Functional analysis of MPK3 and MPK6, two mitogen-activated protine kinases in Arabidopsis thaliana

Abstract only availableMitogen-activated protein kinase (MAPK) cascades are major pathways involved in the transduction of extracellular signals into intracellular responses. A MAPK cascade consists of three kinases; MAPK, MAPK kinase (MAPKK or MEK) and MAPKK kinase (MAPKKK or MEKK). MAPKKK is at the top of this three-tier cascade. Upon its activation by a receptor/sensor, MAPKKK phosphorylates MAPKK, which in turn phosphorylates MAPK and activates it. The activated MAPK can then phosphorylate other protein kinases or be translocated to the nucleus where it can phosphorylate transcription factors and activate gene expression. About 20 MAPKs were identified in the fully sequenced Arabidopsis genome. To study the function of MPK3 and MPK6, the two most closely related MAPKs in Arabidopsis, we isolated the corresponding T-DNA mutants from mutant libraries generated at Wisconsin Arabidopsis Knockout Facility and Salk Institute Genomic Analysis Laboratory. No morphological or developmental phenotypes were observed in the MPK3-/- and MPK6-/- single mutants. In order to determine if MPK3 and MPK6 have overlapping functions, we crossed the two single mutants (MPK3-/- and MPK6-/-) to generate double mutants. Among the 172 F2 plants that we genotyped, no double homozygous (MPK3-/-/MPK6-/-) mutant plants was identified, indicating that this genotype is lethal. We further observed that plants with the MPK3+/-/MPK6-/- genotype are a little smaller and sterile. Reciprocal back cross to wild type plants demonstrated that MPK3+/-/MPK6-/- plants are female sterile. The resilience of the pollens from such plants is still under investigation. In contrast to MPK3+/-/MPK6-/- plants, MPK3-/-/MPK6+/- plants are fertile and apparently normal. Together with the normal phenotype of MPK3-/- and MPK6-/- single mutants, we conclude that MPK3 and MPK6 perform overlapping but not identical roles in the reproduction and development of Arabidopsis thaliana.EXPRESS Progra

Functional analysis of MAP kinases in Arabidopsis thaliana: Fully rescuing the mpk3/mpk6 mutant phenotypes

Abstract only availableMitogen Activated Protein Kinase (MAPK) cascades are three-stage modules involved in signal transduction. MAPKs function at the lower tier of these cascades and they phosphorylate transcription factors and other protein kinases upon activation, ultimately leading to cellular responses. Twenty genes coding for MAPKs were identified in the fully sequenced Arabidopsis genome. MPK3 and MPK6 are the most closely related. Analysis of T-DNA insertional lines revealed no phenotype in the mpk3 and mpk6 single mutants; however, female sterility is observed in MPK3+/-/MPK6-/- plants and embryo lethality results from knocking out both genes. This indicates overlapping function of MPK3 and MPK6. To better understand the function of these two kinases, an attempt was made to rescue these phenotypes by introducing a Dexamethasone (DEX) inducible: MPK6 transgene. This construct led to only partial rescue of the lethal double mutants, and no signs of fertility were evident in MPK3+/-/MPK6-/- plants. In an attempt to attain complete rescue of these phenotypes, new MPK3 and MPK6 constructs were engineered with the following features: • Transgenes regulated by endogenous promoters were used in order to maintain normal cell/tissue specific expression of the protein, which may be essential for normal plant function. • The transgene products were tagged with Yellow Florescent Protein and Green Florescent Protein in order to ascertain their expression patterns. • Genomic DNA, as opposed to complementary DNA, was used as the coding regions in order to ensure the presence of introns, which may be significant for gene function. Currently, T1 generation transgenic plants have been isolated and transgenic lines with good expression of the transgene proteins, in vivo, will be identified by Western Blot analysis. Indication of a full rescue will be verified in the T2 generation. Failure to observe completely rescued lines may indicate protein tag interference, and untagged constructs will then be attempted.MU Monsanto Undergraduate Research Fellowshi

FAK Promotes Early Osteoprogenitor Cell Proliferation by Enhancing mTORC1 Signaling

