Recombinase Polymerase Amplification and Target-Triggered CRISPR/Cas12a Assay for Sensitive and Selective Hepatitis B Virus DNA Analysis Based on Lanthanide Tagging and Inductively Coupled Plasma Mass Spectrometric Detection.

Herein, we report a target-triggered CRISPR/Cas12a assay by coupling lanthanide tagging and inductively coupled plasma mass spectrometry (ICP-MS) for highly sensitive elemental detection. Hepatitis B virus (HBV) DNA was chosen as a model analyte, and recombinase polymerase amplification (RPA) was used for target amplification. The double-stranded RPA amplicons containing a 5' TTTG PAM sequence can be recognized by Cas12a through a specific CRISPR RNA, activating the trans-cleavage activity of CRISPR/Cas12a and nonspecific cleavage of terbium (Tb)-ssDNA modified on magnetic beads (MBs). Following magnetic separation and acid digestion, the released Tb3+ ions were quantitated by ICP-MS and correlated to the concentration of HBV DNA. Taking advantage of the accelerated cleavage of Tb-ssDNA attached to the MB particles, RPA for target amplification, and ICP-MS for highly selective signal readout, this method permits the detection of 1 copy/µL of HBV DNA in serum with high specificity and holds great promise in the early diagnosis of viral infections or tumor development.

An E3 ligase TRIM1 promotes colorectal cancer progression via K63-linked ubiquitination and activation of HIF1α.

Accumulating studies have shown that E3 ligases play crucial roles in regulating cellular biological processes and signaling pathways during carcinogenesis via ubiquitination. Tripartite-motif (TRIM) ubiquitin E3 ligases consist of over 70 members. However, the clinical significance and their contributions to tumorigenesis remain largely unknown. In this study, we analyzed the RNA-sequencing expression of TRIM E3 ligases in colorectal cancer (CRC) and identified 10 differentially expressed genes, among which TRIM1 expression predicted poor prognosis of CRC patients. We demonstrated that TRIM1 expression is positively associated with CRC pathological stages, and higher expression is positively correlated with infiltrating levels of immune cells and immunotherapy biomarkers. TRIM1 expression promotes the proliferation and migration of colorectal cancer cells in vitro and in vivo. Transcriptional analysis showed that TRIM1 is responsible for metabolism promotion and immune suppression. Mechanistically, we found that TRIM1 binds HIF1α and mediates its K63-linked ubiquitination, which is required for HIF1α nuclear translocation and subsequent activation. Ubiquitination occurs at Lys214 in the loop between the two PAS domains of HIF1α, and mutation of Lys214 severely disturbs the function of HIF1α. Besides, HIF1α ubiquitination enhances its binding with proteins involved in cellular trafficking and nucleocytoplasmic transport pathway. Collectively, our results indicate TRIM1's role in predicting prognosis and reveal how TRIM1 functions to upregulate HIF1α expression and promote tumor cell proliferation.

Involvement of B-aat1 and Cbs in regulating mantle pigmentation in the Pacific oyster (Crassostrea gigas).

BACKGROUND: Shell color formation is an important physiological process in bivalves, the molecular genetic basis has potential application in bivalve aquaculture, but there is still remaining unclear about this issue. The cystine/glutamate transporter (Slc7a11) and cystathionine beta-synthase (Cbs) are integral genes in pheomelanin synthesis pathway, which is vital to skin pigmentation. METHODS AND RESULTS: Here, the sequences of b (0, +) -type amino acid transporter 1 (B-aat1) and Cbs in Pacific oyster (Crassostrea gigas) (CgB-aat1, CgCbs) were characterized. Phylogenetically, the deduced amino acid sequences of CgB-aat1 and CgCbs both possessed conserved features. Genes were both ubiquitously expressed in six tested tissues with more abundant expression level in central mantle. Besides, the polyclonal antibodies of CgB-aat1, CgCbs, CgTyr, and CgTyrp2 were successfully prepared. Immunofluorescence analysis revealed that CgB-aat1 and CgCbs proteins were both expressed in gill rudiments of eyed-larvae and concentrated mainly in cytoplasm of epithelial cell and nerve axons in mantle. Additionally, after CgB-aat1 or CgCbs silencing, expressions at mRNA and protein levels of CgB-aat1 and CgCbs involved in pheomelanin synthesis were significantly suppressed, and CgTyr, CgTyrp1 and CgTyrp2 related to eumelanin synthesis were also down-regulated but no apparent differences, respectively. Moreover, micrographic examination found less brown-granules at mantle edge in CgB-aat1 interference group. CONCLUSION: These results implied that pheomelanin synthesis was possible induced by CgB-aat1-CgTyr-CgCbs axis, and it played an essential role on mantle pigmentation in the oysters. These findings provide the useful genetic knowledge and enrich the physiological information for the shell color formation in bivalve aquaculture.

