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The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species.

Background Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by an abnormal accumulation of amyloid-β (Aβ) plaques, neuroinflammation, and impaired neurogenesis. Urolithin A (UA), a gut-microbial metabolite of ellagic acid, has been reported to exert anti-inflammatory effects in the brain. However, it is unknown whether UA exerts its properties of anti-inflammation and neuronal protection in the APPswe/PS1ΔE9 (APP/PS1) mouse model of AD. Methods Morris water maze was used to detect the cognitive function. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to detect neuronal apoptosis. Immunohistochemistry analyzed the response of glia, Aβ deposition, and neurogenesis. The expression of inflammatory mediators were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). The modulating effects of UA on cell signaling pathways were assayed by Western blotting. Results We demonstrated that UA ameliorated cognitive impairment, prevented neuronal apoptosis, and enhanced neurogenesis in APP/PS1 mice. Furthermore, UA attenuated Aβ deposition and peri-plaque microgliosis and astrocytosis in the cortex and hippocampus. We also found that UA affected critical cell signaling pathways, specifically by enhancing cerebral AMPK activation, decreasing the activation of P65NF-κB and P38MAPK, and suppressing Bace1 and APP degradation. Conclusions Our results indicated that UA imparted cognitive protection by protecting neurons from death and triggering neurogenesis via anti-inflammatory signaling in APP/PS1 mice, suggesting that UA might be a promising therapeutic drug to treat AD.

Polypoid species play significant roles in agriculture and food production. Many crop species are polyploid, such as potato, wheat, strawberry, and sugarcane. Genotyping has been a daunting task for genetic studies of polyploid crops, which lags far behind the diploid crop species. Single nucleotide polymorphism (SNP) array is considered to be one of, high-throughput, relatively cost-efficient and automated genotyping approaches. However, there are significant challenges for SNP identification in complex, polyploid genomes, which has seriously slowed SNP discovery and array development in polyploid species. Ploidy is a significant factor impacting SNP qualities and validation rates of SNP markers in SNP arrays, which has been proven to be a very important tool for genetic studies and molecular breeding. In this review, we (1) discussed the pros and cons of SNP array in general for high throughput genotyping, (2) presented the challenges of and solutions to SNP calling in polyploid species, (3) summarized the SNP selection criteria and considerations of SNP array design for polyploid species, (4) illustrated SNP array applications in several different polyploid crop species, then (5) discussed challenges, available software, and their accuracy comparisons for genotype calling based on SNP array data in polyploids, and finally (6) provided a series of SNP array design and genotype calling recommendations. This review presents a complete overview of SNP array development and applications in polypoid crops, which will benefit the research in molecular breeding and genetics of crops with complex genomes.

Sugarcane smut caused by Sporisorium scitamineum is a critical fungal disease in the sugarcane industry. However, molecular mechanistic studies of pathological response of sugarcane to S. scitamineum are scarce and preliminary. Here, transcriptome analysis of sugarcane disease induced by S. scitamineum at 24, 48 and 120 h was conducted, using an S. scitamineum-resistant and -susceptible genotype (Yacheng05-179 and \"ROC\"22). The reliability of Illumina data was confirmed by real-time quantitative PCR. In total, transcriptome sequencing of eight samples revealed gene annotations of 65,852 unigenes. Correlation analysis of differentially expressed genes indicated that after S. scitamineum infection, most differentially expressed genes and related metabolic pathways in both sugarcane genotypes were common, covering most biological activities. However, expression of resistance-associated genes in Yacheng05-179 (24-48 h) occurred earlier than those in \"ROC\"22 (48-120 h), and more transcript expressions were observed in the former, suggesting resistance specificity and early timing of these genes in non-affinity sugarcane and S. scitamineum interactions. Obtained unigenes were related to cellular components, molecular functions and biological processes. From these data, functional annotations associated with resistance were obtained, including signal transduction mechanisms, energy production and conversion, inorganic ion transport and metabolism, and defense mechanisms. Pathway enrichment analysis revealed that differentially expressed genes are involved in plant hormone signal transduction, flavonoid biosynthesis, plant-pathogen interaction, cell wall fortification pathway and other resistance-associated metabolic pathways. Disease inoculation experiments and the validation of in vitro antibacterial activity of the chitinase gene ScChi show that this sugarcane chitinase gene identified through RNA-Seq analysis is relevant to plant-pathogen interactions. In conclusion, expression data here represent the most comprehensive dataset available for sugarcane smut induced by S. scitamineum and will serve as a resource for finally unraveling the molecular mechanisms of sugarcane responses to S. scitamineum.

