Ribonuclease 4 functions as an intestinal antimicrobial protein to maintain gut microbiota and metabolite homeostasis.

Antimicrobial proteins contribute to host-microbiota interactions and are associated with inflammatory bowel disease (IBD), but our understanding on antimicrobial protein diversity and functions remains incomplete. Ribonuclease 4 (Rnase4) is a potential antimicrobial protein with no known function in the intestines. Here we find that RNASE4 is expressed in intestinal epithelial cells (IEC) including Paneth and goblet cells, and is detectable in human and mouse stool. Results from Rnase4-deficient mice and recombinant protein suggest that Rnase4 kills Parasutterella to modulate intestinal microbiome, thereby enhancing indoleamine-2,3-dioxygenase 1 (IDO1) expression and subsequently kynurenic and xanthurenic acid production in IECs to reduce colitis susceptibility. Furthermore, deceased RNASE4 levels are observed in the intestinal tissues and stool from patients with IBD, correlating with increased stool Parasutterella. Our results thus implicate Rnase4 as an intestinal antimicrobial protein regulating gut microbiota and metabolite homeostasis, and as a potential diagnostic biomarker and therapeutic target for IBD.

Activation of angiogenin expression in macrophages by lipopolysaccharide via the TLR4/NF-κB pathway in colitis.

Inflammatory bowel disease (IBD) is a debilitating condition that can lead to life-threatening complications. Macrophages are crucial in IBD management because they secrete various cytokines and regulate tissue repair. Macrophage-derived angiogenin (ANG) has been shown to be essential for limiting colonic inflammation, but its upstream regulatory pathway and role in macrophages remain unclear. Here we show that ANG expression is up-regulated in macrophages during colitis treatment or upon lipopolysaccharides (LPS) treatment. Mechanistically, LPS activates Toll-like receptor 4 (TLR4) to initiate NF-κB translocation from the cytoplasm to the nucleus, where it binds to the ANG promoter and enhances its transcriptional activity, leading to increased ANG expression. Interestingly, our data also reveal that the deletion of ANG in macrophages has no adverse effect on key macrophage functions, such as phagocytosis, chemotaxis, and cell survival. Our findings establish a "LPS-TLR4-NF-κB-ANG" regulatory axis in inflammatory disorders and confirm that ANG controls inflammation in a paracrine manner, highlighting the importance of ANG as a key mediator in the complex network of inflammatory processes.

Low-temperature plasma jet suppresses bacterial colonisation and affects wound healing through reactive species.

An argon-based low-temperature plasma jet (LTPJ) was used to treat chronically infected wounds in Staphylococcus aureus-laden mice. Based on physicochemical property analysis and in vitro antibacterial experiments, the effects of plasma parameters on the reactive nitrogen and oxygen species (RNOS) content and antibacterial capacity were determined, and the optimal treatment parameters were determined to be 4 standard litre per minute and 35 W. Additionally, the plasma-treated activation solution had a bactericidal effect. Although RNOS are related to the antimicrobial effect of plasma, excess RNOS may be detrimental to wound remodelling. In vivo studies demonstrated that medium-dose LTPJ promoted MMP-9 expression and inhibited bacterial growth during the early stages of healing. Moreover, LTPJ increased collagen deposition, reduced inflammation, and restored blood vessel density and TGF-ß levels to normal in the later stages of wound healing. Therefore, when treating chronically infected wounds with LTPJ, selecting the medium dose of plasma is more advantageous for wound recovery. Overall, our study demonstrated that low-temperature plasma jets may be a potential tool for the treatment of chronically infected wounds.

Single-Ended Eddy Current Micro-Displacement Sensor with High Precision Based on Temperature Compensation.

