Diagnosis and classification of intestinal diseases with urine by surface-enhanced Raman spectroscopy.

Intestinal Disease (ID) is often characterized by clinical symptoms such as malabsorption, intestinal dysfunction, and injury. If treatment is not timely, it will increase the risk of cancer. Early diagnosis of ID is the key to cure it. There are certain limitations of the conventional diagnostic methods, such as low sensitivity and specificity. Therefore, development of a highly sensitive, non-invasive diagnostic method for ID is extremely important. Urine samples are easier to collect and more sensitive to changes in biomolecules than other pathological diagnostic samples such as tissue and blood. In this paper, a diagnostic method of ID with urine by surface-enhanced Raman spectroscopy (SERS) is proposed. A classification model between ID patients and healthy controls (HC) and a classification model between different pathological types of ID (i.e., benign intestinal disease (BID) and colorectal cancer (CRC)) are established. Here, 830 urine samples, including 100 HC, 443 BID, and 287 CRC, were investigated by SERS. The ID/HC classification model was developed by analyzing the SERS spectra of 150 ID and 100 HC, while BID/CRC classification model was built with 300 BID and 150 CRC patients by principal component analysis (PCA)-support vector machines (SVM). The two established models were internally verified by leave-one-out-cross-validation (LOOCV). Finally, the BID/CRC classification model was further evaluated by 143 BID and 137 CRC patients as an external test set. It shows that the accuracy of the classification model validated by the LOOCV for ID/HC and BID/CRC is 86.4% and 85.56%, respectively. And the accuracy of the BID/CRC classification model with external test set is 82.14%. It shows that high accuracy can be achieved with these two established classification models. It indicates that ID patients in the general population can be identified and BID and CRC patients can be further classified with measuring urine by SERS. It shows that the proposed diagnostic method and established classification models provide valuable information for clinicians to early diagnose ID patients and analyze different stages of ID.

Cancer-associated fibroblasts undergoing neoadjuvant chemotherapy suppress rectal cancer revealed by single-cell and spatial transcriptomics.

Neoadjuvant chemotherapy (NAC) for rectal cancer (RC) shows promising clinical response. The modulation of the tumor microenvironment (TME) by NAC and its association with therapeutic response remain unclear. Here, we use single-cell RNA sequencing and spatial transcriptome sequencing to examine the cell dynamics in 29 patients with RC, who are sampled pairwise before and after treatment. We construct a high-resolution cellular dynamic landscape remodeled by NAC and their associations with therapeutic response. NAC markedly reshapes the populations of cancer-associated fibroblasts (CAFs), which is strongly associated with therapeutic response. The remodeled CAF subsets regulate the TME through spatial recruitment and crosstalk to activate immunity and suppress tumor progression through multiple cytokines, including CXCL12, SLIT2, and DCN. In contrast, the epithelial-mesenchymal transition of malignant cells is upregulated by CAF_FAP through MIR4435-2HG induction, resulting in worse outcomes. Our study demonstrates that NAC inhibits tumor progression and modulates the TME by remodeling CAFs.

Photo-crosslinking hydrogel for wound healing in a pilonidal sinus patient after open surgery.

Pilonidal sinus is a chronic infectious disease with large incision and high risk of relapse after surgical management. Therefore, effective intervention strategies are urgently needed to reduce the relapse and shorten the wound healing time. Hydrogels have been widely used in regenerative medicine for its great biocompatibility, however, it remains challenging to integrate the material with wound tissues. Here, we reported a case of pilonidal sinus patient using a novel tissue integration material, Photo-crosslinking hydrogel after open surgery. A 38-year-old man with a pilonidal sinus for ˃5 years underwent open surgery. When the surgery was finished, the wound was injected with hydrogel that was irradiated with a ultraviolet light source until covered and solidified completely. Hydrogel needed to be changed 1-2 times per week. We evaluated the healing time as primary outcome and then followed up for ˃1 year to observe the relapse. The wound healed completely in 46 days after open surgery, which was shorter than that reported in other studies. Meanwhile, no recurrence was detected during follow-up. Photo-crosslinking hydrogel effectively promoted wound healing and has the potential to be easily applied in Pilonidal sinus patients after open surgery.

