Integrated transcriptome and proteome analysis reveals molecular responses of soybean anther under high-temperature stress.

It is well documented that high temperature (HT) severely affects the development of soybean male reproductive organs. However, the molecular mechanism of thermo-tolerance in soybean remains unclear. To explore the candidate genes and regulatory mechanism of soybean response to HT stress and flower development, here, the anthers of two previously identified HT-tolerant (JD21) and HT-sensitive (HD14) varieties were analyzed by RNA-seq. In total, 219 (172 upregulated and 47 downregulated), 660 (405 upregulated and 255 downregulated), and 4,854 (2,662 upregulated and 2,192 downregulated) differentially expressed genes (DEGs) were identified between JD21 anthers treated with HT stress vs. JD21 anthers in the natural field conditions (TJA vs. CJA), HD14 anthers treated with HT stress vs. HD14 anthers in the natural field conditions (THA vs. CHA), and JD21 vs. HD14 anthers treated with HT stress (TJA vs. THA), respectively. The results showed that there were more DEGs upregulated in JD21; this might be the reason why JD21 was more HT-resistant than the HT-sensitive variety HD14. GO annotation and KEGG enriched analysis showed that many DEGs are mainly involved in defense response, response to biological stimuli, auxin-activated signaling pathway, plant hormone signal transduction, MAPK signaling pathway-plant, starch and sucrose metabolism, etc. The conjoint analysis of RNA-seq and previous iTRAQ results found that there were 1, 24, and 54 common DEGs/DAPs showing the same expression pattern and 1, 2, and 13 common DEGs/DAPs showing the opposite pattern between TJA vs. CJA, THA vs. CHA, and TJA vs. THA at the protein and gene level, respectively, among which HSPs, transcription factor, GSTU, and other DEGs/DAPs participated in the response to HT stress and flower development. Notably, the qRT-PCR analysis and physiological index change results coincided with the sequencing results of RNA-seq and iTRAQ. In conclusion, the HT-tolerant cultivar performed better under stress than the HT-sensitive cultivar through modulation of HSP family proteins and transcription factors, and by keeping key metabolic pathways such as plant hormone signal transduction normal. This study provided important data and some key candidate genes to better study the effect and molecular basis of HT on anther in soybean at a transcription and translation level.

Garmultin-A Incites Apoptosis in CB3 Cells Through miR-17-5p by Attenuating Poly (ADP-Ribose) Polymerase-1.

Background: Leukemia accounts for a large number of deaths, worldwide, every year. Treating this ailment is always a challenging job. Recently, oncogenic miRNA leading to apoptosis are highly promising targets of many natural products. In this study, Garmultin-A (GA), isolated from the bark of Garcinia multiflora, was elucidated for its anti-leukemic effect in CB3 cells. Methods: The effect of the compound on CB3 cell viability was detected by MTT assay and apoptosis by FITC Annexin V/PI and Hochest 33258 staining. The western blot analysis assessed the BAX, BCL2, cMYC, pERK, and PARP-1 protein levels. Autodock analysis predicted the ligand-protein interactions. q-RT-PCR quantified the miR-17-5p expression. Luciferase assay confirmed the interaction between PARP-1 and miR-17-5p. Results: We uncover that GA leads to apoptosis by inducing overexpression of miR-17-5p and significantly downregulate PARP-1 protein levels in CB3 cells. The overexpression of miR-17-5p promotes apoptosis, and the miR-17-5p antagomirs restore GA-triggered apoptosis. Notably, we disclose that PARP-1 is a direct target of miR-17-5p. Increased pro-apoptotic and reduced anti-apoptosis protein levels were also observed in GA-treated CB3 cells. Conclusion: These results provide critical insights that GA could induce apoptosis in CB3 cells through targeting miR-17-5p by attenuating PARP-1. Thus, GA could act as a novel therapeutic agent for erythroleukemia.

Germacranolide sesquiterpenes from Carpesium cernuum and their anti-leukemia activity.

In this study, three new germacranolide sesquiterpenes (1-3), together with six related known analogues (4-9) were isolated from the whole plant of Carpesium cernuum. Their structures were established by a combination of extensive NMR spectroscopic analysis, HR-ESIMS data, and ECD calculations. The anti-leukemia activities of all compounds towards three cell lines (HEL, KG-1a, and K562) were evaluated in vitro. Compounds 1-3 exhibited moderate cytotoxicity with IC50 values ranging from 1.59 to 5.47 µmol·L-1. Mechanistic studies indicated that 2 induced apoptosis by decreasing anti-apoptotic protein Bcl-2 and activating the caspase family in K562 cells. These results suggest that compound 2 is a potential anti-leukemia agent.

