High-throughput visualization mutation screening technology to enhance the specificity of CadR based whole-cell cadmium biosensor.

As a heavy metal pollutant, Cd2+ often enters the human body through the food chain causing great harm to human health. Whole-cell biosensor is an emerging technology for rapid on-site detection of heavy metals with the advantages of inexpensive, fast to mass-produce, and strong in anti-interference resistance, but suffering from insatisfactory specificity. In this study, a strategy of Adjacent Site Saturation Mutation (ASSM) was designed to improve the specificity of transcription factor CadR, which acted as the recognition element and determined the specificity of whole cell Cd2+ biosensors. A specific saturated library was constructed using the strategy of adjacent mutation. After two rounds of high-throughput visual screening, a whole-cell biosensor with good response to Cd2+, and with significant weakened Hg2+ interference was obtained. The optimized whole-cell biosensor showed a linear dynamic concentration range from 500 nM to 100 µM, a detection limit of 0.079 µM, and has satisfactory specificity and anti-interference. The ASSM strategy proposed in this study can provide a new method for the application of synthetic biology in food safety detection, indicating the importance of whole-cell biosensors for the detection of heavy metals.

Replacing manual operation with bio-automation: A high-throughput evolution strategy to construct an integrated whole-cell biosensor for the simultaneous detection of methylmercury and mercury ions without manual sample digestion.

Methylmercury is primarily responsible for most food mercury pollution cases. However, most biosensors developed for mercury pollution analysis can only detect mercury ions. Although oxidative strong-acid digestion or microwave-assisted digestion can convert methylmercury into mercury ions, it is unsuitable for on-site detection. This study designed a bio-digestion gene circuit and integrated it into a mercury ion whole-cell biosensor，creating a novel on-site methylmercury detection method. Five alkyl mercury lyases from different bacterial genomes were screened via bioinformatics analysis, of which goMerB from Gordonia otitis showed the highest catalytic biological digestion efficiency. The goMerB site-specific saturation and random mutation libraries were constructed. After two rounds of high-throughput visualization screening, the catalytic activity of the mutant increased 2.5-fold. The distance between the three crucial amino acid sites and methylmercury changed in the mutant, which likely contributed to the enhanced catalytic efficiency. The optimized whole-cell biosensor showed a linear dynamic concentration range of 100 nM to 100 µM (R2 =0.991), satisfactory specificity, and interference resistance. The detection limit of the goMerBt6-MerR-RFP biosensor was 0.015 µM, while the limit of quantitation was 0.049 µM. This study demonstrated the application of synthetic biology for food safety detection and highlighted the future potential of "Lab in a Cell" for hazard analysis.

Survival Outcomes and Prognostic Factors of Dermatofibrosarcoma Protuberans: A Population-Based Retrospective Cohort Analysis.

BACKGROUND: Limited information is available regarding survival outcomes and risk factors of dermatofibrosarcoma protuberans (DFSP). OBJECTIVE: To investigate the clinicopathologic characteristics and survival outcomes of DFSP. MATERIALS AND METHODS: The study cohort (7,567 patients) was selected from the Surveillance, Epidemiology, and End Results Program (2000-2018). Demographic and clinicopathologic variables, survival outcomes, and prognostic factors were analyzed. RESULTS: A total of 5,640 (74.53%) and 1,927 (25.47%) tumors were located in the skin and soft tissue, respectively. The median follow-up duration was 92 months. Median follow-up times were similar between patients with lymph node (107 months) and distant (102 months) metastases; the median survival time of the 89 patients (1.18%) who died of DFSP was significantly short (41 months, p < .001). Independent risk factors for cancer-specific mortality included age at diagnosis, histologic grade, and tumor size. Patients with tumors ≥10 cm in size or histologic grade III had significantly higher DFSP-specific mortality (7.07% and 10.08%, respectively, p < .001). Tumor locations and surgical procedures did not significantly influence survival. CONCLUSION: Dermatofibrosarcoma protuberans has a favorable survival prognosis, even in patients with node-positive or distant metastases. Dermatofibrosarcoma protuberans-specific mortality is significantly higher in patients with grade III or large (≥10 cm) tumors.

Advances in Synthetic-Biology-Based Whole-Cell Biosensors: Principles, Genetic Modules, and Applications in Food Safety.

