Lens differentiation is controlled by the balance between PDGF and FGF signaling

How multiple receptor tyrosine kinases coordinate cell fate determination is yet to be elucidated. We show here that the receptor for platelet-derived growth factor (PDGF) signaling recruits the p85 subunit of Phosphoinositide 3-kinase (PI3K) to regulate mammalian lens development. Activation of PI3K signaling not only prevents B-cell lymphoma 2 (BCL2)-Associated X (Bax)- and BCL2 Antagonist/Killer (Bak)-mediated apoptosis but also promotes Notch signaling to prevent premature cell differentiation. Reducing PI3K activity destabilizes the Notch intracellular domain, while the constitutive activation of Notch reverses the PI3K deficiency phenotype. In contrast, fibroblast growth factor receptors (FGFRs) recruit Fibroblast Growth Factor Receptor Substrate 2 (Frs2) and Rous sarcoma oncogene (Src) Homology Phosphatase 2 (Shp2) to activate Mitogen-Activated Protein Kinase (MAPK) signaling, which induces the Notch ligand Jagged 1 (Jag1) and promotes cell differentiation. Inactivation of Shp2 restored the proper timing of differentiation in the p85 mutant lens, demonstrating the antagonistic interaction between FGF-induced MAPK and PDGF-induced PI3K signaling. By selective activation of PI3K and MAPK, PDGF and FGF cooperate with and oppose each other to balance progenitor cell maintenance and differentiation

Crk proteins transduce FGF signaling to promote lens fiber cell elongation

Specific cell shapes are fundamental to the organization and function of multicellular organisms. Fibroblast Growth Factor (FGF) signaling induces the elongation of lens fiber cells during vertebrate lens development. Nonetheless, exactly how this extracellular FGF signal is transmitted to the cytoskeletal network has previously not been determined. Here, we show that the Crk family of adaptor proteins, Crk and Crkl, are required for mouse lens morphogenesis but not differentiation. Genetic ablation and epistasis experiments demonstrated that Crk and Crkl play overlapping roles downstream of FGF signaling in order to regulate lens fiber cell elongation. Upon FGF stimulation, Crk proteins were found to interact with Frs2, Shp2 and Grb2. The loss of Crk proteins was partially compensated for by the activation of Ras and Rac signaling. These results reveal that Crk proteins are important partners of the Frs2/Shp2/Grb2 complex in mediating FGF signaling, specifically promoting cell shape changes

Phagocytic Cup Assembly During Rod Outer Segment Phagocytosis By The Retinal Pigment Epithelium

Diurnal phagocytosis of spent photoreceptor outer segment tips (POS) by retinal pigment epithelial (RPE) cells is essential for life-long retinal function. Clearance of POS is triggered by engagement of &agr;vβ5 integrin and Mer tyrosine kinase (MerTK) surface receptors of the RPE by their corresponding extracellular ligands. POS engulfment requires F-actin phagocytic cup formation. These studies presented in this dissertation sought to identify Rho GTPases family proteins that are involved in the regulation of this F-actin phagocytic cup formation during POS phagocytosis by RPE cells. I found that Rac1 is activated while RhoA-ROCK is inhibited during RPE phagocytosis. The activation of Rac1 is a necessary response to POS stimulation preceding POS engulfment. Rac1 is not activated by POS in RPE lacking &agr;vβ5 but occurs normally in RPE lacking MerTK in vivo and in culture. In contrast, RhoA-ROCK activity is down-regulated during phagocytosis by wt RPE but not by MerTK-deficient RPE. Lack of MerTK or its ligand also results in failure of phagocytic cup formation and engulfment. I then tested the dependence of F-actin recruitment to bound particles on Rac1 and the RhoA-ROCK pathway when MerTK signaling pathway is silent. To this end, I investigated the F-actin distribution and phagocytic activity of RPE cells in culture fed with POS with or without ligand supplementation and activating or inhibiting Rac1 and RhoA/ROCK either genetically or pharmacologically. Surface-bound POS failed to recruit F-actin and were not engulfed in the absence of MerTK signaling unless either Rac1 was constitutively and in excess active or RhoA/ROCK was inhibited. ROCK inhibition did not restore F-actin recruitment or engulfment abolished by expression of dominant-negative Rac1 and, conversely, constitutively-active Rac1 failed to restore F-actin recruitment or engulfment inhibited by activation of RhoA. Neither pathway manipulation affected the activity of the other pathway. Taken together, these results demonstrate that activation of Rac1 and inhibition of RhoA-ROCK must both contribute to productive F-actin dynamics that is required for POS engulfment by RPE cells

