage developed into a dozen substrains and coordinated downregulation in the catabolism of aromatic amino acids (AAAs) in their host bacteria which govern the production of immune-active AAA derivates. The metabolic alterations confirmed based on metabolomic assays cooccurred with symptom remission. Our PRM-based analysis provides an easy approach for deciphering the dynamics of the strain-level human gut virome in the context of entire microbiota. Close temporal correlations among virome alteration, microbial metabolism, and disease remission suggest a potential mechanism for how bacterial phages in microbiota are intimately related to human health. IMPORTANCE The vast populations of viruses or bacteriophages in human gut flora remain mysterious. However, poor annotation and abundance estimation remain obstacles to strain-level analysis and clarification of their roles in microbiome ecology and metabolism associated with human health and diseases. We demonstrate that a personal reference metagenome (PRM)-based approach provides strain-level resolution for analyzing the gut microbiota-associated virome. When applying such an approach to longitudinal samples collected from a 2-year-old boy who has experienced a 2-month flare-up of atopic eczema, we observed thriving substrains of a lytic crAssphage, showing temporal correlation with downregulated catabolism of aromatic amino acids, lower production of immune-active metabolites, and remission of the disease. The PRM-based approach is practical and powerful for strain-centric analysis of the human gut virome, and the underlying mechanism of how strain-level virome dynamics affect disease deserves further investigation.

Anionic liposomes prepared without organic solvents for effective siRNA delivery.

Currently, organic solvents are necessary for the preparation of anionic liposomes for siRNA delivery. The removal of organic solvent is time-consuming and the residual organic solvent is not only a hidden danger, but also affects the stability of anionic liposomes. Glycerol, which is physiologically compatible and does not need to be removed, is used to promote the dispersion of lipids and the formation of anionic liposomes. Additionally, the preparation process is simple and not time-consuming. The results showed that anionic liposomes, which were typically spherical with a particle size of 188.9 nm were successfully prepared with glycerol. And with the help of Ca2+ , siRNA was encapsulated in anionic liposomes. The highest encapsulation efficiency at 2.4 mM Ca2+ reached 91%. And the formation of calcium phosphate could promote the endosomal escape of siRNA effectively. The results from cell viability showed that the anionic liposomes had no obvious cytotoxicity. It was also verified that anionic liposomes could improve the resistance of siRNA against degradation. Additionally, siRNA delivered by anionic liposomes could play an effective role in knockout. Therefore, anionic liposomes prepared with glycerol will be a safe and effective delivery platform for siRNA and even other nucleic acid drugs.

Left ventricular myocardial work index and short-term prognosis in patients with light-chain cardiac amyloidosis: a retrospective cohort study.

Background: Reports show that the left ventricular myocardial work index (LVMWI) is a novel parameter for evaluating cardiac function. Decompensated heart failure leads to a high rate of early mortality in advanced patients with light-chain cardiac amyloidosis (AL-CA) and prevents them from a relatively delayed response to chemotherapy. This study aimed to assess the association of the LVMWI with short-term outcomes and to construct a simple model for risk stratification. Methods: A total of 79 patients with an initial diagnosis of AL-CA were included in this retrospective cohort study. LVMWI was calculated by integrating brachial artery cuff blood pressure and left ventricular longitudinal strain (LVLS). The short-term outcome was defined as 6-month all-cause mortality. Receiver operating characteristic (ROC), logistic regression, and Kaplan-Meier analysis were used in this study. Results: The median follow-up time was 21 months (3-36 months), and 23 (29%) patients died in the first 6 months. The time-dependent ROC and the area under the curve (AUC) showed that the LVMWI had the best predictive potential at the 6-month time point [AUC =0.805; 95% confidence interval (CI): 0.690-0.920]. A bivariate prognostic model based on the LVMWI was constructed, and D-dimer showed a synergistic effect with optimum predicted potential (AUC =0.877; 95% CI: 0.791-0.964). Kaplan-Meier analysis demonstrated that patients with two, one, and none of the variates beyond the cut-off value bore a different risk of 6-month all-cause mortality (accumulated mortality was 86%, 30%, 3%, respectively; log-rank, P<0.001). Multivariate nested logistic regression showed that the level of D-dimer provided an incremental prognostic value (Δχ2=10.3; P=0.001) to the value determined from New York Heart Association (NYHA) classification and the LVMWI. Conclusions: The LVMWI is associated with the short-term outcome of patients with AL-CA. The D-dimer test provides additional prognostic information for the LVMWI.

Molecular profiles of single circulating tumor cells from early breast cancer patients with different lymph node statuses.

