Specific functions of single pistil S-RNases in S-gene homozygous Pyrus germplasm.

Gametophytic self-incompatibility (SI) is regulated by S-allele recognition; that is, pollen in a style with the same S-genotype will undergo programmed cell death and stop growing so that it is unable to complete double fertilization, ultimately resulting in the SI response. S-RNase is the female determinant of SI in pear (Pyrus). In the Pyrus genome, there are two different S-RNase alleles at the S-locus, which generate two different S-RNase products in the pistil. The extracted S-glycoprotein is actually a protein complex. In this study, artificial self-pollination was conducted at the bud stage to overcome SI in 'Huanghua' (S1S2) pear. Seven plants homozygous for S1-RNase and four homozygous for S2-RNase were selected from the selfed progeny of 'Huanghua' by S-gene molecular identification biotechnology. We investigated the function of single S-RNases isolated from the pistils of S-gene homozygous Pyrus germplasm. The pollen of 'Huanghua' could smoothly pass through the style of the S-gene homozygous germplasm and complete fertilization. S-RNases were extracted from flower styles of different genotypes and used to treat different types of pollen. The S-RNase from 'Huanghua' completely inhibited the growth of S1S2, S1S1, and S2S2 pollen, while the S-RNase from homozygous germplasm allowed some S1S2 pollen and different single genotypes of pollen to continue growing. These results further validate the core events of SI including cytoskeleton depolymerization and programmed cell death. By iTRAQ-based proteomic analysis of style proteins, a total of 13 S-RNase-related proteins were identified. In summary, we have created reliable S-RNase gene homozygous germplasm, which will play a crucial role in further research on SI in pear and in the development of the pear industry.

Rapid Detection of Alternaria Species Involved in Pear Black Spot Using Loop-Mediated Isothermal Amplification.

Alternaria species are the most important fungal pathogens that attack various crops as well as fruit trees such as pear and cause black spot disease. Here, a loop-mediated isothermal amplification (LAMP) assay is developed for the detection of Alternaria species. A. alternata cytochrome b (cyt-b) gene was used to design two pairs of primers and amplified a 229-bp segment of Aacyt-b gene. The results showed that LAMP assay is faster and simpler than polymerase chain reaction (PCR). LAMP assay is highly sensitive method for the detection of about 1 pg of genomic DNA of A. alternata by using optimized concentration of MgCl2 (4 mM) in final LAMP reaction. In contrast, the limit of detection was 1 ng of target DNA via conventional PCR. Among the genomic DNA of 46 fungal species, only the tubes containing DNA of Alternaria spp. except A. porri, A. solani, and A. infectoria changed color from orange to yellowish green with SYBR Green I including the main pathogens of pear black spot. The yellowish green color was indicative of DNA amplification. Moreover, LAMP assay was used for testing infected tissues among 22 healthy and diseased pear tissues; the orange color changed to yellowish green for infected tissues only. Altogether, we conclude that cyt-b gene can be used for the detection of Alternaria spp. via LAMP assay, which is involved in pear black spot disease.

The pomegranate (Punica granatum L.) genome and the genomics of punicalagin biosynthesis.

Pomegranate (Punica granatum L.) is a perennial fruit crop grown since ancient times that has been planted worldwide and is known for its functional metabolites, particularly punicalagins. We have sequenced and assembled the pomegranate genome with 328 Mb anchored into nine pseudo-chromosomes and annotated 29 229 gene models. A Myrtales lineage-specific whole-genome duplication event was detected that occurred in the common ancestor before the divergence of pomegranate and Eucalyptus. Repetitive sequences accounted for 46.1% of the assembled genome. We found that the integument development gene INNER NO OUTER (INO) was under positive selection and potentially contributed to the development of the fleshy outer layer of the seed coat, an edible part of pomegranate fruit. The genes encoding the enzymes for synthesis and degradation of lignin, hemicelluloses and cellulose were also differentially expressed between soft- and hard-seeded varieties, reflecting differences in their accumulation in cultivars differing in seed hardness. Candidate genes for punicalagin biosynthesis were identified and their expression patterns indicated that gallic acid synthesis in tissues could follow different biochemical pathways. The genome sequence of pomegranate provides a valuable resource for the dissection of many biological and biochemical traits and also provides important insights for the acceleration of breeding. Elucidation of the biochemical pathway(s) involved in punicalagin biosynthesis could assist breeding efforts to increase production of this bioactive compound.

The Synergetic Effect of 3D Printing and Electrospinning Techniques in the Fabrication of Bone Scaffolds.