Focal adhesion kinase (FAK) has important functions in bone homeostasis but its role in early osteoprogenitor cells is unknown. We show herein that mice lacking FAK in Dermo1- expressing cells exhibited low bone mass and decreased osteoblast number. Mechanistically, FAK- deficient early osteoprogenitor cells had decreased proliferation and significantly reduced mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, a central regulator of cell growth and proliferation. Furthermore, our data showed that the pharmacological inhibition of FAK kinase- dependent function alone was sufficient to decrease the proliferation and compromise the mineralization of early osteoprogenitor cells. In contrast to the Fak deletion in early osteoprogenitor cells, FAK loss in Col3.6 Cre- targeted osteoblasts did not cause bone loss, and Fak deletion in osteoblasts did not affect proliferation, differentiation, and mTORC1 signaling but increased the level of active proline- rich tyrosine kinase 2 (PYK2), which belongs to the same non- receptor tyrosine kinase family as FAK. Importantly, mTORC1 signaling in bone marrow stromal cells (BMSCs) was reduced if FAK kinase was inhibited at the early osteogenic differentiation stage. In contrast, mTORC1 signaling in BMSCs was not affected if FAK kinase was inhibited at a later osteogenic differentiation stage, in which, however, the concomitant inhibition of both FAK kinase and PYK2 kinase reduced mTORC1 signaling. In summary, our data suggest that FAK promotes early osteoprogenitor cell proliferation by enhancing mTORC1 signaling via its kinase- dependent function and the loss of FAK in osteoblasts can be compensated by the upregulated active PYK2. © 2020 American Society for Bone and Mineral Research.Schematic model of the differential roles of FAK in the cells of osteoblast lineage. The model depicts the mechanisms of FAK action at three distinct stages of osteoblast lineage in which the roles of FAK have been addressed by genetic and pharmacological approaches as well as the respective Cre transgenes used to target Fak, including Dermo1- Cre (this study), Osterix- Cre,(10) Col3.6- Cre (this study), and Col2.3- Cre.(9) Red - indicates that the loss of FAK in osteoblasts can be compensated by the upregulated active PYK2.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162813/3/jbmr4029-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162813/2/jbmr4029_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162813/1/jbmr4029.pd

Contribution of Hepatitis B Virus Infection to the Aggressiveness of Primary Liver Cancer: A Clinical Epidemiological Study in Eastern China

Background and aims: The contribution of hepatitis B virus (HBV) infection to the aggressiveness of primary liver cancer (PLC) remains controversial. We aimed to characterize this in eastern China.Methods: We enrolled 8,515 PLC patients whose specimens were reserved at the BioBank of the hepatobiliary hospital (Shanghai, China) during 2007–2016. Of those, 3,124 who received primary radical resection were involved in survival analysis. A nomogram was constructed to predict the survivals using preoperative parameters.Results: Hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular cholangiocarcinoma (CHC) accounted for 94.6, 3.7, and 1.7%, respectively. The rates of HBV infection were 87.5, 49.2, and 80.6%, respectively. HBV infection was significantly associated with 10-year earlier onset, more cirrhosis, higher α-fetoprotein, higher carbohydrate antigen 19-9 (CA19-9), more microvascular invasion (MVI), lower neutrophil-to-lymphocyte ratio (NLR), and lower platelet-to-lymphocyte ratio (PLR) in HCC. HBV infection was also associated with 7-year earlier onset, more cirrhosis, higher α-fetoprotein, more MVI, and lower PLR in ICC. In the multivariate Cox analysis, high circulating HBV DNA, α-fetoprotein, CA19-9, NLR, tumor size, number, encapsulation, Barcelona Clinic Liver Cancer (BCLC) stage, and MVI predicted an unfavorable prognosis in HCC; only CA19-9 and BCLC stage, rather than HBV-related parameters, had prognostic values in ICC. A nomogram constructed with preoperative HBV-related parameters including HBV load, ultrasonic cirrhosis, and α-fetoprotein perform better than the current staging systems in predicting postoperative survival in HCC.Conclusion: HBV promotes the aggressiveness of HCC in Chinese population. The contributions of HBV to ICC and other etiological factors to HCC might be indirect via arousing non-resolving inflammation

Synchronous multimode ultrasound for assessing right-to-left shunt: a prospective clinical study

