Identification of a Novel Chitinase from Bacillus paralicheniformis: Gene Mining, Sequence Analysis, and Enzymatic Characterization.

In this study, a novel strain for degrading chitin was identified as Bacillus paralicheniformis HL37, and the key chitinase CH1 was firstly mined through recombinant expression in Bacillus amyloliquefaciens HZ12. Subsequently, the sequence composition and catalytic mechanism of CH1 protein were analyzed. The molecular docking indicated that the triplet of Asp526, Asp528, and Glu530 was a catalytic active center. The enzymatic properties analysis revealed that the optimal reaction temperature and pH was 65 °C and 6.0, respectively. Especially, the chitinase activity showed no significant change below 55 °C and it could maintain over 60% activity after exposure to 85 °C for 30 min. Moreover, the optimal host strain and signal peptide were obtained to enhance the expression of chitinase CH1 significantly. As far as we know, it was the first time finding the highly efficient chitin-degrading enzymes in B. paralicheniformis, and detailed explanations were provided on the catalytic mechanism and enzymatic properties on CH1.

Production, physico-chemical properties and antioxidant activity of melanin from Annulohypoxylon stygium (Lév.) Y.M. Ju, J.D. Rogers and H.M. Hsieh.

To obtain higher melanin production in liquid culture, culture conditions of Annulohypoxylon stygium (Lév.) Y.M. Ju, J.D. Rogers and H.M. Hsieh were optimised. The results showed that using single factor experiment and orthogonal test, the optimised production of melanin reached 2.20 g/L, which was 2.06 times higher than that of the control group. In addition, it was speculated that A. stygium melanin (AsM) was 3,4-dihydroxyphenylalanine (DOPA) melanin and showed an amorphous irregular structure. Moreover, it had good solubility in alkaline solution. AsM showed good antioxidant activity at a concentration of 500 mg/L, with DPPH, ABTS and OH radicals scavenging activities of 90.83%, 75.36% and 70.90%, respectively. AsM prevented alcohol-induced oxidative damage and oxidative stress in HepG2 cells by inhibiting the decrease of antioxidant key enzyme activity under alcohol stimulation. It was proved to have a great potential for application as a natural antioxidant and a substitute for synthetic pigments.

Polysaccharide from Pleurotus tuber-regium mycelium improves DSS-induced colitis in mice by regulating inflammatory cytokines, oxidative stress and gut microbiota.

Pleurotus tuber-regium (PTR) has been proved to have obvious pharmacological properties. In this study, a polysaccharide was extracted from the mycelium of PTR and administered to DSS-induced colitis mice to clarify the protective effect and mechanism of the PTR polysaccharide (PTRP) on colitis. The results showed that PTRP significantly improved the clinical symptoms and intestinal tissue damage caused by colitis and inhibited the secretion of pro-inflammatory cytokines and myeloperoxidase activity, while the levels of oxidative stress factors in mice decreased and the antioxidant capacity increased. The 16S rRNA sequencing of the mouse cecum content showed that PTRP changed the composition of gut microbiota, and the diversity and abundance of beneficial bacteria increased. In addition, PTRP also enhanced the production of short-chain fatty acids by regulating gut microbiota. In conclusion, our study shows that PTRP has the potential to relieve IBD symptoms and protect intestinal function by regulating inflammatory cytokines, oxidative stress and gut microbiota.

Stress events and stress symptoms in Chinese secondary school students: gender and academic year characteristics of the relationship.

Objective: To explore the relationship between stress events and stress symptoms and their gender and academic year characteristics in Chinese secondary school students. Methods: 4,995 secondary school students were investigated by the Adolescent Self-rating Life Events Checklist (ASLEC) and the Calgary Symptoms of Stress Inventory (C-SOSI). Results: First, there were significant differences in all dimensions and total scores of stress events and stress symptoms between boys and girls in secondary school and between junior high school students and senior high school students. Second, the dimensions and total scores of stress events in secondary school students are positively correlated with the dimensions and total scores of stress symptoms. Third, the influence of punishment on the stress symptoms of secondary school boys is the most obvious, and the influence of punishment, adaption, relationship stress, and learning stress on the stress symptoms of secondary school girls is the most obvious. The influence of punishment on the stress symptoms of junior high school students is the most obvious, and the influence of punishment and relationship on the stress symptoms of senior high school students is the most obvious. Conclusion: Stress events and stress symptoms of Chinese secondary school students have significant differences in gender and academic year. The same stress event has different influence mechanisms on the stress symptoms of Chinese secondary school students of different genders and different academic years.

