Synthesis and in†vitro Anti-Inflammatory Activity of Novel Dendrobine Amide/Sulfonamide Derivatives.

A traditional Chinese medicine ingredient, dendrobine, has been demonstrated to have anti-inflammatory properties. However, due to its poor anti-inflammatory properties, its clinical use is limited. Consequently, we have designed and synthesized 32 new amide/sulfonamide dendrobine derivatives and screened their anti-inflammatory activities in vitro. Experiments showed that nitric oxide (NO) generation in lipopolysaccharide (LPS)-induced RAW264.7 cells was strongly reduced by derivative 14, with an IC50 of 2.96 µM. Western blot research revealed that 14 decreased the concentration-dependent expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS). Molecular docking was used to predict the binding of the inflammation-associated proteins COX-2 and INOS to compound 14.

Structurally Diverse Coumarins from Peucedanum praeruptorum and their Anti-Inflammatory Activities via NF-κB Signaling Pathway.

The phytochemical study of Peucedanum praeruptorum led to the isolation of twenty-five coumarins (1-25). Of which, (±) praeruptol A (±1), one pair of previous undescribed seco-coumarin enantiomers were obtained. Their structures were established according to HR-ESI-MS, NMR, X-ray single crystal diffraction analysis, as well as ECD calculation. All compounds were tested for anti-inflammatory activity in the RAW264.7 macrophage model, and eight compounds (7-10, and 13-16) exhibited significant inhibitory effects with IC50 values ranging from 9.48 to 34.66 µM. Among them, compound 7 showed the strongest inhibitory effect, which significantly suppressed the production of IL-6, IL-1ß, and TNF-α, as well as iNOS and COX-2 in a concentration-dependent manner. Further investigated results showed that compound 7 exerted an anti-inflammatory effect via the NF-κB signaling pathway.

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail.

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

An unconventional regulatory circuitry involving Aurora B controls anaphase onset and error-free chromosome segregation in trypanosomes.

Accurate chromosome segregation during mitosis requires that all chromosomes establish stable bi-oriented attachments with the spindle apparatus. Kinetochores form the interface between chromosomes and spindle microtubules and as such are under tight control by complex regulatory circuitry. As part of the chromosomal passenger complex (CPC), the Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint, a feedback control system that delays the onset of anaphase by inhibiting the anaphase-promoting complex/cyclosome. Intriguingly, Aurora B is conserved even in kinetoplastids, an evolutionarily divergent group of eukaryotes, whose kinetochores are composed of a unique set of structural and regulatory proteins. Kinetoplastids do not have a canonical spindle checkpoint and it remains unclear how their kinetochores are regulated to ensure the fidelity and timing of chromosome segregation. Here, we show in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness, that inhibition of Aurora B using an analogue-sensitive approach arrests cells in metaphase, with a reduction in properly bi-oriented kinetochores. Aurora B phosphorylates several kinetochore proteins in vitro, including the N-terminal region of the divergent Bub1-like protein KKT14. Depletion of KKT14 partially overrides the cell cycle arrest caused by Aurora B inhibition, while overexpression of a non-phosphorylatable KKT14 protein results in a prominent delay in the metaphase-to-anaphase transition. Finally, we demonstrate using a nanobody-based system that re-targeting the catalytic module of the CPC to the outer kinetochore is sufficient to promote mitotic exit but causes massive chromosome mis-segregation in anaphase. Our results indicate that the CPC and KKT14 are involved in an unconventional pathway controlling mitotic exit and error-free chromosome segregation in trypanosomes.

Limonoids from the Branches and Leaves of Aglaia lawii and Their Cytotoxic Activities.

