Physiological, Biochemical, and Molecular Analyses Reveal Dark Heartwood Formation Mechanism in Acacia melanoxylon.

Acacia melanoxylon is highly valued for its commercial applications, with the heartwood exhibiting a range of colors from dark to light among its various clones. The underlying mechanisms contributing to this color variation, however, have not been fully elucidated. In an effort to understand the factors that influence the development of dark heartwood, a comparative analysis was conducted on the microstructure, substance composition, differential gene expression, and metabolite profiles in the sapwood (SW), transition zone (TZ), and heartwood (HW) of two distinct clones, SR14 and SR25. A microscopic examination revealed that heartwood color variations are associated with an increased substance content within the ray parenchyma cells. A substance analysis indicated that the levels of starches, sugars, and lignin were more abundant in SP compared to HW, while the concentrations of phenols, flavonoids, and terpenoids were found to be higher in HW than in SP. Notably, the dark heartwood of the SR25 clone exhibited greater quantities of phenols and flavonoids compared to the SR14 clone, suggesting that these compounds are pivotal to the color distinction of the heartwood. An integrated analysis of transcriptome and metabolomics data uncovered a significant accumulation of sinapyl alcohol, sinapoyl aldehyde, hesperetin, 2', 3, 4, 4', 6'-peptahydroxychalcone 4'-O-glucoside, homoeriodictyol, and (2S)-liquiritigenin in the heartwood of SR25, which correlates with the up-regulated expression of CCRs (evm.TU.Chr3.1751, evm.TU.Chr4.654_667, evm.TU.Chr4.675, evm.TU.Chr4.699, and evm.TU.Chr4.704), COMTs (evm.TU.Chr13.3082, evm.TU.Chr13.3086, and evm.TU.Chr7.1411), CADs (evm.TU.Chr10.2175, evm.TU.Chr1.3453, and evm.TU.Chr8.1600), and HCTs (evm.TU.Chr4.1122, evm.TU.Chr4.1123, evm.TU.Chr8.1758, and evm.TU.Chr9.2960) in the TZ of A. melanoxylon. Furthermore, a marked differential expression of transcription factors (TFs), including MYBs, AP2/ERFs, bHLHs, bZIPs, C2H2s, and WRKYs, were observed to be closely linked to the phenols and flavonoids metabolites, highlighting the potential role of multiple TFs in regulating the biosynthesis of these metabolites and, consequently, influencing the color variation in the heartwood. This study facilitates molecular breeding for the accumulation of metabolites influencing the heartwood color in A. melanoxylon, and offers new insights into the molecular mechanisms underlying heartwood formation in woody plants.

Comparative physiological, biochemical, metabolomic, and transcriptomic analyses reveal the formation mechanism of heartwood for Acacia melanoxylon.

Acacia melanoxylon is well known as a valuable commercial tree species owing to its high-quality heartwood (HW) products. However, the metabolism and regulatory mechanism of heartwood during wood development remain largely unclear. In this study, both microscopic observation and content determination proved that total amount of starches decreased and phenolics and flavonoids increased gradually from sapwood (SW) to HW. We also obtained the metabolite profiles of 10 metabolites related to phenolics and flavonoids during HW formation by metabolomics. Additionally, we collected a comprehensive overview of genes associated with the biosynthesis of sugars, terpenoids, phenolics, and flavonoids using RNA-seq. A total of ninety-one genes related to HW formation were identified. The transcripts related to plant hormones, programmed cell death (PCD), and dehydration were increased in transition zone (TZ) than in SW. The results of RT-PCR showed that the relative expression level of genes and transcription factors was also high in the TZ, regardless of the horizontal or vertical direction of the trunk. Therefore, the HW formation took place in the TZ for A. melanoxylon from molecular level, and potentially connected to plant hormones, PCD, and cell dehydration. Besides, the increased expression of sugar and terpenoid biosynthesis-related genes in TZ further confirmed the close connection between terpenoid biosynthesis and carbohydrate metabolites of A. melanoxylon. Furthermore, the integrated analysis of metabolism data and RNA-seq data showed the key transcription factors (TFs) regulating flavonoids and phenolics accumulation in HW, including negative correlation TFs (WRKY, MYB) and positive correlation TFs (AP2, bZIP, CBF, PB1, and TCP). And, the genes and metabolites from phenylpropanoid and flavonoid metabolism and biosynthesis were up-regulated and largely accumulated in TZ and HW, respectively. The findings of this research provide a basis for comprehending the buildup of metabolites and the molecular regulatory processes of HW formation in A. melanoxylon.