RS-5645 attenuates inflammatory cytokine storm induced by SARS-CoV-2 spike protein and LPS by modulating pulmonary microbiota.

An inflammatory cytokine storm is considered an important cause of death in severely and critically ill COVID-19 patients, however, the relationship between the SARS-CoV-2 spike (S) protein and the host's inflammatory cytokine storm is not clear. Here, the qPCR results indicated that S protein induced a significantly elevated expression of multiple inflammatory factor mRNAs in peripheral blood mononuclear cells (PBMCs), whereas RS-5645 ((4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone) attenuated the expression of the most inflammatory factor mRNAs. RS-5645 also significantly reduced the cellular ratios of CD45+/IFNγ+, CD3+/IFNγ+, CD11b+/IFNγ+, and CD56+/IFNγ+ in human PBMCs. In addition, RS-5645 effectively inhibited the activation of inflammatory cells and reduced inflammatory damage to lung tissue in mice. Sequencing results of 16S rRNA v3+v4 in mouse alveolar lavage fluid showed that there were 494 OTUs overlapping between the alveolar lavage fluid of mice that underwent S protein+ LPS-combined intervention (M) and RS-5645-treated mice (R), while R manifested 64 unique OTUs and M exhibited 610 unique OTUs. In the alveoli of group R mice, the relative abundances of microorganisms belonging to Porphyromonas, Rothia, Streptococcus, and Neisseria increased significantly, while the relative abundances of microorganisms belonging to Psychrobacter, Shimia, and Sporosarcina were significantly diminished. The results of KEGG analysis indicated that the alveolar microbiota of mice in the R group can increase translation and reduce the activity of amino acid metabolism pathways. COG analysis results indicated that the abundance of proteins involved in ribosomal structure and biogenesis related to metabolism was augmented in the alveolar microbiota of the mice in the R group, while the abundance of proteins involved in secondary metabolite biosynthesis was significantly reduced. Therefore, our research results showed that RS-5645 attenuated pulmonary inflammatory cell infiltration and the inflammatory storm induced by the S protein and LPS by modulating the pulmonary microbiota.

Quyu Shengxin Decoction Alleviates DSS-Induced Ulcerative Colitis in Mice by Suppressing RIP1/RIP3/NLRP3 Signalling.