Background Alcohol dehydrogenases (ADHs) in plants are encoded by a multigene family. ADHs participate in growth, development, and adaptation in many plant species, but the evolution and function of the ADH gene family in sugarcane is still unclear. Results In the present study, 151 ADH genes from 17 species including 32 ADH genes in Saccharum spontaneum and 6 ADH genes in modern sugarcane cultivar R570 were identified. Phylogenetic analysis demonstrated two groups of ADH genes and suggested that these genes underwent duplication during angiosperm evolution. Whole-genome duplication (WGD)/segmental and dispersed duplications played critical roles in the expansion of ADH family in S. spontaneum and R570, respectively. ScADH3 was cloned and preferentially expressed in response to cold stress. ScADH3 conferred improved cold tolerance in E. coli cells. Ectopic expression showed that ScADH3 can also enhance cold tolerance in transgenic tobacco. The accumulation of reactive oxygen species (ROS) in leaves of transgenic tobacco was significantly lower than in wild-type tobacco. The transcript levels of ROS-related genes in transgenic tobacco increased significantly. ScADH3 seems to affect cold tolerance by regulating the ROS-related genes to maintain the ROS homeostasis. Conclusions This study depicted the size and composition of the ADH gene family in 17 species, and investigated their evolution pattern. Comparative genomics analysis among the ADH gene families of S. bicolor , R570 and S. spontaneum revealed their close evolutionary relationship. Functional analysis suggested that ScADH3 , which maintained the steady state of ROS by regulating ROS-related genes, was related to cold tolerance. These findings will facilitate research on evolutionary and functional aspects of the ADH genes in sugarcane, especially for the understanding of ScADH3 under cold stress.

Objective To evaluate the efficacy and safety of anlotinib plus temozolomide (TMZ) as a first-line treatment for recurrent/Residual glioma. Methods Thirty eligible patients who either relapsed after the standard chemoradiotherapy regimen (TMZ plus radiotherapy) or had macroscopic residual tumors due to involvement of eloquent brain areas were enrolled between March 2018 and January 2021. Patients received anlotinib (12 mg once daily, 14 days on/7 days off) in combination with TMZ (200 mg/m², 5 days on/23 days off) until disease progression or unacceptable toxicity. The efficacy was evaluated using the Response Assessment in Neuro-Oncology(RANO) criteria for high-grade gliomas. Safety was assessed using the NCI-CTCAE 4.0. Survival outcomes were estimated with the Kaplan-Meier method and compared using the log-rank test. Results By April 2023, the median follow-up time was 28.1 ± 5.07 months(95% CI:18.20-38.06). The median overall survival (OS, measured from recurrence to death or last follow-up) was 17.87 ± 4.29 months (95% CI: 9.46–26.28). 1-year, 1.5 year, 2-year OS rates were 60.0%, 46.7%, and 36.7%, respectively. The median progression-free survival (PFS, measured from initiation of Anlotinib plus TMZ) was 7.83 ± 0.82 months(95% CI, 6.22–9.44). The 6-month, 1-year PFS rates were 63.3% and 36.7%, respectively. Univariate analysis showed that patients with WHO grade 2 gliomas had significantly longer median OS and PFS compared with those with grade 3 or 4 gliomas (grade 2: 30.45 ± 4.32, grade 3: 15.07 ± 5.72, grade 4: 9.21 ± 1.90, P  = 0.035). Patients aged ≤ 55 years had significantly longer median PFS than those > 55 years (12.97 ± 1.71 vs. 7.33 ± 1.95. p  = 0.010), although the difference in OS did not reach statistical difference (19.90 ± 6.29 m vs. 10.53 ± 1.68 m, p  = 0.106). Additionally, sex, 1p/19q and IDH mutations were not significant negative predictors of OS or PFS. Multivariate analysis further indicated that age, pathological grade, and IDH mutation were not independent predictors of PFS or OS in recurrent glioma. Conclusion Anlotinib combined with TMZ demonstrated potential efficacy for recurrent glioma, as reflected in observed OS and PFS outcomes, and demonstrated an acceptable tolerability profile in this patient cohort. Larger randomized controlled trials are warranted to confirm these findings and further define the role of anlotinib plus TMZ for the management of recurrent glioma.

