We previously reported that dickkopf WNT signaling pathway inhibitor 3 (DKK3) expression is correlated with poorer prognosis in head and neck squamous cell carcinoma (HNSCC). Here we investigated DKK3 expression by using The Cancer Genome Atlas (TCGA) public database and bioinformatic analyses.
We used the RNA sequence data and divided the tumor samples into \"DKK3-high\" and \"DKK3-low\" groups according to median DKK3 expression. The correlations between DKK3 expression and the clinical data were investigated. Differentially expressed genes (DEGs) were detected using DESEq2 and analyzed by ShinyGO 0.77. A gene set enrichment analysis (GSEA) was also performed using GSEA software. The DEGs were also analyzed with TargetMine to establish the protein-protein interaction (PPI) network.
DKK3 expression was significantly increased in cancer samples, and a high DKK3 expression was significantly associated with shorter overall survival. We identified 854 DEGs, including 284 up-regulated and 570 down-regulated. Functional enrichment analyses revealed several Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with extracellular matrix remodeling. The PPI network identified COL8A1, AGTR1, FN1, P4HA3, PDGFRB, and CEP126 as the key genes.
These results suggested the cancer-promoting ability of DKK3, the expression of which is a promising prognostic marker and therapeutic target for HNSCC.

Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are a new class of medications for managing renal anemia in patients with chronic kidney disease (CKD). In addition to their erythropoietic activity, HIF-PHIs exhibit multifaceted effects on iron and glucose metabolism, mitochondrial metabolism, and angiogenesis through the regulation of a wide range of HIF-responsive gene expressions. However, the systemic biological effects of HIF-PHIs in CKD patients have not been fully explored. In this prospective, single-center study, we comprehensively investigated changes in plasma metabolomic profiles following the switch from an erythropoiesis-stimulating agent (ESA) to an HIF-PHI, daprodustat, in 10 maintenance hemodialysis patients. Plasma metabolites were measured before and three months after the switch from an ESA to an HIF-PHI. Among 106 individual markers detected in plasma, significant changes were found in four compounds (erythrulose, n-butyrylglycine, threonine, and leucine), and notable but non-significant changes were found in another five compounds (inositol, phosphoric acid, lyxose, arabinose, and hydroxylamine). Pathway analysis indicated decreased levels of plasma metabolites, particularly those involved in phosphatidylinositol signaling, ascorbate and aldarate metabolism, and inositol phosphate metabolism. Our results provide detailed insights into the systemic biological effects of HIF-PHIs in hemodialysis patients and are expected to contribute to an evaluation of the potential side effects that may result from long-term use of this class of drugs.

Molecular oxygen is essential for all multicellular life forms. In humans, the hypoxia-inducible factor (HIF) prolyl hydroxylase domain-containing enzymes (PHDs) serve as important oxygen sensors by regulating the activity of HIF, the master regulator that mediates cellular oxygen homeostasis, in an oxygen-dependent manner. In normoxia, PHDs catalyze the prolyl hydroxylation of HIF, which leads to its degradation and prevents cellular hypoxic response to be triggered. PHDs are current inhibition targets for the potential treatments of a number of diseases. In this chapter, we discuss in vitro and cell-based methods to study the modulation of PHD2, the most important human PHD isoform in normoxia and mild hypoxia. These include the production and purification of recombinant PHD2, the use of mass spectrometry to follow PHD2-catalyzed reactions and the studies of HIF stabilization in cells by immunoblotting.

To analyze the incisional hernia recurrence rate at a long-term follow-up using a biosynthetic long-term absorbable mesh in patients with a higher risk of surgical infection in a contaminated surgical field.
This was a retrospective multicentric study. All patients undergoing incisional hernia repair between 2016 and 2018 at 6 participating university centers were included. Patients were classified according to the Ventral Hernia Working Group (VHWG). All consecutive patients who underwent abdominal wall repair using biosynthetic long-term absorbable mesh (Phasix®) in contaminated fields (grade 3 and 4 of the VHWG classification) were included. Patients were followed-up until September 2021. Preoperative, operative, and postoperative data were collected. All patients\' surgical site infections (SSIs) and surgical site occurrences (SSOs) were recorded. The primary outcome of interest was the clinical incisional hernia recurrence rate.
One hundred and eight patients were included: 77 with VHWG grade 3 (71.3%) and 31 with VHWG grade 4 (28.7%). Median time follow-up was 41 months [24; 63]. Twenty-four patients had clinical recurrence during the follow-up (22.2%). The SSI and SSO rates were 24.1% and 36.1%, respectively. On multivariate analysis, risk factors for incisional hernia recurrence were previous recurrence, mesh location, and postoperative enterocutaneous fistula.
At the 3 year follow-up, the recurrence rate with a biosynthetic absorbable mesh (Phasix®) for incisional hernia repair in high-risk patients (VHWG grade 3 and 4) seemed to be suitable (22.2%). Most complications occurred in the first year, and SSI and SSO rates were low despite high-risk VHWG grading.