Three undescribed limonoids (1-3), named aglaians G-I, and one new natural product azedaralide (4), together with nine known analogues (5-13) were isolated from the branches and leaves of Aglaia lawii by RP C18 column, silica gel column, Sephadex LH-20 column chromatography and preparative HPLC. The structures of the new compounds were elucidated by IR, HRESIMS, 1D, 2D NMR, electronic circular dichroism (ECD) calculations and X-ray crystallography diffraction analysis. The results of bioassay showed that the compound 12 exhibited potential inhibitory activity against six human tumor cell lines (MDA-MB-231, MCF-7, Ln-cap, A549, HeLa and HepG-2) with IC50 values as 8.0-18.6 µM.

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases.

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites-short linear motifs found in MAPK substrates, regulators, and scaffolds-can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

Unusual triterpenoids and steroids from Cipadessa baccifera and their biological activities.

Five undescribed triterpenoids and steroids (1-5), as well as ten known compounds, were purified from the branches and leaves of Cipadessa baccifera. Notably, 1 and 2 are rare cipadesin-type limonoids with an unusual 8,30-epoxide ring and 1,8-ether linkage, respectively. Compound 5 possessed pregnane steroid skeleton with an uncommon 5/6/6/6/5-fused ring system. Their structures were constructed by extensive spectroscopic analysis (NMR, IR, UV, and HRESIMS), and their absolute configurations were confirmed by ECD calculations and quantum chemical calculations. All the isolates were in vitro assayed for their antimicrobial potentials against 6 pathogenic microorganisms and antiproliferation activities against five human cancer cell lines. As a result, compounds 5, 12, 13, and 14 exhibited moderate antibacterial activities (MIC: 25-50 µg/mL). Moreover, 5 showed cytotoxicity against five cancer cell lines with IC50 values ranging from 8.0 to 19.9 µM.

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail.

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but the aspects of cofilin functionality driving this conservation are not clear. Here, we screened a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and subsequent biochemical analysis of individual variants revealed distinct sequence requirements for actin binding and regulation by LIM kinase. While the presence of a serine, rather than threonine, phosphoacceptor residue was essential for phosphorylation by LIM kinase, the native cofilin N-terminus was otherwise a suboptimal LIM kinase substrate. This circumstance was not due to sequence requirements for actin binding and severing, but rather appeared primarily to maintain the capacity for phosphorylation to inactivate cofilin. Overall, the individual sequence requirements for cofilin function and regulation were remarkably loose when examined separately, but collectively restricted the N-terminus to sequences found in natural cofilins. Our results illustrate how a regulatory phosphorylation site can balance potentially competing sequence requirements for function and regulation.

Setdb1 -loss induces type-I interferons and immune clearance of melanoma.

Despite recent advances in the treatment of melanoma, many patients with metastatic disease still succumb to their disease. To identify tumor-intrinsic modulators of immunity to melanoma, we performed a whole-genome CRISPR screen in melanoma and identified multiple components of the HUSH complex, including Setdb1 , as hits. We found that loss of Setdb1 leads to increased immunogenicity and complete tumor clearance in a CD8+ T-cell dependent manner. Mechanistically, loss of Setdb1 causes de-repression of endogenous retroviruses (ERVs) in melanoma cells and triggers tumor-cell intrinsic type-I interferon signaling, upregulation of MHC-I expression, and increased CD8+ T-cell infiltration. Furthermore, spontaneous immune clearance observed in Setdb1 -/- tumors results in subsequent protection from other ERV-expressing tumor lines, supporting the functional anti-tumor role of ERV-specific CD8+ T-cells found in the Setdb1 -/- microenvironment. Blocking the type-I interferon receptor in mice grafted with Setdb1 -/- tumors decreases immunogenicity by decreasing MHC-I expression, leading to decreased T-cell infiltration and increased melanoma growth comparable to Setdb1 wt tumors. Together, these results indicate a critical role for Setdb1 and type-I interferons in generating an inflamed tumor microenvironment, and potentiating tumor-cell intrinsic immunogenicity in melanoma. This study further emphasizes regulators of ERV expression and type-I interferon expression as potential therapeutic targets for augmenting anti-cancer immune responses.