PURPOSE: To study the therapeutic effect of Quyu (QY) Shengxin (SX) decoction (QYSXD) in mice with dextran sulfate sodium- (DSS-) induced ulcerative colitis and to investigate the effects of QYSXD on the regulation of the receptor-interacting protein kinase 1 (RIP1)/receptor-interacting protein kinase 3 (RIP3)/nucleotide-binding oligomerization domain-like receptor family pyrin domain protein 3 (NLRP3) signaling pathway. METHOD: Thirty-six mice were randomly divided into the following 6 groups: the experimental group (QYSX group), the model group (DSS group), the positive control group (5-aminosalicylic acid (5-ASA) group), the control group, the first component group (QY group), and the second component group (SX group). Each group included 6 mice. Ulcerative colitis (UC) was induced in the mice by providing 3.5% DSS in drinking water. The mice were weighed every day to evaluate the disease activity index (DAI). After 7 days, the mice were sacrificed, and colonic tissues were obtained for colon length measurement. The morphological changes in the colon and the pathological scores of the mice in each group were observed by hematoxylin-eosin (HE) staining. The messenger ribonucleic acid (mRNA) and protein expression levels of RIP1, RIP3, dynamin-related protein 1 (Drp1), NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1), interleukin (IL)-1ß, and IL-18 in the colon tissues of the mice in each group were detected and compared by real-time quantitative reverse transcription PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). The levels of RIP1, RIP3, NLRP3, IL-1ß, and IL-8 in the colonic mucosa were detected by ELISA. Western blotting was used to compare the protein expression of Drp1, caspase-1, mitochondrial fission protein 1 (FIS1), and mitophagy-associated protein light chain 3a/b (LC3a/b) among groups. The levels of reactive oxygen species (ROS) in the colonic mucosal cells were compared by immunofluorescence. RESULTS: Compared with those in the DSS group, the mice with DSS-induced colitis in the QYSX group exhibited clearly higher body weights (P < 0.05) and DAI scores (P < 0.05). The colon lengths of the mice in the QYSX group were longer than those in the DSS group (P < 0.05), and the pathological score of the QYSX group was lower than that of the DSS group (P < 0.05). The RIP1, RIP3, Drp1, IL-1ß, IL-18, and caspase-1 mRNA levels in the QYSX, 5-ASA, SX, and QY groups were significantly lower than those in the DSS group (P < 0.05), but there were no differences between the QYSX group and the 5-ASA group. The levels of RIP1, RIP3, NLRP3, IL-1ß, and IL-18 in the QYSX group were lower than those in the DSS group (P < 0.01). The levels of Drp1, caspase-1, FIS1, and LC3a/b in the QYSX group and the 5-ASA group were lower than those in the DSS group (P < 0.05). The levels of ROS in the colonic mucosal cells in the QYSX, 5-ASA, and QY groups were lower than those in the DSS group (P < 0.05). CONCLUSION: QYSXD has certain therapeutic effects on DSS-induced colitis in mice and may be as effective as 5-ASA. QY and SX decoctions also have certain effects on colitis; however, these decoctions are not as beneficial as QYSXD. QYSXD may ameliorate colitis by inhibiting the expression of RIP1/RIP3/NLRP3 pathway-related proteins and reversing mitochondrial dysfunction to control inflammation.

Astragaloside IV alleviates mouse slow transit constipation by modulating gut microbiota profile and promoting butyric acid generation.

Gut microbiota and short-chain fatty acids (SCFAs) are associated with the development of various human diseases. In this study, we examined the role of astragaloside IV in modulating mouse gut microbiota structure and the generation of SCFAs, as well as in slow transit constipation (STC). An STC model was established by treating mice with loperamide, in which the therapeutic effects of astragaloside IV were evaluated. The microbiota community structure and SCFA content were analysed by 16S rRNA gene sequencing and gas chromatography-mass spectrometry, respectively. The influence of butyrate on STC was assessed using a mouse model and Cajal cells (ICC). Astragaloside IV promoted defecation, improved intestinal mobility, suppressed ICC loss and alleviated colonic lesions in STC mice. Alterations in gut microbiota community structure in STC mice, such as decreased Lactobacillus reuteri diversity, were improved following astragaloside IV treatment. Moreover, astragaloside IV up-regulated butyric acid and valeric acid, but decreased isovaleric acid, in STC mouse stools. Butyrate promoted defecation, improved intestinal mobility, and enhanced ICC proliferation by regulating the AKT-NF-κB signalling pathway. Astragaloside IV promoted intestinal transit in STC mice and inhibited ICC loss by regulating the gut microbiota community structure and generating butyric acid.

Modified Stapled Transanal Rectal Resection Procedure for Limited Rectal Prolapse: A Viable Alternative to the Delorme Operation.

Purpose. The optimal surgical approach for full-thickness rectal prolapse (FTRP) remains controversial. In China, patients with limited FTRP (<5 cm in length) are usually managed by perineal surgery. We retrospectively assessed the outcome of Delorme's procedure and compared it with modified stapled transanal rectal resection (STARR). Methods. The study was conducted in 2 public tertiary referral centers in China with modified STARR or Delorme's procedure performed by experienced surgeons. Outcomes assessed recurrence, operative times, blood loss, complications, length of hospital stay, and continence and constipation scoring. Results. Between December 2012 and May 2019, 65 patients were assessed, including 48 with modified STARR (group 1) and 17 with Delorme's procedure (group 2). The median follow-up was 22 months (range, 3-86 months). The mean operative time for group 1 was 37.4 ± 17.5 minutes vs 74.3 ± 30.6 minutes for group 2 (P < .001). The blood loss for group 1 was significantly lower than that for group 2 (17.4 ± 15.9 mL vs 27.8 ± 16.7 mL, respectively; P = .028). There was no significant difference between groups in recurrence (group 1 18.8% vs group 2 23.5%; P = .944) with no effect of operation type. Both procedures showed improvement in constipation and continence scoring with a similar impact. Conclusions. Modified STARR and the Delorme operation are comparable in managing limited FTRP with superior results in operative time and blood loss for STARR.