A machine vision-assisted Argonaute-mediated fluorescence biosensor for the detection of viable Salmonella in food without convoluted DNA extraction and amplification procedures.

The precise identification viable pathogens hold paramount significance in the prevention of foodborne diseases outbreaks. In this study, we integrated machine vision and learning with single microsphere to develop a phage and Clostridium butyricum Argonaute (CbAgo)-mediated fluorescence biosensor for detecting viable Salmonella typhimurium (S. typhimurium) without convoluted DNA extraction and amplification procedures. Phage and lysis buffer was utilized to capture and lyse viable S. typhimurium, respectively. Subsequently, CbAgo can cleave the bacterial DNA to obtain target DNA that guides a newly targeted cleavage of fluorescent probes. After that, the resulting fluorescent signal accumulates on the streptavidin-modified single microsphere. The overall detection process is then analyzed and interpreted by machine vision and learning algorithms, achieving highly sensitive detection of S. typhimurium with a limit of detection at 40.5 CFU/mL and a linear range of 50-107 CFU/mL. Furthermore, the proposed biosensor demonstrates standard recovery rates and coefficients of variation at 93.22% - 106.02% and 1.47% - 12.75%, respectively. This biosensor exhibits exceptional sensitivity and selectivity, presenting a promising method for the rapid and effective detection of foodborne pathogens. ENVIRONMENTAL IMPLICATION: Bacterial pathogens exist widely in the environment and seriously threaten the safety of human life. In this study, we developed a phage and Clostridium butyricum Argonaute-mediated fluorescence biosensor for the detection of viable Salmonella typhimurium in environmental water and food samples. Compared with other Salmonella detection methods, this method does not need complex DNA extraction and amplification steps, which reduces the use of chemical reagents and experimental consumables in classic DNA extraction kit methods.

Functional characterization of a catalase gene PtCat associated with sclerotia formation in Pleurotus tuber-regium.

Pleurotus tuber-regium (Fr.) Sing. can evade oxygen by forming sclerotia under oxidative stress, consequently averting the development of hyperoxidative state, during which the expression level of catalase gene (PtCat) is significantly up-regulated. To investigate the relationship between the catalase gene and sclerotia formation, over-expression and interference strains of the PtCat gene were obtained by Agrobacterium tumefaciens-mediated transformation for phenotypic analysis. In the absence of hydrogen peroxide (H2O2) stress, a minor difference was observed in the mycelial growth rate and the activity of antioxidant enzymes between the over-expression and interference strains. However, when exposed to 1-2 mM H2O2, the colony diameter of the over-expression strain was approximately 2-3× that of the interference strain after 8 days of culturing. The catalase activity of the over-expression strain increased by 1000 U/g under 2 mM H2O2 stress, while the interference strain increased by only 250 U/g. After one month of cultivation, the interference strain formed an oval sclerotium measuring 3.5 cm on the long axis and 2 cm on the short axis, while the over-expression strain did not form sclerotia. Therefore, it is concluded that catalase activity regulates the formation of sclerotia in P. tuber-regium.

Hepatoprotective Effect of Annulohypoxylon stygium Melanin on Acute Alcoholic Liver Injury in Mice.

Annulohypoxylon stygium melanin (AsM) has various functional properties such as antioxidant and anti-radiation, but its biological activity in vivo has not been fully investigated. In this study, we researched the effects of AsM on the protection against acute liver injury in mice and its mechanism. The results showed that AsM had no significant effect on body weight in mice but reduced the liver index. It was able to significantly decrease the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the contents of triglyceride (TG) and total cholesterol (TC) in mice. Simultaneously, it raised the levels of superoxide dismutase (SOD), peroxidase (CAT), and glutathione peroxidase (GSH-Px), which obviously exceeded those of the EtOH group. AsM could significantly lower the levels of inflammatory factors, with inhibition rates of 68.30%, 29.0%, and 19.50% for IL-1ß, IL-6, and TNF-α, respectively. H&E and Oil red O staining also showed that AsM ameliorated liver damage and lipid accumulation in mice. The protective mechanism of AsM may be associated to the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant signaling pathway, which could activate the downstream antioxidant enzymes heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC). These findings confirmed that AsM had an alleviating effect on alcoholic liver injury and provided new thoughts for the development of natural product.