A whole-cell biosensor based on synthetic biology provides a promising new method for the on-site detection of food contaminants. The basic components of whole-cell biosensors include the sensing elements, such as transcription factors and riboswitches, and reporting elements, such as fluorescence, gas, etc. The sensing and reporting elements are coupled through gene expression regulation to form a simple gene circuit for the detection of target substances. Additionally, a more complex gene circuit can involve other functional elements or modules such as signal amplification, multiple detection, and delay reporting. With the help of synthetic biology, whole-cell biosensors are becoming more versatile and integrated, that is, integrating pre-detection sample processing, detection processes, and post-detection signal calculation and storage processes into cells. Due to the relative stability of the intracellular environment, whole-cell biosensors are highly resistant to interference without the need of complex sample preprocessing. Due to the reproduction of chassis cells, whole-cell biosensors replicate all elements automatically without the need for purification processing. Therefore, whole-cell biosensors are easy to operate and simple to produce. Based on the above advantages, whole-cell biosensors are more suitable for on-site detection than other rapid detection methods. Whole-cell biosensors have been applied in various forms such as test strips and kits, with the latest reported forms being wearable devices such as masks, hand rings, and clothing. This paper examines the composition, construction methods, and types of the fundamental components of synthetic biological whole-cell biosensors. We also introduce the prospect and development trend of whole-cell biosensors in commercial applications.

Applications of the Whole-Cell System in the Efficient Biosynthesis of Heme.

Heme has a variety of functions, from electronic reactions to binding gases, which makes it useful in medical treatments, dietary supplements, and food processing. In recent years, whole-cell system-based heme biosynthesis methods have been continuously explored and optimized as an alternative to the low-yield, lasting, and adverse ecological environment of chemical synthesis methods. This method relies on two biosynthetic pathways of microbial precursor 5-aminolevulinic acid (C4, C5) and three known downstream biosynthetic pathways of heme. This paper reviews the genetic and metabolic engineering strategies for heme production in recent years by optimizing culture conditions and techniques from different microorganisms. Specifically, we summarized and analyzed the possibility of using biosensors to explore new strategies for the biosynthesis of heme from the perspective of synthetic biology, providing a new direction for future exploration.

A hypoxia risk score for prognosis prediction and tumor microenvironment in adrenocortical carcinoma.

Background: Adrenocortical carcinoma (ACC) is a rare malignant endocrine tumor derived from the adrenal cortex. Because of its highly aggressive nature, the prognosis of patients with adrenocortical carcinoma is not impressive. Hypoxia exists in the vast majority of solid tumors and contributes to invasion, metastasis, and drug resistance. This study aimed to reveal the role of hypoxia in Adrenocortical carcinoma and develop a hypoxia risk score (HRS) for Adrenocortical carcinoma prognostic prediction. Methods: Hypoxia-related genes were obtained from the Molecular Signatures Database. The training cohorts of patients with adrenocortical carcinoma were downloaded from The Cancer Genome Atlas, while another three validation cohorts with comprehensive survival data were collected from the Gene Expression Omnibus. In addition, we constructed a hypoxia classifier using a random survival forest model. Moreover, we explored the relationship between the hypoxia risk score and immunophenotype in adrenocortical carcinoma to evaluate the efficacy of immune check inhibitors (ICI) therapy and prognosis of patients. Results: HRS and tumor stage were identified as independent prognostic factors. HRS was negatively correlated with immune cycle activity, immune cell infiltration, and the T cell inflammatory score. Therefore, we considered the low hypoxia risk score group as the inflammatory immunophenotype, whereas the high HRS group was a non-inflammatory immunophenotype. In addition, the HRS was negatively related to the expression of common immune checkpoint molecules such as PD-L1, CD200, CTLA-4, and TIGIT, suggesting that patients with a lower hypoxia risk score respond better to immunotherapy. Conclusion: We developed and validated a novel hypoxia risk score to predict the immunophenotype and response of patients with adrenocortical carcinoma to immune check inhibitors therapy. These findings not only provide fresh prognostic indicators for adrenocortical carcinoma but also offer several promising treatment targets for this disease.

Extracellular vesicles delivering nuclear factor I/C for hard tissue engineering: Treatment of apical periodontitis and dentin regeneration.

Apical periodontitis (AP) causes arrest of tooth root development, which is associated with impaired odontoblastic differentiation of stem cells from apical papilla (SCAPs), but the underlying mechanism remains unclear. Here, we investigated roles of extracellular vesicle (EV) in AP and odontoblastic differentiation of SCAPs, moreover, a novel nuclear factor I/C (NFIC)-encapsulated EV was developed to promote dentin regeneration. We detected a higher expression of EV marker CD63 in inflamed apical papilla, and found that EVs from LPS-stimulated dental pulp cells suppressed odontoblastic differentiation of SCAPs through downregulating NFIC. Furthermore, we successfully constructed the NFIC-encapsulated EV by overexpressing NFIC in HEK293FT cells, which could upregulate cellular NFIC level in SCAPs, promoting the proliferation and migration of SCAPs, as well as dentinogenesis both in vitro and in vivo. Collectively, based on pathological roles of EV in AP, our study provides a novel strategy for dentin regeneration by exploiting EV to deliver NFIC.