BACKGROUND: Characterization of early breast cancer circulating tumor cells (CTCs) may provide valuable information on tumor metastasis. METHODS: We used immunomagnetic nanospheres to capture CTCs from the peripheral blood of eight early breast cancer patients and then performed single-cell RNA sequencing using our proposed bead-dd-seq method. RESULTS: CTCs displayed obvious tumor cell characteristics, such as the activation of oxidative stress, proliferation, and promotion of metastasis. CTCs were clustered into two subtypes significantly correlated with the lymph node metastasis status of patients. CTCs in subtype 1 showed a strong metastatic ability because these CTCs have the phenotype of partial epithelial-mesenchymal transition and enriched transcripts, indicating breast cancer responsiveness and proliferation. Furthermore, DNA damage repair pathways were significantly upregulated in subtype 1. We performed in vitro and in vivo investigations, and found that cellular oxidative stress and further DNA damage existed in CTCs. The activated DNA damage repair pathway in CTCs favors resistance to cisplatin. A checkpoint kinase 1 inhibitor sensitized CTCs to cisplatin in mouse models of breast cancer metastasis. CONCLUSION: The present study dissects the molecular characteristics of CTCs from early-stage breast cancer, providing novel insight into the understanding of CTC behavior in breast cancer metastasis.

Implications of oral streptococcal bacteriophages in autism spectrum disorder.

Growing evidence suggests altered oral and gut microbiota in autism spectrum disorder (ASD), but little is known about the alterations and roles of phages, especially within the oral microbiota in ASD subjects. We enrolled ASD (n = 26) and neurotypical subjects (n = 26) with their oral hygiene controlled, and the metagenomes of both oral and fecal samples (n = 104) are shotgun-sequenced and compared. We observe extensive and diverse oral phageome comparable to that of the gut, and clear signals of mouth-to-gut phage strain transfer within individuals. However, the overall phageomes of the two sites are widely different and show even less similarity in the oral communities between ASD and control subjects. The ASD oral phageome exhibits significantly reduced abundance and alpha diversity, but the Streptococcal phages there are atypically enriched, often dominating the community. The over-representation of Streptococcal phages is accompanied by enriched oral Streptococcal virulence factors and Streptococcus bacteria, all exhibiting a positive correlation with the severity of ASD clinical manifestations. These changes are not observed in the parallel sampling of the gut flora, suggesting a previously unknown oral-specific association between the excessive Streptococcal phage enrichment and ASD pathogenesis. The findings provide new evidence for the independent microbiome-mouth-brain connection, deepen our understanding of how the growth dynamics of bacteriophages and oral microbiota contribute to ASD, and point to novel effective therapeutics.

A Biocultural Study on Gaoligongshan Pig (Sus scrofa domesticus), an Important Hog Landrace, in Nujiang Prefecture of China.

Over 80% proteins consumed by the local people in Nujiang Prefecture of Southwest China, a remote and mountainous area in the Eastern Himalayas, are from pork, or Gaoligongshan pig (a landrace of Sus scrofa domestica Brisson). Previous research on the Gaoligongshan pig has focused on nutritional composition, production performance, and genetic resource characteristics, but neglected the reasons behind the local people's practice. From 2019 to 2022, we have used ethnobiological research methods to comprehensively document the traditional rearing and management patterns and the traditional culture associated with Gaoligongshan pigs. The results show that Gaoligongshan pigs graze in mixed herds with cattle and sheep during the day and prefer to eat 23 wild plant species, in which 17 species have medicinal values. At night, the pigs are artificially fed and rest in the pigsty. The local Bai and Lisu people have developed a creative food culture, rituals, and festivals culture associated with Gaoligongshan pigs. Overall, the biocultural diversity of Gaoligongshan pig contributes to the in situ conservation of genetic diversity of this important hog landrace, and supports rural development in this remote area.

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection.

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and ß within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Identification of genes and key pathways underlying the pathophysiological association between nonalcoholic fatty liver disease and atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is one of the most prevalent sustained cardiac arrhythmias. The latest studies have revealed a tight correlation between nonalcoholic fatty liver disease (NAFLD) and AF. However, the exact molecular mechanisms underlying the association between NAFLD and AF remain unclear. The current research aimed to expound the genes and signaling pathways that are related to the mechanisms underlying the association between these two diseases. MATERIALS AND METHODS: NAFLD- and AF- related differentially expressed genes (DEGs) were identified via bioinformatic analysis of the Gene Expression Omnibus (GEO) datasets GSE63067 and GSE79768, respectively. Further enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), the construction of a protein-protein interaction (PPI) network, the identification of significant hub genes, and receiver operator characteristic curve analysis were conducted. The gene-disease interactions were analyzed using the Comparative Toxicogenomics Database. In addition, the hub genes were validated by quantitative Real-Time PCR (qRT-PCR) in NAFLD cell model. RESULTS: A total of 45 co-expressed differentially expressed genes (co-DEGs) were identified between the NAFLD/AF and healthy control individuals. GO and KEGG pathway analyses revealed that the co-DEGs were mostly enriched in neutrophil activation involved in the immune response and cytokine-cytokine receptor interactions. Moreover, eight hub genes were selected owing to their high degree of connectivity and upregulation in both the NAFLD and AF datasets. These genes included CCR2, PTPRC, CXCR2, MNDA, S100A9, NCF2, S100A12, and S100A8. CONCLUSIONS: In summary, we conducted the gene differential expression analysis, functional enrichment analysis, and PPI analysis of DEGs in AF and NAFLD, which provides novel insights into the identification of potential biomarkers and valuable therapeutic leads for AF and NAFLD.