The primary goal of bone tissue engineering is to restore and rejuvenate bone defects by using a suitable three-dimensional scaffold, appropriate cells, and growth hormones. Various scaffolding methods are used to fabricate three-dimensional scaffolds, which provide the necessary environment for cell activity and bone formation. Multiple materials may be used to create scaffolds with hierarchical structures that are optimal for cell growth and specialization. This study examines a notion for creating an optimal framework for bone regeneration using a combination of the robocasting method and the electrospinning approach. Research indicates that the integration of these two procedures enhances the benefits of each method and provides a rationale for addressing their shortcomings via this combination. The hybrid approach is anticipated to provide a manufactured scaffold that can effectively replace bone defects while possessing the necessary qualities for bone regeneration.

Analysis of shield tunnel response to bilateral pit excavation with a focus on perimeter pressure and deformation mechanisms.

This study aims to investigate the responses of shield tunnel structures subjected to disturbances caused by bilateral pit excavation, and it systematically reveals for the first time the impact mechanism of bilateral pit excavation on the distribution of perimeter pressure and deformation patterns of shield tunnels. Using a bilateral pit excavation project in Nanjing as a case study, this research establishes methods for calculating longitudinal displacement and circumferential pressure of tunnels under bilateral pit excavation conditions, employing the image source method for analysis. A refined three-ring segment model is developed, and the load structure method is used to analyze the impact of deep foundation excavation on the tunnel located between the two excavation sites. The results indicate that, compared to unilateral excavation, bilateral excavation significantly increases the perimeter pressure at the top and bottom of the tunnel, with a smaller increase in pressure at the arch waist. The deformation pattern is characterized by contraction at the top and bottom and expansion at the waist, forming a transverse elliptical deformation. The maximum vertical convergence values of the middle segment ring are 25.00 mm at the top and 25.88 mm at the bottom, with a vertical absolute convergence value of 44.5 mm and a convergence ratio (ΔDt/Dt) of 0.72%. As the foundation coefficient increases, the perimeter pressure at the top and bottom of the tunnel also increases. When the tunnel is closer to the foundation pits (Sp decreases), the perimeter pressure at the bottom of the tunnel increases. Conversely, as the distance between the two foundation pits (S) increases, the impact of excavation on the tunnel shifts from the upper part to the lower part, resulting in decreased upper perimeter pressure and increased lower perimeter pressure. The research findings provide important references for similar engineering projects.

Effects on stone cell development and lignin deposition in pears by different pollinators.

Introduction: The pear pulp is formed by the development of the ovary wall, which is the somatic cell of the female parent, and its genetic traits are identical to those of the female parent, so that its phenotypic traits should also be identical to those of the female parent. However, the pulp quality of most pears, especially the stone cell clusters (SCCs) number and degree of polymerization (DP), were significantly affected by the paternal type. Stone cells are formed by the deposition of lignin in parenchymal cell (PC) walls. Studies on the effect of pollination on lignin deposition and stone cell formation in pear fruit have not been reported. Methods: In this study, 'Dangshan Su' (P. bretschneideri Rehd.) was selected as the mother tree, while 'Yali' (P. bretschneideri Rehd.) and 'Wonhwang' (P. pyrifolia Nakai.) were used as the father trees to perform cross-pollination. We investigated the effects of different parents on SCCs number and DP, and lignin deposition by microscopic and ultramicroscopic observation. Results and Discussion: The results showed that the formation of SCCs proceeds was consistent in DY and DW, but the SCC number and DP in DY were higher than that in DW. Ultramicroscopy revealed that the lignification process of DY and DW were all from corner to rest regions of the compound middle lamella and the secondary wall, with lignin particles deposited along the cellulose microfibrils. They were alternatively arranged until they filled up the whole cell cavity to culminate in the formation of stone cells. However, the compactness of the wall layer of cell wall was significantly higher in DY than in DW. We also found that the pit of stone cell was predominantly single pit pair, they transported degraded material from the PCs that were beginning to lignify out of the cells. Stone cell formation and lignin deposition in pollinated pear fruit from different parents were consistent, but the DP of SCCs and the compactness of the wall layer were higher in DY than that in DW. Therefore, DY SCC had a higher ability to resist the expansion pressure of PC.

Silencing ApoC3 alleviates LPS-induced acute lung injury by inhibiting TLR signaling pathway.