Correction: Zhang et al. Genotype-Environment Interaction and Horizontal and Vertical Distributions of Heartwood for Acacia melanoxylon R.Br. Genes 2023, 14, 1299.

In the original publication [...].

Plasma-derived exosomal immunoglobulins IGHV4-4 and IGLV1-40 as new non-small cell lung cancer biomarkers.

Exosomal proteins represent valuable research directions in the liquid biopsy of lung cancer (LC). Immunoglobulin subtypes, immunoglobulin molecules with different domains in variable regions, are products of B cell responses to different tumor antigens and are associated with tumor incidence and development. The plasma of patients with LC should theoretically contain a large number of B cell-derived exosomes that specifically recognize tumor antigens. This paper intended to assess the value of the proteomic screening of plasma exosomal immunoglobulin subtypes for diagnosing non-small cell LC (NSCLC). The plasma exosomes of NSCLC patients and healthy control participants (HCs) were isolated using ultracentrifugation. Label-free proteomics was employed to assess the differentially expressed proteins (DEPs), while the biological characteristics of the DEPs were analyzed using GO enrichment. The immunoglobulin content in the top two fold change (FC) values of the DEPs and the immunoglobulin with the lowest P-value were verified using an enzyme-linked immunosorbent assay (ELISA). The differentially expressed immunoglobulin subtypes verified via ELISA were selected to statistically analyze the receiver operating characteristic curve (ROC), after which the diagnostic values of the NSCLC immunoglobulin subtypes were determined via the ROC area under the curve (AUC). The plasma exosomes of the NSCLC patients contained 38 DEPs, of which 23 were immunoglobulin subtypes, accounting for 60.53%. The DEPs were mainly related to the binding between immune complexes and antigens. The ELISA results showed significant differences between the immunoglobulin heavy variable 4-4 (IGHV4-4) and immunoglobulin lambda variable 1-40 (IGLV1-40) in the LC patients and HCs. Compared with the HCs, the AUCs of IGHV4-4, IGLV1-40, and a combination of the two in diagnosing NSCLC were 0.83, 0.88, and 0.93, respectively, while the AUCs for non-metastatic cancer were 0.80, 0.85, and 0.89. Moreover, their diagnostic values for metastatic cancer compared to non-metastatic cancer displayed AUCs of 0.71, 0.74, and 0.83, respectively. When IGHV4-4 and IGLV1-40 were combined with serum CEA to diagnose LC, the AUC value increased, exhibiting values of 0.95, 0.89, and 0.91 for the NSCLC, non-metastatic, and metastatic groups, respectively. Plasma-derived exosomal immunoglobulins containing IGHV4-4 and IGLV 1-40 domains can provide new biomarkers for diagnosing NSCLC and metastatic patients.

Genotype-Environment Interaction and Horizontal and Vertical Distributions of Heartwood for Acacia melanoxylon R.Br.

Acacia melanoxylon (blackwood) is a valuable wood with excellent-quality heartwood extensively utilized worldwide. The main aim of this study was to confirm the horizontal and vertical variation and provide estimated values of genetic gains and clonal repeatabilities for improving breeding program of A. melanoxylon. Six blackwood clones at 10 years old were analyzed in Heyuan and Baise cities in China. Stem trunk analysis was conducted for sample trees to explore the differences between heartwood and sapwood. The heartwood radius (HR), heartwood area (HA), and heartwood volume (HV) in heartwood properties decreased as tree height (H) in growth traits increased, and the HV = 1.2502 DBH (diameter at breast height)1.7009 model can accurately estimate the heartwood volume. Furthermore, G × E analysis showed that the heritabilities of the eleven indices, including DBH, DGH (diameter at ground height), H, HR, SW (sapwood width), BT (bark thickness), HA, SA (sapwood area), HV, HRP (heartwood radius percentage), HAP (heartwood area percentage), and HVP (heartwood volume percentage) were between 0.94 and 0.99, and repeatabilities of the eleven indices were between 0.74 and 0.91. Clonal repeatability of DBH (0.91), DGH (0.88), and H (0.90) in growth traits, HR (0.90), HVP (0.90), and HV (0.88) in heartwood properties were slightly higher than for SA (0.74), SW (0.75), HAP (0.75), HRP (0.75), and HVP (0.75). These data also implied that the growth characteristics of heartwood and sapwood of blackwood clones were less affected by the environment and had substantial heritability.