To measure the micro-displacement reliably with high precision, a single-ended eddy current sensor based on temperature compensation was studied in detail. At first, the principle of the eddy current sensor was introduced, and the manufacturing method of the probe was given. The overall design plan for the processing circuit was induced by analyzing the characteristics of the probe output signal. The variation in the probe output signal was converted to pulses with different widths, and then it was introduced to the digital phase discriminator along with a reference signal. The output from the digital phase discriminator was processed by a low-pass filter to obtain the DC component. At last, the signal was amplified and compensated to reduce the influence of temperature. The selection criteria of the frequency of the exciting signal and the design of the signal conditioning circuit were described in detail, as well as the design of the temperature-compensating circuit based on the digital potentiometer with an embedded temperature sensor. Finally, an experimental setup was constructed to test the sensor, and the results were given. The results show that nonlinearity exists in the single-ended eddy current sensor with a large range. When the range is 500 µm, the resolution can reach 46 nm, and the repeatability error is ±0.70% FR. Within the temperature range from +2 °C to +58 °C, the voltage fluctuation in the sensor is reduced to 44 mV after temperature compensation compared to the value of 586 mV before compensation. The proposed plan is verified to be feasible, and the measuring range, precision, and target material should be considered in real-world applications.

Immunoassay System Based on the Technology of Time-Resolved Fluorescence Resonance Energy Transfer.

To enhance the specificity and sensitivity, cut the cost, and realize joint detection of multiple indicators, an immunoassay system based on the technology of time-resolved fluorescence resonance energy transfer (TR-FRET) was studied. Due to the FRET of the reagent, the donor probe and acceptor probe emitted specific fluorescence to enhance specificity. Long-lifetime specific fluorescence from the acceptor probe was combined with time-resolved technology to enhance sensitivity. A xenon flash lamp and a photomultiplier tube (PMT) were selected as the light source and detector, respectively. A filter-switching mechanism was placed in the light path, so the fluorescence signal from the donor and acceptor was measured alternately. The instrument's design is given, and some specificI parts are described in detail. Key technical specifications of the instrument and procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6(IL-6) were tested, and the test results were presented subsequently. The CV value of the self-designed counting module is better than 0.01%, and the instrument noises for 620 nm and 665 nm are 41.44 and 10.59, respectively. When set at 37 °C, the temperature bias (B) is 0.06 °C, and the temperature fluctuation is 0.10 °C. The CV and bias are between ±3% and 5%, respectively, when pipetting volumes are between 10 µL and 100 µL. Within the concentration range of 0.01 nM to 10 nM, the luminescence values exhibit linear regression correlation coefficients greater than 0.999. For PCT detection, when the concentration ranges from 0.02 ng/mL to 50 ng/mL, the correlation coefficient of linear fitting exceeds 0.999, and the limit of quantification is 0.096 ng/mL. For CRP and IL-6, the detection concentration ranges from 0 ng/mL to 500 ng/mL and 0 ng/mL to 20 ng/mL, respectively, with limits of quantification of 2.70 ng/mL and 2.82 ng/mL, respectively. The experimental results confirm the feasibility of the technical and instrumental solutions.

Protocol for electrotaxis of large epithelial cell sheets.

Here, we present a protocol for electrotaxis of large epithelial cell sheets without compromising the integrity of cell epithelia in a high-throughput customized directed current electrotaxis chamber. We describe the fabrication and use of polydimethylsiloxane stencils to control the size and shape of human keratinocyte cell sheets. We detail cell tracking, cell sheet contour assay, and particle image velocimetry to reveal the spatial and temporal motility dynamics of cell sheets. This approach is applicable to other collective cell migration studies. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.

Highly Regioselective Propargylation/Allenylation of Organolanthanum Reagents with Aldehydes.

The metal-mediated propargylation or allenylation of carbonyl compounds is well-adapted to the preparation of homopropargylic or allenylic alcohols, which are multifunctional intermediates in synthetic chemistry. However, the regioselectivity of reactions using propargyl or allenyl metal reagents is difficult to control, owing to the equilibrium between the two species. In our study, propargyl or allenyl organolanthanum reagents were prepared using trimethylsilylpropyne or prop-1-yn-1-ylbenzene substrates. The treatment of the organolanthanum reagents with aldehydes yielded the regioselective products, respectively. This study provides a better understanding of structural specificity and the special chemoselectivity of rare earth metal reagents.

