X-ray Structure-Guided Discovery of a Potent Benzimidazole Glutaminyl Cyclase Inhibitor That Shows Activity in a Parkinson's Disease Mouse Model.

The secretory glutaminyl cyclase (sQC) and Golgi-resident glutaminyl cyclase (gQC) are responsible for N-terminal protein pyroglutamation and associated with various human diseases. Although several sQC/gQC inhibitors have been reported, only one inhibitor, PQ912, is currently undergoing clinic trials for the treatment of Alzheimer's disease. We report an X-ray crystal structure of sQC complexed with PQ912, revealing that the benzimidazole makes "anchor" interactions with the active site zinc ion and catalytic triad. Structure-guided design and optimization led to a series of new benzimidazole derivatives exhibiting nanomolar inhibition for both sQC and gQC. In a MPTP-induced Parkinson's disease (PD) mouse model, BI-43 manifested efficacy in mitigating locomotor deficits through reversing dopaminergic neuronal loss, reducing microglia, and decreasing levels of the sQC/gQC substrates, α-synuclein, and CCL2. This study not only offers structural basis and new leads for drug discovery targeting sQC/gQC but also provides evidence supporting sQC/gQC as potential targets for PD treatment.

High-Resolution Depth-Discrete Analysis of PFAS Distribution and Leaching for a Vadose-Zone Source at an AFFF-Impacted Site.

The long-term leaching of polyfluoroalkyl substances (PFAS) within the vadose zone of an AFFF application site for which the depth to groundwater is approximately 100 m was investigated by characterizing the vertical distribution of PFAS in a high spatial resolution. The great majority (99%) of PFAS mass resides in the upper 3 m of the vadose zone. The depths to which each PFAS migrated, quantified by moment analysis, is an inverse function of molar volume, demonstrating chromatographic separation. The PFAS were operationally categorized into three chain-length groups based on the three general patterns of retention observed. The longest-chain (>∼335 cm3/mol molar volume) PFAS remained within the uppermost section of the core, exhibiting minimal leaching. Conversely, the shortest-chain (<∼220 cm3/mol) PFAS accumulated at the bottom of the interval, which coincides with the onset of a calcic horizon. PFAS with intermediate-chain lengths were distributed along the length of the core, exhibiting differential magnitudes of leaching. The minimal or differential leaching observed for the longest- and intermediate-chain-length PFAS, respectively, demonstrates that retention processes significantly impacted migration. The accumulation of shorter-chain PFAS at the bottom of the core is hypothesized to result from limited deep infiltration and potential-enhanced retention associated with the calcic horizon.

Modified Composite Biodegradable Mulch for Crop Growth and Sustainable Agriculture.

Using biodegradable films as a substitute for conventional polyolefin films has emerged as a crucial technology to combat agricultural white pollution. To address the shortcomings in the tensile strength, water vapor barrier properties, and degradation period of PBAT-based biodegradable films, this investigation aimed to create a composite film that could improve the diverse properties of PBAT films. To achieve this, a PBAT/PLA-PPC-PTLA ternary blend system was introduced in the study. The system effectively fused PBAT with PLA and PPC, as evidenced by electron microscopy tests showing no apparent defects on the surface and cross-section of the blended film. The developed ternary blend system resulted in a 58.62% improvement in tensile strength, a 70.33% enhancement in water vapor barrier properties, and a 30-day extension of the functional period compared to pure PBAT biodegradable films. Field experiments on corn crops demonstrated that the modified biodegradable film is more suitable for agricultural production, as it improved thermal insulation and moisture retention, leading to a 5.45% increase in corn yield, approaching the yield of traditional polyolefin films.

Two new withanolides from Physalis minima L.

Two new withanolides named physaminilides L (1) and M (2), together with four known ones (3-6) were isolated from the Physalis minima L. The structures were established by analysis of the HR ESIMS, IR and NMR spectroscopic data. The absolute configurations were determined through NOESY and ECD spectra. For compounds 1-5 assayed at 20 µM and compound 6 at 10 µM, inhibition rates of hepatic fibrosis were 22.19%, 15.29%, 37.07%, 9.27%, 12.45%, and 37.03%, respectively.

Uric acid-lowering effect of harpagoside and its protective effect against hyperuricemia-induced renal injury in mice.

