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Bacillus strains are widely used in food fermentation and plant disease control. An efficient transformation method is crucial for genetic manipulation in these organisms. To enhance the transformation efficiency of three Bacillus strains— Bacillus amyloliquefaciens YN-J3 (B.a YN-J3), Bacillus velezensis JN-Y2 (B.v JN-Y2) and  Bacillus subtilis S-16 (B.s S-16), we optimized transformation conditions using orthogonal experiments combined with response surface analysis. Additionally, we tested various cell wall agents to improve competence. Our results showed that the optimal transformation parameters for B.a YN-J3 and B.v JN-Y2 had an OD 600 of 0.70, a competent cell volume of 91 μL, a plasmid concentration of 1040 ng·μL⁻ 1 , and a field strength of 18.1 kV·cm⁻ 1 . For B.s S-16, the optimal conditions were an OD 600 of 0.71, a competent cell volume of 92 μL, a plasmid concentration of 1052 ng·μL⁻ 1 , and a field strength of 18.2 kV·cm⁻ 1 . Under these optimal conditions, the transformation efficiencies for B.a YN-J3, B.v JN-Y2, and B.s S-16 were 22,198.33 CFU·μg⁻ 1 DNA, 24,498.67 CFU·μg⁻ 1 DNA, and 23,305.00 CFU·μg⁻ 1 DNA, respectively. Screening of cell wall agents revealed that 50 mg/mL glycine significantly boosted transformation efficiency by 40, 36, and 24 times for B.a YN-J3, B.v JN-Y2, and B.s S-16, respectively. These findings demonstrate that combining glycine treatment with optimized transformation conditions provides an efficient approach for the genetic manipulation of Bacillus strains. Key points • The electroporation transformation parameters of three Bacillus were optimized by combining orthogonal experiments with response surface methodology • A stable and efficient electroporation transformation system suitable for three types of Bacillus was established. • 3. 50 mg/mL glycine solution can increase the transformation efficiency by 40, 36 and 24 times, respectively.

Background Endophytic bacteria serve as important resources for the development of novel biocontrol agents for sustainable agriculture. This study provides a detailed characterization of a newly isolated oat endophyte, Bacillus amyloliquefaciens Ba. YN.J3, using an integrated analysis of phenotypic, genomic, and comparative genomic data to explore its biocontrol and plant growth-promoting (PGP) potential. Results The current findings indicate that Ba. YN.J3 possessed efficient PGP and biocontrol potential both in vitro and in planta . Additionally, Ba. YN.J3 showed broad-spectrum antifungal activity against six major phytopathogens and was found to produce multiple cell wall-degrading enzymes (CWDEs) and siderophores, significantly increasing the growth of several crop species and regulating defense enzymes in oats. The complete 4.06 Mb genome of Ba. YN.J3 contains numerous gene clusters encoding vital secondary metabolites (e.g., surfactin, fengycin), CWDEs, and proteins associated with PGP functions and chemotaxis. The genome also harbors a robust set of genes related to abiotic stress tolerance, suggesting its potential to survive and function effectively in challenging field environments. Furthermore, comparative genomic analysis revealed 830 strain-specific genes, including two unique gene families potentially associated with chemotaxis (flagellar rod protein FlgC) and nitrogen metabolism and assimilation (regulatory protein YutI). Conclusions This integrated study provides genomic insights into the dual function of Ba. YN.J3 and its unique genetic determinants. The flgC and yutI gene families, in particular, offer potential genomics insights into its targeted antagonism and nutritional self-sufficiency. Hence, the current findings highlight Ba. YN.J3 as a promising candidate for the development of effective biopesticides and biofertilizers.

To evaluate the validity of two abbreviated protocols (AP) of MRI in breast cancer screening of dense breast tissue. This was a retrospective study in 356 participants with dense breast tissue and negative mammography results. The study was approved by the Nanjing Medical University Ethics Committee. Patients were imaged with a full diagnostic protocol (FDP) of MRI. Two APs (AP-1 consisting of the first post-contrast subtracted [FAST] and maximum-intensity projection [MIP] images, and AP-2 consisting of AP-1 combined with diffusion-weighted imaging [DWI]) and FDP images were analyzed separately, and the sensitivities and specificities of breast cancer detection were calculated. Of the 356 women, 67 lesions were detected in 67 women (18.8%) by standard MR protocol, and histological examination revealed 14 malignant lesions and 53 benign lesions. The average interpretation time of AP-1 and AP-2 were 37 seconds and 54 seconds, respectively, while the average interpretation time of the FDP was 3 minutes and 25 seconds. The sensitivities of the AP-1, AP-2, and FDP were 92.9, 100, and 100%, respectively, and the specificities of the three MR protocols were 86.5, 95.0, and 96.8%, respectively. There was no significant difference among the three MR protocols in the diagnosis of breast cancer ( > 0.05). However, the specificity of AP-1 was significantly lower than that of AP-2 ( = 0.031) and FDP ( = 0.035), while there was no difference between AP-2 and FDP ( > 0.05). The AP may be efficient in the breast cancer screening of dense breast tissue. FAST and MIP images combined with DWI of MRI are helpful to improve the specificity of breast cancer detection.