Is it necessary for young patients with recurrent implantation failure to undergo preimplantation genetic testing for aneuploidy?

Objective: To determine whether preimplantation genetic testing for aneuploidy (PGT-A) can improve the pregnancy outcomes of patients aged under 38 years who have a history of recurrent implantation failure(RIF). Design: Retrospective cohort study. Methods: We retrospectively studied the pregnancy outcomes of RIF patients aged under 38 years from January 2017 to December 2021.178 patients were divided into two groups according to whether they underwent PGT-A: the PGT-A group(n=59)and the control group(n=119).In the PGT-A group, we compared the euploidy rate of the different quality and developmental rate blastocysts. In both groups,the patients were the first frozen-thaw single blastocysts transfer after the diagnosis of RIF. Among the pregnancy outcomes, the clinical pregnancy rate was assessed as the primary outcome. The spontaneous abortion rate and ongoing pregnancy rate were the secondry outcomes. The generalized estimation equation was used to adjust for the blastocysts derived from the same patients. Multivariate logistic analysis models were used to compare the pregnancy outcomes between the two groups. Results: In the PGT-A group, 293 blastocysts obtained from59 patients underwent PGT-A. The proportions of euploidy, aneuploidy and mosaic blastocysts were 56.31%, 25.60% and 18.09%, respectively. A comparison of the euploidy rates of different quality blastocysts showed that the rate of good-quality blastocysts was significantly higher than that of poor-quality blastocysts (67.66% vs 46.88%; odds ratio [OR], 2.203; 95%confidence interval[CI], 0.943-3.612; P=0.002). However, no significant difference was observed in the different developmental rates blastocysts. Compared with Day 5 blastocysts, the euploidy rates of Day 6 and Day 7 blastocysts were not significantly different(61.54%vs51.91%; OR,0.945; 95%CI, 0.445-2.010; P=0.884; and 61.54%vs47.37%; OR, 1.106; 95%CI, 0.774-1.578; P=0.581, respectively).As for the pregnancy outcomes, the clinical pregnancy rate was significantly increase after the use of PGT-A compared with the control group(71.19%vs56.30%; OR, 0.538; 95%CI, 0.262-1.104; P=0.039). However, the spontaneous abortion rates and ongoing pregnancy rates were not significantly different between the control and PGT-A groups (21.43% vs 19.40%; aOR,0.727; 95%CI,0.271-1.945; P=0.525; and55.93% vs 45.38%; aOR, 0.649; 95%CI, 0.329-1.283; P = 0.214,respectively). Conclusion: PGT-A improved the clinical pregnancy rate after blastocyst transfer in RIF patients aged under 38 years.

Proteome-wide screening for mitogen-activated protein kinase docking motifs and interactors.

Essential functions of mitogen-activated protein kinases (MAPKs) depend on their capacity to selectively phosphorylate a limited repertoire of substrates. MAPKs harbor a conserved groove located outside of the catalytic cleft that binds to short linear sequence motifs found in substrates and regulators. However, the weak and transient nature of these "docking" interactions poses a challenge to defining MAPK interactomes and associated sequence motifs. Here, we describe a yeast-based genetic screening pipeline to evaluate large collections of MAPK docking sequences in parallel. Using this platform, we analyzed a combinatorial library based on the docking sequences from the MAPK kinases MKK6 and MKK7, defining features critical for binding to the stress-activated MAPKs JNK1 and p38α. Our screen of a library consisting of ~12,000 sequences from the human proteome revealed multiple MAPK-selective interactors, including many that did not conform to previously defined docking motifs. Analysis of p38α/JNK1 exchange mutants identified specific docking groove residues that mediate selective binding. Last, we verified that docking sequences identified in the screen functioned in substrate recruitment in vitro and in cultured cells. Together, these studies establish an approach to characterize MAPK docking sequences and provide a resource for future investigation of signaling downstream of p38 and JNK.