Real-time imaging of RNA polymerase I activity in living human cells.

RNA polymerase I (Pol I) synthesizes about 60% of cellular RNA by transcribing multiple copies of the ribosomal RNA gene (rDNA). The transcriptional activity of Pol I controls the level of ribosome biogenesis and cell growth. However, there is currently a lack of methods for monitoring Pol I activity in real time. Here, we develop LiveArt (live imaging-based analysis of rDNA transcription) to visualize and quantify the spatiotemporal dynamics of endogenous ribosomal RNA (rRNA) synthesis. LiveArt reveals mitotic silencing and reactivation of rDNA transcription, as well as the transcriptional kinetics of interphase rDNA. Using LiveArt, we identify SRFBP1 as a potential regulator of rRNA synthesis. We show that rDNA transcription occurs in bursts and can be altered by modulating burst duration and amplitude. Importantly, LiveArt is highly effective in the screening application for anticancer drugs targeting Pol I transcription. These approaches pave the way for a deeper understanding of the mechanisms underlying nucleolar functions.

Long-term monitoring of intravital biological processes using fluorescent protein-assisted NIR-II imaging.

High spatial resolution, low background, and deep tissue penetration have made near-infrared II (NIR-II) fluorescence imaging one of the most critical tools for in vivo observation and measurement. However, the relatively short retention time and potential toxicity of synthetic NIR-II fluorophores limit their long-term application. Here, we report the use of infrared fluorescent proteins (iRFPs) as in vitro and in vivo NIR-II probes permitting prolonged continuous imaging (up to 15 months). As a representative example, iRFP713 is knocked into the mouse genome to generate a transgenic model to allow temporal and/or spatial expression control of the probe. To demonstrate its feasibility in a genuine diagnostic context, we adopt two liver regeneration models and successfully track the process for a week. The performance and monitoring efficacy are comparable to those of µCT and superior to those of indocyanine green dye. We are also able to effectively observe the pancreas, despite its deep location, under both physiological and pathological conditions. These results indicate that the iRFP-assisted NIR-II fluorescence system is suitable for monitoring various tissues and in vivo biological processes, providing a powerful noninvasive long-term imaging platform.

Low Temperature Plasma Jet Affects Acute Skin Wounds in Diabetic Mice Through Reactive Components.

As a common complication of diabetes, diabetic foot ulcers serious affect the life quality even lead to amputation if it's not properly treated. In this paper, we developed a Low Temperature Plasma Jet (LTPJ) system for treating diabetic foot ulcers on streptozotocin-induced diabetic mice. This system generates time-dependent reactive nitrogen and oxygen species (RNOS), which have temperature below 40°C. The wound area of normal mice was significantly reduced after LTPJ treatment. Histological and immunohistochemistry analysis showed faster deposition of collagen and more vessel formation both in plasma-treated normal and diabetic mice on Day 3. However, diabetic wounds showed poor collagen deposition and angiogenesis on Day 8, which might be the reason of slow wound healing. Reactive nitrogen species (RNS) that generated by LTPJ can promote endogenous nitric oxide (NO) production in diabetic wounds, thus promoting inflammation, stromal deposition, angiogenesis, cell proliferation and remodeling, while excess reactive oxygen species (ROS) will exacerbate oxidative stress in wound tissues of diabetic mice. In conclusion, LTPJ improved acute wound healing in normal mice, increased collagen deposition and angiogenesis in initial diabetic wound healing, but had no significant effect on diabetic wound healing rate.

Propagation dynamics of electrotactic motility in large epithelial cell sheets.

