LTA and PGN from Bacillus siamensis can alleviate soybean meal-induced enteritis and microbiota dysbiosis in Lateolabrax maculatus.

An eight-week feeding trial was designed to assess which component of commensal Bacillus siamensis LF4 can mitigate SBM-induced enteritis and microbiota dysbiosis in spotted seabass (Lateolabrax maculatus) based on TLRs-MAPKs/NF-кB signaling pathways. Fish continuously fed low SBM (containing 16 % SBM) and high SBM (containing 40 % SBM) diets were used as positive (FM group) and negative (SBM group) control, respectively. After feeding high SBM diet for 28 days, fish were supplemented with B. siamensis LF4-derived whole cell wall (CW), cell wall protein (CWP), lipoteichoic acid (LTA) or peptidoglycan (PGN) until 56 days. The results showed that a high inclusion of SBM in the diet caused enteritis, characterized with significantly (P < 0.05) decreased muscular thickness, villus height, villus width, atrophied and loosely arranged microvillus. Moreover, high SBM inclusion induced an up-regulation of pro-inflammatory cytokines and a down-regulation of occludin, E-cadherin, anti-inflammatory cytokines, apoptosis related genes and antimicrobial peptides. However, dietary supplementation with CW, LTA, and PGN of B. siamensis LF4 could effectively alleviate enteritis caused by a high level of dietary SBM. Additionally, CWP and PGN administration increased beneficial Cetobacterium and decreased pathogenic Plesiomonas and Brevinema, while dietary LTA decreased Plesiomonas and Brevinema, suggesting that CWP, LTA and PGN positively modulated intestinal microbiota in spotted seabass. Furthermore, CW, LTA, and PGN application significantly stimulated TLR2, TLR5 and MyD88 expressions, and inhibited the downstream p38 and NF-κB signaling. Taken together, these results suggest that LTA and PGN from B. siamensis LF4 could alleviate soybean meal-induced enteritis and microbiota dysbiosis in L. maculatus, and p38 MAPK/NF-κB pathways might be involved in those processes.

Paraprobiotic and postbiotic forms of Bacillus siamensis improved growth, immunity, liver and intestinal health in Lateolabrax maculatus fed soybean meal diet.

Live commensal Bacillus siamensis LF4 showed reparative potentials against high SM-induced negative effects, but whether its paraprobiotic (heat-killed B. siamensis, HKBS) and postbiotic (cell-free supernatant, CFS) forms had reparative functions and potential mechanisms are not yet known. In this study, the reparative functions of HKBS and CFS were investigated by establishing an injured model of spotted seabass (Lateolabrax maculatus) treated with dietary high soybean meal (SM). The results showed that HKBS and CFS effectively mitigated growth suppression, immune deficiency, and liver injury induced by dietary high SM. Simultaneously, HKBS and CFS application positively shaped intestinal microbiota by increased the abundance of beneficial bacteria (Fusobacteria, Firmicutes, Bacteroidota, and Cetobacterium) and decreased harmful bacteria (Proteobacteria and Plesiomonasare). Additionally, HKBS and CFS improved SM-induced intestinal injury by restoring intestinal morphology, upregulating the expression of tight junction proteins, anti-inflammatory cytokines, antimicrobial peptides, downregulating the expression of pro-inflammatory cytokines and apoptotic proteins. Furthermore, HKBS and CFS intervention significantly activated TLR2, TLR5 and MyD88 signaling, and eventually inhibited p38 and NF-κB pathways. In conclusion, paraprobiotic (HKBS) and postbiotic (CFS) from B. siamensis LF4 can improve growth, immunity, repair liver and intestinal injury, and shape intestinal microbiota in L. maculatus fed high soybean meal diet, and TLRs/p38 MAPK/NF-κB signal pathways might be involved in those processes. These results will serve as a base for future application of paraprobiotics and postbiotics to prevent and repair SM-induced adverse effects in fish aquaculture.

Probiotic components of Bacillus siamensis LF4 mitigated ß-conglycinin caused cell injury via modulating TLR2/MAPKs/NF-κB signaling in Lateolabrax maculatus.