Triplophysa wulongensis, a new species of cave-dwelling loach (Teleostei, Nemacheilidae) from Chongqing, Southwest China.

We describe a new species of cave-dwelling loach, Triplophysa wulongensis sp. nov., based on specimens collected in a subterranean pool in a cave in Wulong County, Chongqing, Southwest China. The pool is connected to the Wujiang River drainage. Triplophysa wulongensis differs from its congeners by the following combination of characters: eyes present, caudal fin with 18 branched rays; posterior chamber of the air bladder degenerate; stomach U-shaped; intestine without bends or loops immediately posterior to stomach; body smooth and scaleless, and lateral line complete. The mitochondrial cytochrome b sequence differs from those of other published sequences of species of Triplophysa by 14.9-24.9% in K2P distance. Phylogenetic analysis based on cytochrome b gene sequences recovered T. wulongensis as sister taxon to all other cave-dwelling species of Triplophysa.

Triplophysa sanduensis, a new loach species of nemacheilid (Teleostei: Cypriniformes) from South China.

A new species of Nemacheilidae, Triplophysa sanduensis sp. nov., is described here based on specimens collected from a small cave in Dengguang Village, Zhonghe Town, Sandu County, Guizhou Province, China. The new species can be distinguished from its congeners by the following combination of characters: branched dorsal-fin rays 8-9; branched pectoral-fin rays 8-9; branched pelvic-fin rays 5; branched anal-fin rays 5; vertebrae 4+37; snout length 45.5-50.1% of head length; postdorsal length 39.0-42.8% of standard length; caudal-peduncle depth/caudal-peduncle length range from 56.4% to 71.9%; the posterior chamber of the gas bladder degenerated; body elongated, covered by scattered scales; eyes present and normal in size, not reduced or absent; and a complete lateral line. A phylogenetic analysis based on the mitochondrial cytochrome oxidase subunit 1 (COI) gene resolved Triplophysa sanduensis as sister to Triplophysa longliensis. Kimura's 2-parameter genetic distance of the COI gene sequences between these two species was 1.9%.

Comparative study of breast cancer with or without concomitant Paget disease: An analysis of the SEER database.

BACKGROUND: Most mammary Paget disease (MPD) is associated with underlying in situ or invasive breast cancer. The objective of this study was to compare the clinicopathological characteristics and survival outcomes between breast cancer with Paget disease (PD) and breast cancer alone. METHODS: From the Surveillance, Epidemiology, and End Results (SEER) database, 2000-2015, of the US National Cancer Institute, we identified 1569 women who had PD with invasive ductal carcinoma (PD-IDC) and 1489 women who had PD with ductal carcinoma in situ (PD-DCIS). Independent demographic and clinicopathological variables as well as survival outcomes of these patients were compared to patients with the corresponding breast cancer without concomitant PD. RESULTS: PD-IDC and PD-DCIS both had worse survival outcomes and poorer tumor characteristics than the corresponding disease without PD. Contrary to in the breast cancer alone groups, in the breast cancer with PD groups, the HR status (P = 0.182 in PD-IDC and P = 0.371 in PD-DCIS), HER2 status (P = 0.788 in PD-IDC and P = 0.643 in PD-DCIS), and combined molecular subtype (P = 0.196 in PD-IDC and P = 0.853 in PD-DCIS) were not found to affect disease prognosis. After matching tumor characteristics and treatment approaches, PD-IDC as well as PD-DCIS exhibited no significant difference in disease prognosis with corresponding IDC and DCIS. Finally, by comparative analysis, a kind of PD-DCIS (ICD-O-3 code 8543/3) showed many invasive behaviors (31.8% of 8543/3 patients had stage I-III cancer) and was associated with worse survival outcomes than the other type of PD-DCIS. CONCLUSIONS: Breast cancer with concomitant PD was associated with more aggressive tumor characteristics and worse survival outcomes. The HR status, HER2 status, and combined molecular subtype could not affect the prognosis of breast cancer with PD. Moreover, a portion of the PD-DCIS cases were invasive breast cancer cases that required special treatment.

Real-time monitoring of auxin vesicular exocytotic efflux from single plant protoplasts by amperometry at microelectrodes decorated with nanowires.