Background Tailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering. Results In this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UAS F-E-C -Core1 minimal promoter as a library template and determined its Kozak motif (K 0 ). Next, we randomly mutated the K 0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K 0 control. The best one named K 528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UAS F-E-C -Core1 and the strong native constitutive promoter P TDH3 , respectively. Subsequently, we chose three representative strong chimeric promoters (K 540 , K 536 , and K 528 ) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K 528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K 0 control (3.1 mg/L). Conclusions All these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

The digital content industry is developing rapidly worldwide, with the Yangtze River Delta (YRD) region emerging as China’s representative hub through its comprehensive industrial chain, enterprise clusters, and innovation capacity. The coordinated development of the digital content industry in the YRD is crucial for promoting regional integration and enhancing industry competitiveness. While industrial policies play a vital guiding role, there is a lack of systematic evaluation from a regional perspective. This study uses the Policy Modeling Consistency (PMC) Index model to systematically evaluate 54 Digital Content Industry Policies (DCIPs) in the YRD to assess policy characteristics, gaps, and regional coordination foundations. The results indicate that the region has established a relatively mature policy framework that includes various policy types, evidence-based decision-making processes, and well-defined objectives. It also faces challenges, such as homogeneous types of policy instruments, vague incentive measures, and insufficient regional policy synergy. From the perspective of inter-provincial differentiation comparison, Shanghai achieves the most balanced and effective policy formulation, Jiangsu excels in balancing policy quantity and quality, Zhejiang stands out for its forward-looking approach but lacks innovation support, while Anhui falls behind in both policy coverage and effectiveness. The research proposes actionable recommendations, including establishing coordinated mechanisms to promote specialized industrial divisions, fostering a collaborative policy community, optimizing policy instruments, and implementing a dynamic policy adjustment mechanism. Together, they aim to enhance the coordinated development and competitiveness of the digital content industry in the YRD.

Background Cold is a major abiotic stress limiting the production of tropical and subtropical crops in new production areas. Sugarcane ( Saccharum spp.) originates from the tropics but is cultivated primarily in the sub-tropics where it frequently encounters cold stress. Besides regulating plant growth, miRNAs play an important role in environmental adaption. Results In this study, a total of 412 sugarcane miRNAs, including 261 known and 151 novel miRNAs, were obtained from 4 small RNA libraries through the Illumina sequencing method. Among them, 62 exhibited significant differential expression under cold stress, with 34 being upregulated and 28 being downregulated. The expression of 13 miRNAs and 12 corresponding targets was validated by RT-qPCR, with the majority being consistent with the sequencing data. GO and KEGG analysis indicated that these miRNAs were involved in stress-related biological pathways. To further investigate the involvement of these miRNAs in tolerance to abiotic stresses, sugarcane miR156 was selected for functional analysis. RT-qPCR revealed that miR156 levels increased in sugarcane during cold, salt and drought stress treatments. Nicotiana benthamiana plants transiently overexpressing miR156 exhibited better growth status, lower ROS levels, higher anthocyanin contents as well as the induction of some cold-responsive genes, suggesting its positive role in the plant cold stress response. Conclusions This study provides a global view of the association of miRNA expression with the sugarcane response to cold stress. The findings have enriched the present miRNA resource and have made an attempt to verify the involvement of miR156 in plant response to cold stress.

Circular RNAs (circRNAs) are a new group of endogenous RNAs recently found to be involved in the development of various diseases, including their confirmed involvement in the progression of several types of cancers. Unluckily, the abnormal expression and functions of circRNAs in breast cancer shall be further investigated. This work aims to elucidate the action and molecular mechanism of circHSDL2 in the malignant progression of breast cancer. Differential expression profiles of circRNAs in breast cancer tissues relative to normal breast tissues and in the exosomes of breast cancer patients compared to healthy women were analyzed from databases to identify potentially functional circRNAs. CircHSDL2 was selected for further investigation. Cell proliferation, migration and invasion assays were done to assess the effect of circHSDL2 overexpression on breast cancer cells. Bioinformatics test and dual-luciferase reporter experiments were done to explore the interaction between circHSDL2 and miRNA. Downstream target genes were further investigated through proteomics analysis and Western blotting. The influence of circHSDL2 on breast cancer in vivo was evaluated through xenograft experiments in nude mice. Functional analysis demonstrated circHSDL2 overexpression promoted the division, movement, and invasion of breast cancer cells both in vivo and in vitro. Mechanistically, circHSDL2 acted as a sponge for miR-7978 to affect ZNF704 expression and thereby regulate the Hippo pathway in breast cancer cells. In conclusion, circHSDL2 regulates the Hippo pathway through the miR-7978/ZNF704 axis to facilitate the malignancy of breast cancer. This may be a potential biomarker and treatment target.