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1.
Plant J ; 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33595875

RESUMO

Gray blight (GB) is one of the most destructive diseases of tea plants causing considerable damage and productivity losses. However, the dynamic roles of defense genes during pathogen infection remain largely unclear. To explore the numerous molecular interactions associated with GB stress in tea plants, we employed transcriptome, sRNAome, and degradome sequencing from 1 to 13 days post inoculation (dpi) at 3-day intervals. The transcriptomics results showed that differentially expressed genes (DEGs) related to flavonoid synthesis, like chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL), were particularly induced at 4 dpi. Consistent with this, the contents of catechins (especially gallocatechin), which are the dominant flavonoids of tea plants, also increased in GB-infected tea leaves. Combined analysis of the sRNAome and degradome revealed that miRNAs could mediate tea plant immunity by regulating DEG expression at the post-transcriptional level. Co-expression network analysis demonstrated that miR530b-ethylene responsive factor 96 (ERF96) and miRn211-thaumatin-like protein (TLP) play crucial roles in GB response. Accordingly, gene-specific antisense oligonucleotides assays suggested that suppressing ERF96 decreased ROS levels, whereas suppressing TLP increased the levels of ROS. Furthermore, ERF96 was induced, but TLP was suppressed in susceptible tea cultivars. Our results collectively demonstrate that ERF96 was a negative regulator, while TLP was a positive regulator in the tea plant's response to GB. Taken together, our comprehensive integrated analysis revealed a dynamic regulatory network linked to GB stress in tea plants and provides candidate genes for tea plant improvement.

2.
Ophthalmic Res ; 2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33540418

RESUMO

PURPOSE: The aim of this study was to evaluate the short-term efficacy and safety of ultrasound cycloplasty (UCP) procedure in Chinese glaucoma patients. METHODS: As a single-centre, prospective, non-comparative study, 23 eyes of 23 patients suffering from glaucoma with uncontrolled intraocular pressure (IOP) ≥ 21 mmHg underwent a multi-dose UCP treatment with the activations of 6, 8 or10 sectors. Types of glaucoma include primary open-angle glaucoma (POAG) (7/23), primary angle closure glaucoma (PACG) (9/23) and secondary glaucoma (SG) (7/23). A complete ophthalmic examination including intraocular pressure (IOP) measurements was performed before UCP procedure and at 1 day, 1 month, 3months and 6 months after the procedure. An IOP reduction of ≥20% and IOP > 5 mmHg without increasing hypotensive medication at the follow-up visit was defined as therapeutic success. The post-operative complications were also recorded and compared to baseline for safety evaluation. RESULTS: The mean baseline IOP of 23 treated eyes was 37.2 ± 12.1 mmHg. The IOP reduction after UCP procedure were 23%, 49%, 33% and 34% at 1 day, 1 month, 3 months and 6 months, respectively. Thus, the corresponding overall therapeutic success rates reached 61% (14/23), 83% (19/23), 65% (15/23) and 61% (14/23), respectively. Baseline IOPs of 8 and 10 sectors group (37.0 ± 9.9 mmHg and 50.1 ± 12.2 mmHg) were significantly higher than that of 6 sectors group (30.1 ± 8.2 mmHg). Therapeutic success rates of 6, 8 and 10 sectors groups reached 44% (4/9), 56% (5/9) and 100% (5/5), respectively. There were the highest percentage of IOP reduction (50% and 41%) and therapeutic success rate (6/7; 86% and 7/9; 78%) in SG group and PACG groups respectively. In addition, pre-operative ocular pain symptoms of four patients were all disappeared within one week after UCP. No serious intra-operative or post-operative complications occurred. CONCLUSION: UCP procedure is an effective and well-tolerated treatment to reduce IOP in Chinese glaucoma patients, which offered a novel alternative for glaucoma treatment.