This study aims to confirm whether apolipoprotein C3 (ApoC3) can regulate the inflammatory response and tissue damage in acute lung injury (ALI) and explore its regulatory pathway. ALI mouse model was established by intraperitoneal injection of lipopolysaccharide (LPS). ApoC3 levels were detected by real-time quantitative polymerase chain reaction, immunohistochemistry, and western blot assays. The levels of various inflammatory factors were detected by enzyme-linked immunosorbent assay and western blot analysis. Finally, the expression of toll-like receptor (TLR)/nuclear factor kappa B (NF-κB) signaling pathway-related protein [TLR2, myeloid differentiation primary response protein 88 (MyD88), IL-1 receptor-associated kinase 1 (IRAK1), NF-κB p65, and inhibitor of kappa B alpha (IκBα)], SLP adaptor and CSK interacting membrane protein (SCIMP), spleen tyrosine kinase (Syk), and phosphorylated (p)-Syk was detected by western blot analysis. ApoC3 was overexpressed in ALI mouse lung tissue and cell inflammation model. Silencing ApoC3 reduced inflammatory factors and alleviated lung tissue damage in ALI mice. Silencing ApoC3 reduced inflammatory factors and downregulated the expression of TLR2, MyD88, IRAK1, NF-κB p65, and increased IκBα expression in LPS-treated RAW264.7 cells. Moreover, co-transfection of si-TLR2 and shApoC3 further enhanced the inhibitory effects on the levels of inflammatory factors induced by silencing ApoC3. ApoC3 overexpression increased the levels of inflammatory factors and protein expression of SCIMP and p-Syk, while silencing TLR2 reversed the promotive effects of ApoC3 overexpression on above factors. In LPS-induced ALI mouse model and inflammatory cell model, downregulation of ApoC3 reduced inflammatory factors and relieved tissue damage. This process might be achieved through the TLR pathway.

[Penehyclidine hydrochloride regulates angiopoietin 2/vascular endothelial cadherin (Ang2/VE-cadherin) pathway to alleviate LPS induced lung injury in rats].

Objective To explore the effect and mechanism of penehyclidine hydrochloride (PHCD) on vascular endothelial injury in septic rats. Methods Fifty male SD rats were randomly divided into control group, lipopolysaccharide (LPS) induced sepsis group (model group), low dose PHCD (0.3 mg/kg) group, medium dose PHCD (1.0 mg/kg) group and high dose PHCD (3.0 mg/kg) groups, ten mice for each group. Normal saline was injected into the tail vein of the control group, and 10 mg/kg lipopolysaccharide (LPS) was injected into the tail vein of the rats in other groups to prepare the sepsis rat models. After the models were successfully established, low, medium and high doses (0.3, 1.0, 3.0 mg/kg) of PHCD solution were injected into the tail vein of the rats of corresponding groups. Wet/dry mass ratio (W/D) of lung tissue of rats in each group was measured, and ELISA was used to assay interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 content and rat plasma angiopoietin 2 (Ang2) content in bronchoalveolar lavage fluid (BALF). HE staining was used to observe the pathological changes of lung tissues. Immunohistochemical staining was used to observe the expression of Ang2 in the right lung tissues. Western blot analysis was performed to detect Ang2 and vascular endothelial cadherin (VE-cadherin) protein in lung tissues. Results Compared with the control group, the W/D ratio of the lung tissues of rats in the model group and the contents of IL-1ß, IL-6 and TNF-α in BALF were significantly increased; the lung tissues showed obvious pathological damage, with up-regulation of Ang2 expression and down-regulation of VE-Cadherin expression. Compared with the model group, the W/D ratio of the lung tissues of rats in three PHCD treatment groups and the contents of IL-1ß, IL-6 and TNF-α in BALF were significantly reduced; the pathological damage of lung tissue was significantly reduced, with down-regulation of Ang2 expression and up-regulation of VE-cadherin expression. Conclusion PHCD can reduce LPS-induced lung inflammation in rats with sepsis by regulating the Ang2/VE-Cadherin pathway, thereby improving vascular endothelial injury.

Inhibition of microRNA-665 Alleviates Septic Acute Kidney Injury by Targeting Bcl-2.

Sepsis can easily cause acute kidney injury (AKI) and seriously endanger human health. This article aims to investigate and study the role of microRNA-665 (miR-665) in septic AKI and the underlying molecular mechanism. Lipopolysaccharide (LPS) was used to construct cell and animal models of septic AKI. The expression of miR-665 in cells and kidney tissues was detected by quantitative reverse-transcription polymerase chain reaction (RT-PCR). The contents of inflammatory factors (TNF-α, IL-1ß, and IL-6) in the cell supernatant were detected using commercial kits. Renal tissue damage was observed by hematoxylin-eosin (HE) staining. Kidney function was assessed by serum Cr, serum BUN, and urine NAG levels. The apoptosis of HK-2 cells was analyzed by flow cytometry and TUNEL staining. Luciferase activity assay was performed for the verification of the target of miR-665. The expression of miR-665 was increased in the cell model and animal model of septic AKI constructed by LPS. By transfecting miR-665 inhibitor in HK-2 cells and injecting miR-665 antagomir (antagomiR-665) through the tail vein of rats, the expression of miR-665 in HK-2 cells and rat kidneys was remarkably reduced. Silencing miR-665 dramatically inhibited the expression of inflammatory factors (TNF-α, IL-1ß, and IL-6) in LPS-induced HK-2 cells and reduced LPS-induced apoptosis in HK-2 cells. At the same time, the levels of serum Cr, serum BUN, and urine NAG decreased markedly, and the damage of the kidney was also alleviated. Finally, luciferase reporter experiments demonstrated that miR-665 directly targets Bcl-2. We revealed that miR-665 expression was increased in septic AKI, and silencing miR-665 could inhibit LPS-induced inflammation and apoptosis of the kidney by targeting Bcl-2, thereby improving renal function.