Acute RhoA/Rho Kinase Inhibition Is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE. We show here that mutant RPE lacking MerTK and wild-type RPE deprived of MerTK ligand both fail to form phagocytic cups regardless of Rac1 activation. However, in wild-type RPE in vivo, a decrease in RhoA activity coincides with the daily phagocytosis burst, while RhoA activity in MerTK-deficient RPE is constant. Elevating RhoA activity blocks phagocytic cup formation and phagocytosis by wild-type RPE. Conversely, inhibiting RhoA effector Rho kinases (ROCKs) rescues both F-actin assembly and POS internalization of primary RPE if MerTK or its ligand are lacking. Most strikingly, acute ROCK inhibition is sufficient to induce the formation and acidification of endogenous POS phagosomes by MerTK-deficient RPE ex vivo. Altogether, RhoA pathway inactivation is a necessary and sufficient downstream effect of MerTK phagocytic signaling such that the acute manipulation of cytosolic ROCK activity suffices to restore phagocytic capacity to MerTK-deficient RPE

Coordinated response of milk bacterial and metabolic profiles to subacute ruminal acidosis in lactating dairy cows

Abstract Background Bovine milk is an important source of nutrition for human consumption, and its quality is closely associated with the microbiota and metabolites in it. But there is limited knowledge about the milk microbiome and metabolome in cows with subacute ruminal acidosis. Methods Eight ruminally cannulated Holstein cows in mid lactation were selected for a 3-week experiment. The cows were randomly allocated into 2 groups, fed either a conventional diet (CON; 40% concentrate; dry matter basis) or a high-concentrate diet (HC; 60% concentrate; dry matter basis). Results The results showed that there was a decreased milk fat percentage in the HC group compared to the CON group. The amplicon sequencing results indicated that the alpha diversity indices were not affected by the HC feeding. At the phylum level, the milk bacteria were dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes both in the CON and HC groups. At the genus level, the HC cows displayed an improved proportion of Labrys (P = 0.015) compared with the CON cows. Results of both the principal components analysis and partial least squares of discriminant analysis of milk metabolome revealed that samples of the CON and HC groups clustered separately. A total of 31 differential metabolites were identified between the two groups. Of these, the levels of 11 metabolites decreased (α-linolenic acid, prostaglandin E2, L-lactic acid, L-malic acid, 3-hydroxysebacic acid, succinyladenosine, guanosine, pyridoxal, L-glutamic acid, hippuric acid, and trigonelline), whereas the levels of the other 20 metabolites increased in the HC group with respect to the CON group (P < 0.05). Conclusion These results suggested that subacute ruminal acidosis less impacted the diversity and composition of milk microbiota, but altered the milk metabolic profiles, which led to the decline of the milk quality

Fecal microbial gene transfer contributes to the high-grain diet-induced augmentation of aminoglycoside resistance in dairy cattle

ABSTRACTA high-grain (HG) diet can rapidly lower the rumen pH and thus modify the gastrointestinal microbiome in dairy cattle. Although the prevalence of antibiotic resistance is strongly linked with the gut microbiome, the influences of HG diet on animals’ gut resistome remain largely unexplored. Here, we examined the impact and mechanism of an HG diet on the fecal resistome in dairy cattle by metagenomically characterizing the gut microbiome. Eight lactating Holstein cattle were randomly allocated into two groups and fed either a conventional (CON) or HG diet for 3 weeks. The fecal microbiome and resistome were significantly altered in dairy cattle from HG, demonstrating an adaptive response that peaks at day 14 after the dietary transition. Importantly, we determined that feeding an HG diet specifically elevated the prevalence of resistance to aminoglycosides (0.11 vs 0.24 RPKG, P < 0.05). This diet-induced resistance increase is interrelated with the disproportional propagation of microbes in Lachnospiraceae, indicating a potential reservoir of aminoglycosides resistance. We further showed that the prevalence of acquired resistance genes was also modified by introducing a different diet, likely due to the augmented frequency of lateral gene transfer (LGT) in microbes (CON vs HG: 254 vs 287 taxa) such as Lachnospiraceae. Consequently, we present that diet transition is associated with fecal resistome modification in dairy cattle and an HG diet specifically enriched aminoglycosides resistance that is likely by stimulating microbial LGT.IMPORTANCEThe increasing prevalence of antimicrobial resistance is one of the most severe threats to public health, and developing novel mitigation strategies deserves our top priority. High-grain (HG) diet is commonly applied in dairy cattle to enhance animals’ performance to produce more high-quality milk. We present that despite such benefits, the application of an HG diet is correlated with an elevated prevalence of resistance to aminoglycosides, and this is a combined effect of the expansion of antibiotic-resistant bacteria and increased frequency of lateral gene transfer in the fecal microbiome of dairy cattle. Our results provided new knowledge in a typically ignored area by showing an unexpected enrichment of antibiotic resistance under an HG diet. Importantly, our findings laid the foundation for designing potential dietary intervention strategies to lower the prevalence of antibiotic resistance in dairy production