OBJECTIVE: Trefoil factor 3 (TFF3) is a gut mucosal protective molecule that is secreted by intestinal goblet cells. The dimeric structure of TFF3 enables it to function in intestinal mucosal repair and to maintain its own stability. Protein disulfide isomerase a1 (PDIA1) can directly catalyze the formation, isomerization and reduction of disulfide bonds in proteins and may play an important role in the formation of TFF3 dimer. In this study, we focused on the specific molecular mechanism of TFF3 dimerization by PDIA1 and the changes during sepsis.
METHODS: We examined the changes of PDIA1 and TFF3 in sepsis rats and cell models and used a variety of experimental techniques to investigate the specific molecular mechanism of PDIA1-catalyzed TFF3 dimerization.
RESULTS: We found that PDIA1 can directly catalyze the dimerization of TFF3. Our MD model proposed that two TFF3 monomers form hydrogen bonds with the region b\' of PDIA1 through two stepwise reactions. Furthermore, we propose that the Cys24-Cys27 active site at the region a\' of PDIA1 mediates disulfide bond formation between the Cys79 residues of each of the two TFF3 monomers via deprotonation and nucleophilic attack. During sepsis, PDIA1 is downregulated and the excessive release of nitric oxide (NO) promoted PDIA1 nitrosylation. This modification reduced PDIA1 activity, which resulted in the corresponding decrease of TFF3 dimerization and compromised TFF3 dimer function.
CONCLUSIONS: Our study revealed a novel mechanism for the inhibition of intestinal mucosal repair during sepsis and revealed novel targets for the prevention and treatment of sepsis.

Phthalates are commonly included as ingredients in personal care products such as cosmetics, shampoos and perfumes. Diethyl phthalate (DEP) has been found to be anti-androgenic and linked with adverse reproductive effects on males, but effects on females are poorly understood. We designed an integrative and translational study to experimentally examine the effects of DEP exposure at a human-equivalent dose on the mammary transcriptome in rats and to subsequently examine the DEP gene signature in breast tissues (both pre-malignant and tumor) from a population study. In Sprague-Dawley rats treated orally with DEP from birth to adulthood, we identified a signature panel of 107 genes predominantly down-regulated by DEP exposure. Univariate analysis of this 107 DEP gene signature in pre-malignant breast tissues revealed that six genes (P4HA1, MPZL3, TMC4, PLEKHA6, CA8, AREG) were inversely associated with monoethyl phthalate (MEP; the urinary metabolite of DEP) concentration (p < 0.05) among postmenopausal women; all six genes loaded on to one of seven factors identified by factor analysis. Transcription factor enrichment analysis revealed that genes in this factor were enriched for androgen receptor binding sites. These six genes were also significantly down-regulated in pre-malignant adjacent tissues compared to the corresponding tumor tissues in pair-wise analyses (p < 0.05). Results from our translational study indicate that low level exposure to diethyl phthalate results in measurable genomic changes in breast tissue with implications in breast carcinogenesis.

PHD fingers represent one of the largest families of epigenetic readers capable of decoding post-translationally modified or unmodified histone H3 tails. Because of their direct involvement in human pathologies they are increasingly considered as a potential therapeutic target. Several PHD/histone-peptide structures have been determined, however relatively little information is available on their dynamics. Studies aiming to characterize the dynamic and energetic determinants driving histone peptide recognition by epigenetic readers would strongly benefit from computational studies. Herein we focus on the dynamic and energetic characterization of the PHD finger subclass specialized in the recognition of histone H3 peptides unmodified in position K4 (H3K4me0). As a case study we focused on the first PHD finger of autoimmune regulator protein (AIRE-PHD1) in complex with H3K4me0. PCA analysis of the covariance matrix of free AIRE-PHD1 highlights the presence of a \"flapping\" movement, which is blocked in an open conformation upon binding to H3K4me0. Moreover, binding free energy calculations obtained through Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology are in good qualitative agreement with experiments and allow dissection of the energetic terms associated with native and alanine mutants of AIRE-PHD1/H3K4me0 complexes. MM/PBSA calculations have also been applied to the energetic analysis of other PHD fingers recognizing H3K4me0. In this case we observe excellent correlation between computed and experimental binding free energies. Overall calculations show that H3K4me0 recognition by PHD fingers relies on compensation of the electrostatic and polar solvation energy terms and is stabilized by non-polar interactions.