ß-conglycinin is a recognized factor in leading to intestinal inflammation and limiting application of soybean meal in aquaculture. Our previous study reported that heat-killed B. siamensis LF4 could effectively mitigate inflammatory response and apoptosis caused by ß-conglycinin in spotted seabass (Lateolabrax maculatus) enterocytes, but the mechanisms involved are not fully understood. In the present study, therefore, whole cell wall (CW), peptidoglycan (PG) and lipoteichoic acid (LTA) and cell-free supernatant (CFS) have been collected from B. siamensis LF4 and their mitigative function on ß-conglycinin-induced adverse impacts and mechanisms underlying were evaluated. The results showed that ß-conglycinin-induced cell injury, characterized with significantly decreased cell viability and increased activities of lactate dehydrogenase, glutamic oxaloacetic transaminase, glutamic propylic transaminase (P < 0.05), were reversed by subsequent heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS treatment. Enterocytes co-cultured with heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS (especially PG) significantly increased expressions of anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1), tight junction proteins (ZO-1, occludin and claudin-b) and antimicrobial peptides (ß-defensin, hepcidin-1, NK-lysin and piscidin-5), and decreased expressions of pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis-related genes (caspase 3, caspase 8 and caspase 9) (P < 0.05), indicating their excellent mitigation effects on ß-conglycinin-induced cell damages. In addition, heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS significantly increased TLR2 mRNA level (especially in PG treatment), and decreased MAPKs (JNK, ERK, p38 and AP-1) and NF-κB related genes expressions. In conclusion, heat-killed B. siamensis LF4 and its CW, LTA, PG and CFS could modulating TLR2/MAPKs/NF-κB signaling and alleviating ß-conglycinin-induced enterocytes injury in spotted seabass (L. maculatus), and PG presented the best potential.

Commensal Bacillus siamensis LF4 ameliorates ß-conglycinin induced inflammation in intestinal epithelial cells of Lateolabrax maculatus.

ß-conglycinin and glycinin, two major heat-stable anti-nutritional factors in soybean meal (SM), have been suggested as the key inducers of intestinal inflammation in aquatic animals. In the present study, a spotted seabass intestinal epithelial cells (IECs) were used to compare the inflammation-inducing effects of ß-conglycinin and glycinin. The results showed that IECs co-cultured with 1.0 mg/mL ß-conglycinin for 12 h or 1.5 mg/mL glycinin for 24 h significantly decreased the cell viability (P < 0.05), and overstimulated inflammation and apoptosis response by significantly down-regulating anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1) expressions and significantly up-regulated pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis genes (caspase 3, caspase 8 and caspase 9) expressions (P < 0.05). Subsequently, a ß-conglycinin based inflammation IECs model was established and used for demonstrating whether commensal probiotic B. siamensis LF4 can ameliorate the adverse effects of ß-conglycinin. The results showed ß-conglycinin-induced cell viability damage was completely repaired by treated with 109 cells/mL heat-killed B. siamensis LF4 for ≥12 h. At the same time, IECs co-cultured with 109 cells/mL heat-killed B. siamensis LF4 for 24 h significantly ameliorated ß-conglycinin-induced inflammation and apoptosis by up-regulating anti-inflammatory genes (IL-2, IL-4, IL-10 and TGF-ß1) expressions and down-regulated pro-inflammatory genes (IL-1ß, IL-8 and TNF-α) and apoptosis genes (caspase 3, caspase 8 and caspase 9) expressions (P < 0.05). In summary, both ß-conglycinin and glycinin can lead to inflammation and apoptosis in spotted seabass IECs, and ß-conglycinin is more effective; commensal B. siamensis LF4 can efficiently ameliorate ß-conglycinin induced inflammation and apoptosis in IECs.

Autochthonous Bacillus subtilis and Enterococcus faecalis improved liver health, immune response, mucosal microbiota and red-head disease resistance of yellow drum (Nibea albiflora).