Hyperuricemia (HUA) is caused by increased synthesis and/or insufficient excretion of uric acid (UA). Long-lasting HUA may lead to a number of diseases including gout and kidney injury. Harpagoside (Harp) is a bioactive compound with potent anti-inflammatory activity from the roots of Scrophularia ningpoensis. Nevertheless, its potential effect on HUA was not reported. The anti-HUA and nephroprotective effects of Harp on HUA mice were assessed by biochemical and histological analysis. The proteins responsible for UA production and transportation were investigated to figure out its anti-HUA mechanism, while proteins related to NF-κB/NLRP3 pathway were evaluated to reveal its nephroprotective mechanism. The safety was evaluated by testing its effect on body weight and organ coefficients. The results showed that Harp significantly reduced the SUA level and protected the kidney against HUA-induced injury but had no negative effect on safety. Mechanistically, Harp significantly reduced UA production by acting as inhibitors of xanthine oxidase (XOD) and adenosine deaminase (ADA) and decreased UA excretion by acting as activators of ABCG2, OAT1 and inhibitors of GLUT9 and URAT1. Moreover, Harp markedly reduced infiltration of inflammatory cells and down-regulated expressions of TNF-α, NF-κB, NLRP3 and IL-1ß in the kidney. Harp was a promising anti-HUA agent.

Actor-Critic With Synthesis Loss for Solving Approximation Biases.

Approximation biases of value functions are considered a key problem in reinforcement learning (RL). In particular, existing RL algorithms are hindered by overestimation and underestimation biases, i.e., value mismatching between RL's actual returns and action-value approximations limits the performance of RL algorithms. In this article, we first develop a new synthesis loss function for RL's action-value estimation integrating a regularization term and a modified "clipped double Q -learning" structure for solving overestimation and underestimation biases. To minimize the differences between action-value estimations and actual returns in RL, we develop a new discrepancy function to determine the type and magnitude of approximation biases. Then, two coefficients embedded in the synthesis loss are automatically tuned by minimizing the discrepancy function during training to minimize approximation biases. We further design a new actor-critic (AC) algorithm, named AC with synthesis loss (ACSL), by integrating the synthesis loss function and an error-controlled mechanism. Experimental results on continuous control tasks illustrate that the proposed ACSL algorithm outperforms other cutting-edge RL methods in many tasks and that the proposed synthesis loss function is easily implemented into other algorithms and significantly reduces approximation biases while improving performance. The proposed method can successfully handle many complex continuous control tasks and can greatly outperform other state-of-the-art algorithms on most tasks.

Exploration of the genetic influence of MYOT and MB genes on the plumage coloration of Muscovy ducks.

Plumage color, a pivotal attribute delineating diverse Muscovy duck strains, assumes considerable significance within the field of Muscovy duck breeding research. This study extends the existing research by delving into the hereditary aspects of genes associated with plumage coloration in Muscovy ducks. The principal objective is to discern marker genes conducive to targeted breeding strategies based on plumage color, thereby furnishing indispensable technical foundations for the development of novel Muscovy duck varieties. Our investigation focused on scrutinizing the impact of MYOT and MB genes on the genetic expression of plumage color at both the RNA and protein levels in Muscovy ducks. The results elucidate that black Muscovy ducks manifest markedly elevated mRNA and protein expression levels of MYOT and MB genes in comparison to their white counterparts, indicating that both genes may play a constructive regulatory role in the context of plumage coloration in Muscovy ducks. The outcomes of this study delineate a discernible correlation between MYOT and MB genes and the plumage coloration in Muscovy ducks. Employing gene expression analysis, we successfully identified candidate genes that may be intricately linked to the determination of plumage color in these ducks.

13-Oxyingenol-dodecanoate inhibits the growth of non-small cell lung cancer cells by targeting ULK1.

Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers. Euphorbia kansui yielded 13-oxyingenol-dodecanoate (13OD), an ingenane-type diterpenoid, which had a strong cytotoxic effect on NSCLC cells. The underlying mechanism and potential target, however, remained unknown. The study found that 13OD effectively inhibited the cell proliferation and colony formation of NSCLC cells (A549 and H460 cells), with less toxicity in normal human lung epithelial BEAS-2B cells. Moreover, 13OD can cause mitochondrial dysfunction, and apoptosis in NSCLC cells. Mechanistically, the transcriptomics results showed that differential genes were mainly enriched in the mTOR and AMPK signaling pathways, which are closely related to cellular autophagy, the related indicators were subsequently validated. Additionally, bafilomycin A1 (Baf A1), an autophagy inhibitor, reversed the mitochondrial damage caused by 13OD. Furthermore, the Omics and Text-based Target Enrichment and Ranking (OTTER) method predicted ULK1 as a potential target of 13OD against NSCLC cells. This hypothesis was further confirmed using molecular docking, the cellular thermal shift assay (CETSA), and Western blot analysis. Remarkably, ULK1 siRNA inhibited 13OD's toxic activity in NSCLC cells. In line with these findings, 13OD was potent and non-toxic in the tumor xenograft model. Our findings suggested a possible mechanism for 13OD's role as a tumor suppressor and laid the groundwork for identifying targets for ingenane-type diterpenoids.

Harnessing developmental dynamics of spinal cord extracellular matrix improves regenerative potential of spinal cord organoids.

Neonatal spinal cord tissues exhibit remarkable regenerative capabilities as compared to adult spinal cord tissues after injury, but the role of extracellular matrix (ECM) in this process has remained elusive. Here, we found that early developmental spinal cord had higher levels of ECM proteins associated with neural development and axon growth, but fewer inhibitory proteoglycans, compared to those of adult spinal cord. Decellularized spinal cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserved these differences. DNSCM promoted proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs) and facilitated axonal outgrowth and regeneration of spinal cord organoids more effectively than DASCM. Pleiotrophin (PTN) and Tenascin (TNC) in DNSCM were identified as contributors to these abilities. Furthermore, DNSCM demonstrated superior performance as a delivery vehicle for NPCs and organoids in spinal cord injury (SCI) models. This suggests that ECM cues from early development stages might significantly contribute to the prominent regeneration ability in spinal cord.

LncRNA HOTAIR accelerates free fatty acid-induced inflammatory response in HepG2 cells by recruiting SRSF1 to stabilize MLXIPL mRNA.

LncRNA HOTAIR has been reported to be associated with metabolic diseases of the liver. However, the effect of HOTAIR on non-alcoholic fatty liver disease (NAFLD) inflammation and its potential mechanism have not been reported. Genes and proteins expression were detected by qRT-PCR and Western blot respectively. The level of inflammatory cytokines was assessed by ELISA. HepG2 cell viability was detected by MTT assay. TG level and lipid accumulation were measured by Assay Kit and Oil red O staining, respectively. Direct binding relationship between HOTAIR and Serine/arginine splicing factor 1 (SRSF1), SRSF1 and MLX interacting protein like (MLXIPL) were confirmed by RNA-pull down and RIP assay. HOTAIR was highly expressed in free fatty acids (FFA)-treated HepG2 cells. HOTAIR knockdown alleviated FFA-induced inflammation of HepG2 cells. Then further analysis showed that HOTAIR and SRSF1 had a mutual binding relationship, and HOTAIR maintained MLXIPL mRNA stability via recruiting SRSF1 in HepG2 cells. Moreover, the inhibitory effect of HOTAIR knockdown on FFA-induced inflammation in HepG2 cells was reversed by MLXIPL overexpression. HOTAIR accelerates inflammation of FFA-induced HepG2 cells by recruiting SRSF1 to stabilize MLXIPL mRNA, which will help to find new effective strategies for NAFLD therapy. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00614-x.

4 mC site recognition algorithm based on pruned pre-trained DNABert-Pruning model and fused artificial feature encoding.

DNA 4 mC plays a crucial role in the genetic expression process of organisms. However, existing deep learning algorithms have shortcomings in the ability to represent DNA sequence features. In this paper, we propose a 4 mC site identification algorithm, DNABert-4mC, based on a fusion of the pruned pre-training DNABert-Pruning model and artificial feature encoding to identify 4 mC sites. The algorithm prunes and compresses the DNABert model, resulting in the pruned pre-training model DNABert-Pruning. This model reduces the number of parameters and removes redundancy from output features, yielding more precise feature representations while upholding accuracy.Simultaneously, the algorithm constructs an artificial feature encoding module to assist the DNABert-Pruning model in feature representation, effectively supplementing the information that is missing from the pre-trained features. The algorithm also introduces the AFF-4mC fusion strategy, which combines artificial feature encoding with the DNABert-Pruning model, to improve the feature representation capability of DNA sequences in multi-semantic spaces and better extract 4 mC sites and the distribution of nucleotide importance within the sequence. In experiments on six independent test sets, the DNABert-4mC algorithm achieved an average AUC value of 93.81%, outperforming seven other advanced algorithms with improvements of 2.05%, 5.02%, 11.32%, 5.90%, 12.02%, 2.42% and 2.34%, respectively.