The aim of this study was to evaluate and compare the colour stabilities of three types of orthodontic clear aligners exposed to staining agents in vitro. Sixty clear orthodontic aligners produced by three manufacturers （Invisalign, Angelalign, and Smartee） were immersed in three staining solutions （coffee, black tea, and red wine） and one control solution （distilled water）. After 12-h and 7-day immersions, the aligners were washed in an ultrasonic cleaner and measured with a colourimeter. The colour changes （△E＊） were calculated on the basis of the Commission Internationale de I＇Eclairage L＊a＊b＊ colour system （CIE L＊a＊b＊）, and the results were then converted into National Bureau of Standards （NBS） units. Fourier transformation infrared （FT-IR） spectroscopy and scanning electron microscopy （SEM） were conducted to observe the molecular and morphologic alterations to the aligner surfaces, respectively. The three types of aligners exhibited slight colour changes after 12 h of staining, with the exception of the Invisalign aligners stained with coffee. The Invisalign aligners exhibited significantly higher AE＊ values （ranging from 0.30 to 27.81） than those of the Angelalign and Smartee aligners （AE＊ values ranging from 0.33 to 1.89 and 0.32 to 1.61, respectively, P〈O.05）. IFT-IR analysis confirmed that the polymer-based structure of aligners did not exhibit significant chemical differences before and after the immersions. The SEM results revealed different surface alterations to the three types of aligner materials after the 7-day staining. The three types of aesthetic orthodontic appliances exhibited colour stability after the 12-h immersion, with the exception of the Invisalign aligners stained by coffee. The Invisalign aligners were more prone than the Angelalign and Smartee aligners to pigmentation. Aligner materials may be improved by considering aesthetic colour stability properties.

•An optimized FAPα-targeted DNA vaccine was constructed.•DNA sequence homology between optimized FAPα and original FAPα was only 76%.•The vaccine induced more FAPα-specific CD8+ T cells.•Clearance of CAFs decreased the infiltration of MDSCs in tumor.•Mouse FAPα vaccine and human FAPα vaccine had similar anti-tumor effects. Cancer-associated fibroblasts (CAFs), major components of the tumor microenvironment (TME), promote tumor growth and metastasis and inhibit the anti-tumor immune response. We previously constructed a DNA vaccine expressing human FAPα, which is highly expressed by CAFs, to target these cells in the TME, and observed limited anti-tumor effects in the 4T1 breast cancer model. When the treatment time was delayed until tumor nodes formed, the anti-tumor effect of the vaccine completely disappeared. In this study, to improve the safety and efficacy, we constructed a new FAPα-targeted vaccine containing only the extracellular domain of human FAPα with a tissue plasminogen activator signal sequence for enhanced antigen secretion and immunogenicity. The number of CAFs was more effectively reduced by CD8+ T cells induced by the new vaccine. This resulted in decreases in CCL2 and CXCL12 expression, leading to a significant decrease in the ratio of myeloid-derived suppressor cells in the TME. Moreover, when mice were treated after the establishment of tumors, the vaccine could still delay tumor growth. To facilitate the future application of the vaccine in clinical trials, we further optimized the gene codons and reduced the homology between the vaccine and the original sequence, which may be convenient for evaluating the vaccine distribution in the human body. These results indicated that the new FAPα-targeted vaccine expressing an optimized secreted human FAPα induced enhanced anti-tumor activity by reducing the number of FAPα+ CAFs and enhancing the recruitment of effector T cells in the 4T1 tumor model mice.

Background Although mitochondrial dysfunction is an established hallmark of ventricular remodeling, the molecular mechanisms governing this process remain incompletely characterized. This study systematically investigates the regulatory role of Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) in AMPK-mediated mitochondrial dysfunction and its pathological consequences in ventricular remodeling. Methods Ventricular remodeling was induced by angiotensin II (Ang II) or isoproterenol (ISO) in vivo and in vitro, with assessment of hypertrophy and fibrosis using echocardiography, molecular analyses and histopathology. Bulk RNA-seq demonstrated that SHP099, an SHP2 inhibitor, modulated the AMPK pathway. LC-MS/MS and Co-IP confirmed the interaction between AMPK and SHP2. We constructed SHP2 mutant plasmids and employed AAV9-mediated cardiac SHP2 overexpression together with AMPK activator A769662 administration to validate the functional significance of this interaction. Results SHP2 expression is upregulated in Ang II- or ISO-induced ventricular remodeling models. Both SHP099 (pharmacological inhibition) or siSHP2 (genetic knockdown) ameliorated cardiac hypertrophy and mitochondrial dysfunction, whereas SHP2 overexpression exacerbated these pathological changes. Mechanistically, SHP2 directly interacts with AMPK via its protein tyrosine phosphatase (PTP) domain at cysteine 459, dephosphorylating AMPK at Thr172. AAV9-mediated SHP2 overexpression aggravated ventricular remodeling, which was rescued by AMPK activator A769662. Conclusions This study demonstrates that SHP2 acts as a key phosphatase directly dephosphorylates AMPK, thereby triggering mitochondrial dysfunction and exacerbating ventricular remodeling. Our findings provide novel mechanistic insights into heart failure progression and highlight SHP2 as a potential therapeutic target for heart failure treatment. Graphical Abstract Plain english summary SHP2 drives Ang II- or ISO-induced myocardial hypertrophy and fibrosis by dephosphorylating AMPK T172 and impairing mitochondrial function, establishing its role as a therapeutic target for ventricular remodeling.

