Human endothelial cells and fibroblasts express and produce the coagulation proteins necessary for thrombin generation

In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV–FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin–antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

Superior Technique for the Production of Agarose Dressing Containing Sericin and Its Wound Healing Property

Finding a simple and eco-friendly production technique that matches to the natural agent and results in a truly valuable natural scaffold production is still limited amongst the intensively competitive natural scaffold development. Therefore, the purpose of this study was to develop natural scaffolds that were environmentally friendly, low cost, and easily produced, using natural agents and a physical crosslinking technique. These scaffolds were prepared from agarose and sericin using the freeze-drying method (D) or freeze-thawing together with the freeze-drying method (TD). Moreover, plasticizers were added into the scaffold to improve their properties. Their physical, mechanical, and biological properties were investigated. The results showed that scaffolds that were prepared using the TD method had stronger bonding between sericin and other compounds, leading to a low swelling ratio and low protein release of the scaffolds. This property may be applied in the development of further material as a controlled drug release scaffold. Adding plasticizers, especially glycerin, into the scaffolds significantly increased elongation properties, leading to an increase in elasticity of the scaffold. Moreover, all scaffolds could activate cell migration, which had an advantage on wound healing acceleration. Accordingly, this study was successful in developing natural scaffolds using natural agents and simple and green crosslinking methods.

Loading Effect of Chitosan Derivative Nanoparticles on Different Antigens and Their Immunomodulatory Activity on Dendritic Cells

Drug carrier nanoparticles (NPs) were prepared by the polyelectrolyte method, with chitosan sulfate, with different substituents and quaternary ammonium chitosan, including C236-HACC NPs, C36-HACC NPs, and C6-HACC NPs. To evaluate whether the NPs are suitable for loading different antigens, we chose bovine serum albumin (BSA), ovalbumin (OVA), and myoglobin (Mb) as model antigens to investigate the encapsulation effect of the NPs. The characteristics (size, potential, and encapsulation efficiency) of the NPs were measured. Moreover, the NPs with higher encapsulation efficiency were selected for the immunological activity research. The results showed that chitosan derivative NPs with different substitution sites had different loading effects on the three antigens, and the encapsulation rate of BSA and OVA was significantly better than that of Mb. Moreover, the NPs encapsulated with different antigens have different immune stimulating abilities to DCS cells, the immune effect of OVA-coated NPs was significantly better than that of BSA-coated NPs and blank NPs, especially C236-HACC-OVA NPs. Furthermore, we found that C236-HACC-OVA NPs could increase the phosphorylation level of intracellular proteins to activate cell pathways. Therefore, C236-HACC NPs are more suitable for the loading of antigens similar to the OVA structure.

NADase and now Ca2+ channel, what else to learn about plant NLRs?

Plant intracellular immune receptors known as NLR (Nucleotide-binding Leucine-rich repeat, NB-LRR) proteins confer resistance and cause cell death upon recognition of cognate effector proteins from pathogens. Plant NLRs contain a variable N-terminal domain: a Toll/interleukin-1 receptor (TIR) domain or a coiled-coil (CC) domain or an RPW8 (Resistance to Powdery Mildew 8)-like CC (CCR) domain. TIR-NLR, CC-NLR and CCR-NLR are known as TNL, CNL and RNL, respectively. TNLs and CNLs recognize pathogen effectors to activate cell death and defense responses, thus are regarded as sensor NLRs. RNLs are required downstream of TNLs to activate cell death and defense responses, thus are regarded as helper NLRs. Previous studies show that some TNLs form tetrameric resistosome as NAD+ cleaving enzymes to transduce signal, while some CNLs form pentameric resistosome with undefined biochemical function. Two recent breakthrough studies show that activated CNL and RNL function as Ca2+ channel to cause cell death and defense responses and provide a completely new insight into the downstream signaling events of CNL and TNL pathways.

Lineage-specific control of convergent differentiation by a Forkhead repressor

During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell-type specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.