The oxygen sensing pathway modulates erythropoietin expression. In normal cells, intracellular oxygen tensions are directly sensed by prolyl hydroxylase domain (PHD)-containing proteins. PHD2 isozyme has a key role in tagging hypoxia-inducible factor (HIF)-α subunits for polyubiquitination and proteasomal degradation. Erythrocytosis-associated PHD2 mutations reduce hydroxylation of HIF-α. The investigation of 67 patients with isolated erythrocytosis, either sporadic or familial, allowed the identification of three novel mutations in the catalytic domain of the PHD2 protein. All new mutations are germ-line, heterozygous and missense, and code for a predicted full length mutant PHD2 protein. Identification of the disease-causing genes will be of critical importance for a better classification of familial and acquired erythrocytosis, offering additional insight into the erythropoietin regulating oxygen sensing pathway.

OBJECTIVE: To detect the expression of type I collagen, type III collagen, prolyl-4-hydroxylases (PH4) and matrix metalloproteinase 1 (MMP-1) in sacral ligament fibroblasts under stress, to understand the collagen synthesis and metabolism in stress situations change.
METHODS: Eight patients who underwent abdominal hysterectomy for uterine benign disease were enrolled in this study. Primary sacral ligament fibroblasts were isolated by explant. After mechanical loading, gene expression of type I, III collagen, PH4 and MMP-1 were measured.
RESULTS: Stress of 8% continuing for 24 hours, collagen I (1.13 ± 0.24), collagen III (1.05 ± 0.31) mRNA expression and PH4 expression (1.11 ± 0.31) compared with static groups (1) showed increasing trends;when the stress were 4% and 12%, collagen I (0.86 ± 0.26 and 0.85 ± 0.25), collagen III showed increasing trends (0.74 ± 0.29 and 0.83 ± 0.38) mRNA expression were decreased. After removal of the stress, in the stress of 4% for 1 hour, collagen I (0.79 ± 0.40, 0.97 ± 0.24 and 1.46 ± 0.75), collagen III (0.86 ± 0.40, 0.99 ± 0.60 and 1.59 ± 0.82) and PH4 (1.11 ± 0.51, 1.17 ± 0.54 and 1.37 ± 0.39) mRNA expression increased gradually. In 8% stress group, collagen I mRNA expression (1.16 ± 0.62, 1.01 ± 0.51 and 1.05 ± 0.80) reached the peak in day 1, and collagen III (0.99 ± 0.69, 1.59 ± 0.55 and 1.03 ± 0.91) and PH4 (1.05 ± 0.31, 1.07 ± 0.80 and 0.85 ± 0.31) mRNA expression reached the peak in day 2, then decreased. 4% and 8% of the stress with time after the change, MMP-1 mRNA expression have peaked at day 1.
CONCLUSIONS: Moderate stress could contribute to pelvic floor collagen synthesis, too much or too little stress is not conducive to the synthesis of collagen. Collagen I and collagen III on the stress response may be different, the former have faster reaction than the latter. PH4 were involved in the synthesis of collagen, while MMP-1 may play a role in collagen degradation.

Thirty-six unrelated cases with erythrocytosis of unknown origin were investigated. Exons 5-8 of the erythropoietin receptor gene (EPOR), the von Hippel-Lindau gene, and the prolyl hydroxylase domain protein 2 gene (PHD2) were screened by direct DNA sequencing. The Janus kinase 2 mutation, JAK2 (Val617Phe), was screened by allele specific PCR. In this study, three new mutations of EPOR causing deletions in exon 8 were found: the first led directly to a stop codon [g.5957_5958delTT (p.Phe424X)], the second to a stop codon after one residue [g.5828_5829delCC (p.Pro381GlnfsX1)] and the third to a stop codon following a frameshift sequence of 23 residues [g.5971delC (p.Leu429TrpfsX23)]. One patient had a previously reported EPOR mutation [g.6146A>G (p.Asn487Ser)] and another, a silent one (g.5799G>A). All were heterozygotes. In addition, 2 patients were positive for JAK2 (Val617Phe), and 2 reported elsewhere, were mutated in the PHD2 gene [c.606delG (p.Met202IlefsX71).