Fibroblast activation protein α (FAPα) is a tumor stromal antigen overexpressed by cancer-associated fibroblasts (CAFs). CAFs are genetically more stable compared with the tumor cells and immunosuppressive components of the tumor microenvironment, rendering them excellent targets for cancer immunotherapy. DNA vaccines are widely applied due to their safety. To specifically destroy CAFs, we constructed and examined the immunogenicity and anti-tumor immune mechanism of a DNA vaccine expressing human FAPα. This vaccine successfully reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses which could kill CAFs, and the decrease in FAPα-expressing CAFs resulted in markedly attenuated expression of collagen I and other stromal factors that benefit the tumor progression. Based on these results, a DNA vaccine targeting human FAPα may be an attractive and effective cancer immunotherapy strategy.

Fibroblast activation protein α (FAPα) is expressed in cancer-associated fibroblasts (CAFs) of more than 90% of malignant epithelia carcinomas. CAFs are the main type of cells in the tumor microenvironment which offer nutrition and protection to the tumor and regulate immunosuppression. To eliminate CAFs, a vaccine targeting FAPα may be used with a heterologous prime-boost strategy to enhance the FAPα-specific cellular immunity. Here, a FAP vaccine using a recombinant adenovirus (rAd) vector was constructed as well as a DNA vaccine reported in our previous work. Although the DNA prime-rAd boost strategy enhanced FAPα-specific immune responses, improvement of anti-tumor immunity effects was not observed. Examination of immunosuppressive factors revealed that high expression of the IL-10 cytokine was considered the main cause of the failure of the prime-boost strategy. However, heterologous vaccination in combination with a low-dose of cyclophosphamide (CY), which was reported to reduce IL-10 production and promote a shift from immunosuppression to immunopotentiation, resulted in enhanced effects in terms of numbers of effector T cells and tumor growth inhibition rates, compared to the CY alone or DNA alone group. Tumor growth was inhibited markedly when the prime-boost strategy was combined with CY in both the prophylactic and therapeutic settings and the survival time of 4T1 tumor bearing mice was also prolonged significantly. With the reduction of IL-10, enhancement of the anti-tumor effect by the prime-boost strategy was observed. These results suggest that FAPα-targeted rAd boosting in combination with CY is an attractive approach to overcoming immunosuppression in cancer vaccines.

IMP （inosine 5＇-monophosphate） is a compound that enhances the flavor of poultry meat. IMP has become a new breeding trait to improve poultry meat quality. We tried to identify several potential regulatory genes, and construct their predicted regulatory relationships. Time series gene expression profiles of thigh muscle tissues of Rugao chicken, a famous indigenous breed in China, were performed for analysis of genes that are co-expressed or correlated with the concentration of IMP. We found 15 crucial co-expression genes, which are Hspa2, Pten, Gabpa, Bpi, Mkll, Srf,, Cd34, Hspa4, EtvS, Bmpr2, Gdel, IgfbpS, Cd28, Pecam1 and Gja1, that may directly or indirectly regulate IMP metabolism. Eventually, we computed the correlation coefficient between 19 IMP Genes and 15 CGs （15 co-expression genes）, and we identified and constructed a predicted regulation network. In conclusion, variation of IMP concentration was primarily connected with the muscle development process. During this process, 15 CGs were identified that may have significant influence on IMP metabolism. In particular, Bmpr2, Pten and co-expression genes correlated with Entpd8 might play important roles in regulating IMP de novo synthesis, decomposition and salvage synthesis.

The springback of tailor rolled blanks with quenching and partitioning steels was investigated.In order to find out the springback behavior and related influence factors for the novel sheets,both experimental and simulation methods have been used to compare and analyze the springback characteristics of equal thickness blanks and tailor rolled blanks in U-channel forming.From the results,the overall springback angles of tailor rolled blanks at thin and thick sides are respectively 106.79° and 99.705°,which are both lower than those of the corresponding equal thickness blanks.Due to the existence of the thickness transition zone,the stress distribution in thin and thick sides of blanks is changed.The location of dangerous region in thin side of tailor rolled blanks is closer to the end of side,and the thick side moved to the middle of straight wall,which are different with the equal thickness blanks.Afterwards,the released quantitles of tangential stress and strain per unit section of blank are adopted to calculate relative springback angles and give novel evaluation criteria for qualitatively analyzing the amount of springback angles.By comparing the results,it shows that the tangential strain method is more suitable for the actual situation.