Self-compatibility of 'Zaoguan' (Pyrus bretschneideri Rehd.) is associated with style-part mutations.

The pear cultivar 'Zaoguan' (S(4)S(34)) is the a self-compatible descendant of 'Yali' (S(21)S(34)) × 'Qingyun'(S(4)S(9)). Two self-incompatible cultivars 'Xinya' and 'Yaqing', also S-genotyped as S(4)S(34) for the S-RNase gene, were used as controls. Field pollination data revealed that 'Zaoguan' displayed SC, whereas 'Xinya' and 'Yaqing' showed self-incompatibility (SI) upon self-pollination. Reciprocal pollinations between the varieties showed that most of the 'Zaoguan' flowers pollinated with 'Xinya' or 'Yaqing' pollen set fruits but that few of the 'Xinya' or 'Yaqing' flowers set fruit when pollinated with 'Zaoguan' pollen. The pollen performance was monitored with fluorescence microscopy, and we observed that 'Zaoguan' accepted self-pollen as well as 'Xinya' or 'Yaqing' pollen, whereas 'Xinya' or 'Yaqing' rejected self-pollen and 'Zaoguan' pollen. The S(34)-RNase but not the S(4)-RNase could be detected in all selfed progeny of 'Zaoguan'. Comparisons of the 2D-PAGE profiles of the stylar extracts from the three cultivars showed that the S(4)-RNase protein expressed normally, but the S(34)-RNase of 'Zaoguan' was not found. Thus, we concluded that the stylar S(34) products were defective in 'Zaoguan' and that the S (4)-allele functioned normally. The nucleotide sequences of the S(4)- and S(34)-RNase of 'Zaoguan' showed no differences from those of 'Xinya' or 'Yaqing', and they transcribed normally. These results indicate that SC in 'Zaoguan' was due to the loss of the S(34)-RNase caused by unknown post-transcriptional factors.

Diversity of metabolite accumulation patterns in inner and outer seed coats of pomegranate: exploring their relationship with genetic mechanisms of seed coat development.

The expanded outer seed coat and the rigid inner seed coat of pomegranate seeds, both affect the sensory qualities of the fruit and its acceptability to consumers. Pomegranate seeds are also an appealing model for the study of seed coat differentiation and development. We conducted nontarget metabolic profiling to detect metabolites that contribute to the morphological differentiation of the seed coats along with transcriptomic profiling to unravel the genetic mechanisms underlying this process. Comparisons of metabolites in the lignin biosynthetic pathway accumulating in seed coat layers at different developmental stages revealed that monolignols, including coniferyl alcohol and sinapyl alcohol, greatly accumulated in inner seed coats and monolignol glucosides greatly accumulated in outer seed coats. Strong expression of genes involved in monolignol biosynthesis and transport might explain the spatial patterns of biosynthesis and accumulation of these metabolites. Hemicellulose constituents and flavonoids in particular accumulated in the inner seed coat, and candidate genes that might be involved in their accumulation were also identified. Genes encoding transcription factors regulating monolignol, cellulose, and hemicellulose metabolism were chosen by coexpression analysis. These results provide insights into metabolic factors influencing seed coat differentiation and a reference for studying seed coat developmental biology and pomegranate genetic improvement.

Identification of protein complexes associated with myocardial infarction using a bioinformatics approach.

Myocardial infarction (MI) is a leading cause of mortality and disability worldwide. Determination of the molecular mechanisms underlying the disease is crucial for identifying possible therapeutic targets and designing effective treatments. On the basis that MI may be caused by dysfunctional protein complexes rather than single genes, the present study aimed to use a bioinformatics approach to identifying complexes that may serve important roles in the development of MI. By investigating the proteins involved in these identified complexes, numerous proteins have been reported that are related to MI, whereas other proteins interacted with MI­related proteins, which implied that these protein complexes may indeed be related to the development of MI. The protein complexes detected in the present study may aid in our understanding of the molecular mechanisms that underlie MI pathogenesis.