3.
Artigo em Inglês | MEDLINE | ID: mdl-33558290

RESUMO

Polyketide synthase 13 (Pks13) is an important enzyme found in Mycobacterium tuberculosis (M. tuberculosis) that condenses two fatty acyl chains to produce α-alkyl ß-ketoesters, which in turn serve as the precursors for the synthesis of mycolic acids that are essential building blocks for maintaining the cell wall integrity of M. tuberculosis Coumestan derivatives have recently been identified in our group as a new chemotype that exert their antitubercular effects via targeting of Pks13. These compounds were active on both drug-susceptible and drug-resistant strains of M. tuberculosis as well as showing low cytotoxicity to healthy cells and a promising selectivity profile. No cross-resistance was found between the coumestan derivatives and first-line TB drugs. Here we report that treatment of M. tuberculosis bacilli with 15 times the MIC of compound 1, an optimized lead coumestan compound, resulted in a colony forming unit (CFU) reduction from 6.0 log10 units to below the limit of detection (1.0 log10 units) per mL culture, demonstrating a bactericidal mechanism of action. Single dose (10 mg/kg) pharmacokinetic studies revealed favorable parameters with a relative bioavailability of 19.4%. In a mouse infection and chemotherapy model, treatment with 1 showed dose-dependent mono-therapeutic activity, whereas treatment with 1 in combination with rifampin showed clear synergistic effects. Together these data suggest that coumestan derivatives are promising agents for further TB drug development.

4.
BMC Complement Med Ther ; 21(1): 52, 2021 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-33549076

RESUMO

BACKGROUND: Ursolic acid (UA) is an anti-cancer herbal compound. In the present study, we observed the effects of UA on anchorage-dependent and -independent growth of human colorectal cancer (CRC) RKO cells. METHODS: RKO cells were cultured in conventional and detached condition and treated with UA. Cell viability was evaluated by CCK-8 assay. Cell cycle was analyzed by flow cytometry. Apoptosis was identified by Hoechst 33258 staining and flow cytometry analysis. Activities of caspases were measured by commercial kits. Reactive oxygen species (ROS) was recognized by DCFH-DA fluorescent staining. Anoikis was identified by EthD-1 fluorescent staining and flow cytometry analysis. Expression and phosphorylation of proteins were analyzed by western blot. RESULTS: UA inhibited RKO cell viability in both a dose- and time-dependent manner. UA arrested the cell cycle at the G0/G1 phase, and induced caspase-dependent apoptosis. UA inhibited Bcl-2 expression and increased Bax expression. In addition, UA up-regulated the level of ROS that contributed to UA activated caspase-3, - 8 and - 9, and induced apoptosis. Furthermore, UA inhibited cell growth in a detached condition and induced anoikis in RKO cells that was accompanied by dampened phosphorylation of FAK, PI3K and AKT. UA also inhibited epithelial-mesenchymal transition (EMT) as indicated by the down-regulation of N-Cad expression and up-regulation of E-Cad expression. CONCLUSIONS: UA induced caspase-dependent apoptosis, and FAK/PI3K/AKT singling and EMT related anoikis in RKO cells. UA was an effective anti-cancer compound against both anchorage-dependent and -independent growth of RKO cells.

5.
Eur J Med Chem ; 213: 113202, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33516983

RESUMO

We previously reported a series of coumestans-a naturally occurring tetracyclic scaffold containing a δ-lactone-that effectively target the thioesterase domain of polyketide synthase 13 (Pks13) in Mycobacterium tuberculosis (Mtb), resulting in superior anti-tuberculosis (TB) activity. Compared to the corresponding 'open-form' ethyl benzofuran-3-carboxylates, the enhanced anti-TB effects seen with the conformationally restricted coumestan series could be attributed to the extra π-π stacking interactions between the benzene ring of coumestans and the phenyl ring of F1670 residue located in the Pks13-TE binding domain. To further probe this binding feature, novel tetracyclic analogues were synthesized and evaluated for their anti-TB activity against the Mtb strain H37Rv. Initial comparison of the 'open-form' analogueues against the tetracyclic counterparts again showed that the latter is superior in terms of anti-TB activity. In particular, the δ-lactam-containing 5H-benzofuro [3,2-c]quinolin-6-ones gave the most promising results. Compound 65 demonstrated potent activity against Mtb H37Rv with MIC value between 0.0313 and 0.0625 µg/mL, with high selectivity to Vero cells (64-128 fold). The thermal stability analysis supports the notion that the tetracyclic compounds bind to the Pks13-TE domain as measured by nano DSF, consistent with the observed SAR trends. Compound 65 also showed excellent selectivity against actinobacteria and therefore unlikely to develop potential drug resistance to nonpathogenic bacteria.

