Breast cancer (BC) is the most common malignancy in women worldwide, and more effective biomarkers are urgently needed for the prevention and treatment of BC. Our study aimed to investigate the role of the HOXC gene family (HOXCs) and its relationship with the immune response in BC. The differential expression of HOXCs and its clinical prognostic significance in BC were explored using bioinformatics analysis, and the cBioPortal database was used to evaluate the genetic mutation profile of the HOXCs in BC. The results indicated that the expression levels of HOXC4, 10, 11, 12, and 13 were significantly increased in BC tissues compared with the normal tissues, and expressions of these genes were closely associated with BC stage, among them, high expression levels of HOXC10 and HOXC13 predicted poor outcome in BC patients. In addition, to elucidate the essential role of HOXCs in the tumor microenvironment and immunotherapeutic response of BC, the impact of HOXCs on the regulation of immune infiltration in BC was comprehensively assessed. The result showed that HOXC10 and HOXC13 expressions were significantly positively linked with the infiltration levels of CD8+T cell and M1 macrophage, while they were negatively related to Mast and Natural killer cells, suggesting the important influence of HOXCs on regulating tumor immunity in BC patients. Lastly, the RT-qPCR assay was employed to validate HOXCs expression in samples of BC patients. In conclusion, HOXCs may be a promising prognostic indicator and could regulate the immune infiltration in BC patients, thus being a promising targeted immunotherapy for BC.

Utilizing publicly available RNA-seq data to screen for ideal reference genes is more efficient and accurate than traditional methods. Previous studies have identified optimal reference genes in various chicken tissues, but none have specifically focused on the oviduct (including the infundibulum, magnum, isthmus, uterus, and vagina), which is crucial for egg production. Identifying stable reference genes in the oviduct is essential for improving research on gene expression levels. This study investigated genes with consistent expression patterns in the chicken oviduct, encompassing both individual oviduct tract tissues and the entire oviduct, by utilizing multiple RNA-seq datasets. The screening results revealed the discovery of 100 novel reference genes in each segment of oviduct tissues, primarily associated with cell cycle regulation and RNA binding. Moreover, the majority of housekeeping genes (HKGs) showed inconsistent expression levels across distinct samples, suggesting their lack of stability under varying conditions. The stability of the newly identified reference genes was assessed in comparison to previously validated stable reference genes in chicken oviduct and commonly utilized HKGs, employing traditional reference gene screening methods. HERPUD2, CSDE1, VPS35, PBRM1, LSM14A, and YWHAB were identified to be suitable novel reference gene for different parts of the oviduct. HERPUD2 and YWHAB were reliable for gene expression normalization throughout the oviduct tract. Furthermore, overexpression and interference assays in DF1 cells showed LSM14A and YWHAB play a crucial role in cell proliferation, highlighting the importance of these newly reference genes for further research. Overall, this study has expanded the options for reference genes in RT-qPCR experiments in different segments of the chicken oviduct and the entire oviduct.

Dracocephalum moldavica is widely used as an ornamental, medicine, and perfume in industry. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) is widely and accurately utilized for gene expression evaluations. Selecting optimal reference genes is essential for normalizing RT-qPCR results. However, the identification of suitable reference genes in D. moldavica has not been documented. A total of 12 reference genes in D. moldavica were identified by PEG6000 (15%) treatment under hypertonia conditions in different tissues (roots, stem, leaves, flower, seeds and sepal) and during three stages of flower development, then used to validate the expression stability. There were four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper) used to analyze the stability. Finally, the RefFinder program was employed to evaluate the candidate reference genes\' stability. The results showed that ACTIN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and EF1α (elongation factor-1α) were stable reference genes under the PEG6000 treatment. Heat shock protein 70 (HSP70) was the most stable gene across different flower development stages. ADP-ribosylation factor (ARF) was the most stable gene in different tissues and total samples. This study provides reliable gene expression studies for future research in D. moldavica.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus that undergoes rapid mutation. Based on viral whole genome sequencing analysis in Hebei Province, China, we identified several essential single nucleotide variants (SNVs) on primer-probe regions accumulating within some Omicron variants\' genomes. In this study, we focused on three SNVs, C28290T, T28297C, and C28311T emerging on 2019-nCoV-N1 (CDC-N1) primer-probe regions, recommended by CDC in 2020, and two SNVs, C26270T, A26275G emerging on E (Charité-E) primer-probe regions recommended by Charité, Germany. Our findings revealed that the presence of one or two SNVs in the primer or probe region affected the sensitivity of reverse transcription-quantitative polymerase chain reaction and droplet digital PCR to varying extents. This discovery underscores the importance of continuously monitoring the whole genome sequences of SARS-CoV-2 variants, especially the primer-probe targeting regions, and correspondingly updating commercial test kits or recommended primer-probe sequence sets.
OBJECTIVE: The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in a growing number of mutations in its genome, presenting new challenges for the diagnosis of SARS-CoV-2 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (RT-ddPCR) methods. There is an urgent need to develop refined methods for modifying primers and probes to improve the detection of these emerging variants. In this study, our focus was on the SNVs that have emerged in the CDC-N1 and Charité-E primer-probe regions. Our research has confirmed that the presence of these SNVs in the primer or probe region can significantly affect the results of coronavirus disease 2019 tests. we have developed and validated a modified detection method that can provide higher sensitivity and specificity. This study emphasizes the importance of refining the primer-probe sets to ensure the diagnostic accuracy of RT-qPCR and RT-ddPCR detection.