Transmembrane TNF-α as a Novel Biomarker for the Diagnosis of Cytokine Storms in a Mouse Model of Multiple Organ Failure.

A cytokine storm (CS) is an out-of-control inflammatory response closely associated with the progression of diseases, such as multiple organ failure (MOF), severe sepsis, and severe or critical COVID-19. However, there is currently a lack of reliable diagnostic markers to distinguish CS from normal inflammatory responses. Tumor necrosis factor-α (TNF-α) includes transmembrane TNF-α (tmTNF-α) and secreted TNF-α (sTNF-α). The MOF mouse model in this study showed that the tmTNF-α expression changes in the neutrophils differed from the serum TNF-α and serum IL-18, INF-γ, IL-4, and IL-6. Furthermore, tmTNF-α, instead of serum TNF-α, IL-18, INF-γ, IL-4, and IL-6, reflected liver and kidney tissue damage and increased with the aggravation of these injuries. Analysis of the ROC results showed that tmTNF-α effectively distinguished between inflammatory responses and CS and efficiently differentiated between surviving and dead mice. It also significantly improved the diagnostic value of the traditional CRP marker for CS. These results indicated that the tmTNF-α expressed in the neutrophils could be used to diagnose CS in MOF mice, providing an experimental basis to further develop tmTNF-α for diagnosing CS patients.

Influences of pulsed light-UV treatment on the storage period of dry-cured meat and shelf life prediction by ASLT method.

In order to prolong the shelf life of Chinese traditional dry-cured meat products, a pulsed ultraviolet light (PL-UV) irradiation method was adopted to treat meat products according to the following parameters: pulse energy of 8 J, 300 pulses, and an effective exposure distance of 10 cm; the UV light irradiation power of 6 W, an effective exposure distance of 11 cm, and an exposure period of 5 min. After a pulsed ultraviolet irradiation, total bacterial count in dry-cured meat decreased from 6.89 to 4.53 lg (CFU/g). The number of Micrococcus and Staphylococcus in samples decreased from 6.49 to 4.10 lg (CFU/g) and the number of molds and yeasts decreased from 5.45 to 4.28 lg (CFU/g). The number of Lactic acid bacteria increased from 3.97 to 4.55 lg (CFU/g) and Escherichia coli was not detected. Total colonies, target bacteria, peroxide value, thiobarbituric acid-reactive substances, water activity, T 2 relaxation time, pH, color difference, total volatile basic nitrogen, and the sensory evaluations of dry-cured meat products after PL-UV treatments were determined in a 30-d storage experiment. The shelf life of dry-cured meat treated with PL-UV irradiation at 20 °C was predicted to reach to 294 d by applying of shelf life testing method accelerated. The quality and safety of dry-cured meat treated with PL-UV irradiation was better than that of untreated samples.

Optimization of Processing Conditions of Chinese Smoke-cured Bacon (Larou) with a New Natural Coating Solution during Storage Period.

The study aims to determine the optimum sterilization rate and water activity of Chinese traditional smoke-cured bacon product (Larou) in the preservation with natural coating solution. With the response surface methodology (RSM), we analyzed 3 factors of processing conditions (the concentration of lysozyme, concentration of sodium alginate, and concentration of chitosan) and 2 response factors (sterilization rate and water activity). Sterilization rate and water activity of Larou were largely affected by the concentration of lysozyme, concentration of sodium alginate, and concentration of chitosan. The final optimum concentrations of lysozyme, sodium alginate, and chitosan were 0.09, 1.40, and 1.60% and realized the high sterilization rate. Water activity of sliced Larou was significantly correlated with the sterilization rate. Low-field nuclear magnetic resonance analysis verified the optimum processing conditions. The coating resulted in 99.69% rate of reducing bacteria after 30-day storage. The data of the total number of colony, peroxidation value, moisture content, pH, and sensory evaluation provided the theoretical basis for extending the shelf life of Chinese traditional smoke-cured bacon product (Larou) with natural coating solution.

In vitro immunosuppressive and cytotoxic activities of Tripterygium wilfordii extract.