BackgroundRight-to-left shunt (RLS) is associated with several conditions and causes morbidity. In this study, we aimed to evaluate the effectiveness of synchronous multimode ultrasonography in detecting RLS.MethodsWe prospectively enrolled 423 patients with high clinical suspicion of RLS and divided them into the contrast transcranial Doppler (cTCD) group and synchronous multimode ultrasound group, in which both cTCD and contrast transthoracic echocardiography (cTTE) were performed during the same process of contrast-enhanced ultrasound imaging. The simultaneous test results were compared with those of cTCD alone.ResultsThe positive rates of grade II (22.0%:10.0%) and III (12.7%:10.8%) shunts and the total positive rate (82.1748%) in the synchronous multimode ultrasound group were higher than those in the cTCD alone group. Among patients with RLS grade I in the synchronous multimode ultrasound group, 23 had RLS grade I in cTCD but grade 0 in synchronous cTTE, whereas four had grade I in cTCD but grade 0 in synchronous cTTE. Among patients with RLS grade II in the synchronous multimode ultrasound group, 28 had RLS grade I in cTCD but grade II in synchronous cTTE. Among patients with RLS grade III in the synchronous multimode ultrasound group, four had RLS grade I in cTCD but grade III in synchronous cTTE. Synchronous multimode ultrasound had a sensitivity of 87.5% and specificity of 60.6% in the patent foramen ovale (PFO) diagnosis. Binary logistic regression analyses showed that age (odds ratio [OR] = 1.041) and risk of paradoxical embolism score ≥ 7 (OR = 7.798) were risk factors for stroke recurrence, whereas antiplatelets (OR = 0.590) and PFO closure with antiplatelets (OR = 0.109) were protective factors.ConclusionSynchronous multimodal ultrasound significantly improves the detection rate and test efficiency, quantifies RLS more accurately, and reduces testing risks and medical costs. We conclude that synchronous multimodal ultrasound has significant potential for clinical applications

ДВУХСТОРОННИЙ ГНОЙНЫЙ КЕРАТОСКЛЕРИТ, ВЫЗВАННЫЙ СИНЕГНОЙНОЙ ПАЛОЧКОЙ, У ПАЦИЕНТКИ В КОМЕ

The article describes a clinical case of bilateral keratoscleritis caused by Ps. aeroginosa in a female patient suffering from occlusive hydrocephaly and intracranial hypertension with duration of coma and artificial pulmonary ventilation (APV) for 20 days. Materials and methods. At the moment of examination (in 7 days after purulent keratoscleritis started and rapidly progressed) the patient had lagophthalmos, purulent corneal ulcer, purulent xerotic sclera of OU, keratorrhexis of OS. On the same day directly in the intensive care unit, emergency penetrating sclerokeratoplasty was performed on OU in order to save eyes as organs. Forced instillations of antibiotics and antiseptics were used in the post-operative period. In 2 years 2 penetrating keratoplasties were performed in OD for optic purposes. Results. Emergency theurapetical penetrating sclerokeratoplasty with instillation of anterior segment and intraocular administration of a high dilution antibiotic were the only chance to save vision in OD. OS, where keratorrhexis developed due to advanced purulent sclerokeratitis and purulent iridocyclitis was lost despite all the efforts. After three surgeries (sclerokeratoplasty and 2 keratoplasties) vision acuity in the only right eye makes 0.1, which can be regarded as a satisfactory outcome. Conclusions. A malicious course of the disease, peracute progress and extremely severe consequences are typical of the advanced corneal ulcer caused by Ps. aeroginosa. Special attention is to be paid to the eyes of patients being in an intensive care unit in a coma and having continuous artificial pulmonary ventilation due to the high risk of developing purulent corneal ulcer and eye loss. Представлено клиническое наблюдение развитого двухстороннего синегнойного склерокератита, развившегося у пациентки с окклюзионной гидроцефалией и внутричерепной гипертензией с длительностью пребывания в коме и искусственной вентиляции легких (ИВЛ) 20 дней. Материалы и метод. На момент осмотра (спустя 7 дней после начала гнойного склерокератита и бурного прогрессирования заболевания) у больной определялись лагофтальм, развитая гнойная язва роговицы, гнойное расплавление склеры OU, перфорация роговицы на OS. В тот же день непосредственно в отделении реанимации и интенсивной терапии с целью спасения глаза как органа была проведена операция неотложной сквозной склерокератопластики на OU. В послеоперационном периоде применяли форсированные инстилляции антибиотиков и антисептиков. Спустя 2 года на OD было проведено 2 ре-СКП (сквозные кератопластики) c оптической целью. Результаты. Безотлагательно выполненная операция лечебной сквозной склерокератопластики (ССКП) с орошением переднего отрезка глаза и внутриглазным введением высокого разведения антибиотика явилась единственным шансом спасения зрения на OD. На OS, где имелась перфорация на фоне развитого гнойного склерокератита и гнойного иридоциклита, несмотря на все предпринятые меры, спасти глаз не удалось. Острота зрения после проведенных трех операций (ССКП и 2 ре-СКП) на единственном правом глазу составляет 0,1, что можно считать удовлетворительным результатом. Выводы. Развитая язва роговицы, вызванная синегнойной палочкой, характеризуется злокачественным течением, молниеносным прогрессированием и исключительно тяжелыми последствиями. Состоянию глаз пациентов, находящихся в отделении реанимации и интенсивной терапии в состоянии комы и при проведении длительной ИВЛ, следует уделять особое внимание в связи с высоким риском развития гнойной язвы роговицы и гибели глаза.