Yellow drum (Nibea albiflora), a commercially important fish species in the coastal regions of southeast China, is highly susceptible to red-head disease caused by Vibrio harveyi B0003. Probiotics have been shown to enhance disease resistance in fish, but whether commensal probiotics could improve of the resistance to red-head disease in yellow drum and possible mechanisms has yet not been reported. A six-week feeding trial was conducted to investigate the red-head disease resistance potentials of five probiotic candidates (Bacillus megaterium B1M2, B. subtilis B0E9, Enterococcus faecalis AT5, B. velezensis DM5 and B. siamensis B0E14), and the liver health, serum and skin immunities, gut and skin mucosal microbiota of yellow drum were determined to illustrate the possible mechanisms. The results showed that autochthonous B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5, P < 0.05) effectively improved red-head disease resistance in yellow drum. Furthermore, B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5) efficiently improve liver health by improving liver morphology and decreasing serum glutamic oxaloacetic transaminase and glutamic propylic transaminase activities pre and post challenged with V. harveyi B0003 (P < 0.05). B. subtilis B0E9 and E. faecalis AT5 led to significant improvement (P < 0.05) in the serum complement 3 content (un-detected after challenged with V. harveyi B0003), lysozyme activity and skin mucosal immunity (such as IL-6, IL-10 and lysozyme expression) pre and post challenged with V. harveyi B0003, which was generally consistent with the cumulative mortality after challenged with V. harveyi B0003. This induced activations of serum and skin mucosal immunities were consistent with the microbiota data showing that B. subtilis B0E9 and E. faecalis AT5 modulated the overall structure of intestinal and skin mucosal microbiota, and in particular, the relative abundance of potentially pathogenic Achromobacter decreased while beneficial Streptococcus, Rothia, and Lactobacillus increased in fish fed with B. subtilis B0E9 and E. faecalis AT5. Overall, autochthonous B. subtilis B0E9 and E. faecalis AT5 (particularly E. faecalis AT5) can improve liver health, serum and skin immunities (especially up-regulated lysozyme activity and inflammation-related genes expression), positively shape gut and skin mucosal microbiota, and enhance red-head disease resistance of yellow drum.

Commensal Bacillus siamensis LF4 induces antimicrobial peptides expression via TLRs and NLRs signaling pathways in intestinal epithelial cells of Lateolabrax maculatus.

Antimicrobial peptides (AMPs) play an important role in modulating intestinal microbiota, and our previous study showed that autochthonous Baccilus siamensis LF4 could shape the intestinal microbiota of spotted seabass (Lateolabrax maculatus). In the present study, a spotted seabass intestinal epithelial cells (IECs) model was used to investigate whether autochthonous B. siamensis LF4 could modulate the expression of AMPs in IECs. And then, the IECs were treated with active, heat-inactivated LF4 and its supernatant to illustrate their AMPs inducing effects and the possible signal transduction mechanisms. The results showed that after 3 h of incubation with 108 CFU/mL B. siamensis LF4, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic propylic transaminase (GPT) activities in supernatant decreased significantly and obtained minimum values, while supernatant alkaline phosphatase (AKP) activity, ß-defensin protein level and IECs Na+/K+-ATPase activity, AMPs (ß-defensin, hepcidin-1, NK-lysin, piscidin-5) genes expression increased significantly and obtained maximum values (P < 0.05). Further study demonstrated that the active, heat-inactivated LF4 and its supernatant treatments could effectively decrease the LDH, GOT, and GPT activities in IECs supernatant, increase AKP activity and ß-defensin (except LF4 supernatant treatment) protein level in IECs supernatant and Na+/K+-ATPase and AMPs genes expression in IECs. Treatment with active and heat-inactivated B. siamensis LF4 resulted in significantly up-regulated the expressions of TLR1, TLR2, TLR3, TLR5, NOD1, NOD2, TIRAP, MyD88, IRAK1, IRAK4, TRAF6, TAB1, TAB2, ERK, JNK, p38, AP-1, IKKα, IKKß and NF-κB genes. Treatment with B. siamensis LF4 supernatant also resulted in up-regulated these genes, but not the genes (ERK, JNK, p38, and AP-1) in MAPKs pathway. In summary, active, heat-inactivated and supernatant of B. siamensis LF4 can efficiently induce AMPs expression through activating the TLRs/NLRs-MyD88-dependent signaling, active and heat-inactivated LF4 activated both the downstream MAPKs and NF-κB pathways, while LF4 supernatant only activated NF-κB pathway.