Mesophilic Argonaute-Mediated Polydisperse Droplet Biosensor for Amplification-Free, One-Pot, and Multiplexed Nucleic Acid Detection Using Deep Learning.

Detection of nucleic acids from a single multiplexed and amplification-free test is critical for ensuring food safety, clinical diagnostics, and environmental monitoring. In this study, we introduced a mesophilic Argonaute protein from Clostridium butyricum (CbAgo), which exhibits nucleic acid endonuclease activity, to achieve a programmable, amplification-free system (PASS) for rapid nucleic acid quantification at ambient temperatures in one pot. By using CbAgo-mediated binding with specific guide DNA (gDNA) and subsequent targeted cleavage of wild-type target DNAs complementary to gDNA, PASS can detect multiple foodborne pathogen DNA (<102 CFU/mL) simultaneously. The fluorescence signals were then transferred to polydisperse emulsions and analyzed by using deep learning. This simplifies the process and increases the suitability of polydisperse emulsions compared to traditional digital PCR, which requires homogeneous droplets for accurate detection. We believe that PASS has the potential to become a next-generation point-of-care digital nucleic acid detection method.

Transcriptome analysis reveals the underlying mechanism for over-accumulation of alkaline protease in Bacillus licheniformis.

AIMS: Bacillus licheniformis AQ is an industrial strain with high production of alkaline protease (AprE), which has great industrial application value. However, how to regulate the production of AprE in the process of industrial fermentation is still not completely clear. Therefore, it is important to understand the metabolic process of AprE production in the industrial fermentation medium. METHODS AND RESULTS: In this study, transcriptome sequencing of the whole fermentation course was performed to explore the synthesis and regulation mechanism of AprE in B. licheniformis AQ. During the fermentation process, the AprE got continuously accumulated, reaching a peak of 42 020 U/mL at the fermentation endpoint (48 h). Meanwhile, the highly expressed genes were observed. Compared with the fermentation endpoint, there were 61 genes in the intersection of differentially expressed genes, functioning as catabolic processes, peptidases and inhibitors, chaperones, and folding catalysts. Furthermore, the protein-protein interactions network of AprE was constructed. CONCLUSION: This study provides important transcriptome information for B. licheniformis AQ and potential molecular targets for further improving the production of AprE.

PriA is involved in Pleurotus ostreatus development and defense against Pseudomonas tolaasii.

Pleurotus ostreatus is a crucial commercial mushroom widely cultivated for diverse uses. Scientists have worked on breeding disease-resistant and high-yielding varieties to secure food supply. Studies on the molecular genetic mechanism of growth and development can provide valuable information to facilitate crop breeding programs by genetic engineering. Aegerolysins are pore-forming proteins widely distributed in both prokaryotes and eukaryotes, which are reported to have haemolytic activity and be involved in the early stages of fructification. The present study aimed to explore biological function of a differential expressed aegerolysin gene PriA in P. ostreatus. The expression level of PriA gene was higher in primordium and fruiting body than that in mycelium. The PriA expression in overexpression (OE) and RNAi interference (RNAi) strains was detected by qRT-PCR. The RNAi strains grew at slightly slower rates and advanced producing yellow pigments than the wild type, while OE strains showed no prominent phenotypic characteristics. Furthermore, Pseudomonas tolaasii infection assays showed that the PriA OE strains could enhance mycelia and caps resistance to P. tolaasii. These data demonstrate PriA from P. ostreatus play an essential role in mycelial development and increase antagonism against P. tolaasii. Our study provides some reference information on interactions between edible fungi and pathogenic bacteria and offers a new resistance-conferring gene for breeding.

The Effect of Mushroom Dietary Fiber on the Gut Microbiota and Related Health Benefits: A Review.