Immune-Related Prognostic Model in Colon Cancer: A Gene Expression-Based Study.

Mounting evidence supports that the malignant phenotypes of cancers are defined not only by the intrinsic activity of tumor cells but also by immune cells that are recruited and activated in tumor-related microenvironment. Here, we developed a diagnostic and prognostic model for colon cancer, based on expression profiles of immune-related genes and immune cell component. As a result, we found that B cell infiltration ratio, CD4+ T cells, as well as immune-related genes of TRIB3, CHGA, CASP7, LGALS4, LEP, NOX4, IL17A, and HSPD1 may be highly relevant with clinical outcome of colon cancer.

A novel prognostic model based on multi-omics features predicts the prognosis of colon cancer patients.

BACKGROUND: As a common malignant tumor in the colon, colon cancer (CC) has high incidence and recurrence rates. This study is designed to build a prognostic model for CC. METHODS: The gene expression dataset, microRNA-seq dataset, copy number variation (CNV) dataset, DNA methylation dataset, and transcription factor (TF) dataset of CC were downloaded from UCSC Xena database. Using limma package, the differentially methylated genes (DMGs), and differentially expressed genes (DEGs) and miRNAs (DEMs) were identified. Based on random forest method, prognostic model for each omics dataset were constructed. After the omics features related to prognosis were selected using logrank test, the prognostic model based on multi-omics features was built. Finally, the clinical phenotypes correlated with prognosis were screened using Kaplan-Meier survival analysis, and the nomogram model was established. RESULTS: There were 1625 DEGs, 268 DEMs, and 386 DMGs between the tumor and normal samples. A total of 105, 29, 159, five, and six genes/sites significantly correlated with prognosis were identified in the gene expression dataset (GABRD), miRNA-seq dataset (miR-1271), CNV dataset (RN7SKP247), DNA methylation dataset (cg09170112 methylation site [located in SFSWAP]), and TF dataset (SIX5), respectively. The prognostic model based on multi-omics features was more effective than those based on single omics dataset. The number of lymph nodes, pathologic_M stage, and pathologic_T stage were the clinical phenotypes correlated with prognosis, based on which the nomogram model was constructed. CONCLUSION: The prognostic model based on multi-omics features and the nomogram model might be valuable for the prognostic prediction of CC.

A 6 lncRNA-Based Risk Score System for Predicting the Recurrence of Colon Adenocarcinoma Patients.

Colon adenocarcinoma (COAD) is a common type of colon cancer, and post-operative recurrence and metastasis may occur in COAD patients. This study is designed to build a risk score system for COAD patients. The Cancer Genome Atlas (TCGA) dataset of COAD (the training set) was downloaded, and GSE17538 and GSE39582 (the validation sets) from Gene Expression Omnibus database were obtained. The differentially expressed RNAs (DERs) were analyzed by limma package. Using survival package, the independent prognosis-associated long non-coding RNAs (lncRNAs) were selected for constructing risk score system. After the independent clinical prognostic factors were screened out using survival package, a nomogram survival model was constructed using rms package. Furthermore, competitive endogenous RNA (ceRNA) regulatory network and enrichment analyses separately were performed using Cytoscape software and DAVID tool. Totally 404 DERs between recurrence and non-recurrence groups were identified. Based on the six independent prognosis-associated lncRNAs (including H19, KCNJ2-AS1, LINC00899, LINC01503, PRKAG2-AS1, and SRRM2-AS1), the risk score system was constructed. After the independent clinical prognostic factors (Pathologic M, pathologic T, and RS model status) were identified, the nomogram survival model was built. In the ceRNA regulatory network, there were three lncRNAs, four miRNAs, and 77 mRNAs. Additionally, PPAR signaling pathway and hedgehog signaling pathway were enriched for the mRNAs in the ceRNA regulatory network. The risk score system and the nomogram survival model might be used for predicting COAD recurrence. Besides, PPAR signaling pathway and hedgehog signaling pathway might affect the recurrence of COAD patients.