Microplitis bicoloratus parasitism promotes cyclophilin D-p53 interaction to induce apoptosis of hemocytes in Spodoptera litura.

Microplitis bicoloratus parasitism can induce apoptosis of hemocytes in the M. bicolortus host, Spodoptera litura. However, it is unclear how M. bicolortus parasitism regulates host signaling pathways to induce apoptosis. Expression of cyclophilin D (CypD) and p53 was significantly upregulated in S. litura hemocytes at 6 days postparasitization. In the parasitized hemocytes, there was mitochondrial membrane potential (△Ψm ) loss, cytochrome c (Cyt C) release from mitochondria, and caspase-3 activation. These occurred while hemocytes were undergoing upregulation of CypD and p53. Parasitism also promoted the interaction between CypD and p53. CypD silencing could rescue the apoptotic phenotypes induced by parasitism, but had no effect on apoptosis in unparasitized S. litura. These findings suggest that the CypD-p53 pathway may be an important component of the parasitism-induced immunosuppressive response and establish a basis for further studies of parasitoid/host interactions.

Insights to the superoxide dismutase genes and its roles in Hevea brasiliensis under abiotic stress.

The superoxide dismutase (SOD) protein significantly influences the development and growth of plants and their reaction to abiotic stresses. However, little is known about the characteristics of rubber tree SOD genes and their expression changes under abiotic stresses. The present study recognized 11 SOD genes in the rubber tree genome, including 7 Cu/ZnSODs, 2 MnSODs, and 2 FeSODs. Except for HbFSD1, SODs were scattered on five chromosomes. The phylogenetic analysis of SOD proteins in rubber trees and a few other plants demonstrated that the SOD proteins contained three major subgroups. Moreover, the genes belonging to the same clade contained similar gene structures, which confirmed their classification further. The extension of the SOD gene family in the rubber tree was mainly induced by the segmental duplication events. The cis-acting components analysis showed that HbSODs were utilized in many biological procedures. The transcriptomics data indicated that the phosphorylation of the C-terminal domain of RNA polymerase II might control the cold response genes through the CBF pathway and activate the SOD system to respond to cold stress. The qRT-PCR results showed that the expression of HbCSD1 was significantly downregulated under drought and salt stresses, which might dominate the adaption capability to different stresses. Additionally, salt promoted the expression levels of HbMSD1 and HbMSD2, exhibiting their indispensable role in the salinity reaction. The study results will provide a theoretical basis for deep research on HbSODs in rubber trees. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03328-7.

Preventive and reparative functions of host-associated probiotics against soybean meal induced growth, immune suppression and gut injury in Japanese seabass (Lateolabraxjaponicus).

