Small integral membrane protein 20/phoenixin (SMIM20/PNX) and its receptor GPR173 (G Protein-Coupled Receptor 173) play a role in the regulation of the hypothalamic–pituitary–gonadal axis (HPG). The aim of the study was to determine PNX, FSH, LH, and 17β-estradiol association in women with endometriosis, and the expression of SMIM20/PNX signaling via GPR173. Serum PNX, FSH, LH, and 17β-estradiol concentrations were measured by enzyme and electrochemiluminescence immunoassay. SMIM20/PNX and GPR173 expression in the eutopic and ectopic endometrium was assessed by qPCR and immunohistochemistry. Reduced PNX level, increased LH/FSH ratio and elevated 17β-estradiol concentration were found in patients with endometriosis. No differences in SMIM20 expression were observed between the studied endometria. GPR173 expression was lower in ectopic than in eutopic endometria. SMIM20 expression was mainly restricted to stroma. GPR173 was detected in some eutopic and ectopic stromal cells and in eutopic glandular epithelial cells. Discriminant analysis indicates the diagnostic relevance of PNX and LH/FSH ratio in patients with endometriosis. In women with endometriosis, reduced PNX levels and GPR173 expression may be responsible for HPG axis dysregulation. These new insights may contribute to a better understanding of the pathophysiology of endometriosis and provide the basis for a new strategy for diagnosis and treatment of endometriosis.

Purpose: Mitochondrial DNA (mtDNA) abnormalities were previously found to be causative in the pathogenesis of various diseases. Here, comprehensive mitochondrial and nuclear sequence and transcript analyses, along with analyses of the methylation aspects of nuclear genes related to mitochondrial function, were performed in patients with keratoconus (KTCN) to evaluate their contribution to the KTCN pathogenesis. Methods: Blood mtDNA of 42 KTCN and 51 non-KTCN individuals was Sanger sequenced and analyzed along with the previously obtained corneal RNA-sequencing data of 20 KTCN and 21 non-KTCN individuals. In addition, the expression and methylation of mtDNA genes and 1223 mitochondria-related nuclear genes were evaluated. Results: The mtDNA sequence alterations detected in blood coincided with variants identified in transcripts of the matched corneal tissues. In KTCN corneas, 97 mitochondria-related genes were deregulated, including TGFB1, P4HB, and BCL2, which are involved in the extracellular matrix (ECM) organization, collagen formation, and focal adhesion pathways. No changes in the expression of mtDNA transcripts and no differentially methylated genes among the assessed mitochondrial–nuclear gene sets were found. Conclusions: The absence of corneal-specific mtDNA variants indicates that there is no direct relationship between mitochondrial sequence variability and KTCN phenotype in the studied individuals. However, the identified KTCN-specific transcriptomic alterations of the nuclear genes directly related to the mitochondria functioning point to their possible involvement in the ECM organization, collagen formation, and focal adhesion. Translational Relevance: The identification of abnormalities within nuclear genes regulating ECM formation, collagen synthesis, and/or focal adhesion may form the basis of future treatment strategies or predict the progression of corneal changes in KTCN.

Keratoconus (KTCN) is a non-inflammatory, usually bilateral disorder of the eye which results in the conical shape and the progressive thinning of the cornea. Several studies have suggested that genetic factors play a role in the etiology of the disease. Several loci were previously described as possible candidate regions for familial KTCN; however, no causative mutations in any genes have been identified for any of these loci. The purpose of this study was to evaluate role of the collagen genes collagen type IV, alpha-1 (COL4A1) and collagen type IV, alpha-2 (COL4A2) in KTCN in Ecuadorian families. COL4A1 and COL4A2 in 15 Ecuadorian KTCN families were examined with polymerase chain reaction amplification, and direct sequencing of all exons, promoter and intron-exon junctions was performed. Screening of COL4A1 and COL4A2 revealed numerous alterations in coding and non-coding regions of both genes. We detected three missense substitutions in COL4A1: c.19G>C (Val7Leu), c.1663A>C (Thr555Pro), and c.4002A>C (Gln1334His). Five non-synonymous variants were identified in COL4A2: c.574G>T (Val192Phe), c.1550G>A (Arg517Lys), c.2048G>C (Gly683Ala), c.2102A>G (Lys701Arg), and c.2152C>T (Pro718Ser). None of the identified sequence variants completely segregated with the affected phenotype. The Gln1334His variant was possibly damaging to protein function and structure. This is the first mutation screening of COL4A1 and COL4A2 genes in families with KTCN and linkage to a locus close to these genes. Analysis of COL4A1 and COL4A2 revealed no mutations indicating that other genes are involved in KTCN causation in Ecuadorian families.