L20, a Calothrixin B analog, induces intrinsic apoptosis on HEL cells through ROS/γ-H2AX/p38 MAPK pathway.

Erythroleukemia is a malignant disease in the blood system. Quinones consists of a class of antitumor agents. Calothrixin B is a carbazole-1,4-quinone alkaloid isolated from Calothrix cyanobacteria with a unique indolo[3,2-j] phenanthridine framework. This study aimed to investigate the anti-leukemic effect of the new Calothrixin B derivative, L20, and to dig up the underlying mechanisms. Cytotoxicity analysis of L20 has revealed that it shows significant IC50 concentrations in HEL cells at low doses (1.10 ± 0.05 µM) than in K562, and KG-1a (5.46 ± 3.09, and 1.82 ± 1.08 µM respectively). The study even revealed that the L20 could induce a dose and time-dependent cellular death in HEL cells. The L20 increased expression of phospho-γ-H2A.X and phospho-p38 in HEL cells, causing DNA damage and nuclear alterations due to the G2/M phase cell cycle arrest. The HEL cells even lost the mitochondrial membrane potential (MMP) and resulted in the release of reactive oxygen species (ROS). Additionally, L20 inhibited the proliferation of HEL cells by inducing apoptosis through the mitochondrial pathway, depending on the caspase family. The study even established this may be due to the upregulation of the p-P38MAPK and downregulation of p-ERK. Pretreatment with P38/ERK inhibitors, SB203580, and U0126, decreased L20-induced apoptosis. These findings indicated that L20 induced mitochondrial mediated-apoptosis and G2/M arrest through DNA damage and modulation of p38 MAPK pathways. Thus, the study suggests L20, a chemical analog of Calothrixin B, as a novel chemotherapeutic agent against erythroleukemia.

A natural small molecule induces megakaryocytic differentiation and suppresses leukemogenesis through activation of PKCδ/ERK1/2 signaling pathway in erythroleukemia cells.

Kaempferol-3-O-rhamnoside (KOR) has multiple potency involved in anti-cancer, anti-inflammatory and antibacterial actions. However, the potential roles of KOR and the analogues isolated from the leaves of Cyclocarya paliurus in anti-erythroleukemia remain unclear. In the present study, KOR and the two analogues (Kaempferol-3-O-(4″-O-acetyl-a-L-rhamnopyranoside) (KLR) and (kaempferol-3-O-α-L-(4″-E-p-coumaroyl) rhamnoside) (KCR) were isolated from leaves of Cyclocarya paliurus. Cell viability assay showed that KCR exerted an excellent anti-erythroleukemia activity. We observed that KCR not only significantly increased the percentage of G2 phase and apoptotic cells compared with control group, but also induced megakaryocytic differentiation in HEL and K562 cells by flow cytometry, indicating that KCR might inhibit cell proliferation through inducing differentiation-mediated apoptosis and cell cycle arrest. Mechanism investigation revealed that KCR treatment obviously increased phosphorylation levels of PKCδ and ERK1/2 as well as GATA1 expression. Taken together, these findings demonstrate that KCR induces megakaryocytic differentiation and suppresses leukemogenesis at least partly through activation of PKCδ/ERK1/2 signaling pathway in erythroleukemia cells. KCR may also serve as a promising natural compound for human erythroleukemia treatment.

Novel racemosin B derivatives as new therapeutic agents for aggressive breast cancer.

Carbazole derivatives show anti-cancer activity and are of great interest for drug development. In this study, we synthesized and analyzed several new alkylamide derivatives of racemocin B, a natural indolo[3,2-a]carbazole molecule originally isolated from the green alga Caulerpa racemose. Several alkylamide derivatives were found to exhibit moderate to strong growth inhibition against human breast cancer cell lines. They induced G2/M cell cycle arrest and apoptosis in the aggressive triple-negative breast cancer cell line MDA-MB-231. Among these derivatives, compound 25 with the lowest IC50 induced cell death by suppressing autophagy. This was accompanied by inhibition of autophagic flux and accumulation of autophagy protein 1 light chain 3, LC3II, and p62. The novel alkylamide derivative offers a potential new treatment for human breast cancer.

Heterologous expression of antifreeze protein gene AnAFP from Ammopiptanthus nanus enhances cold tolerance in Escherichia coli and tobacco.