Foxa1 mediates eccrine sweat gland development through transcriptional regulation of Na-K-ATPase expression.

Eccrine sweat glands (ESGs) perform critical functions in temperature regulation in humans. Foxa1 plays an important role in ESG maturation and sweat secretion. Its molecular mechanism, however, remains unknown. This study investigated the expression of Foxa1 and Na-K-ATPase (NKA) in rat footpads at different development stages using immunofluorescence staining, qRT-PCR, and immunoblotting. Also, bioinformatics analysis and Foxa1 overexpression and silencing were employed to evaluate Foxa1 regulation of NKA. The results demonstrated that Foxa1 was consistently expressed during the late stages of ESGs and had a significant role in secretory coil maturation during sweat secretion. Furthermore, the mRNA abundance and protein expression of NKA had similar accumulation trends to those of Foxa1, confirming their underlying connections. Bioinformatics analysis revealed that Foxa1 may interact with these two proteins via binding to conserved motifs in their promoter regions. Foxa1 gain-of-function and loss-of-function experiments in Foxa1-modified cells demonstrated that the activities of NKA were dependent on the presence of Foxa1. Collectively, these data provided evidence that Foxa1 may influence ESG development through transcriptional regulation of NKA expression.

Autophagy, not apoptosis, plays a role in lumen formation of eccrine gland organoids.

BACKGROUND: Sweat secreted by eccrine sweat glands is transported to the skin surface through the lumen. The eccrine sweat gland develops from the initial solid bud to the final gland structure with a lumen, but how the lumen is formed and the mechanism of lumen formation have not yet been fully elucidated. This study aimed to investigate the mechanism of lumen formation of eccrine gland organoids (EGOs). METHODS: Human eccrine sweat glands were isolated from the skin for tissue culture, and the primary cultured cells were collected and cultured in Matrigel for 14 days in vitro. EGOs at different development days were collected for hematoxylin and eosin (H&E) staining to observe morphological changes and for immunofluorescence staining of proliferation marker Ki67, cellular motility marker filamentous actin (F-actin), and autophagy marker LC3B. Western blotting was used to detect the expression of Ki67, F-actin, and LC3B. Moreover, apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit, and the expression of poly (ADP-ribose) polymerase and Caspase-3 was detected by Western blot. In addition, 3-methyladenine (3MA) was used as an autophagy inhibitor to detect whether the formation of sweat glands can be effectively inhibited. RESULTS: The results showed that a single gland cell proliferated rapidly and formed EGOs on day 4. The earliest lumen formation was observed on day 6. From day 8 to day 14, the rate of lumen formation in EGOs increased significantly. The immunofluorescence and Western blot analyses showed that the expression of Ki67 gradually decreased with the increase in days, while the F-actin expression level did not change. Notably, the expression of autophagy marker LC3B was detected in the interior cells of EGOs as the apoptosis signal of EGOs was negative. Compared with the control group, the autophagy inhibitor 3MA can effectively limit the formation rate of the lumen and reduce the inner diameter of EGOs. CONCLUSION: Using our model of eccrine gland 3D-reconstruction in Matrigel, we determined that autophagy rather than apoptosis plays a role in the lumen formation of EGOs.

Foxa1 mediates eccrine sweat gland development through transcriptional regulation of Na-K-ATPase expression

Eccrine sweat glands (ESGs) perform critical functions in temperature regulation in humans. Foxa1 plays an important role in ESG maturation and sweat secretion. Its molecular mechanism, however, remains unknown. This study investigated the expression of Foxa1 and Na-K-ATPase (NKA) in rat footpads at different development stages using immunofluorescence staining, qRT-PCR, and immunoblotting. Also, bioinformatics analysis and Foxa1 overexpression and silencing were employed to evaluate Foxa1 regulation of NKA. The results demonstrated that Foxa1 was consistently expressed during the late stages of ESGs and had a significant role in secretory coil maturation during sweat secretion. Furthermore, the mRNA abundance and protein expression of NKA had similar accumulation trends to those of Foxa1, confirming their underlying connections. Bioinformatics analysis revealed that Foxa1 may interact with these two proteins via binding to conserved motifs in their promoter regions. Foxa1 gain-of-function and loss-of-function experiments in Foxa1-modified cells demonstrated that the activities of NKA were dependent on the presence of Foxa1. Collectively, these data provided evidence that Foxa1 may influence ESG development through transcriptional regulation of NKA expression.

Serum lipidomic biomarkers for non-small cell lung cancer in nonsmoking female patients.