There are immunological consequences to the method by which neutrophils undergo cell death. Neutrophil apoptosis, called silent death, leads to the resolution of inflammation, while NETosis deepens and prolongs the inflammatory response and is associated with a worse prognosis of severe infections, e.g., sepsis. Besides nociceptive inhibition, local anaesthetics modulate leukocyte functions, even at low, clinically relevant concentrations. There is currently no data on ropivacaine NETosis, and this study aimed to evaluate the impact of clinical concentrations of ropivacaine (0.0007, 0.007 and 1.4 mmol/L) and lidocaine (0.002, 0.02 and 4 mmol/L) on apoptosis and NETosis of adult peripheral blood neutrophils after 2 h of incubation. Neutrophil identification, apoptosis and NETosis were evaluated by flow cytometry using forward and side scatter characteristics and fluorescent labelling: CD15 for neutrophils identification; Annexin V and propidium iodide for apoptosis and citrullinated histone H3 and myeloperoxidase for NETosis. Lidocaine (4 mmol/L) and ropivacaine (1.4 mmol/L) induced early apoptosis in resting but not in stimulated neutrophils. Low doses of ropivacaine (0.0007 and 0.007 mmol/L) decreased the number of late apoptotic neutrophils, and the lowest dose slightly increased their viability. None of the drugs induced NETosis in resting neutrophils but decreased NETosis at clinical concentrations compared to PMA-stimulated 4 mM lidocaine, PMA-stimulated control, and 1.4 mM ropivacaine. The effect of lidocaine and ropivacaine on apoptosis and NETosis depended on neutrophil stimulation and drug concentrations. Ropivacaine tends to be cytoprotective at concentrations observed in plasma under local anaesthesia. Lidocaine enhanced NETosis at high concentration only in stimulated neutrophils. Thus, both drugs have the ability to change the course of inflammation.

Endometriosis is one of the most common gynecologic diseases in women of reproductive age. The pathophysiology of endometriosis is still not fully understood. Phoenixin (PNX-14) is a newly discovered neuropeptide that regulates the hypothalamo-pituitary-gonadal (HPG) axis and reproductive functions. Recently, we reported that PNX-14, its precursor protein and receptor were expressed in human endometrium. Moreover, PNX-14 serum levels in endometriosis were reduced. This study aimed to evaluate the in vitro biological functions of physiological PNX-14 concentrations on the ectopic endometrium Z12 cells. The proliferation and migration of Z12 cells were assessed using the xCELLigence® RTCA DP system following 72 h of stimulation with 0.05 and 0.2 nM of PNX-14. GPR173 and small integral membrane protein 20 (SMIM20) gene expression was evaluated using quantitative polymerase chain reaction (qPCR) and the protein levels of GPR173 were analyzed using Western blot analysis. PNX-14 at the concentration observed in the serum of patients with endometriosis (0.05 nM) reduced GPR173 and increased SMIM20 expression, while protein levels of GPR173 remained unchanged. Cell proliferation was increased by the 0.02 nM PNX-14- the concentration found in healthy subjects. The 0.2 nM of PNX-14 decreased SMIM20 expression with no change to GPR173 expression and reduced ectopic epithelial cell proliferation during the first 5 h after stimulation. However, at 72 h, the proliferation increased. This study shows that PNX-14 at endometriosis specific concentration desensitized ectopic epithelium to PNX-14, and increased the expression of SMIM20 to restore the physiological levels of PNX-14.