Ginkgo biloba L. exocarp petroleum ether extract inhibits methicillin-resistant Staphylococcus aureus by modulating ion transport, virulence, and biofilm formation in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: As reported in the Ancient Chinese Medicinal Books, Ginkgo biloba L. fruit has been used as a traditional Chinese medicine for the treatment asthma and cough or as a disinfectant. Our previous study demonstrated that G. biloba exocarp extract (GBEE), an extract of a traditional Chinese herb, inhibits the formation of methicillin-resistant Staphylococcus aureus (MRSA) biofilms. However, GBEE is a crude extract that contains many components, and the underlying mechanisms of purified GBEE fractions extracted with solvents of different polarities are unknown. AIM OF THE STUDY: This study aimed to investigate the different components in GBEE fractions extracted with solvents of different polarities and their antibacterial effects and mechanisms against MRSA and Staphylococcus haemolyticus biofilms both in vitro and in vivo. METHODS: The components in different fractions were detected by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). Microbroth dilution assays and time growth curves were used to determine the antibacterial effects of the fractions on 15 clinical bacterial isolates. Crystal violet staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were utilized to identify the fractions that affected bacterial biofilm formation. The potential MRSA targets of the GBEE fraction obtained with petroleum ether (PE), denoted GBEE-PE, were screened by transcriptome sequencing, and the gene expression profile was verified by quantitative polymerase chain reaction (qPCR). RESULTS: HPLC-HRMS analysis revealed that the four GBEE fractions (extracted with petroleum ether, ethyl acetate, n-butanol, and water) contained different ginkgo components, and the antibacterial effects decreased as the polarity of the extraction solvent increased. The antibacterial activity of GBEE-PE was greater than that of the GBEE fraction extracted with ethyl acetate (EA). GBEE-PE improved H. illucens survival and reduced MRSA colonization in model mouse organs. Crystal violet staining and SEM and TEM analyses revealed that GBEE-PE inhibited MRSA and S. haemolyticus biofilm formation. Transcriptional analysis revealed that GBEE-PE inhibits MRSA biofilms by altering ion transport, cell wall metabolism and virulence-related gene expression. In addition, the LO2 cell viability and H. illucens toxicity assay data showed that GBEE-PE at 20 mg/kg was nontoxic. CONCLUSION: The GBEE fractions contained different components, and their antibacterial effects decreased with increases in the polarity of the extraction solvent. GBEE-PE limited MRSA growth and biofilm formation by affecting ion transport, cell wall synthesis, and virulence-related pathways. This research provides a more detailed overview of the mechanism by which GBEE-PE inhibits MRSA both in vitro and in vivo and suggests that GBEE-PE is a new prospective antimicrobial with the potential to be used in MRSA therapeutics in the future.

TBK1, a prioritized drug repurposing target for amyotrophic lateral sclerosis: evidence from druggable genome Mendelian randomization and pharmacological verification in vitro.