Mushroom dietary fiber is a type of bioactive macromolecule derived from the mycelia, fruiting bodies, or sclerotia of edible or medicinal fungi. The use of mushroom dietary fiber as a prebiotic has recently gained significant attention for providing health benefits to the host by promoting the growth of beneficial microorganisms; therefore, mushroom dietary fiber has promising prospects for application in the functional food industry and in drug development. This review summarizes methods for the preparation and modification of mushroom dietary fiber, its degradation and metabolism in the intestine, its impact on the gut microbiota community, and the generation of short-chain fatty acids (SCFAs); this review also systematically summarizes the beneficial effects of mushroom dietary fiber on host health. Overall, this review aims to provide theoretical guidance and a fresh perspective for the prebiotic application of mushroom dietary fiber in the development of new functional foods and drugs.

Enhancing activity of food protein-derived peptides: An overview of pretreatment, preparation, and modification methods.

Food protein-derived peptides have garnered considerable attention due to their potential bioactivities and functional properties. However, the limited activity poses a challenge in effective utilization aspects. To overcome this hurdle, various methods have been explored to enhance the activity of these peptides. This comprehensive review offers an extensive overview of pretreatment, preparation methods, and modification strategies employed to augment the activity of food protein-derived peptides. Additionally, it encompasses a discussion on the current status and future prospects of bioactive peptide applications. The review also addresses the standardization of mass production processes and safety considerations for bioactive peptides while examining the future challenges and opportunities associated with these compounds. This comprehensive review serves as a valuable guide for researchers in the food industry, offering insights and recommendations to optimize the production process of bioactive peptides.

An Efficient Prephenate Dehydrogenase Gene for the Biosynthesis of L-tyrosine: Gene Mining, Sequence Analysis, and Expression Optimization.

L-tyrosine is a key precursor for synthesis of various functional substances, but the microbial production of L-tyrosine faces huge challenges. The development of new microbial chassis cell and gene resource is especially important for the biosynthesis of L-tyrosine. In this study, the optimal host strain Bacillus amyloliquefaciens HZ-12 was firstly selected by detecting the production capacity of L-tyrosine. Subsequently, the recombinant expression of 15 prephenate dehydrogenase genes led to the discovery of the best gene, Bao-tyrA from B. amyloliquefaciens HZ-12. After the overexpression of Bao-tyrA, the L-tyrosine yield of the recombinant strain HZ/P43-Bao-tyrA reach 411 mg/L, increased by 42% compared with the control strain (HZ/pHY300PLK). Moreover, the nucleic acid sequence and deduced amino acid sequence of the gene Bao-tyrA were analyzed, and their conservative sites and catalytic mechanisms were proposed. Finally, the expression of Bao-tyrA was regulated through a promoter and 5'-UTR sequence to obtain the optimal expression elements. Thereby, the maximum L-tyrosine yield of 475 mg/L was obtained from HZ/P43-UTR3-Bao-tyrA. B. amyloliquefaciens was applied for the first time to produce L-tyrosine, and the optimal prephenate dehydrogenase gene Bao-tyrA and corresponding expression elements were obtained. This study provides new microbial host and gene resource for the construction of efficient L-tyrosine chassis cells, and also lays a solid foundation for the production of various functional tyrosine derivatives.

Relationship between antioxidant enzymes and sclerotial formation of Pleurotus tuber-regium under abiotic stress.

In order to explore the relationship between sclerotial formation and antioxidant enzymes under abiotic stresses, the effects of abiotic stresses including temperature, pH value, osmotic pressure, limited nitrogen, and hydrogen peroxide (H2O2) on the activities of antioxidant enzymes, ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in Pleurotus tuber-regium were studied. Meanwhile, the sclerotial formation under these abiotic stress conditions was also investigated. It was found that low temperature, weak alkaline, appropriate osmotic stress, and H2O2 can promote sclerotial formation, and sclerotial formation always tended to occur when the activities of antioxidant enzymes were at a high value. During the prolonged low temperature stress, SOD acted mainly in the early stage of stress, while POD and CAT had higher activity in the middle and late stage. Moreover, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) results showed that SOD.193 and POD.535 were significantly down-regulated in sclerotia, and CAT.1115 and POD.401 were up-regulated instead. These antioxidant enzyme genes played an important role in the sclerotial formation under low temperature stress. It is strongly suggested that antioxidant enzymes and abiotic stresses are closely related to sclerotial formation in P. tuber-regium. KEY POINTS: • Low temperature and H2O2 can promote sclerotial formation. • Sclerotia are more likely to form under high antioxidant enzyme activity. • POD.401, POD.535, SOD.193, and CAT.1115 are important for sclerotial formation.