Recent biochemical results suggest that auxin (IAA) efflux is mediated by a vesicular cycling mechanism, but no direct detection of vesicular IAA release from single plant cells in real-time has been possible up to now. A TiC@C/Pt-QANFA micro-electrochemical sensor has been developed with high sensitivity in detection of IAA, and it allows real-time monitoring and quantification of the quantal release of auxin from single plant protoplast by exocytosis.

On-chip cryopreservation: a novel method for ultra-rapid cryoprotectant-free cryopreservation of small amounts of human spermatozoa.

Cryopreservation of human spermatozoa free from cryoprotectant can avoid toxicity caused by highly concentrated cryoprotectant and a series of specific carriers have been previously explored, except for PDMS chip. Our study is aimed at exploring a novel device for ultra-rapid cryopreservation of small numbers of spermatozoa without cryoprotectant based on polydimethylsiloxane (PDMS) chips. Spermatozoa from 25 healthy men were involved in this study, comparing on-chip cryopreservation with different micro-channel height (group A: 10 µm height, group B: 50 µm height, group C: 100 µm height) and conventional freezing (group D) in liquid nitrogen for 72 h. The viability, motility, DNA integrity by comet assay and acrosome integrity by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining of frozen-thawed spermatozoa of each group were compared. The motility and viability of post-thawed spermatozoa was significantly decreased than that of pre-freezing spermatozoa. There was no difference of viability and motility of frozen-thawed spermatozoa between group A and D, while viability and motility of group B and C decreased compared to group A. Comet assay showed that no matter for group A or D, there was no difference of CR, TL, TD and OTM between pre-frozen and post-thawed spermatozoa. There was no difference of CR, TL, TD and OTM of post-thawed spermatozoa between group A and group D neither, while spermatozoa DNA damage was more serious in group B and group C with increasing height of micro-channel compared with group A. The proportion of intact acrosome of post-thawed spermatozoa in group A was the highest when compared with group B and group C, though similar to that of group D. In conclusion, PDMS chip with 10 µm height micro-channel is ideal for ultra-rapid cryopreservation of small quantity of spermatozoa without cryoprotectant.

Vascular lumen simulation and highly-sensitive nitric oxide detection using three-dimensional gelatin chip coupled to TiC/C nanowire arrays microelectrode.

Reproducing the physiological environment of blood vessels for the in vitro investigation of endothelial cell functions is very challenging. Here, we describe a vascular-like structure based on a three-dimensional (3D) gelatin chip with good compatibility and permeability which is also cost-effective and easy to produce. The controllable lumen diameter and wall thickness enable close mimicking of blood vessels in vitro. The 3D gelatin matrix between adjacent lumens is capable of generating soluble-factor gradients inside, and diffusion of molecules with different molecular weights through the matrix is studied. The cultured human umbilical vein endothelial cells proliferate on the gelatin lumen linings to form a vascular lumen. The hemodynamic behavior including adhesion, alignment of endothelial cells (ECs) under shear stress and pulsatile stretch is studied. Furthermore, a microelectrode comprising TiC/C nanowire arrays is fabricated to detect nitric oxide with sub-nM detection limits and NO generation from the cultured ECs is monitored in real time. This vascular model reproduces the surrounding parenchyma of endothelial cells and mimics the hemodynamics inside blood vessels very well, thereby enabling potential direct investigation of hemodynamics, angiogenesis, and tumor metastasis in vitro.

Integrated microdevice for long-term automated perfusion culture without shear stress and real-time electrochemical monitoring of cells.

Electrochemical techniques based on ultramicroelectrodes (UMEs) play a significant role in real-time monitoring of chemical messengers' release from single cells. Conversely, precise monitoring of cells in vitro strongly depends on the adequate construction of cellular physiological microenvironment. In this paper, we developed a multilayer microdevice which integrated high aspect ratio poly(dimethylsiloxane) (PDMS) microfluidic device for long-term automated perfusion culture of cells without shear stress and an independently addressable microelectrodes array (IAMEA) for electrochemical monitoring of the cultured cells in real time. Novel design using high aspect ratio between circular "moat" and ring-shaped micropillar array surrounding cell culture chamber combined with automated "circular-centre" and "bottom-up" perfusion model successfully provided continuous fresh medium and a stable and uniform microenvironment for cells. Two weeks automated culture of human umbilical endothelial cell line (ECV304) and neuronal differentiation of rat pheochromocytoma (PC12) cells have been realized using this device. Furthermore, the quantal release of dopamine from individual PC12 cells during their culture or propagation process was amperometrically monitored in real time. The multifunctional microdevice developed in this paper integrated cellular microenvironment construction and real-time monitoring of cells during their physiological process, and would possibly provide a versatile platform for cell-based biomedical analysis.