BACKGROUND: There is a lack of effective therapeutic strategies for amyotrophic lateral sclerosis (ALS); therefore, drug repurposing might provide a rapid approach to meet the urgent need for treatment. METHODS: To identify therapeutic targets associated with ALS, we conducted Mendelian randomization (MR) analysis and colocalization analysis using cis-eQTL of druggable gene and ALS GWAS data collections to determine annotated druggable gene targets that exhibited significant associations with ALS. By subsequent repurposing drug discovery coupled with inclusion criteria selection, we identified several drug candidates corresponding to their druggable gene targets that have been genetically validated. The pharmacological assays were then conducted to further assess the efficacy of genetics-supported repurposed drugs for potential ALS therapy in various cellular models. RESULTS: Through MR analysis, we identified potential ALS druggable genes in the blood, including TBK1 [OR 1.30, 95%CI (1.19, 1.42)], TNFSF12 [OR 1.36, 95%CI (1.19, 1.56)], GPX3 [OR 1.28, 95%CI (1.15, 1.43)], TNFSF13 [OR 0.45, 95%CI (0.32, 0.64)], and CD68 [OR 0.38, 95%CI (0.24, 0.58)]. Additionally, we identified potential ALS druggable genes in the brain, including RESP18 [OR 1.11, 95%CI (1.07, 1.16)], GPX3 [OR 0.57, 95%CI (0.48, 0.68)], GDF9 [OR 0.77, 95%CI (0.67, 0.88)], and PTPRN [OR 0.17, 95%CI (0.08, 0.34)]. Among them, TBK1, TNFSF12, RESP18, and GPX3 were confirmed in further colocalization analysis. We identified five drugs with repurposing opportunities targeting TBK1, TNFSF12, and GPX3, namely fostamatinib (R788), amlexanox (AMX), BIIB-023, RG-7212, and glutathione as potential repurposing drugs. R788 and AMX were prioritized due to their genetic supports, safety profiles, and cost-effectiveness evaluation. Further pharmacological analysis revealed that R788 and AMX mitigated neuroinflammation in ALS cell models characterized by overly active cGAS/STING signaling that was induced by MSA-2 or ALS-related toxic proteins (TDP-43 and SOD1), through the inhibition of TBK1 phosphorylation. CONCLUSIONS: Our MR analyses provided genetic evidence supporting TBK1, TNFSF12, RESP18, and GPX3 as druggable genes for ALS treatment. Among the drug candidates targeting the above genes with repurposing opportunities, FDA-approved drug-R788 and AMX served as effective TBK1 inhibitors. The subsequent pharmacological studies validated the potential of R788 and AMX for treating specific ALS subtypes through the inhibition of TBK1 phosphorylation.

Withanolides from the active extract of Physalis angulate and their anti-hepatic fibrosis effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalis angulata L., a traditional Chinese medicine called "Kuzhi" in China, was used traditionally to treat liver diseases (eg. icterus, hepatitis) as well as malaria, asthma, and rheumatism. AIM OF THE STUDY: Our study aimed to investigate the withanolides with anti-hepatic fibrosis effect from P. angulate. MATERIALS AND METHODS: Withanolides were obtained from the EtOH extract of P. angulate by bioassay-molecular networking analysis-guided isolation using column chromatography and normal/reversed-phase semipreparative HPLC. The structures of new withanolides were elucidated by combinations of spectroscopic techniques with NMR and ECD calculations. MTT cell viability assay, AO/EB staining method, cell wound healing assay, ELISA and Western blot experiments were employed to evaluate the anti-hepatic fibrosis activity and to uncover related mechanism. Molecular docking analysis and cellular thermal shift assay were used to evaluate and verify the interaction between the active withanolides and their potential targets. RESULTS: Eight unreported withanolides, withagulides A-H (1-8), along with twenty-eight known ones were obtained from P. angulate. Withanolides 6, 9, 10, 24, 27, and 29-32 showed marked anti-hepatic fibrosis effect with COL1A1 expression inhibition above 50 %. Physalin F (9), the main component in the active fraction, significantly decreased the TGF ß1-stimulated expressions of collagen I and α-SMA in LX-2 cells. Mechanism study revealed that physalin F exerted its anti-hepatic fibrosis effect via the PI3K/AKT/mTOR signaling pathway. CONCLUSION: This study suggested that withanolides were an important class of natural products with marked anti-hepatic fibrosis effect. The main withanolide physalin F might be a promising candidate for hepatic fibrosis treatment. The work provided experimental foundation for the use of P. angulate to treat hepatic fibrosis.

Lithium response in bipolar disorder is associated with focal adhesion and PI3K-Akt networks: a multi-omics replication study.

Lithium is the gold standard treatment for bipolar disorder (BD). However, its mechanism of action is incompletely understood, and prediction of treatment outcomes is limited. In our previous multi-omics study of the Pharmacogenomics of Bipolar Disorder (PGBD) sample combining transcriptomic and genomic data, we found that focal adhesion, the extracellular matrix (ECM), and PI3K-Akt signaling networks were associated with response to lithium. In this study, we replicated the results of our previous study using network propagation methods in a genome-wide association study of an independent sample of 2039 patients from the International Consortium on Lithium Genetics (ConLiGen) study. We identified functional enrichment in focal adhesion and PI3K-Akt pathways, but we did not find an association with the ECM pathway. Our results suggest that deficits in the neuronal growth cone and PI3K-Akt signaling, but not in ECM proteins, may influence response to lithium in BD.