Sources, chemical synthesis, functional improvement and applications of food-derived protein/peptide-saccharide covalent conjugates: a review.

Proteins/peptides and saccharides are two kinds of bioactive substances in nature. Recently, increasing attention has been paid in understanding and utilizing covalent interactions between proteins/peptides and saccharides. The products obtained through covalent conjugation of proteins/peptides to saccharides are shown to have enhanced functional attributes, such as better gelling property, thermostability, and water-holding capacity. Additionally, food-derived protein/peptide-saccharide covalent conjugates (PSCCs) also have biological activities, such as antibacterial, antidiabetic, anti-osteoporosis, anti-inflammatory, anti-cancer, immune regulatory, and other activities that are widely used in the functional food industry. Moreover, PSCCs can be used as packaging or delivery materials to improve the bioavailability of bioactive substances, which expands the development of food-derived protein and saccharide resources. Thus, this review was aimed to first summarize the current status of sources, classification structures of natural PSCCs. Second, the methods of chemical synthesis, reaction conditions, characterization and reagent formulations that improve the desired functional characteristics of food-derived PSCCs were introduced. Third, functional properties such as emulsion, edible films/coatings, and delivery of active substance, bio-activities such as antioxidant, anti-osteoporosis, antidiabetic, antimicrobial of food-derived PSCCs were extensively discussed.

Novel bioorthogonal reaction-mediated particle counting sensing platform using phage for rapid detection of Salmonella.

Bacterial detection in foods have to overcome the hindrance of complex matrices, resulting in more sophisticated pre-treatment steps and low sensitivity and reproductivity. Therefore, it remains a challenge to develop simple, rapid, accurate, and low-cost pathogen detection methods which were applicable for detection in turbid or complex food matrices. In this study, a novel bioorthogonal reaction-amplified microparticle counting sensing based on phages has been developed for the rapid detection of viable Salmonella in different foods. Phage not only acted as a bioreceptor to recognize viable Salmonella, but also used as an effective carrier for signal amplifiers due to its large specific surface area. The bioorthogonal reaction was introduced to further amplify the signal, the presence of Salmonella can specifically induce a quantitative change in the functionalized polystyrene microspheres, and thus can be monitored by the microporous resistance particle counter. Under the optimized conditions, this sensor could achieve a limit of detection of 33.58 CFU/mL and a linear range from 102 CFU/mL to 106 CFU/mL. This phage-mediated microporous resistance particle sensor can specifically recognize viable Salmonella in 1 h, providing a powerful platform for rapid and low-cost Salmonella quantification.

Enhancement on antioxidant, anti-hyperglycemic and antibacterial activities of blackberry anthocyanins by processes optimization involving extraction and purification.

This research aimed to recover anthocyanin-rich extracts from blackberry (Rubus spp. Hull cultivar) by optimizing the processing conditions, and to characterize anthocyanin individuals and determine influences of optimization on enhancement of antioxidant and anti-hyperglycemic activities of anthocyanins as natural supplements. The ethanol concentration of 69.87%, HCl dosage of 0.53%, solid-to-liquid ratio of 1:19.06 at 47.68°C for 17.04 h were optimal to obtain the highest extraction yield of anthocyanins at 0.72 mg/g. By using AB-8 macroporous resins, the anthocyanin concentration of 3.0 mg/mL, ethanol concentration of 90%, and elution rate of 2.0 mL/min were selected to boost the anthocyanin purity up to be 60.11%. Moreover, the purified anthocyanin extracts from blackberry contained nine main pigments which could be divided into three aglycone-based forms, and cyanidin-3-O-glucoside was the most abundant among them. Due to the successive processes of extraction and purification, the blackberry purified anthocyanin extracts (BA-PAE) showed much higher bioactive capacities than the blackberry crude anthocyanin extracts (BA-CAE) and blackberry fruit slurry extracts (BA-FSE), e.g., DPPH and ABTS radical scavenging activities (EC50 = 0.08 and 0.04, 0.32 and 0.24, and 1.31 and 0.41 mg/mL), oxygen radical absorbance capacity (1.60, 0.59, and 0.15 mmol TEAC/g), cytoprotective effects against oxidative stress in PC12 cells (1.69-, 1.58-, and 1.50-fold cell viability compared to oxidative group), α-amylase and α-glucosidase inhibitory activities (IC50 = 0.10 and 0.06, 0.56 and 0.32, and 3.98 and 2.16 mg/mL), and antibacterial activity (93.23, 40.85, and 80.42% reduced biofilm).