A 56-day feeding trial was conducted to examine the preventive and reparative functions of host-associated probiotics against high soybean meal (SM)-induced negative effects in Japanese seabass (Lateolabrax japonicus). Fish continuously fed low SM (containing 16% SM) and high SM (containing 40% SM) diets were named as positive (PC) and negative (C) control, respectively. Preventive functions of probiotics were evaluated by continuously feeding diets LF3 (Lactococcus petauri LF3 supplemented in high SM diet, group PLF3) and LF4 (Bacillus siamensis LF4 supplemented in high SM diet, group PLF4), while reparative functions were estimated by feeding the high SM diet during 0-28 days, then feeding diets LF3 (group RLF3) and LF4 (group RLF4) until day 56. Compared with the group PC, suppressed growth and immunity, and damaged intestinal health were observed in the group C on days 28 and 56. Fish in groups PLF3 and PLF4, rather than in groups RLF3 and RLF4, showed higher growth compared with the group C and displayed similar immune status to the group PC, indicating that the initial and continued application of probiotic LF3 and LF4 can efficiently improve high SM induced growth and immune deficiency in Japanese seabass, but probiotics had limited reparative benefits when they were administrated at the middle of the feeding trial (28 d). Furthermore, probiotics showed good preventive functions and limited reparative functions on gut health via improving intestinal morphology and inflammation markers, for example, decreasing diamine oxidase activity and d-lactate content, while up-regulating anti-inflammatory TGF-ß1 expression and down-regulating pro-inflammatory TNF-α, IL-1ß, and IL-8 expressions. Moreover, dietary supplementation of probiotics (especially on day 56) could effectively shape the gut microbiota, such as significantly decreasing abundances of opportunistic pathogens (phylum Actinobacteria, genera Pseudomonas and Moheibacter on day 28, phylum Proteobacteria, genus Plesiomonas on day 56), significantly increasing gut microbial diversity and abundances of possible beneficial bacteria (phylum Bacteroidetes and genus Lactobacillus on day 28, phyla Firmicutes, Bacteroidetes and Cyanobacteria, genera Bacillus, Lactobacillus and Bacteroides on day 56). In conclusion, we evidenced for the first time that host-associated L. petauri LF3 and B. siamensis LF4 can provide effectively preventive and certain reparative functions against high SM-induced adverse effects in L. japonicus.

Delivery of sorafenib by myofibroblast-targeted nanoparticles for the treatment of renal fibrosis.

Fibrosis is an excessive accumulation of the extracellular matrix within solid organs in response to injury and a common pathway that leads functional failure. No clinically approved agent is available to reverse or even prevent this process. Herein, we report a nanotechnology-based approach that utilizes a drug carrier to deliver a therapeutic cargo specifically to fibrotic kidneys, thereby improving the antifibrotic effect of the drug and reducing systemic toxicity. We first adopted in vitro-in vivo combinatorial phage display technology to identify peptide ligands that target myofibroblasts in mouse unilateral ureteral obstruction (UUO)-induced fibrotic kidneys. We then engineered lipid-coated poly(lactic-co-glycolic acid) nanoparticles (NPs) with fibrotic kidney-homing peptides on the surface and sorafenib, a potent antineoplastic multikinase inhibitor, encapsulated in the core. Sorafenib loaded in the myofibroblast-targeted NPs significantly reduced the infiltration of α-smooth muscle actin-expressing myofibroblasts and deposition of collagen I in UUO-treated kidneys and enhanced renal plasma flow measured by Technetium-99m mercaptoacetyltriglycine scintigraphy. This study demonstrates the therapeutic potential of the newly identified peptide fragments as anchors to target myofibroblasts and represents a strategic advance for selective delivery of sorafenib to treat renal fibrosis. SIGNIFICANCE STATEMENT: Renal fibrosis is a pathological feature accounting for the majority of issues in chronic kidney disease (CKD), which may progress to end-stage renal disease (ESRD). This manuscript describes a myofibroblast-targeting drug delivery system modified with phage-displayed fibrotic kidney-homing peptides. By loading the myofibroblast-targeting nanoparticles (NPs) with sorafenib, a multikinase inhibitor, the NPs could suppress collagen synthesis in cultured human myofibroblasts. When given intravenously to mice with UUO-induced renal fibrosis, sorafenib loaded in myofibroblast-targeting NPs significantly ameliorated renal fibrosis. This approach provides an efficient therapeutic option to renal fibrosis. The myofibroblast-targeting peptide ligands and nanoscale drug carriers may be translated into clinical application in the future.

Autochthonous Probiotics Alleviate the Adverse Effects of Dietary Histamine in Juvenile Grouper (Epinephelus coioides).