Lung cancer (Lca) is one of the malignant tumors with the fastest morbidity and mortality increase and the greatest threat to human health and life. The incidence of non-small cell lung cancer (NSCLC) in the nonsmoking female has increased recently. However, its pathogenesis is still unclear, and there is an urgent need for clinical diagnostic biomarkers, especially for early diagnosis. A nontargeted lipidomic approach based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS), as well as two machine learning approaches (genetic algorithm and binary logistic regression) was used to screen candidate discriminating lipids and define a combinational lipid biomarker in serum samples to distinguish female patients with NSCLC from healthy controls. Moreover, the candidate biomarkers were verified by using an external validation sample set. Our result revealed that fatty acid (FA) (20:4), FA (22:0) and LPE (20:4) can serve as a combinational biomarker for distinguishing female patients with NSCLC from healthy control with good sensitivity and specificity. Furthermore, this combinational biomarker also showed good performance in distinguishing early-stage NSCLC female patients from a healthy control. We observed that levels of unsaturated fatty acids clearly decreased, while saturated fatty acids and lysophosphatidylethanolamines pronouncedly increased in Lca patients, compared with the healthy controls, which revealed significant disturbance of lipid metabolism in NSCLC females. Our results not only provide hints to the pathological mechanism of NSCLC in nonsmoking female but also supply a combinational lipid biomarker to aid the diagnosis of NSCLC at early-stage.

EGFR inhibitor AG1478 blocks the formation of 3D structures mainly through ERK signaling pathway in Matrigel-induced 3D reconstruction of eccrine sweat gland-like structures.

EGFR signaling plays important roles in the development of eccrine sweat glands. We previously demonstrate that Matrigel induces eccrine sweat gland cells to reconstruct the three-dimensional (3D) structures of eccrine sweat glands, but the mechanisms are still unknown. In the study, eccrine sweat gland cells were cultured within a 3D Matrigel, and EGFR inhibitor AG1478, or MEK1/2 inhibitor U0126, were added to the medium respectively. The morphology of the 3D-reconstructed eccrine sweat gland-like structures was observed, the localization of phospho-EGFR was detected, and protein levels of EGFR, phospho-EGFR, phospho-JAK, phospho-AKT and phospho-ERK were examined. The results showed that cells treatment with AG1478 from Day 0 of 3D cultures blocked formation of spheroid-like structures. AG1478 administration caused reduced phospho-EGFR, concomitant with downregulation of phospho-ERK1/2, but not phospho-JAK or phospho-AKT. Phospho-EGFR and phospho-ERK were reduced, and only a small number of 3D-structures were formed following treatment with U0126. We conclude that EGFR plays important roles in Matrigel-induced 3D structures of eccrine sweat gland-like structures, and ERK1/2 signaling is responsible, at least in part, for the effect of EGFR.

Melatonin ameliorates the fertilization capacity of oocytes exposed to 17α-ethynylestradiol.

17α-ethynylestradiol (EE2), a synthetic hormone that derives from the natural hormone estradiol, has been reported to alter the sex determination, sexual maturity and secondary sexual characteristics of exposed organisms. However, the adverse effects of EE2 on the oocyte quality have not fully determined. Here, we found that EE2 exposure compromised the fertilization capacity of mouse oocytes, while treatment of melatonin remarkably elevated the fertilization rate. Specifically, we observed that EE2 exposure led to the abnormal distribution and premature exocytosis of ovastacin, leading to the reduced number of sperm binding to the EE2-exposed oocytes. In addition, we found that the abundance of Juno, the sperm receptor on the oocyte membrane, was also diminished, which might be another potential cause leading to the fertilization failure of EE2-exposed oocytes. Finally, we demonstrated that melatonin improved the fertilization ability of EE2-exposed oocytes through eliminating the excessive ROS and inhibiting apoptosis.

K31 as a novel marker for clear secretory cells in human eccrine sweat glands.

K31 was previously considered as one of the hair keratins. During a study on differential markers between hair follicles and eccrine sweat glands, we observed that K31 was expressed in eccrine sweat gland cells in a scattered pattern, similar to the distribution of dark or clear secretory cells. To investigate the precise cell localization of K31 in human eccrine sweat glands and find new marker for eccrine sweat gland cells, human skin samples were fixed, paraffined and sectioned. The serial sections were stained for K31, dark secretory cell marker gross cystic disease fluid protein 15 (GCDFP15) and clear secretory cell marker carbonic anhydrase II (CAII). The exact cell localization of K31 was detected by double immunofluorescence staining of K31 and a serial of cell-specific markers, and further by dual stain using a combination of periodic acid-Schiff (PAS) and immunofluorescence for K31 and GCDFP15. The expression pattern of K31-positive cells was similar to that of CAII-positive cells but was different from that of GCDFP15-positive staining in serial sections. Double immunofluorescent staining showed that K31-positive cells co-expressed K7 and CAII, but not S100P, α-SMA or GCDFP15. Dual stain by combined PAS and immunofluorescence showed that K31-positive cells are negative for PAS staining. We conclude that K31 is a previously unreported eccrine clear cell marker that allows for distinction between clear and dark secretory cells, as well as between secretory coils and ducts of eccrine sweat glands in human eccrine sweat glands.