Melanin of fungi: from classification to application.

Melanin is a secondary metabolite composed of complex heterogeneous polymers. Fungal melanin is considered to be a sustainable and biodegradable natural pigment and has a variety of functional properties and biological activities. On one hand, due to its own specific properties it can play the role of antioxidant, anti-radiation, adsorption, and photoprotection. On the other hand, it has good biological activities such as hepatoprotective effect, hypolipidemic effect and anti-cancer. Therefore, it is widely used in various fields of daily life, including dyeing, food, biomedical and commercial industry. It is conducive to environmental protection and human health. However, the insolubility of fungal melanin in water, acids and organic solvents has been an obstacle to its commercial applications. Thus, the chemical modification methods of fungal melanin are summarized to increase its solubility and expand the application fields. Although fungal melanin has been used in many industries, as the structure and function of fungal melanin and modified melanin are further studied, more functional properties and bioactivities are expected to be discovered for a wide range of applications in the future.

Invasive Lobular Carcinoma Has Worse Outcome Compared with Invasive Ductal Carcinoma in Stage IV Breast Cancer with Bone-Only Metastasis.

Background: Invasive lobular carcinoma (ILC) is more likely to have bone metastasis than invasive ductal carcinoma (IDC). However, the prognosis for bone metastasis in ILC and IDC is barely known. So, the aim of this study was to investigate the difference of prognosis between ILC and IDC accompanied by bone metastasis. Methods: We evaluated the women with bone-only metastasis of defined IDC or ILC reported to the Surveillance, Epidemiology and End Results program from 2010 to 2016. Pearson's χ2 test was used to compare the differences of clinicopathologic factors between IDC and ILC. Univariate and multivariate analyses were performed to verify the effects of histological types (IDC and ILC) and other clinicopathologic factors on the overall survival (OS) and cancer-specific survival (CSS). Results: Overall, 3,647 patients with IDC and 945 patients with ILC met the inclusion criteria and were analyzed in our study. The patients with ILC were more likely to be older and to have lower histological grade and a higher proportion of the HR*/HER2- subtype. However, less treatment was administered to ILC than IDC, such as surgery of the breast, radiation, and chemotherapy. Compared to patients with IDC, patients with ILC showed worse OS (median OS, 36 and 42 months, respectively, p < 0.001) and CSS (median CSS, 39 and 45 months, respectively, p < 0.001), especially in subgroups with HR*/HER2- subtype (OS, hazard ratio: 1.501, 95% CI 1.270-1.773, p < 0.001; CSS, hazard ratio: 1.529, 95% CI 1.281-1.825, p < 0.001), lower histological grade (I-II) (OS, hazard ratio: 1.411, 95% CI 1.184-1.683, p < 0.001; CSS, hazard ratio: 1.488, 95% CI 1.235-1.791, p < 0.001), or tumor burden, such as T0-2 (OS, hazard ratio: 1.693, 95% CI 1.368-2.096, p < 0.001; CSS, hazard ratio: 1.76, 95% CI 1.405-2.205, p < 0.001) and N1-2 (OS, hazard ratio: 1.451, 95% CI 1.171-1.799, p = 0.001; CSS, hazard ratio: 1.488, 95% CI 1.187-1.865, p = 0.001). Furthermore, older age, black race, unmarried status, higher tumor burden (T3-4 and N3), triple-negative subtype, and higher histological grade were independent risk factors for both OS and CSS. Surgery of the breast and chemotherapy could significantly improve the prognosis for patients. Conclusion: Patients with ILC have worse outcomes compared to those with IDC when associated with bone-only metastasis, especially in subgroups with lower histological grade or tumor burden. More effective treatment measures may be needed for ILC, such as cyclin-dependent kinase 4/6 inhibitors, new targeted drugs, etc.