Directional migration initiated at the wound edge leads epithelia to migrate in wound healing. How such coherent migration is achieved is not well understood. Here, we used electric fields to induce robust migration of sheets of human keratinocytes and developed an in silico model to characterize initiation and propagation of epithelial collective migration. Electric fields initiate an increase in migration directionality and speed at the leading edge. The increases propagate across the epithelial sheets, resulting in directional migration of cell sheets as coherent units. Both the experimental and in silico models demonstrated vector-like integration of the electric and default directional cues at free edge in space and time. The resultant collective migration is consistent in experiments and modeling, both qualitatively and quantitatively. The keratinocyte model thus faithfully reflects key features of epithelial migration as a coherent tissue in vivo, e.g. that leading cells lead, and that epithelium maintains cell-cell junction.

Ribonuclease 4 is associated with aggressiveness and progression of prostate cancer.

Prostate specific antigen screening has resulted in a decrease in prostate cancer-related deaths. However, it also has led to over-treatment affecting the quality of life of many patients. New biomarkers are needed to distinguish prostate cancer from benign prostate hyperplasia (BPH) and to predict aggressiveness of the disease. Here, we report that ribonuclease 4 (RNASE4) serves as such a biomarker as well as a therapeutic target. RNASE4 protein level in the plasma is elevated in prostate cancer patients and is positively correlated with disease stage, grade, and Gleason score. Plasma RNASE4 level can be used to predict biopsy outcome and to enhance diagnosis accuracy. RNASE4 protein in prostate cancer tissues is enhanced and can differentiate prostate cancer and BPH. RNASE4 stimulates prostate cancer cell proliferation, induces tumor angiogenesis, and activates receptor tyrosine kinase AXL as well as AKT and S6K. An RNASE4-specific monoclonal antibody inhibits the growth of xenograft human prostate cancer cell tumors in athymic mice.

Angiogenin promotes angiogenesis via the endonucleolytic decay of miR-141 in colorectal cancer.

Mature microRNA (miRNA) decay is a key step in miRNA turnover and gene expression regulation. Angiogenin (ANG), the first human tumor-derived angiogenic protein and also a member of the RNase A superfamily, can promote tumor growth and metastasis by regulating rRNA biogenesis and tiRNA production. However, its effect on miRNA has not been explored. In this study, we find that ANG exclusively downregulates mature miR-141 in human umbilical endothelial cells (HUVECs) via its ribonuclease activity and preferably cleaves single-stranded miR-141 at the A5/C6, U7/G8, and U14/A15 sites via endonucleolytic digestion. By downregulating miR-141, ANG promotes HUVECs proliferation, migration, tube formation, and angiogenesis both in vitro and in vivo. Conversely, downregulated ANG inhibits ANG-mediated miR-141 decay, thus decreasing the angiogenesis process of HUVECs. We also find an inverse correlation between ANG and miR-141 expression in colorectal cancer (CRC) tissues. Our study indicates that ANG regulates CRC progression by disrupting miR-141 and its regulation on angiogenesis-related target genes, not only revealing a new mechanism of ANG action but also newly identifying miR-141 as a substrate of ANG. This study suggests that targeting ANG nuclease activity might be valuable in treating angiogenesis-related diseases through coordinately regulating the metabolism of rRNA, tiRNA, and miRNA.

Bacterial Characteristics of Intestinal Tissues From Patients With Crohn's Disease.