Clinical outcome analysis of frozen-thawed embryo transfer on Day 7.

Objective: To investigate the clinical outcomes of Day 7 (D7) frozen-thawed embryo transfer (FET) and to provide a reference value for clinical work. Methods: This was a retrospective cohort study. Patients undergoing FET cycles in the Reproductive Medicine Center of the Third Affiliated Hospital of Zhengzhou University between December 2015 and January 2021 were included. According to the developmental stage of the embryos at transfer, the embryos were divided into three groups: Day (D) 5, D6 and D7 blastocysts. Group D7 was compared with Groups D5 and D6. Simultaneously, the preimplantation genetic testing (PGT) and non-PGT cycles in Group D7 were analyzed and compared. The main outcomes were the clinical pregnancy, live birth and miscarriage rates. The secondary outcomes were the implantation and euploidy rates. Results: In total, 5945, 4094 and 137 FET cycles were included in the D5, D6 and D7 groups, respectively. The clinical pregnancy rate was significantly lower in Group D7 than in Groups D5 (13.9% vs 62.9%, P <0.001) and D6 (13.9% vs 51.4%, P <0.001). Additionally, the live birth rate was significantly lower in Group D7 than in Groups D5 (7.3% vs 50.7%, P <0.001) and D6 (7.3% vs 40.5%, P <0.001). However, the miscarriage rate was significantly higher in Group D7 than in Groups D5 (47.4% vs 18.2%, P =0.001) and D6 (47.4% vs 20.6%, P =0.004). The clinical pregnancy and live birth rates for D7 blastocysts were significantly higher in the PGT group than in the non-PGT group (41.7% vs 13.9%, P=0.012; 33.3% vs 7.3%, P =0.003). Conclusions: D7 blastocyst transfer can yield a live birth rate that is lower than that for D5 and D6 blastocysts but has value for transfer. PGT for D7 blastocysts may reduce the number of ineffective transfers and improve the outcome of D7 blastocyst transfer, which can be performed according to a patient's situation.

Cipacinerasins A-K, structurally diverse limonoids from Cipadessa baccifera.

Eleven undescribed limonoids, cipacinerasins A-K, involving of four diverse carbon skeletal types, along with fifteen known analogues, were isolated from the branches and leaves of Cipadessa baccifera. Within them, cipacinerasins A and B feature a rearranged tetrahydropyranyl ring B formed between C-8 and C-30, are unusual miscellaneous-type limonoids. Cipacinerasins E and F are rare trijugin-type limonoids, of which the D-ring δ-lactone is cleaved. Their structures were elucidated on the basis of extensive spectroscopic data (HRESIMS, NMR, UV and IR), electronic circular dichroism (ECD) calculations, and single-crystal X-ray diffraction analysis. All compounds were evaluated in vitro cytotoxicity against five human tumor cell lines (K562, HeLa, PC3, LN-Cap and Hell), and cipacinerasin E showed moderate antitumor activity with IC50 values ranging from 8.0 to 24.8 µM.

Effect of Blastocyst Morphology and Developmental Rate on Euploidy and Live Birth Rates in Preimplantation Genetic Testing for Aneuploidy Cycles With Single-Embryo Transfer.