SAMD9L autoinflammatory or ataxia pancytopenia disease mutations activate cell-autonomous translational repression

Sterile α motif domain-containing protein 9-like (SAMD9L) is encoded by a hallmark interferon-induced gene with a role in controlling virus replication that is not well understood. Here, we analyze SAMD9L function from the perspective of human mutations causing neonatal-onset severe autoinflammatory disease. Whole-genome sequencing of two children with leukocytoclastic panniculitis, basal ganglia calcifications, raised blood inflammatory markers, neutrophilia, anemia, thrombocytopaenia, and almost no B cells revealed heterozygous de novo SAMD9L mutations, p.Asn885Thrfs*6 and p.Lys878Serfs*13. These frameshift mutations truncate the SAMD9L protein within a domain a region of homology to the nucleotide-binding and oligomerization domain (NOD) of APAF1, ∼80 amino acids C-terminal to the Walker B motif. Single-cell analysis of human cells expressing green fluorescent protein (GFP)-SAMD9L fusion proteins revealed that enforced expression of wild-type SAMD9L repressed translation of red fluorescent protein messenger RNA and globally repressed endogenous protein translation, cell autonomously and in proportion to the level of GFP-SAMD9L in each cell. The children’s truncating mutations dramatically exaggerated translational repression even at low levels of GFP-SAMD9L per cell, as did a missense Arg986Cys mutation reported recurrently as causing ataxia pancytopenia syndrome. Autoinflammatory disease associated with SAMD9L truncating mutations appears to result from an interferon-induced translational repressor whose activity goes unchecked by the loss of C-terminal domains that may normally sense virus infection.

Human Endothelial Cells And Fibroblasts Express And Produce The Coagulation Proteins Necessary For Thrombin Generation

In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV-FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin-antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and g-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

Targeting Adaptation to Cancer Treatment by Drug Combinations

ABSTRACTAdaptation of tumors to therapeutic interventions contributes to dismal long-term patient outcomes. Adaptation to therapy involves co-action of functionally related proteins that together activate cell survival programs and compensate for the therapeutic impact. Oncogenic dependencies to such adaptive events, however, can generate new therapeutic vulnerabilities that can be targeted with drug combinations. The precision medicine approaches in which targeted drugs are matched to pre-existing genomic aberrations fail to address the adaptive responses and resulting vulnerabilities. Here, we provide the mathematical formulation, implementation and validation of the TargetScore method. The TargetScore identifies collective adaptive responses to targeted interventions as concurrent changes of phospho-proteins that are connected within a signaling network. Based on the adaptive responses, the method predicts drug-induced vulnerabilities. Using TargetScore, we inferred the adaptive responses with short-term (i.e., days) stress and long-term (i.e., months) acquired resistance to inhibitors of anti-apoptotic mediators, MCL1 and BCL2. With experiments guided by the predictions, we identified synergistic interactions between inhibitors of PARP, SHP2, and MCL1 in breast cancer cells. TargetScore is readily applicable to existing precision oncology efforts by matching targeted drug combinations to emerging molecular signatures under therapeutic stress.

DDIT4 is an Innate Protector in Psoriatic Keratinocytes and 1,25(OH)2D3 Exerts Anti-Psoriasis Through Promoting DDIT4 Inducing Macro-Autophagy and Proliferation Inhibition

Psoriasis vulgaris is a chronic inflammatory skin disorder. Its pathogenesis is now still unelucidated and the treatment is far from satisfied. DNA Damage-Inducible Transcript 4 (DDIT4) is a widely expressed protein in different tissue, which can activate cell macro-autophagy through mTORC1 passway. Vitamin D3 and analogues is a classic topical reagent for psoriasis vulgaris for more than 30 years, but its exact mechanism is not fully clear. In this study, we intend to verify whether vitamin D3 also exerts anti-psoriasis through promoting DDIT4 inducing macro-autophagy and proliferation inhibition in psoriasis vulgaris. Results showed DDIT4 was over-expressed in psoriatic tissue, and probably acted as an innate protector during psoriasis pathogenesis. 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) could promote DDIT4 expression in a rough linear correlation and subsequently activate macro-autophagy and inhabit cell proliferation, especially at the high concentration of 100nM. In terms of our understanding, this is the first time to reveal the interactions between 1,25(OH)2D3, DDIT4 and macro-autophagy in psoriasis vulgaris. DDIT4 is also probably a potential therapeutic target in future psoriatic treatments.

Ceramides: focus on obesity

It is generally known that obesity increases the risk of developing cardiovascular disease. A pathological increase in the mass of adipose tissue leads to a violation of the control of lipid accumulation at the molecular level, abnormal lipid metabolism with the formation of metabolites, which are critical for the development of these pathologies against the background of obesity. Ceramides are one of these metabolites. Ceramides perform many physiological functions, but under pathological conditions they induce insulin resistance, uncouple cellular respiration and phosphorylation, activate cell apoptosis, and play an important role in the induction of adipose tissue dysfunction. Altering ceramide biosynthesis through dysregulation of key enzymes leads to the formation and accumulation of ceramides, which block insulin signaling and induce adipose tissue inflammation.This review highlights the metabolism of ceramides, the reasons for their ectopic deposition in tissues in obesity, as well as potential intracellular signaling pathways that modulate ceramide activity.