Antifreeze proteins are a class of polypeptides produced by certain animals, plants, fungi and bacteria that permit their survival under the subzero environments. Ammopiptanthus nanus is the unique evergreen broadleaf bush endemic to the Mid-Asia deserts. It survives at the west edge of the Tarim Basin from the disappearance of the ancient Mediterranean in the Tertiary Period. Its distribution region is characterized by the arid climate and extreme temperatures, where the extreme temperatures range from -30 °C to 40 °C. In the present study, the antifreeze protein gene AnAFP of A. nanus was used to transform Escherichia coli and tobacco, after bioinformatics analysis for its possible function. The transformed E. coli strain expressed the heterologous AnAFP gene under the induction of isopropyl ß-D-thiogalactopyranoside, and demonstrated significant enhancement of cold tolerance. The transformed tobacco lines expressed the heterologous AnAFP gene in response to cold stress, and showed a less change of relative electrical conductivity under cold stress, and a less wilting phenotype after 16 h of -3 °C cold stress and thawing for 1h than the untransformed wild-type plants. All these results imply the potential value of the AnAFP gene to be used in genetic modification of commercially important crops for improvement of cold tolerance.

[Research on TLC identification and anti-coagulant activity quantitatively methods of Scolopendra subspinipes mutilans].

OBJECTIVE: To improve the quality standard of Scolopendra subspinipes mutilans by researching the methods of the TLC identification and anti-coagulant activity quantitatively. METHODS: Identified the free arginine (Arg) and serine (Ser) in scolopendra by TLC, screened the samples preparation process and developed solvent systems; Determined the anti-coagulant activity by method of titration with thrombin and screened the pretreatment methods. RESULTS: When medicinal materials was extracted by formic acid and 95% ethanol (1:1) with ultrasonic method and developed by n-butanol-acetic acid-water (12:5:4), the spots of Arg and Ser were well separated. Ultrasonic method was suitable for preparation of the anti-coagulant components in Scolopendra subspinipes mutilans and their anti-coagulant activity was determined by method of titration with thrombin could get a well reproducibility, the anti-thrombin activity of testing sample was (14.00 +/- 1.53) U/g and those of three different batch were (13.00 +/- 0.58) U/g, (17.00 +/- 1.15) U/g, (15.67 +/- 1.53) U/g respectively. CONCLUSION: The methods of TLC identification and anti-coagulant activity quantitatively could be used as a basis for improving the quality standard of Scolopendra subspinipes mutilans.

[Application of flap or musculocutaneous flap primary transplantation in Pilon fractures of type III].

OBJECTIVE: To discuss the value of one stage flap or musculocutaneous flap to repair the soft tissue defect in Pilon fracture. METHODS: Twelve cases with Pilon fracture included 9 male and 3 female with an average age of 39.2 years ranging from 21 to 61 years. All fractures were type III according to Rüedi and Allgöwer. According to Gustilo sysytem, III a were in 4 cases, III b were in 8. A thorough debridement was made before internal fixation. After the internal fixation implanted, the tourniquet was released and a thorough debridement was made again. Be sure of the wound cleansed of all dead and foreign material, the wound was covered with local flap, musculocutaneous or gastrocnemius flaps depending on the size and localization of wound. RESULTS: All the patients were followed up from 6 to 36 months, 18 months in average. The function of ankle was assessed according Mazur system. The results were excellent in 6 cases, good in 3, fair in 2 and poor in 1. No infection or necrosis happened on flaps. Although necrosis happened in the wound margin of two patients, they all healled up by conservative methods. CONCLUSION: Primary closure of soft tissue defect in Pilon fracture using flap or musculocutaneous flap have ability to shorten the treating time and recover the function of ankle. It is important to have a thorough debridement of the dead tissue and free bone.

Application of flap or musculocutaneous flap primary transplantation in Pilon fractures of type III / 中国骨伤

<p><b>OBJECTIVE</b>To discuss the value of one stage flap or musculocutaneous flap to repair the soft tissue defect in Pilon fracture.</p><p><b>METHODS</b>Twelve cases with Pilon fracture included 9 male and 3 female with an average age of 39.2 years ranging from 21 to 61 years. All fractures were type III according to Rüedi and Allgöwer. According to Gustilo sysytem, III a were in 4 cases, III b were in 8. A thorough debridement was made before internal fixation. After the internal fixation implanted, the tourniquet was released and a thorough debridement was made again. Be sure of the wound cleansed of all dead and foreign material, the wound was covered with local flap, musculocutaneous or gastrocnemius flaps depending on the size and localization of wound.</p><p><b>RESULTS</b>All the patients were followed up from 6 to 36 months, 18 months in average. The function of ankle was assessed according Mazur system. The results were excellent in 6 cases, good in 3, fair in 2 and poor in 1. No infection or necrosis happened on flaps. Although necrosis happened in the wound margin of two patients, they all healled up by conservative methods.</p><p><b>CONCLUSION</b>Primary closure of soft tissue defect in Pilon fracture using flap or musculocutaneous flap have ability to shorten the treating time and recover the function of ankle. It is important to have a thorough debridement of the dead tissue and free bone.</p>