Exogenous pyruvate facilitates ultraviolet B-induced DNA damage repair by promoting H3K9 acetylation in keratinocytes and melanocytes.

Ultraviolet radiation (UVR) is the major cause of numerous skin diseases, including sunburn, skin aging, and skin cancers. Pyruvate is a key intermediate in cellular metabolic pathways, which has shown protective effects against oxidative stress and apoptosis, but its role in UV protection remains unclear. Here we established human and mice in vivo model and found that pyruvate protects both human and mouse skin from UVB-induced DNA damage. Moreover, assays in primary keratinocytes and melanocytes further supported the protective role of exogenous pyruvate against UVB-induced DNA damage. Mechanically, pyruvate stimulates the activation of Histone H3 Lysine 9 (H3K9) acetylation, which is an essential step for nucleotide excision repair (NER) pathway. In conclusion, our results suggest that treatment of pyruvate might be an effective strategy to prevent UVB-induced skin diseases.

Association of a novel seven-gene expression signature with the disease prognosis in colon cancer patients.

Older patients who are diagnosed with colon cancer face unique challenges, specifically regarding to cancer treatment. The aim of this study was to identify prognostic signatures to predicting prognosis in colon cancer patients through a detailed transcriptomic analysis. RNA-seq expression profile, miRNA expression profile, and clinical phenotype information of all the samples of TCGA colon adenocarcinoma were downloaded and differentially expressed mRNAs (DEMs), differentially expressed lncRNAs (DELs) and differentially expressed miRNAs (DEMis) were identified. A competing endogenous RNA (ceRNA) network was constructed further and DEMs related with prognosis in the ceRNA network was screened using Cox regression analysis. Risk score models for predicting the prognosis of colon cancer patients were built using these DEMs. A total of 1476 DEMs, 9 DELs, and 243 DEMis between the tumor and normal samples were identified and functional enrichment analyses showed that the DEMs were significantly enriched in the nervous system development, ribosome biogenesis pathways in eukaryotes, and drug metabolism cytochrome P450. Twelve DEMs related with prognosis were screened from the ceRNA network. Thereafter, the risk score models of prognostic DEMs were obtained, involving seven DEMs (SGCG, CLDN23, SLC4A4, CCDC78, SLC17A7, OTOP3, and SMPDL3A). Additionally, cancer stage was identified as a prognostic clinical factor. This prognostic signature was further validated in two independent datasets. Our study developed a seven-mRNA and one-clinical factor signature that are associated with prognosis in colon cancer patients, which may serve as possible biomarkers and therapeutic targets in the future.

Targeting MC1R depalmitoylation to prevent melanomagenesis in redheads.

Some genetic melanocortin-1 receptor (MC1R) variants responsible for human red hair color (RHC-variants) are consequently associated with increased melanoma risk. Although MC1R signaling is critically dependent on its palmitoylation primarily mediated by the ZDHHC13 protein-acyl transferase, whether increasing MC1R palmitoylation represents a viable therapeutic target to limit melanomagenesis in redheads is unknown. Here we identify a specific and efficient in vivo strategy to induce MC1R palmitoylation for therapeutic benefit. We validate the importance of ZDHHC13 to MC1R signaling in vivo by targeted expression of ZDHHC13 in C57BL/6J-MC1RRHC mice and subsequently inhibit melanomagenesis. By identifying APT2 as the MC1R depalmitoylation enzyme, we are able to demonstrate that administration of the selective APT2 inhibitor ML349 treatment efficiently increases MC1R signaling and represses UVB-induced melanomagenesis in vitro and in vivo. Targeting APT2, therefore, represents a preventive/therapeutic strategy to reduce melanoma risk, especially in individuals with red hair.

