NADPH oxidase 4-mediating oxidative stress contributes to endometriosis.

NADPH oxidase 4 (NOX4) plays an important role in the regulation of oxidative stress, which is associated with endometriosis. This study aims to investigate the effects of NOX4 in endometriosis and its molecular mechanisms. Clinical specimens were collected, and human endometrial stromal cells (HESCs) were isolated. The knockdown of NOX4 cell lines was established on the HESCs and induced by peritoneal fluid. The levels of NOX4 were determined by using immunohistochemistry (IHC) staining, western blotting, and qPCR, respectively. The levels of oxidative stress markers were determined by using western blotting and ELISAs, respectively. The correlation of NOX4 and oxidative stress markers was analyzed by the Pearson correlation coefficient. The levels of NOX4 were dramatically elevated in the ectopic endometrium. Besides, oxidative stress biomarkers were also dysregulated in the ectopic endometrium as compared to the normal endometrium. Pearson's correlation coefficient analysis revealed a relationship between NOX4 and oxidative stress biomarkers in the ectopic endometrium. NOX4 modulated the expressions of oxidative stress markers in endometrial stromal cells stimulated by the peritoneal fluid from endometriosis. The effects of NOX4 on endometriosis are in part by its regulatory effects against oxidative stress.

A high Z-score might increase the positive predictive value of cell-free noninvasive prenatal testing for singleton-pregnant women.

OBJECTIVE: To explore the positive predictive value (PPV) in noninvasive prenatal testing (NIPT)-positive cases and analyze the effect of the Z-score intervals on PPV performance. METHODS: In this retrospective study, 26,667 pregnant women underwent NIPT from November 2014 to August 2022, of which 169 were NIPT-positive cases. NIPT-positive cases were divided into three groups according to the Z-score: 3 ≤ Z < 6, 6 ≤ Z < 10, and Z ≥ 10. RESULTS: The PPVs of NIPT were 91.26% (94/103) for trisomy (T) 21, 80.65% (25/31) for T18, and 36.84% (7/19) for T13. The PPVs for the 3 ≤ Z < 6, 6 ≤ Z < 10, and Z ≥ 10 groups were 50%, 84.62%, and 87.95%, respectively. A higher PPV was found in the NIPT results when the Z-score was larger, with significant differences. The PPVs for T21/T18/T13 were 71.43%/42.86%/25% for 3 ≤ Z < 6, 90.32%/85.71%/57.14% for 6 ≤ Z < 10, and 93.85%/100%/25% for Z ≥ 10. For T21, T18, and T13, the correlations between the Z-score and fetal fraction concentration in true positives were r = 0.85, r = 0.59, and r = 0.71 (all p < .001), respectively. CONCLUSION: Z-score is associated with the PPV performance of NIPT in fetal T13, T18, and T21. The possibility of false positives caused by placental chimerism should be considered when determining whether high Z-values lead to high PPVs.

Immunotherapy: A promising novel endometriosis therapy.

Endometriosis is a common disease of the female reproductive system and has malignant features. Although endometriosis by itself is a benign disease, its erosive growth characteristics lead to severe pelvic pain and female infertility. Unfortunately, several aspects of the pathogenesis of endometriosis are still unclear. Furthermore, the clinical therapeutic methods are unsatisfactory. The recurrence rate of endometriosis is high. Accumulating evidence suggests that the onset and development of endometriosis are closely related to the abnormal function of the female autoimmune system, especially the function of some immune cells such as the aggregation of neutrophils, abnormal differentiation of macrophages, decreased cytotoxicity of NK cells, and abnormal function of T- and B-cell lines. Therefore, immunotherapy is probably a novel therapeutic strategy for endometriosis besides surgery and hormone therapy. However, information regarding the clinical application of immunotherapy in the treatment of endometriosis is very limited. This article aimed to review the effects of existing immunomodulators on the development of endometriosis, including immune cell regulators and immune factor regulators. These immunomodulators clinically or experimentally inhibit the pathogenesis and development of endometriosis lesions by acting on the immune cells, immune factors, or immune-related signaling pathways. Thus, immunotherapy is probably a novel and effective clinical treatment choice for endometriosis. Experimental studies of the detailed mechanism of immunotherapy and large-scale clinical studies about the effectiveness and safety of this promising therapeutic method are required in the future.

Efficacy and safety of pembrolizumab on cervical cancer: A systematic review and single-arm meta-analysis.