Extreme weather poses huge challenges for animals that must adapt to wide variations in environmental temperature and, in many cases, it can lead to the local extirpation of populations or even the extinction of an entire species. Previous studies have found that one element of amphibian adaptation to environmental stress involves changes in mitochondrial gene expression at low temperatures. However, to date, comparative studies of gene expression in organisms living at extreme temperatures have focused mainly on nuclear genes. This study sequenced the complete mitochondrial genomes of five Asian hylid frog species: Dryophytes japonicus, D. immaculata, Hyla annectans, H. chinensis and H. zhaopingensis. It compared the phylogenetic relationships within the Hylidae family and explored the association between mitochondrial gene expression and evolutionary adaptations to cold stress. The present results showed that in D. immaculata, transcript levels of 12 out of 13 mitochondria genes were significantly reduced under cold exposure (p < 0.05); hence, we put forward the conjecture that D. immaculata adapts by entering a hibernation state at low temperature. In H. annectans, the transcripts of 10 genes (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COX1, COX2 and ATP8) were significantly reduced in response to cold exposure, and five mitochondrial genes in H. chinensis (ND1, ND2, ND3, ND4L and ATP6) also showed significantly reduced expression and transcript levels under cold conditions. By contrast, transcript levels of ND2 and ATP6 in H. zhaopingensis were significantly increased at low temperatures, possibly related to the narrow distribution of this species primarily at low latitudes. Indeed, H. zhaopingensis has little ability to adapt to low temperature (4 °C), or maybe to enter into hibernation, and it shows metabolic disorder in the cold. The present study demonstrates that the regulatory trend of mitochondrial gene expression in amphibians is correlated with their ability to adapt to variable climates in extreme environments. These results can predict which species are more likely to undergo extirpation or extinction with climate change and, thereby, provide new ideas for the study of species extinction in highly variable winter climates.

BACKGROUND: AP2/ERF is a large family of plant transcription factor proteins that play essential roles in signal transduction, plant growth and development, and responses to various stresses. The AP2/ERF family has been identified and verified by functional analysis in various plants, but so far there has been no comprehensive study of these factors in Chinese prickly ash. Phylogenetic, motif, and functional analyses combined with transcriptome analysis of Chinese prickly ash fruits at different developmental stages (30, 60, and 90 days after anthesis) were conducted in this study.
RESULTS: The analysis identified 146 ZbAP2/ERF genes that could be classified into 15 subgroups. The motif analysis revealed the presence of different motifs or elements in each group that may explain the functional differences between the groups. ZbERF13.2, ZbRAP2-12, and ZbERF2.1 showed high levels of expression in the early stages of fruit development. ZbRAP2-4, and ZbERF3.1 were significantly expressed at the fruit coloring stage (R2 and G2). ZbERF16 were significantly expressed at fruit ripening and expression level increased as the fruit continued to develop. Relative gene expression levels of 6 representative ZbAP2/ERFs assessed by RT-qPCR agreed with transcriptome analysis results.
CONCLUSIONS: These genes identified by screening can be used as candidate genes that affect fruit development. The results of the analysis can help guide future genetic improvement of Chinese prickly ash and enrich our understanding of AP2/ERF transcription factors and their regulatory functions in plants.

UNASSIGNED: Forest musk deer (FMD, Moschus Berezovskii) is a critically endangered species world-widely, the death of which can be caused by pulmonary disease in the farm. Pulmonary fibrosis (PF) was a huge threat to the health and survival of captive FMD. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been involved in the regulation of immune genes and disease development. However, the regulatory profiles of mRNAs and miRNAs involved in immune regulation of FMD are unclear.
UNASSIGNED: In this study, mRNA-seq and miRNA-seq in blood were performed to constructed coexpression regulatory networks between PF and healthy groups of FMD. The hub immune- and apoptosis-related genes in the PF blood of FMD were explored through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Further, protein-protein interaction (PPI) network of immune-associated and apoptosis-associated key signaling pathways were constructed based on mRNA-miRNA in the PF blood of the FMD. Immune hub DEGs and immune hub DEmiRNAs were selected for experimental verification using RT-qPCR.
UNASSIGNED: A total of 2744 differentially expressed genes (DEGs) and 356 differentially expressed miRNAs (DEmiRNAs) were identified in the PF blood group compared to the healthy blood group. Among them, 42 DEmiRNAs were negatively correlated with 20 immune DEGs from a total of 57 correlations. The DEGs were significantly associated with pathways related to CD molecules, immune disease, immune system, cytokine receptors, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, and NOD-like receptor signaling pathway. There were 240 immune-related DEGs, in which 186 immune-related DEGs were up-regulated and 54 immune-related DEGs were down-regulated. In the protein-protein interaction (PPI) analysis of immune-related signaling pathway, TYK2, TLR2, TLR4, IL18, CSF1, CXCL13, LCK, ITGB2, PIK3CB, HCK, CD40, CD86, CCL3, CCR7, IL2RA, TLR3, and IL4R were identified as the hub immune genes. The mRNA-miRNA coregulation analysis showed that let-7d, miR-324-3p, miR-760, miR-185, miR-149, miR-149-5p, and miR-1842-5p are key miRNAs that target DEGs involved in immune disease, immune system and immunoregulation.
UNASSIGNED: The development and occurrence of PF were significantly influenced by the immune-related and apoptosis-related genes present in PF blood. mRNAs and miRNAs associated with the development and occurrence of PF in the FMD.