High dose (0.3%) of dietary histamine can cause adverse effects on growth performance, innate immunity, and gut health in juvenile grouper (Epinephelus coioides). In the present study, three autochthonous probiotics (Bacillus pumilus SE5, Psychrobacter sp. SE6, and Bacillus clausii DE5) were supplemented separately to diets containing 0.3% of histamine and their effects on growth performance, innate immunity, and gut health of grouper (E. coioides) were evaluated in a 56-day feeding trial. The results showed considerable increase in weight gain, specific growth rate, hepatosomatic index, and decreased feed conversion rate in groupers fed with probiotic-supplemented diets. Supplementation of autochthonous probiotics has improved antioxidant capacity and innate immunity of E. coioides by measuring correlative parameters, such as total antioxidant capacity, superoxide dismutase activity, malondialdehyde content, and so on. Additionally, dietary probiotics have significantly reduced the levels of serum interleukin-1ß (at days 28 and 56), fatty acid-binding protein 2, and intestinal trefoil factor (at day 28), and promoted intestinal integrity following remarkably increased muscle thickness and mucosal fold height at day 56, especially in grouper fed with B. pumilus SE5 containing diet (P < 0.05). On day 56, the gut microbial composition of E. coioides was positively shaped by autochthonous probiotics, the relative abundance of potentially pathogenic Photobacterium decreased while beneficial Lactobacillus increased in fish fed with probiotic strains, especially with B. pumilus SE5 and B. clausii DE5. These results suggest that among the three autochthonous probiotic strains tested, B. pumilus SE5 is showing better efficiency in alleviating the adverse effects of (high levels) dietary histamine by decreasing the expression of inflammatory markers and by improving the growth, innate immunity, and gut health of juvenile grouper E. coioides.

Assistive Navigation Using Deep Reinforcement Learning Guiding Robot With UWB/Voice Beacons and Semantic Feedbacks for Blind and Visually Impaired People.

Facilitating navigation in pedestrian environments is critical for enabling people who are blind and visually impaired (BVI) to achieve independent mobility. A deep reinforcement learning (DRL)-based assistive guiding robot with ultrawide-bandwidth (UWB) beacons that can navigate through routes with designated waypoints was designed in this study. Typically, a simultaneous localization and mapping (SLAM) framework is used to estimate the robot pose and navigational goal; however, SLAM frameworks are vulnerable in certain dynamic environments. The proposed navigation method is a learning approach based on state-of-the-art DRL and can effectively avoid obstacles. When used with UWB beacons, the proposed strategy is suitable for environments with dynamic pedestrians. We also designed a handle device with an audio interface that enables BVI users to interact with the guiding robot through intuitive feedback. The UWB beacons were installed with an audio interface to obtain environmental information. The on-handle and on-beacon verbal feedback provides points of interests and turn-by-turn information to BVI users. BVI users were recruited in this study to conduct navigation tasks in different scenarios. A route was designed in a simulated ward to represent daily activities. In real-world situations, SLAM-based state estimation might be affected by dynamic obstacles, and the visual-based trail may suffer from occlusions from pedestrians or other obstacles. The proposed system successfully navigated through environments with dynamic pedestrians, in which systems based on existing SLAM algorithms have failed.

Complete chloroplast genome sequence and phylogenetic analysis of dragon fruit (Selenicereus undatus (Haw.) D.R.Hunt).

Selenicereus undatus (Haw.) D.R.Hunt is a member of the family Cactaceae. The chloroplast genome of S. undatus was sequenced, assembled, and annotated in the present study. The chloroplast genome was 133,326 bp in length, consisting of a typical quadripartite circle: a large single-copy region of 68,256 bp, two inverted repeat regions of 21,677 bp, and a small single copy region of 21,716 bp. A total of 120 predicted genes were identified, and a maximum likelihood was constructed, placing S. undatus as the sister taxon of Lophocereus schottii and Carnegiea gigantea, other members of the family Cactaceae.

The complete chloroplast genome and phylogenetic analysis of Nephelium lappaceum (rambutan).