Objective: The aim of this study was to investigate whether blastocyst morphology and developmental rate are associated with euploidy and live birth rates (LBRs) in single euploid frozen-thawed embryo transfer (FET) cycles. Design: Retrospective cohort study. Methods: This study included 431 preimplantation genetic testing for aneuploidy (PGT-A) cycles followed by 393 FET cycles performed at our center from June 2017 to March 2021. All cycles were analyzed for euploidy based on blastocyst morphology (good, average and poor), developmental stage (day 5 and 6) and maternal age (< 35 and ≥ 35 years old). Multivariate logistic analysis models were used to identify the independent effects of conventional blastocyst morphology, developmental rate and morphological parameters (degree of blastocoele expansion, and grade of inner cell mass and trophectoderm (TE)) on LBRs. Results: In the group of women aged < 35 years, compared with poor-quality blastocysts, good-quality blastocysts (62.90% vs. 32.46%; odds ratio (OR) 3.163, 95% confidence interval (CI) 2.247-4.451; P < 0.001) and average-quality blastocysts (46.70% vs. 32.46%; OR 1.665, 95% CI 1.287-2.154; P < 0.001) had significantly higher euploidy rates. Additionally, day 5 blastocysts were associated with higher euploidy rates than day 6 blastocysts (49.28% vs. 35.02%; OR 1.506, 95% CI 1.191-1.903; P= 0.001). In the group of women aged ≥ 35 years, euploidy rates were also associated with blastocyst morphology, with 41.86%, 45.65% and 24.39% of good, average and poor-quality embryos, respectively, exhibiting euploidy. However, no relationship was seen between euploidy and blastocyst developmental rate. Multiple logistic regression analysis show that overall blastocyst morphology of euploid embryos was not associated with LBR, only embryos with A-grade TE had significantly higher LBRs than those with C-grade TE (62.71% vs. 45.40%; OR 2.189, 95% CI 1.166-4.109; P=0.015). Similarly, LBRs were significantly higher when day 5 blastocysts were transferred than when day 6 blastocysts were transferred (57.75% vs. 41.67%; OR 2.132, 95% CI 1.370-3.318; P = 0.001). Conclusion: Poor-quality embryos have reduced rates of euploidy. However, blastocyst developmental rate only significantly associates with euploidy rates in women aged younger than 35. Furthermore, only TE grade and blastocyst developmental rate are significantly associated with LBRs following FET cycles.

Novel flavonolignans from the roots of Indigofera stachyodes.

Two pairs of new enantiomeric flavonolignans, ±stachyols A and B (±1 and ± 2), along with two novel isoflavanelignans, stachyols C and D (3 and 4) were isolated from the roots of Indigofera stachyodes. Their chemical structures and absolute configurations were determined using nuclear magnetic resonance and comparison of experimental and theoretical electronic circular dichroism (ECD) spectra, as well as quantum chemical calculations. Of those compounds, 1 and 2 represented the first examples of flavonolignans with 5-deoxyflavonoids adduct phenylpropanoids. Moreover, 3 and 4 possess an unprecedented skeleton with isoflavanes adduct phenylpropanoids. The antioxidant activity was evaluated for all compounds in terms of ABTS+ and DPPH bioassays. Compounds 3 and 4 exhibited significant radical-scavenging activity in the ABTS+ assay, with IC50 values of 15.15 and 5.83 µM, respectively.

Perinatal outcomes of singleton live births after preimplantation genetic testing during single frozen-thawed blastocyst transfer cycles: a propensity score-matched study.

OBJECTIVE: To determine whether singleton pregnancy achieved after preimplantation genetic testing (PGT) is associated with a higher risk of adverse perinatal outcomes than in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) singleton pregnancy. DESIGN: A retrospective cohort study. SETTING: A university-affiliated fertility center. PATIENT(S): This cohort study included singleton live births resulting from PGT (n = 232) and IVF/ICSI singleton pregnancies (n = 2,829) with single frozen-thawed blastocyst transfer. Multiple baseline covariates were used for propensity score matching, yielding 214 PGT singleton pregnancies matched to 617 IVF/ICSI singleton pregnancies. INTERVENTION(S): Trophectoderm biopsy. MAIN OUTCOME MEASURE(S): The primary outcome was gestational hypertension, and various clinical perinatal secondary outcomes related to maternal and neonatal health were measured. RESULT(S): Compared with IVF/ICSI singleton pregnancy, PGT singleton pregnancy was associated with a significantly higher risk of gestational hypertension (adjusted odds ratio, 2.58; 95% confidence interval, 1.32, 5.05). In the matched sample, the risk of gestational hypertension remained higher with PGT singleton pregnancy (odds ratio, 2.33; 95% confidence interval, 1.04, 5.22) than with IVF/ICSI singleton pregnancy. No statistical differences were noted in any other measured outcomes between the groups. CONCLUSION(S): The perinatal outcomes of PGT and IVF/ICSI singleton pregnancies were similar except for the observed potentially higher risk of gestational hypertension with PGT singleton pregnancy. However, because the data on PGT singleton pregnancies are limited, this conclusion warrants further investigation.