Multiplexed and Rapid AST for Escherichia coli Infection by Simultaneously Pyrosequencing Multiple Barcodes Each Specific to an Antibiotic Exposed to a Sample.

Antimicrobial susceptibility testing (AST) is an effective way to guide antibiotic selection. However, conventional culture-based phenotypic AST is time-consuming. The key point to shorten the test is to quantify the small change in the bacterial number after the antibiotic exposure. To achieve rapid AST, we proposed a combination of multiplexed PCR with barcoded pyrosequencing to significantly shorten the time for antibiotic exposure. First, bacteria exposed to each antibiotic were labeled with a unique barcode. Then, the pool of the barcoded products was amplified by PCR with a universal primer pair. Finally, barcodes in the amplicons were individually and quantitatively decoded by pyrosequencing. As pyrosequencing is able to discriminate as low as 5% variation in target concentrations, as short as 7.5 min was enough for cultivation to detect the susceptibility of Escherichia coli to an antibiotic. The barcodes enable more than six kinds of drugs or six kinds of concentrations of a drug to be tested at a time. The susceptibility of 6 antibiotics to 43 E. coli-positive samples from 482 clinical urine samples showed a consistency of 99.3% for drug-resistant samples and of 95.7% for drug-sensitive samples in comparison with the conventional method. In addition, the minimum inhibitory concentration (MIC) of 29 E. coli samples was successfully measured. The proposed AST is dye free (pyrosequencing), multiplexed (six antibiotics), fast (a half-working day for reporting the results), and able to detect the MIC, thus having a great potential for clinical use in quick antibiotic selection.

Galectin-3 enhances trastuzumab resistance by regulating cancer malignancy and stemness in HER2-positive breast cancer cells.

PURPOSE: The aim of this study was to explore the role of galectin-3 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells and the potential mechanism. METHODS: Kaplan-Meier (KM)-plot and The Cancer Genome Atlas (TCGA) databases were used to study the role of galectin-3 in the prognosis of HER2-positive breast cancer. The effects of galectin-3 on cell proliferation, migration, invasion, and colony formation ability in HER2-positive breast cancer cells were examined. The relationship between galectin-3 and important components in the HER2 pathways, including HER2, epidermal growth factor receptor (EGFR), protein kinase B (AKT), and phosphatase and tensin homolog (PTEN), was further studied. Lentivirus and CRISPR/Cas9 were used to construct stable cell lines. Cell counting kit-8 (CCK-8) and apoptosis assays were used to study the relationship between galectin-3 and trastuzumab. The effect of galectin-3 on cell stemness was studied by mammosphere formation assay. The effects of galectin-3 on stemness biomarkers and the Notch1 pathway were examined. Tumorigenic models were used to evaluate the effects of galectin-3 on tumorigenesis and the therapeutic effect of trastuzumab in vivo. RESULTS: HER2-positive breast cancer patients with a high expression level of LGALS3 (the gene encoding galectin-3) messenger RNA (mRNA) showed a poor prognosis. Galectin-3 promoted cancer malignancy through phosphoinositide 3-kinase (PI3K)/AKT signaling pathway activation and upregulated stemness by activating the Notch1 signaling pathway in HER2-positive breast cancer cells. These two factors contributed to the enhancement of trastuzumab resistance in cells. Knockout of LGALS3 had a synergistic therapeutic effect with trastuzumab both in vitro and in vivo. CONCLUSIONS: Galectin-3 may represent a prognostic predictor and therapeutic target for HER2-positive breast cancer.

Lipid membrane anchoring and highly specific fluorescence detection of cancer-derived exosomes based on postfunctionalized zirconium-metal-organic frameworks.

Cancer-derived exosomes carry a variety of important biomarkers specific to the formation, invasion and metastasis of tumor tissue. Dynamic monitoring of exosomes originated from cancer cells has clinical significance. Here we proposed a novel method to employ zirconium-metal-organic frameworks (Zr-MOFs) for extracting and identifying exosomes from blood. At first UiO-66 was magnetically modified as the adsorbent to anchor exosomes by forming Zr-O-P bonds. Then UiO-66-NH2 modified with anti-EpCAM was used to construct the fluorescent probe to recognize the extracted EpCAM-positive exosomes by forming a "MOF-exosome-MOF" structure. The proposed fluorescence detection method was evaluated by quantifying MCF-7 cell-derived exosomes at the concentration as low as 16.72 particles/µl. This method was successfully applied to analyze exosomes in the plasma samples from healthy donors and breast cancer patients, demonstrating that our method might have a great potential in assisting the early diagnosis and in dynamically monitoring the efficacy of cancer treatment. We believe that the method could be extended to the detection of other biomarkers in exosomes derived from cancer cell.