7.
Microb Pathog ; 150: 104724, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33400988

RESUMO

Japaneses encephalitis (JE) is most common zoonoses caused by Japanese encephalitis virus (JEV) with a high mortality and disability rate. To take timely preventive and control measures, early and rapid detection of JE RNA is necessary. But due to characteristic brief and low viraemia, JE RNA detection remains challenging. In this study, a real-time nucleic acid sequence-based amplification (RT-NASBA) was developed for rapid and simultaneous detection of JEV. Four pairs of primer were designed using a multiple genome alignment of all JEV strains from GenBank. NASBA assay established and optimal reaction conditions were confirmed by using primers and probe on ns1 gene of JEV. The specificity and sensitivity of the assay were compared with RT-PCR by using serial RNA and virus cultivation dilutions. The results showed that JEV RT-NASBA assay was established, and robust signals could be observed in 10 min with high specificity. The limit of dectetion of RT-NASBA was 6 copies per reaction. The assay was thus 100 to 1, 000 times more sensitive than RT-PCR. The cross-reaction was performed with other porcine pathogens, and negative amplification results indicated the high specificity of this method. The novel JEV RT-NASBA assay could be used as an efficient molecular biology tool to diagnose JEV, which would facilitate the surveillance of reproductive failure disease in swine and would be beneficial for public health security.

8.
Nat Commun ; 11(1): 6266, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33293512

RESUMO

Grain weight (GW) is one of the component traits of wheat yield. Existing reports have shown that multiple phytohormones are involved in the regulation of GW in different crops. However, the potential role of jasmonic acid (JA) remains unclear. Here, we report that triticale grain weight 1 (tgw1) mutant, with marked reductions in both GW and JA content, is caused by a premature stop mutation in keto-acyl thiolase 2B (KAT-2B) involved in ß-oxidation during JA synthesis. KAT-2B overexpression increases GW in wild type and boosts yield. Additionally, KAT-2B compliments the grain defect in tgw1 and rescues the lethal phenotype of the Arabidopsis kat2 mutant in a sucrose-free medium. Despite the suppression of JA synthesis in tgw1 mutant, ABA synthesis is upregulated, which is accompanied by enhanced expression of SAG3 and reduction of chlorophyll content in leaves. Together, these results demonstrate a role of the JA synthetic gene KAT-2B in controlling GW and its potential application value for wheat improvement.

9.
Lab Invest ; 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33299127

RESUMO

Activating transcription factor 3 (ATF-3), a cyclic AMP-dependent transcription factor, has been shown to play a regulatory role in melanoma, although its function during tumor progression remains unclear. Here, we demonstrate that ATF-3 exhibits tumor suppressive function in melanoma. Specifically, ATF-3 nuclear expression was significantly diminished with melanoma progression from nevi to primary to metastatic patient melanomas, correlating low expression with poor prognosis. Significantly low expression of ATF-3 was also found in cultured human metastatic melanoma cell lines. Importantly, overexpression of ATF-3 in metastatic melanoma cell lines significantly inhibited cell growth, migration, and invasion in vitro; as well as abrogated tumor growth in a human melanoma xenograft mouse model in vivo. RNA sequencing analysis revealed downregulation of ERK and AKT pathways and upregulation in apoptotic-related genes in ATF-3 overexpressed melanoma cell lines, which was further validated by Western-blot analysis. In summary, this study demonstrated that diminished ATF-3 expression is associated with melanoma virulence and thus provides a potential target for novel therapies and prognostic biomarker applications.

10.
J Agric Food Chem ; 68(49): 14438-14451, 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33225708

RESUMO

Biological control of plant pathogens is considered as one of the green and effective technologies using beneficial microorganisms or microbial secondary metabolites against plant diseases, and so microbial natural products have played important roles in the research and development of new and green agrochemicals. To explore the potential applications for natural γ-lactam alkaloids and their derivatives, 26 γ-lactams that have flexible substituent patterns were synthesized and characterized, and their in vitro antifungal activities against eight kinds of plant pathogens belonging to oomycetes, basidiomycetes, and deuteromycetes were fully evaluated. In addition, the high potential compounds were further tested using an in vivo assay against Phytophthora blight of pepper to verify a practical application for controlling oomycete diseases. The potential modes of action for compound D1 against Phytophthora capsici were also investigated using microscopic technology (optical microscopy, scanning electron microscopy, and transmission electron microscopy) and label-free quantitative proteomics analysis. The results demonstrated that compound D1 may be a potential novel fungicidal agent against oomycete diseases (EC50 = 4.9748 µg·mL-1 for P. capsici and EC50 = 5.1602 µg·mL-1 for Pythium aphanidermatum) that can act on steroid biosynthesis, which can provide a certain theoretical basis for the development of natural lactam derivatives as potential antifungal agents.