Background and Aims: It is believed that intestinal bacteria play an indispensable role in promoting intestinal inflammation. However, the characteristics of these tissue-associated bacteria remain elusive. The aim of this study is to explore the bacterial loads, compositions, and structures in the noninflamed mucosa, inflamed mucosa, and creeping fat taken from patients with Crohn's disease (CD). Methods: Noninflamed mucosa, inflamed mucosa, and creeping fat samples were obtained from 10 surgical patients suffering from CD. Total bacterial DNA was extracted in a sterile environment using aseptic techniques. The V3-V4 regions of bacterial 16S rDNA were amplified and analysed using standard microbiological methods. qPCR was used to confirm the change in abundance of specific species in additional 30 independent samples. Results: Inflamed mucosa exhibited the highest bacterial load (3.8 and 12 times more than that of non-inflamed mucosa and creeping fat) and species diversity. The relative abundance of Proteobacteria was dominant in most samples and was negatively associated with Firmicutes. Moreover, the relative abundances of Methylobacterium and Leifsonia in creeping fat significantly increased more than twice as much as other tissue types. The bacterial community structure analysis showed that the bacterial samples from the same individual clustered more closely. Conclusion: This study reveals the significant differences in bacterial load, species diversity, and composition among different intestinal tissue types of CD patients and confirms that the bacterial samples from the same individual are highly correlated. Our findings will shed light on fully revealing the characteristics of tissue-associated bacteria and their roles in CD pathogenesis.

rRNA biogenesis regulates mouse 2C-like state by 3D structure reorganization of peri-nucleolar heterochromatin.

The nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development.

Predicting COVID-19 epidemiological trend by applying population mobility data in two-stage modeling.

:To predict the epidemiological trend of coronavirus disease 2019 (COVID-19) by mathematical modeling based on the population mobility and the epidemic prevention and control measures. : As of February 8，2020，the information of 151 confirmed cases in Yueqing，Zhejiang province were obtained，including patients' infection process，population mobility between Yueqing and Wuhan，etc. To simulate and predict the development trend of COVID-19 in Yueqing, the study established two-stage mathematical models，integrating the population mobility data with the date of symptom appearance of confirmed cases and the transmission dynamics of imported and local cases. : It was found that in the early stage of the pandemic，the number of daily imported cases from Wuhan (using the date of symptom appearance) was positively associated with the number of population travelling from Wuhan to Yueqing on the same day and 6 and 9 days before that. The study predicted that the final outbreak size in Yueqing would be 170 according to the number of imported cases estimated by consulting the population number travelling from Wuhan to Yueqing and the susceptible-exposed-infectious-recovered (SEIR) model; while the number would be 165 if using the reported daily number of imported cases. These estimates were close to the 170，the actual monitoring number of cases in Yueqing as of April 27，2020. : The two-stage modeling approach used in this study can accurately predict COVID-19 epidemiological trend.

Serum angiogenin as a potential biomarker for early detection of colorectal adenomas and colorectal cancer.

Although colorectal cancer (CRC) is one of the most common causes of cancer mortality, early-stage detection dramatically improves survival rate. To explore the feasibility of serum angiogenin (ANG) as a biomarker for early detection of colorectal neoplasia, we collected serum samples from 781 participants, including 369 patients with CRC, 133 with colorectal adenoma and 279 healthy controls. We examined the levels of serum ANG by ELISA, calculated the diagnostic accuracy of ANG by plotted receiver operating characteristic curves (ROCs), and compared it with those obtained by carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9). We also analyzed the relationship between serum ANG level and TNM stage in CRC patients. The results showed that ANG serum levels were significantly elevated in patients with colorectal adenomas and CRC (P < 0.01). The area under the ROC curve (AUC) for ANG in distinguishing CRC patients from healthy controls was 0.740 [95% confidence interval (CI): 0.705-0.744], comparable to that of CEA (0.770; 95% CI: 0.735-0.802; P = 0.26) but significantly higher than that of CA19-9 (0.636; 95% CI: 0.598-0.674, P < 0.01), with much higher sensitivity (67.75%) than CEA (36.86%) or CA19-9 (12.20%). We observed no significant differences in ANG serum levels among CRCs at different TNM stages. Furthermore, sensitivity and specificity of ANG for distinguishing colorectal adenomas patients from healthy controls were 66.20% and 64.90%, respectively. ANG has the potential to serve as a serum biomarker for early detection of colorectal neoplasia.