Sclerosing rhabdomyosarcoma presenting on the knee-joint

Sclerosing rhabdomyosarcoma is a rare pathological diagnosis that easily misdiagnosed. The majority of cases reported the tumor increased rapidly in size and the Ki-67 proliferation index ranged from 10% to 60%. Here, we report the first case of the tumor increased very slowly for 20 years and the Ki-67 proliferation index was lower than 2%, and discuss its histological features and immunohistochemical reactivity with Desmin and Ki-67 and so on

Mechanically Strong, Thermally Healable, and Recyclable Epoxy Vitrimers Enabled by ZnAl-Layer Double Hydroxides

To meet the demand of sustainable development, epoxy vitrimers based on the dynamic transesterification reaction (DTER) have received considerable attention recently due to their reprocessability and repairability. However, they suffer from low mechanical strength and rely heavily on external catalysts to ensure their curing and repair. Herein, we report a facile design of a novel ZnAl-LDH-catalyzed epoxy vitrimer nanocomposite via introducing ZnAl-layered double metal hydroxide (ZnAl-LDH) nanosheets. Our results show that ZnAl-LDH can be well dispersed in the epoxy vitrimer. Notably, ZnAl-LDH has multifunctionality, which can simultaneously catalyze the curing reaction and enhance the mechanical strength and repairable efficiency of the resultant vitrimer. For instance, the peak curing temperature of epoxy vitrimer with 2 wt % ZnAl-LDH is 8 °C lower than that of an epoxy vitrimer under the same loading between Zn2+ of Zn(OAc)2, demonstrating a strong catalytic action. The tensile strength and Young’s modulus of ZnAl-LDH/epoxy resin (ER) increase from 18 and 156 MPa to 42 and 307 MPa, respectively, due to the reinforcing effect of ZnAl-LDH and the increased cross-linking density. The repairable efficiency of ZnAl-LDH/ER can reach 95% after repair at 200 °C for 1 h, which is mainly due to the abundant catalytic sites and large contact areas of the ZnAl-LDH lamella. Hence, this work offers an innovative and scalable strategy for creating epoxy vitrimers combining exceptional mechanical strength and high repairable efficiency, which holds great promise for many practical applications in the industry

Crk proteins transduce FGF signaling to promote lens fiber cell elongation

Specific cell shapes are fundamental to the organization and function of multicellular organisms. Fibroblast Growth Factor (FGF) signaling induces the elongation of lens fiber cells during vertebrate lens development. Nonetheless, exactly how this extracellular FGF signal is transmitted to the cytoskeletal network has previously not been determined. Here, we show that the Crk family of adaptor proteins, Crk and Crkl, are required for mouse lens morphogenesis but not differentiation. Genetic ablation and epistasis experiments demonstrated that Crk and Crkl play overlapping roles downstream of FGF signaling in order to regulate lens fiber cell elongation. Upon FGF stimulation, Crk proteins were found to interact with Frs2, Shp2 and Grb2. The loss of Crk proteins was partially compensated for by the activation of Ras and Rac signaling. These results reveal that Crk proteins are important partners of the Frs2/Shp2/Grb2 complex in mediating FGF signaling, specifically promoting cell shape changes