Tousled-like kinase 2 targets ASF1 histone chaperones through client mimicry.

Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate.

Profiling alkaloids in Aconitum pendulum N. Busch collected from different elevations of Qinghai province using widely targeted metabolomics.

Aconitum pendulum N. Busch (Ranunculaceae) is rich in alkaloids with anti-inflammatory and analgesic activities. Many studies have focused on the identification or quantification of alkaloid components using low-throughput tests. However, the metabolic differences of plants from environmentally distinct regions remain unclear. The present study profiled alkaloid chemical compounds in the rhizomes of A. pendulum from different regions. A total of 80 chemical compounds were identified using a widely targeted ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach. Principal component, hierarchical clustering, and orthogonal partial least squares-discriminant analyses of the chemical compounds indicated that the plants from 6 regions clearly separated into distinct groups. A total of 19 compounds contributed the most to the metabolite differences between collection areas and were identified as potential metabolic markers. The anti-inflammatory activities of the A. pendulum extracts were also evaluated and the potential environmental effects on the regulation of metabolite composition and bioactivity were explored. These results improve our understanding of the variation in chemical composition of plants from different regions and will serve as a reference for evaluating the medicinal value of A. pendulum in different environments.

TAGLN mediated stiffness-regulated ovarian cancer progression via RhoA/ROCK pathway.

BACKGROUND: Ovarian cancer (OC) progression is an unmet medical challenge. Since omental metastases were palpated harder than their primary counterparts during cytoreductive surgery of patients with epithelial ovarian cancer (EOC), we were inspired to investigate OC progression from the perspective of biomechanics. METHODS: Atomic Force Microscope (AFM) was used to measure the Young's modulus of tissues. The collagen-coated polyacrylamide hydrogel (PA gel) system was prepared to mimic the soft and stiff substrates in vitro. The effect of TAGLN was evaluated both in vitro and in vivo using transwell assay, immunofluorescence, western blot analysis and immunohistochemistry. RESULTS: We quantitatively confirmed that omental metastases were stiffer and more abundant in desmoplasia compared with paired primary tumors, and further demonstrated that matrix stiffness could notably regulate OC progression. Remarkably, TAGLN, encoding an actin cross-linking/gelling protein, was identified as a potent mechanosensitive gene that could form a regulation loop with Src activation reacting to environmental stiffness, thus mediating stiffness-regulated OC progression through regulating RhoA/ROCK pathway. CONCLUSIONS: These data demonstrate that targeting extra-cellular matrix (ECM) stiffness could probably hamper OC progression, and of note, targeting TAGLN might provide promising clinical therapeutic value for OC therapy.

PPP6C negatively regulates oncogenic ERK signaling through dephosphorylation of MEK.

Flux through the RAF-MEK-ERK protein kinase cascade is shaped by phosphatases acting on the core components of the pathway. Despite being an established drug target and a hub for crosstalk regulation, little is known about dephosphorylation of MEK, the central kinase within the cascade. Here, we identify PPP6C, a phosphatase frequently mutated or downregulated in melanoma, as a major MEK phosphatase in cells exhibiting oncogenic ERK pathway activation. Recruitment of MEK to PPP6C occurs through an interaction with its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting signaling through the ERK pathway and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation, suggesting that they promote disease at least in part by activating the core oncogenic pathway driving melanoma. Collectively, our studies identify a key negative regulator of ERK signaling that may influence susceptibility to targeted cancer therapies.