11.
J Cardiovasc Pharmacol ; 76(5): 506-513, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33165132

RESUMO

Familial hypercholesterolemia (FH) is a rare autosomal gene deficiency disease with increased low-density lipoprotein cholesterol, xanthoma, and premature coronary heart disease. Calcified aortic valve disease (CAVD) is prevalent in FH patients, resulting in adverse events and heavy health care burden. Aortic valve calcification is currently considered an active biological process, which shares several common risk factors with atherosclerosis, including aging, hypertension, dyslipidemia, and so on. Unfortunately, the pathogenesis and therapy of CAVD in FH are still controversial. There is no pharmacological intervention recommended to delay the development of CAVD in FH, and the only effective treatment for severe CAVD is aortic valve replacement. In this review, we summarize the detailed description of the pathophysiology, molecular mechanism, risk factors, and treatment of CAVD in FH patients.

12.
J Cell Physiol ; 2020 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-33251612

RESUMO

Bone morphogenetic protein (BMP) signaling is well known in bone homeostasis. However, the physiological effects of BMP signaling on mandibles are largely unknown, as the mandible has distinct functions and characteristics from other bones. In this study, we investigated the roles of BMP signaling in bone homeostasis of the mandibles by deleting BMP type I receptor Acvr1 in osteoblast lineage cells with Osterix-Cre. We found mandibular bone loss in conditional knockout mice at the ages of postnatal day 21 and 42 in an age-dependent manner. The decreased bone mass was related to compromised osteoblast differentiation together with enhanced osteoclastogenesis, which was secondary to the changes in osteoblasts in vivo. In vitro study revealed that deletion of Acvr1 in the mandibular bone marrow stromal cells (BMSCs) significantly compromised osteoblast differentiation. When wild type bone marrow macrophages were cocultured with BMSCs lacking Acvr1 both directly and indirectly, both proliferation and differentiation of osteoclasts were induced as evidenced by an increase of multinucleated cells, compared with cocultured with control BMSCs. Furthermore, we demonstrated that the increased osteoclastogenesis in vitro was at least partially due to the secretion of soluble receptor activator of nuclear factor-κB ligand (sRANKL), which is probably the reason for the mandibular bone loss in vivo. Overall, our results proposed that ACVR1 played essential roles in maintaining mandibular bone homeostasis through osteoblast differentiation and osteoblast-osteoclast communication via sRANKL.

13.
J Am Med Dir Assoc ; 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-33097399

RESUMO

OBJECTIVES: High retention rates among direct care workers (DCWs) affect the quality of aged care. However, limited research has explored factors associated with retention in China's aged care industry. This study compared turnover intention among DCWs in Chinese hospitals and long-term care facilities (LTCFs). DESIGN: A cross-sectional survey with the stratified-random sampling method. SETTING AND PARTICIPANTS: A total of 370 DCWs from 7 hospitals and 311 DCWs from 7 LTCFs located in Fujian Province, China, were surveyed. MEASURES: Turnover intention, contractual status, income, sociodemographic characteristics, and other work-related factors were self-reported by surveyed DCWs. RESULTS: Approximately 80% of DCWs in hospitals had employment contracts, whereas only 9% of DCWs were contractual in LTCFs. DCWs from hospitals reported lower turnover intention (20.5% vs. 37.0%) than did DCWs from LTCFs. Contracted DCWs from hospitals had the lowest turnover intention (14.8%). Contracted and noncontracted DCWs in LTCFs were more likely to report turnover intention than hospital contracted DCWs (odds ratio [OR] 3.68, P = .008 and OR = 3.17, P < .001, respectively). CONCLUSIONS AND IMPLICATIONS: Contractual status and facility type were jointly associated with turnover intention of DCWs in China. DCWs in LTCFs were likely to report much higher turnover intention than contracted DCWs in hospitals. The expanded use of employment contracts may lower turnover intention in long-term care DCWs by improving their job stability and security. The labor market of DCWs needs to be regulated by the Chinese Labor Law.