JHBp2 is a peptide purified from Jinhua ham broth with antibacterial activity against Salmonella typhimurium. Untargeted metabolomics and label-free quantitative proteomics were used to analyze metabolic and protein expression changes in S. typhimurium after JHBp2 treatment. Cell wall and membrane damage results indicate that JHBp2 has membrane-disruptive properties, causing leakage of intracellular nucleic acids and proteins. Metabolomics revealed 516 differentially expressed metabolites, involving cofactor biosynthesis, purine metabolism, ABC transporters, glutathione metabolism, pyrimidine metabolism, etc. Proteomics detected 735 differentially expressed proteins, involving pyruvate metabolism, amino acid biosynthesis, purine metabolism, carbon metabolism, glycolysis/gluconeogenesis, etc. RT-qPCR and proteomics results showed a positive correlation, and molecular docking demonstrated stable binding of JHBp2 to some differentially expressed proteins. In summary, JHBp2 could disrupt the S. typhimurium cell wall and membrane structure, interfere with synthesis of membrane-related proteins, trigger intracellular substance leak, and reduce levels of enzymes and metabolites involved in energy metabolism, amino acid anabolism, and nucleotide anabolism.

Controlling the hazard of sclerotia produced by the Sclerotinia sclerotiorum is very complex, and it is urgent to adopt an effective method that is harmonious environmentally to control the disease. Among the six isolates isolated from the rhizosphere of lettuce, the isolate HZA84 demonstrated a high activity in its antagonism towards Sclerotinia sclerotiorum in vitro, and produces siderophore. By amplification of internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and RNA polymerase II subunit (RPB2) genes, the isolate HZA84 was identified as Trichoderma asperellum, which was confirmed by analysis of phylogenetic tree. The Scanning electron microscope monitoring detected that the isolate HZA84 spread over the sclerotial surface, thus, damaging, decomposing, and distorting the globular cells of the outer cortex of the sclerotia. The Real-time polymerase chain reaction (RT-qPCR) analysis disclosed the overexpression of two genes (chit33 and chit37) encoding the endochitinase in addition to one gene (prb1) encoding the proteinase during 4 and 8 days of the parasitism behavior of isolate HZA84 on the sclerotia surface. These enzymes aligned together in the sclerotia destruction by hyperparasitism. On the other hand, the pots trial revealed that spraying of isolate HZA84 reduced the drop disease symptoms of lettuce. The disease severity was decreased by 19.33 and the biocontrol efficiency was increased by 80.67% within the fourth week of inoculation. These findings magnify the unique role of Trichoderma in disrupting the development of plant diseases in sustainable ways.

Iris color is a prominent phenotypic feature of quail. To understand the mechanism of melanin deposition related to quail iris color, iris tissues were selected from Beijing white and Chinese yellow quail for transcriptome analysis. Differentially expressed genes (DEGs) associated with pigmentation were identified using RNA sequencing and validated by quantitative real-time polymerase chain reaction (RT-qPCR). The identified single nucleotide polymorphisms were studied using bioinformatics and iris color correlation analyses. A total of 485 DEGs were obtained, with 223 upregulated and 262 downregulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed. Thirty-two genes were annotated using the GO database. Three important pigment synthesis pathways (Notch signaling, melanogenesis, and tyrosine metabolism) were identified in quail iris tissue (P < 0.05). The expression levels of solute carrier family 45 member 2 (SLC45A2), tyrosinase-related protein 1, vitamin D receptor, opsin 5, and docking protein 5 were significantly different between Beijing white and Chinese yellow quail, as verified by RT-qPCR. The c.1061C>T mutation in SLC45A2, which caused a single amino acid change at position 354 (threonine to methionine), was significantly associated with iris color in Beijing white and Chinese yellow quail, and might be the main reason for the different iris colors between these two quail species.