An integrated analytical modeling framework for determining site-specific soil screening levels for PFAS.

Soils at many contaminated sites have accumulated a significant amount of per- and polyfluoroalkyl substances (PFAS) and may require remediation to mitigate leaching to groundwater. USEPA's current approaches for determining soil screening levels (SSLs) were developed for non-PFAS contaminants. Because many PFAS are interfacially-active with unique leaching behaviors in soils, the current non-PFAS-specific soil screening models may not be applicable. Following USEPA's general methodology, we develop a new modeling framework representing PFAS-specific transport processes for determining site-specific SSLs for PFAS-contaminated sites. We couple a process-based analytical model for PFAS leaching in the vadose zone and a dilution factor model for groundwater in an integrated framework. We apply the new modeling framework to two typical types of contaminated sites. Comparisons with the standard USEPA SSL approach suggest that accounting for the PFAS-specific transport processes may significantly increase the SSL for some PFAS. For the range of soil properties and groundwater recharge rates examined, while SSLs determined with the new model are less than a factor of 2 different from the standard-model values for less interfacially-active shorter-chain PFAS, they are up to two orders of magnitudes greater for more interfacially-active longer-chain PFAS. The new analytical modeling framework provides an effective tool for deriving more accurate site-specific SSLs and improving site characterization and remedial efforts at PFAS-contaminated sites.

Synthesis of gem-Difluoro-3,4-dihydro-2H-pyrans via a TfOH-Catalyzed [4 + 2] Annulation of Difluoroenoxysilanes with α-Cyano Chalcones.

Difluoroenoxysilane, a commonly used difluoroallylating reagent, has attracted considerable attention in recent years. However, its application in the annulation reaction for the construction of fluorinated heterocyclic compounds remains relatively limited. Presented here is the Brønsted acid-catalyzed efficient formal [4 + 2] annulation of difluoroenoxysilanes with α-cyano chalcones. The developed protocol demonstrates tolerance to various substituents under mild reaction conditions, providing a reliable approach to construct gem-difluoro-3,4-dihydro-2H-pyrans in good to excellent yields with high diastereoselectivities.

Application of clustering strategy for automatic segmentation of tissue regions in mass spectrometry imaging.

RATIONALE: Mass spectrometry imaging (MSI) has been widely used in biomedical research fields. Each pixel in MSI consists of a mass spectrum that reflects the molecule feature of the tissue spot. Because MSI contains high-dimensional datasets, it is highly desired to develop computational methods for data mining and constructing tissue segmentation maps. METHODS: To visualize different tissue regions based on mass spectrum features and improve the efficiency in processing enormous data, we proposed a computational strategy that consists of four procedures including preprocessing, data reduction, clustering, and quantitative validation. RESULTS: In this study, we examined the combination of t-distributed stochastic neighbor embedding (t-SNE) and hierarchical clustering (HC) for MSI data analysis. Using publicly available MSI datasets, one dataset of mouse urinary bladder, and one dataset of human colorectal cancer, we demonstrated that the generated tissue segmentation maps from this combination were superior to other data reduction and clustering algorithms. Using the staining image as a reference, we assessed the performance of clustering algorithms with external and internal clustering validation measures, including purity, adjusted Rand index (ARI), Davies-Bouldin index (DBI), and spatial aggregation index (SAI). The result indicated that SAI delivered excellent performance for automatic segmentation of tissue regions in MSI. CONCLUSIONS: We used a clustering algorithm to construct tissue automatic segmentation in MSI datasets. The performance was evaluated by comparing it with the stained image and calculating clustering validation indexes. The results indicated that SAI is important for automatic tissue segmentation in MSI, different from traditional clustering validation measures. Compared to the reports that used internal clustering validation measures such as DBI, our method offers more effective evaluation of clustering results for MSI segmentation. We envision that the proposed automatic image segmentation strategy can facilitate deep learning in molecular feature extraction and biomarker discovery for the biomedical applications of MSI.