Background: According to current research, the objective response rate and overall survival of pembrolizumab in the treatment of several types of solid tumors have been significantly improved. Some high-quality clinical trials have studied the effect of applying pembrolizumab in treating cervical cancer. Multiple clinical trials have been conducted, and some of them have shown good results as expected. Therefore, we performed this meta-analysis on existing studies to reveal the efficacy and safety of pembrolizumab in treating cervical cancer. Methods: PubMed, Embase, Cochrane Library and Web of Science were searched for literatures published until October 31, 2021. Outcomes included complete response (CR), partial response (PR), stable disease (SD), disease progression (PD), objective response rate (ORR), disease control rate (DCR), overall survival (OS), progression-free survival (PFS), the best time to response (TTR), death rate, adverse events (AE). Results: A total of 7 studies with 727 patients were included. The results were as follows: CR (0.027, 95%CI: 0.008-0.053), PR (0.104, 95% CI: 0.074-0.145), SD (0.190, 95% CI: 0.149-0.240), PD (0.541, 95% CI: 0.421-0.661). ORR was 0.155 (95% CI: 0.098-0.236) and DCR was 0.331 (95% CI: 0.277-0.385). OS was 10.23 months (95% CI: 8.96-11.50) and PFS was 4.27 months (95% CI: 1.57-6.96). TTR was 2.10 months (95%CI: 1.69-2.51). The 1-year death rate was 0.388 (95% CI: 0.230-0.574). Main adverse events included abnormal liver function, hypothyroidism, neutropenia, anemia, decreased appetite, fatigue, fever, etc. The total incidence of the adverse events of grade 3 and above was 0.212 (95% CI: 0.065-0.509). Conclusions: Pembrolizumab provides significant benefits in response rate and survival for cervical cancer patients. The results from recent high-quality clinical trials are expected to validate these findings. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42021291723.

A novel splicing variant of proprotein convertase subtilisin/kexin type 9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the most recently identified member of the proprotein convertase family. Genetic and cell biology studies have suggested a critical role of PCSK9 in regulating low-density lipoprotein receptor (LDLR) protein levels and thus modulating plasma LDL cholesterol. Recent data on the molecular basis for PCSK9 action support the model in which PCSK9 is self-cleaved, secreted, and tightly bound to the EGF-A repeat of LDLR extracellular domain. PCSK9 binding to LDLR is essential for the ensuing receptor-mediated endocytosis and is speculated to lock LDLR in a specific conformation that favors degradation in lysosomal compartment instead of recycling back to plasma membrane. We report here a novel human PCSK9 splicing variant, which we named PCSK9sv. PCSK9sv had an in-frame deletion of the eighth exon of 58 amino acids and was expressed in multiple tissues, including liver, small intestine, prostate, uterus, brain, and adipose tissue. Unlike wild-type PCSK9, which is secreted, PCSK9sv expressed in human embryonic kidney HEK293 cells failed to process the prosegment intracellularly and thus was not secreted into the medium. Examination of potential functions revealed that PCSK9sv did not change the LDLR protein levels. Two mutations that have been reported in humans with the associated changes in plasma LDL cholesterol were within exon 8, and thus the expression and function of the two mutants were studied. Both N425S and A443T mutants were processed normally, secreted, and reduced LDLR levels. However, the physiological function of this novel splicing variant of PCSK9 has yet to be determined.

A transforming mutation in the pleckstrin homology domain of AKT1 in cancer.

Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

Derivation and characterization of monoclonal antibodies against human folypolyglutamate synthetase.

Folypolyglutamate synthetase (FPGS) plays a critical role in the cellular retention of both folates and antifolates. Resistance to antifolates is in part related to changes in FPGS enzyme activity and levels of messenger RNA, or in some instances, protein as evaluated by Western blots using polyclonal antisera. The present study was designed to derive a series of monoclonal antibodies (MAb) against the native protein, to characterize them in terms of specificity and epitope mapping, and to determine kinetic constants by Biacore. We report on 3 IgG(1) kappa MAbs-namely, 4-2, 4-3, and 4-18-with epitopes localized to the carboxyl domain of the protein. These antibodies recognize a single band on Western blots of HeLa cell lysates, which is significantly reduced following RNAi knockdown. The recognition of both the native and denatured conformations of FPGS by these MAbs should provide useful reagents for FPGS quantitation in either tumor cell lysates or in tumor biopsies.

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.

Derivation and characterization of a monoclonal antibody against human glycinamide ribonucleotide formyltransferase.

Glycinamide ribonucleotide formyltransferase (GARFT) is a trifunctional enzyme involved in purine biosynthesis. Its central role in folate metabolism has made it an obvious target for the development of GARFT inhibitors, primarily for oncology. While the crystal structure, enzyme kinetics, and mechanism of action of GARFT inhibitors are reasonably well understood, GARFT regulation at the protein level remains unclear. The present study reports the development and characterization of a monoclonal antibody (MAb) specific for human GARFT. This MAb, an IgG1kappa, designated PHR1, recognizes human GARFT by both Western blot and by immunohistochemistry from non-small-cell lung carcinoma and colon adenocarcinoma tissue biopsies, has a KD of 1.14 x 10(10) M, and has been epitope mapped at residues 59-78 of the GARFT functional domain. The ability of PHR1 to recognize both sodium dodecyl sulfate (SDS)-denatured as well as native GARFT should make this MAb an important research tool in determining GARFT protein levels in both normal and neoplastic tissues.