Nephelium lappaceum is a popular tropical fruit belonging to the Sapindaceae family. The plant originated in Malaysia and Indonesia and is commonly called rambutan. Because of its refreshing flavor and exotic appearance, rambutan is widely accepted in the World. Due to its significant medicinal properties, the fruit has also been employed in traditional medicine for centuries. The chloroplast genome of rambutan was sequenced, assembled, and annotated in the present study. The chloroplast genome length was 161,356 bp and contained 132 genes, including 87 protein-coding genes, 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. It possessed the typical quadripartite circle structure with a large single-copy region (86,009 bp), a small single-copy region (18,153 bp), and two inverted repeat regions (28,597 bp). A total of 35 SSR markers were found in the chloroplast genome of Nephelium lappaceum, of which 33 were monomer, 1 was dimer and 1 was tetramer. Phylogenetic analysis based on the complete chloroplast genome sequences of 21 plant species showed that rambutan was closely related to Pometia tomentosa. These results provide a foundation for further phylogenetic and evolutionary studies of the Sapindaceae family.

Integration of Metabolome and Transcriptome Studies Reveals Flavonoids, Abscisic Acid, and Nitric Oxide Comodulating the Freezing Tolerance in Liriope spicata.

Liriope spicata is an evergreen perennial ornamental groundcover with a strong freezing tolerance. However, the molecular mechanism underlying the freezing tolerance in L. spicata remains unclear. In this study, a comprehensive investigation of L. spicata freezing tolerance was conducted at the levels of physiology and biochemistry, metabolite, and transcript during the stress treatment. There were 581 unique differentially expressed metabolites (DEMs) and 10,444 unique differentially expressed genes (DEGs) between freezing treatment and normal cultured plant in leaves. Integrated analysis of metabolomics and transcriptomics showed that flavonoid biosynthesis, carbohydrate metabolism, amino acid metabolism, lipid metabolism, and signal transduction pathways were prominently enriched in response to the freezing stress in L. spicata. Now, we identified genes and metabolites involved in the flavonoid pathway, abscisic acid (ABA) biosynthesis, and the oxidative synthesis pathway of nitric oxide (NO), which may form a regulatory network and play a synergistic effect in osmotic adjustment, reactive oxygen species (ROS) homeostasis, and stomatal closure under freezing stress. These results offer a comprehensive network of flavonoids, ABA, and NO comodulating the freezing tolerance in L. spicata.

The complete chloroplast genome of Hevea camargoana.

Hevea camargoana is a natural latex producing tropical plant and a close relative of H. brasiliensis, the primary commercial source of natural rubber. This study sequenced and analyzed the chloroplast genome of H. camargoana. The circular chloroplast genome of H. camargoana contains 161,291 bp with a GC content of 35.72%. This region contains two inverted repeat regions (26,819 bp), a large single-copy region (89,281 bp), and a small single-copy (18,372 bp) region in the complete chloroplast genome. A total of 134 genes were annotated, including 86 protein-coding genes, 36 transfer RNA genes, 8 ribosomal RNA genes, and 4 pseudogenes. The results showed that H. camargoana and H. brasiliensis were closely related, suggesting that H. camargoana may be used for the future variety improvement of rubber trees.

The complete chloroplast genome sequence of Chrysophyllum cainito, a semidomesticated species.

Chrysophyllum cainito is a semi-domesticated species widely cultivated in tropical regions, such as the Americas and Southeast Asia. In Yunnan, Guangdong, and Fujian Provinces, China, C. cainito is planted as an edible tropical fruit that was introduced from Southeast Asia. In this study, the chloroplast genome sequence of C. cainito was assembled and characterized using Illumina sequencing. The whole chloroplast genome of C. cainito is 158,841 bp long and consists of four regions: a large single-copy region (LSC, 88,256 bp), two inverted repeat regions (IRs, 25,958 bp), and a small single copy (SSC, 18,669 bp) region. The composition of the four bases in the circular chloroplast genome is 31.20% A, 32.00% T, 18.02% G, and 18.78% C, and the GC content of the entire C. cainito chloroplast genome is 36.8%. A total of 129 genes were annotated in the C. cainito chloroplast genome, of which 84 were protein-coding genes, 37 were transfer RNA (tRNA) genes, and eight were ribosomal RNA (rRNA) genes. The phylogenetic analysis indicated that C. cainito was most closely related to Pouteria campechiana. This study provides a foundation for further investigation of chloroplast genome evolution and genetic variation within semi-domesticated species.