14.
Electrophoresis ; 2020 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-33128465

RESUMO

Y chromosome kits are successfully applied in cases where human biological material exists. With the development of genotyping ability, more Y chromosomal markers are needed for finer identification of male individuals and lineages. In this study, a developmental validation of a newly emerged Y chromosome kit that combines two different kinds of markers: 38 Y-STRs and 3 Y-indels are conducted. The results show that this kit has high sensitivity when there is a small amount of DNA (125 pg), more than one male (minor:major = 1:7), or a mixture of males and females (male:female = 125pg:1875pg), inhibited substances (800 µM hematin and more than 1600 ng/µL humic acid). The kit exhibits high precision level with a standard deviation of allele size no more than 0.14 nt. Locus DYS481 shows the largest stutter rate, with three stutters per true allele. Population samples are well identified (MP of 0.001106), and mutations can be observed in father-son pairs (46 mutations in 70 pairs, 10 in locus DYS627). Out of all the population samples, 13.2% belong to haplogroup M117-O2a2b1a1, with their ethnic group being Han Chinese. The results show that this kit can improve the performance of identifying male individuals, obtaining more unique haplotypes (increasing from 894 to 918 of 1000 male samples) and higher discrimination capacity (increasing from 0.942 to 0.955) in this study compared to previous widely used Yfiler Plus kit. Besides, it gives information about their paternal lineages in forensic genetic casework and genealogical database construction.

15.
Macromol Biosci ; : e2000185, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32896072

RESUMO

The pollution of antibiotics in aquaculture environment is increasingly serious, and excessive antibiotics will kill the probiotics in aquaculture feed. How to improve the viability of probiotics in the antibiotics-contaminated environment is of significance. In this study, a new strategy for protecting Saccharomyces cerevisiae cells in situ against antibiotics is constructed based on cell surface engineering technology by putting on wearable protective layers for cells. The protective layer is constructed around cellular surface via the self-assembly of coacervate microdroplets that consist of carboxymethyl chitosan and carboxyl dextran. Without affecting the cell viability, the protective layer can grasp ciprofloxacin and decrease the contact of ciprofloxacin to cells and consequently improve the survival rate of cells when exposing to ciprofloxacin. This work highlights a facile strategy to establish removable artificial cell wall by biodegradable polysaccharides for improving the productivity of probiotics in antibiotic environments.

16.
Appl Opt ; 59(24): 7364-7370, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32902504

RESUMO

An optical sensor that simultaneously measures the concentration of the biochemical oxygen demand (BOD) and temperature in water based on a tapered microfiber is proposed for environmental monitoring. The sensor is characterized by a strong evanescent field, which is more sensitive to liquids with a low refractive index and a low transmission loss. The results show that as the BOD concentration increases, the interference spectrum shifts toward longer wavelengths, the spectral loss decreases, and the sensitivities of the BOD are 12.17 nm/mg/mL and -2.387dB/mg/mL in the range of 0.25-1 mg/mL, which indicates the extent of the water pollution. The detection limit for the BOD concentration is as low as 0.0016 mg/mL. As the ambient temperature increases, the interference spectrum shifts toward shorter wavelengths, the spectral loss decreases, and the temperature sensitivities are -0.339nm/∘C and -0.031dB/∘C in the range of 30°C-60°C. The matrix method can be used to achieve the simultaneous measurement of the BOD concentration and environmental temperature because the spectral interference peaks have different responses to these two parameters. The sensor can not only be used for detecting water pollution in rivers, drinking water, and groundwater but can also be utilized for other types of environmental monitoring. This sensor has great potential to act as a basic sensing unit in fiber-optic sensor networks for multiparameter measurements and intelligent monitoring.