Angiogenin maintains gut microbe homeostasis by balancing α-Proteobacteria and Lachnospiraceae.

OBJECTIVE: Antimicrobial peptides (AMPs) play essential roles in maintaining gut health and are associated with IBD. This study is to elucidate the effect of angiogenin (ANG), an intestine-secreted AMP, on gut microbiota and its relevance with IBD. DESIGN: The effect of ANG on microbiota and its contribution to colitis were evaluated in different colitis models with co-housing and faecal microbiota transplantation. ANG-regulated bacteria were determined by 16S rDNA sequencing and their functions in colitis were analysed by bacterial colonisation. The species-specific antimicrobial activity of ANG and its underlying mechanism were further investigated with microbiological and biochemical methods. ANG level and the key bacteria were characterised in IBD faecal samples. RESULTS: ANG regulated microbiota composition and inhibited intestinal inflammation. Specifically, Ang1 deficiency in mice led to a decrease in the protective gut commensal strains of Lachnospiraceae but an increase in the colitogenic strains of α-Proteobacteria. Direct binding of ANG to α-Proteobacteria resulted in lethal disruption of bacterial membrane integrity, and consequently promoted the growth of Lachnospiraceae, which otherwise was antagonised by α-Proteobacteria. Oral administration of ANG1 reversed the dysbiosis and attenuated the severity of colitis in Ang1-deficient mice. The correlation among ANG, the identified bacteria and IBD status was established in patients. CONCLUSION: These findings demonstrate a novel role of ANG in shaping gut microbe composition and thus maintaining gut health, suggesting that the ANG-microbiota axis could be developed as a potential preventive and/or therapeutic approach for dysbiosis-related gut diseases.

Myeloid cells protect intestinal epithelial barrier integrity through the angiogenin/plexin-B2 axis.

Communication between myeloid cells and epithelium plays critical role in maintaining intestinal epithelial barrier integrity. Myeloid cells interact with intestinal epithelial cells (IECs) by producing various mediators; however, the molecules mediating their crosstalk remain incompletely understood. Here, we report that deficiency of angiogenin (Ang) in mouse myeloid cells caused impairment of epithelial barrier integrity, leading to high susceptibility to DSS-induced colitis. Mechanistically, myeloid cell-derived angiogenin promoted IEC survival and proliferation through plexin-B2-mediated production of tRNA-derived stress-induced small RNA (tiRNA) and transcription of ribosomal RNA (rRNA), respectively. Moreover, treatment with recombinant angiogenin significantly attenuated the severity of experimental colitis. In human samples, the expression of angiogenin was significantly down-regulated in patients with inflammatory bowel disease (IBD). Collectively, we identified, for the first time to our knowledge, a novel mediator of myeloid cell-IEC crosstalk in maintaining epithelial barrier integrity, suggesting that angiogenin may serve as a new preventive agent and therapeutic target for IBD.

YTHDF1-mediated translation amplifies Wnt-driven intestinal stemness.

N6-methyladenosine (m6 A) mRNA methylation has emerged as an important player in many biological processes by regulating gene expression. However, its roles in intestinal stem cell (ISC) homeostasis remain largely unknown. Here, we report that YTHDF1, an m6 A reader, is highly expressed in ISCs and its expression is upregulated by Wnt signaling at the translational level. Whereas YTHDF1 is dispensable for normal intestinal development in mice, genetic ablation of Ythdf1 dramatically blocks Wnt-driven regeneration and tumorigenesis with reduced ISC stemness. Mechanistically, YTHDF1 facilitates the translation of Wnt signaling effectors including TCF7L2/TCF4, while this process is enhanced during Wnt activation to augment ß-catenin activity. Targeting YTHDF1 in ISCs of established tumors leads to tumor shrinkage and prolonged survival. Collectively, our studies unveil YTHDF1 as an amplifier of Wnt/ß-catenin signaling at the translational level, which is required for the maintenance of ISCs during regeneration and tumorigenesis.