Complete chloroplast genome of Hevea benthamiana, a SALB-resistant relative wild species of rubber tree.

Hevea benthamiana is a SALB-resistant wild species of H. brasiliensis, the only source of mass production of high quality natural rubber. This study sequenced and analyzed the chloroplast genome of H. benthamiana. The chloroplast genome of H. benthamiana contains 161,124 bp and consists of 51,495 bp of A (31.96%), 52,022 bp of T (32.29%), 28,915 bp of G (17.95%), and 28,692 bp of C (17.81%). The ring-shaped genome includes four regions: a large single-copy region (LSC, 89,110 bp), a small single copy (SSC, 18,376 bp) region, and two inverted repeat regions (IRs, 26,819 bp). A total of 134 genes were annotated, of which 86 encode proteins; four are pseudogenes; 36 are tRNA genes, and eight are rRNA genes. Phylogenetic analyses showed that H. benthamiana is very closely related to H. Brasiliensis, this result indicates that H. benthamiana is highly valuable for the breeding of SALB-resistant varieties of rubber trees.

[Three-dimensional finite element analysis of different endodontic access methods and full crown restoration in the maxillary central incisor].

OBJECTIVE: This study evaluates the effects of different endodontic access methods and full-ceramic crown on the stress distribution in the maxillary central incisor by using three-dimensional finite element analysis. METHODS: Computed tomography scans of the maxillary central incisor were used to construct a three-dimensional finite element model of the maxillary central incisor. According to the different methods of endodontic and the prosthetic treatments, four models were established, namely, group A (traditional access cavity preparation with resin filling), group B (traditional access cavity preparation restored full-ceramic crown), group C (minimally invasive endodontics with resin filling) and group D (minimally invasive endodontics restored full-ceramic crown). A static force of 100 N and a direction of 45° was applied to the long axis of the tooth at the junction of the incisal section one-third and middle section one-third. The maximum principal stress, the von Mises stress and the modified von Mises stress of the tooth tissue were analyzed using the finite-element analysis software. RESULTS: 1) Stress peaks: the stress peaks of the maximum principal stress, the von Mises stress and the modified von Mises in group A were the largest, except that the stress peak of von Mises stress in group D was slightly lower than that in group C. The stress peaks of the maximum principal stress and the modified von Mises in group C were the lowest. The stress peaks of the maximum principal stress and the modi-fied von Mises stress in group D were lower than those in groups A and B. 2) Stress distribution: compared with group A, the stress distribution of cervical dentin and the area of stress concentration in group C was lower and smaller. In the root dentin, the stress distribution in group C was more uniform than that in group A, and the stress was dispersed to several areas of the root apex. After crown restoration, no significant difference was observed in stress distribution between groups B and D in the root region. The stress distribution state of group B was not significantly different from that of group A. No significant difference was observed in the stress distribution state between groups D and C. CONCLUSIONS: 1) From the perspective of biomechanics, the minimally invasive access was adopted for the maxillary central incisor. 2) Full crown restoration is recommended after traditional access cavity preparation. No obvious advantage is observed in stress analysis for minimally invasive endodontics-restored full-ceramic crown.

Chloroplast genome of tropical and sub-tropical fruit tree Clausena lansium (Rutaceae).

The Clausena lansium, originally native to the southern part of China and has a long history of cultivation, is a tree member of the family Rutaceae. Chloroplast genome sequences play a significant role in the development of molecular markers in plant phylogenetic and population genetic studies. In this study, we report the complete chloroplast genome sequence of C. lansium for the first time (accession number of MH047377). The chloroplast genome is 159,284 bp long and includes 113 genes. It's LSC, SSC, and IR regions are 88,634, 18,896, and 25,877 bp long, respectively. Phylogenetic tree analysis exhibited that C. lansium was clustered with other Rutaceae species with high bootstrap values.