Network Pharmacology-Based Investigation into the Mechanisms of Quyushengxin Formula for the Treatment of Ulcerative Colitis.

OBJECTIVE: Ulcerative colitis (UC) is a chronic idiopathic inflammatory bowel disease whose treatment strategies remain unsatisfactory. This study aims to investigate the mechanisms of Quyushengxin formula acting on UC based on network pharmacology. METHODS: Ingredients of the main herbs in Quyushengxin formula were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Absorption, distribution, metabolism, and excretion properties of all ingredients were evaluated for screening out candidate bioactive compounds in Quyushengxin formula. Weighted ensemble similarity algorithm was applied for predicting direct targets of bioactive ingredients. Functional enrichment analyses were performed for the targets. In addition, compound-target network, target-disease network, and target-pathway network were established via Cytoscape 3.6.0 software. RESULTS: A total of 41 bioactive compounds in Quyushengxin formula were selected out from the TCMSP database. These bioactive compounds were predicted to target 94 potential proteins by weighted ensemble similarity algorithm. Functional analysis suggested these targets were closely related with inflammatory- and immune-related biological progresses. Furthermore, the results of compound-target network, target-disease network, and target-pathway network indicated that the therapeutic effects of Quyushengxin on UC may be achieved through the synergistic and additive effects. CONCLUSION: Quyushengxin may act on immune and inflammation-related targets to suppress UC progression in a synergistic and additive manner.

Targeting the upstream transcriptional activator of PD-L1 as an alternative strategy in melanoma therapy.

Programmed cell death ligand 1 (PD-L1) interacts with programmed cell death protein-1 (PD-1) as an immune checkpoint. Reactivating the immune response by inhibiting PD-L1 using therapeutic antibodies provides substantial clinical benefits in many, though not all, melanoma patients. However, transcriptional suppression of PD-L1 expression as an alternative therapeutic anti-melanoma strategy has not been exploited. Here we provide biochemical evidence demonstrating that ultraviolet radiation (UVR) induction of PD-L1 in skin is directly controlled by nuclear factor E2-related transcription factor 2 (NRF2). Depletion of NRF2 significantly induces tumor infiltration by both CD8+ and CD4+ T cells to suppress melanoma progression, and combining NRF2 inhibition with anti-PD-1 treatment enhanced its anti-tumor function. Our studies identify a critical and targetable PD-L1 upstream regulator and provide an alternative strategy to inhibit the PD-1/PD-L1 signaling in melanoma treatment.

The protective role of DOT1L in UV-induced melanomagenesis.

The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.

Palmitoylation-dependent activation of MC1R prevents melanomagenesis.

The melanocortin-1 receptor (MC1R), a G-protein-coupled receptor, has a crucial role in human and mouse pigmentation. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH) stimulates cAMP signalling and melanin production and enhances DNA repair after ultraviolet irradiation. Individuals carrying MC1R variants, especially those associated with red hair colour, fair skin and poor tanning ability (denoted as RHC variants), are associated with higher risk of melanoma. However, how MC1R activity is modulated by ultraviolet irradiation, why individuals with red hair are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit are unknown. Here we demonstrate a potential MC1R-targeted intervention strategy in mice to rescue loss-of-function MC1R in MC1R RHC variants for therapeutic benefit by activating MC1R protein palmitoylation. MC1R palmitoylation, primarily mediated by the protein-acyl transferase ZDHHC13, is essential for activating MC1R signalling, which triggers increased pigmentation, ultraviolet-B-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-Mc1re/eJ mice, in which endogenous MC1R is prematurely terminated, expressing Mc1r RHC variants, we show that pharmacological activation of palmitoylation rescues the defects of Mc1r RHC variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.