17.
Aging (Albany NY) ; 12(16): 16621-16646, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843583

RESUMO

The Rho-kinases (ROCK) inhibitor Y-27632 has been shown to promote the growth of epidermal cells, however, its potential effects on human dermal fibroblasts (HDFs) need to be clarified. Here we show that prolonged treatment of HDFs with Y-27632 decreased their growth by inducing senescence, which was associated with induction of the senescence markers p16 and p21, and downmodulation of the ERK pathways. The senescent HDFs induced by Y-27632 acquired a cancer-associated-fibroblast (CAF)-like phenotype to promote squamous cell carcinoma (SCC) cell growth in vitro. Expression of a newly identified target of Y-27632 by RNA-seq, insulin growth factor binding protein 5 (IGFBP-5), was dramatically increased after 24 h of treatment with Y-27632. Adding recombinant IGFBP-5 protein to the culture medium produced similar phenotypes of HDFs as did treatment with Y-27632, and knockdown of IGFBP-5 blocked the Y-27632-induced senescence. Furthermore, Y-27632 induced the expression of an IGFBP-5 upstream gene, GATA4, and knockdown of GATA4 also reduced the Y-27632-induced senescence. In summary, these results demonstrate for the first time that Y-27632 promotes cellular senescence in primary HDFs by inducing the expression of IGFBP-5 and that prolonged treatment with Y-27632 potentially transforms primary HDFs into CAF-like cells.

18.
Int J Mol Sci ; 21(17)2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854314

RESUMO

Organogenesis in plants occurs across all stages of the life cycle. Although previous studies have identified many genes as important for either vegetative or reproductive development at the RNA level, global information on translational and post-translational levels remains limited. In this study, six Arabidopsis stages/organs were analyzed using quantitative proteomics and phosphoproteomics, identifying 2187 non-redundant proteins and evidence for 1194 phosphoproteins. Compared to the expression observed in cauline leaves, the expression of 1445, 1644, and 1377 proteins showed greater than 1.5-fold alterations in stage 1-9 flowers, stage 10-12 flowers, and open flowers, respectively. Among these, 294 phosphoproteins with 472 phosphorylation sites were newly uncovered, including 275 phosphoproteins showing differential expression patterns, providing molecular markers and possible candidates for functional studies. Proteins encoded by genes preferentially expressed in anther (15), meiocyte (4), or pollen (15) were enriched in reproductive organs, and mutants of two anther-preferentially expressed proteins, acos5 and mee48, showed obviously reduced male fertility with abnormally organized pollen exine. In addition, more phosphorylated proteins were identified in reproductive stages (1149) than in the vegetative organs (995). The floral organ-preferential phosphorylation of GRP17, CDC2/CDKA.1, and ATSK11 was confirmed with western blot analysis. Moreover, phosphorylation levels of CDPK6 and MAPK6 and their interacting proteins were elevated in reproductive tissues. Overall, our study yielded extensive data on protein expression and phosphorylation at six stages/organs and provides an important resource for future studies investigating the regulatory mechanisms governing plant development.

19.
J Vis Exp ; (161)2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32716369

RESUMO

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.


Assuntos
Amidas/farmacologia , Separação Celular/métodos , Melanócitos , Piridinas/farmacologia , Pele/citologia , Pele/efeitos dos fármacos , Adulto , Células Cultivadas , Meios de Cultura/química , Células Epidérmicas , Humanos , Melanócitos/citologia
20.
Int J Mol Sci ; 21(15)2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32707968

RESUMO

Soybean aphid (Aphis glycines Matsumura) is one of the major limiting factors in soybean production. The mechanism of aphid resistance in soybean remains enigmatic as little information is available about the different mechanisms of antibiosis and antixenosis. Here, we used genome-wide gene expression profiling of aphid susceptible, antibiotic, and antixenotic genotypes to investigate the underlying aphid-plant interaction mechanisms. The high expression correlation between infested and non-infested genotypes indicated that the response to aphid was controlled by a small subset of genes. Plant response to aphid infestation was faster in antibiotic genotype and the interaction in antixenotic genotype was moderation. The expression patterns of transcription factor genes in susceptible and antixenotic genotypes clustered together and were distant from those of antibiotic genotypes. Among them APETALA 2/ethylene response factors (AP2/ERF), v-myb avian myeloblastosis viral oncogene homolog (MYB), and the transcription factor contained conserved WRKYGQK domain (WRKY) were proposed to play dominant roles. The jasmonic acid-responsive pathway was dominant in aphid-soybean interaction, and salicylic acid pathway played an important role in antibiotic genotype. Callose deposition was more rapid and efficient in antibiotic genotype, while reactive oxygen species were not involved in the response to aphid attack in resistant genotypes. Our study helps to uncover important genes associated with aphid-attack response in soybean genotypes expressing antibiosis and antixenosis.

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