Tripterygium wilfordii Hook. f. (TW) is a traditional herbal medicine which has been widely used for the treatment of rheumatoid arthritis and other autoimmune diseases. However, adverse reactions of TW such as hepatotoxicity and nephrotoxicity have been frequently reported in clinic. With the aim to evaluate the potency and toxicity of TW, we collected eleven batches of TW from different localities across Chinese mainland, and investigated the inhibition of their methanol extracts on the proliferation of mouse spleen lymphocytes, normal human hepatocyte (L-02) cells and African green monkey kidney (COS-7) cells. TW extracts with three different concentrations were designed as the experimental groups. Our present findings provided consistent evidence that TW had significant concentration-dependent inhibitory action on lymphocytes, L-02 and COS-7 cells. At the concentrations of 0.75 and 1.5 mg/mL, most TW groups showed statistically significant inhibition of lymphocyte proliferation when compared with the control group (p < 0.01), and the inhibition of TW extract on lymphocytes was almost equal to 1.0 mg/mL aspirin (p > 0.05). In most test groups, significant toxicities were shown on L-02 cells at 0.6 and 3.0 mg/mL (p < 0.01), and on COS-7 cells at 3.0 mg/mL (p < 0.01). At 3.0 mg/mL, almost all TW groups exerted obvious toxicities toward L-02 and COS-7 cells which were equal to or even higher than 1.0 mg/mL aspirin. In view of these results, further studies are needed to elucidate the relations among the effective component, curative effect and toxicity of TW to ensure its effectiveness and safety for human consumption.

Simultaneous quantification of nine major active components in traditional Chinese prescription Mahuang decoction and the influence of herbal compatibility on their contents.

BACKGROUND: Mahuang decoction (MHD), a famous classic traditional Chinese formula, has been extensively applied for treating cold, influenza, asthma, acute bronchitis, and other pulmonary diseases. However, the interaction among four drugs of MHD has not been clearly deciphered from the aspect of molecular composition. OBJECTIVE: To assess the quality of MHD and explore the interplay among different prescription drugs. MATERIALS AND METHODS: A reversed-phase high performance liquid chromatography (RP-HPLC) coupled with diode array detector (DAD) method for the simultaneous separation and determination of nine bioactive components was developed. A somatomedin A (SMA)-phenyl column (4.6 mm × 250 mm, 5 µm) was eluted by a gradient mobile phase contained acetonitrile and 0.05% formic acid-0.05% triethylamine aqueous solution. Four detection wavelengths (210, 252, 278, and 291 nm) were utilized for the quantitative analysis due to the different ultraviolet (UV) spectra of these compounds. RESULTS: Satisfactory separation was obtained for all the components, and the assay was fully validated in respects of linearity, precision, stability, and accuracy. It was found that the calibration curves for all analytes showed good linearity (R (2)≥ 0.9991) within the test ranges. The relative standard deviations (RSDs) for intra- and interday repeatability were not more than 1.70 and 2.66%, respectively. The spike recoveries of nine components varied from 97.50 ± 1.69 to 99.27 ± 1.37%. CONCLUSION: The established method was successfully applied to analyze nine active compounds in decoction samples of various drug compatibilities of MHD. The variations of contents were obvious for different combinations, which hinted the mutual promotion or inhibition of componential dissolution among four herbs of MHD.

[Optimization of ultrasonic extraction conditions of safflower yellow from Carthamus tinctorius by response surface methodology].

OBJECTIVE: To investigate the optimization of extraction conditions of safflower yellow from Cartbamus tirwtorius by response surface methodology. METHODS: Experimental factors and levels were selected by one-factor test, and then according to the central composite experimental design principle, response surface'-methodology with three factors and three levels was used to establish a mathematical model to obtain the optimal extraction conditions with hydroxysafflower yellow A being the target and its extraction yield as response value. RESULTS: The optimal extraction conditions of safflower yellow were as follows: extraction temperature was 55 t, ratio of water to raw material was 16:1 and extraction time was 39 mm for three times. CONCLUSION: Under these conditions, the extraction yield of safflower yellow is 1.798%, and the relative error between the predicted value with actual value is 2.758%. The optimized method can provide reference for the efficient extraction of safflower yellow from Carthomos tinctorius

Hydrogels prepared from unsubstituted cellulose in NaOH/urea aqueous solution.

Novel cellulose hydrogels were synthesized through a "one-step" method from cellulose, which was dissolved directly in NaOH/urea aqueous solution, by using epichlorohydrin as crosslinker. Structure and properties of the hydrogels were characterized by using SEM, NMR, and water absorption testing. The hydrogels are fully transparent and display macroporous inner structure. The equilibrium swelling ratios of the hydrogels in distilled water at 25 degrees C are in the range from 30 to 60 g H(2)O/g dry hydrogel. Moreover, the reswelling water uptake of the hydrogels could be achieved to more than 70% compared with their initial swelling states. This work provided a simple and fast method for preparing eco-friendly hydrogels from unsubstituted cellulose.