GWAS Identifies Novel Susceptibility Loci on 6p21.32 and 21q21.3 for Hepatocellular Carcinoma in Chronic Hepatitis B Virus Carriers

Genome-wide association studies (GWAS) have recently identified KIF1B as susceptibility locus for hepatitis B virus (HBV)–related hepatocellular carcinoma (HCC). To further identify novel susceptibility loci associated with HBV–related HCC and replicate the previously reported association, we performed a large three-stage GWAS in the Han Chinese population. 523,663 autosomal SNPs in 1,538 HBV–positive HCC patients and 1,465 chronic HBV carriers were genotyped for the discovery stage. Top candidate SNPs were genotyped in the initial validation samples of 2,112 HBV–positive HCC cases and 2,208 HBV carriers and then in the second validation samples of 1,021 cases and 1,491 HBV carriers. We discovered two novel associations at rs9272105 (HLA-DQA1/DRB1) on 6p21.32 (OR = 1.30, P = 1.13×$10^{−19}$) and rs455804 (GRIK1) on 21q21.3 (OR = 0.84, P = 1.86×$10^{−8}$), which were further replicated in the fourth independent sample of 1,298 cases and 1,026 controls (rs9272105: OR = 1.25, P = 1.71×$10^{−4}$; rs455804: OR = 0.84, P = 6.92×$10^{−3}$). We also revealed the associations of HLA-DRB1*0405 and 0901*0602, which could partially account for the association at rs9272105. The association at rs455804 implicates GRIK1 as a novel susceptibility gene for HBV–related HCC, suggesting the involvement of glutamate signaling in the development of HBV–related HCC

Dual-Level Regulation of ACC Synthase Activity by MPK3/MPK6 Cascade and Its Downstream WRKY Transcription Factor during Ethylene Induction in Arabidopsis

Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea–induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea–induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction

Irradiation-induced telomerase activity and gastric cancer risk: a case-control analysis in a Chinese Han population

<p>Abstract</p> <p>Background</p> <p>Telomerase expression is one of the characteristics of gastric cancer (GC) cells and telomerase activity is frequently up-regulated by a variety of mechanisms during GC development. Therefore, we hypothesized that elevated levels of activated telomerase might enhance GC risk due to increased propagation of cells with DNA damage, such as induced by γ-radiation.</p> <p>Methods</p> <p>To explore this hypothesis, 246 GC cases and 246 matched controls were recruited in our case-control study. TRAP-ELISA was used to assess the levels of telomerase activity at baseline and after γ-radiation and the γ-radiation-induced telomerase activity (defined as after γ-irradiation/baseline) in cultured peripheral blood lymphocytes (PBLs).</p> <p>Results</p> <p>Our data showed that there was no significant difference for the baseline telomerase activity between GC cases and controls (10.17 ± 7.21 <it>vs. </it>11.02 ± 8.03, <it>p </it>= 0.168). However, after γ-radiation treatment, γ-radiation-induced telomerase activity was significantly higher in the cases than in the controls (1.51 ± 0.93 <it>vs</it>. 1.22 ± 0.66, <it>p </it>< 0.001). Using the median value of γ-radiation-induced telomerase activity in the controls as a cutoff point, we observed that high γ-radiation-induced telomerase activity was associated with a significantly increased GC risk (adjusted odds ratio, 2.45; 95% confidence interval, 1.83-3.18). Moreover, a dose response association was noted between γ-radiation-induced telomerase activity and GC risk. Age, but not sex, smoking and drinking status seem to have a modulating effect on the γ-radiation-induced telomerase activities in both cases and controls.</p> <p>Conclusion</p> <p>Overall, our findings for the first time suggest that the increased γ-radiation-induced telomerase activity in PBLs might be associated with elevated GC risk. Further confirmation of this association using a prospective study design is warranted.</p