[Rapid simultaneous analysis of fluorene, carbazole, benzo[a]pyrene, and perylene by derivative-constant energy synchronous fluorescence spectroscopy].

A new method of derivative constant-energy synchronous fluorescence (CESF) spectroscopy for the simultaneous determination of fluorene, carbazole, benzo[a]pyrene and perylene was developed. The comparison and explanation of its performance are presented. The derivative CESF spectra are apparently more structured than the direct CESF. The scanning of derivative CESF is more effective than derivative constant-wavelength synchronous fluorescence spectroscopy. By using this method, real samples (tap water, seawater and airborne particulates) were determined directly and good results were obtained. The recoveries in tap water, seawater and airborne particulates were 90.0%-108.0%, 90.0%-104.0% and 90.0%-102.0% respectively.

Preparation and Use of a Monoclonal Antibodyagainst Luffin b.

Luffin b is one of the most toxic single chain plant ribosome inactivating proteins. It has been successfully used to prepare an immunotoxin against human melanoma cells. Two strains of hybridomas (1E5 and 2E1) were screened out using cell fusion technique which steadily secreted monoclonal antibodies against luffin b. These antibodies specifically reacted with luffin b. The affinity constants of 1E5 and 2E1 monoclonal antibodies were determined to be 1.1x10(9) mol(-1).L and 7.5x10(8) mol(-)1.L, by RIA,respectively. An immunoaffinity gel composed with the 1E5 monoclonal antibody and Sepharose 4B was prepared. The luffin b was successfully purified by one step immunoaffinity chromatography from the crude extract of Luffa cylindrica seeds. An immunoconjugate 1E5-HRP was also prepared and it was successfully used in Western blotting for detection of recombinant luffin b from E.coli total proteins.

Growth-inhibiting effects of taxol on human liver cancer in vitro and in nude mice.

AIM:To investigate the effects of taxol on SMMC-7721 human hepatoma and its mechanisms.METHODS:In vitro cell growth was assessed by trypan blue exclusion method. Experimental hepatoma model was established by seeding SMMC-7721 cells subcutaneously into Balb/c (nu/nu) nude mice. In vivo tumor growth was determined by measurement of tumor diameter with Vernier calipers. The syntheses of DNA, RNA and protein were analyzed by incorporation of (3)H-thymidine, (3)H-uridine and (3)H-leucine respec-tively. Using light and electron microscopes to observe the morphological changes of cells including mitosis and apoptosis.RESULTS:Taxol was effective against SMMC-7721 human hepatoma cell growth in the ranges of 2.5nmol/L-10nmol/L with mitotic arrest and apoptosis in vitro. DNA, RNA and protein syntheses in cells were also obviously suppressed by in vitro treatment of taxol for 72h. Taxol at 2.5nmol/L reduced (3)H-thymidine uptake to about 34% of the control value (P<0.05). Increasing the dose of taxol to 20nmol/L resulted in a greater decrease in (3)H-thymidine incorporation to 60% of the control value (P < 0.01). At a concentration of 20nmol/L, the (3)H-uridine and (3)H-leucine uptakes were reduced to 52% (P<0.05) and 63% (P<0.01), respectively. In vivo,taxol significantly inhibited SMMC-7721 tumor growth at 10mg/kg, i.p., once daily for 10d. A more than 90% decrease in tumor volume was observed by day 11 (P < 0.01) similarly with mitotic arrest and cell apoptosis.CONCLUSION:Taxol has a marked anticancer activity in SMMC-7721 human hepatoma both in vitro and in nude mice. Its mechanisms might be associated with mitotic arrest, subsequently, apoptosis of the hepatoma cells. No obvious toxicity was observed with in vivo administration of taxol.

In vitro Inhibition of Human Melanoma Cells by Immunotoxin Luffin B-Ng76.

Luffin B, a plan single-chain ribosome inactivating protein, was purified from seeds of Luffa cylindrica by Blue Sepharose CL-6B affinity chromatography. An immunotoxin was constructed with luffin B and Ng76, a monoclonal antibody to human melanoma cell M(21). Luffin B-Ng76 showed 4 000-fold more cytotoxic to target melanoma cells than free luffin B. The IC(50) of luffin B-Ng76 for M(21) cells and non-target HeLa cells was 2.5x10(-11) mol/L and 3.0x10(-8) mol/L, respectively. The results suggest that luffin B is a new potent immunotoxin effector.