Feature Paper from Biomolecules Journal Reviewers

Olive pomace is the main by-product generated by the olive oil production process. Although toxic to the environment, olive pomace is an important source of natural antioxidants due to its high content of phenolic compounds. The aim of the current study is to maximize the extraction yields of the main phenolic compounds present in olive pomace using innovative green technologies. For this purpose, the present work is divided into two parts. The first part is based on a solubility study of targeted phenolic compounds in various ethanol/water ratios at two different temperatures (20 °C and 50 °C). A computational prediction using COSMO-RS software was applied for the calculation of eventual solubility, which was subsequently confirmed by practical experiments. The determination of the optimal extraction conditions of solvent ratio (EtOH/H₂O) (60:40 v/v) and temperature (50 °C) led to the second part of the work, which concerns the intensification of extraction yields. Furthermore, various green extractions using innovative technologies, including accelerated solvent extraction (ASE), ultrasound with its both system (probe (UAE-P) and bath (UAE-B)), bead milling (BM) and microwave (MAE), were carried out and then compared to conventional maceration (CM). Results showed that ASE was the most effective method for extracting phenolic compounds from dried olive pomace powder (5.3 milligrams of tyrosol equivalent (TE) per gram of dried olive pomace powder (DOP)) compared to CM (3.8 mg TE/g DOP). Full article

The formation of droplets of bio-molecular condensates through liquid-liquid phase separation (LLPS) of their component proteins is a key factor in the maintenance of cellular homeostasis. Different protein properties were shown to be important in LLPS onset, making it possible to develop predictors, which try to discriminate a positive set of proteins involved in LLPS against a negative set of proteins not involved in LLPS. On the other hand, the redundancy and multivalency of the interactions driving LLPS led to the suggestion that the large conformational entropy associated with non specific side-chain interactions is also a key factor in LLPS. In this work we build a LLPS predictor which combines the ability to form pi-pi interactions, with an unrelated feature, the propensity to stabilize the $\beta$-pairing interaction mode. The cross-$\beta$ structure is formed in the amyloid aggregates, which are involved in degenerative diseases and may be the final thermodynamically stable state of protein condensates. Our results show that the combination of pi-pi and $\beta$-pairing propensity yields an improved performance. They also suggest that protein sequences are more likely to be involved in phase separation if the main chain conformational entropy of the $\beta$-pairing maintained droplet state is increased. This would stabilize the droplet state against the more ordered amyloid state. Interestingly, the entropic stabilization of the droplet state appears to proceed according to different mechanisms, depending on the fraction of “droplet-driving“ proteins present in the positive set. Full article

Substantial evidence suggests crosstalk between reproductive and gut-axis but mechanisms linking metabolism and reproduction are still unclear. The present study evaluated the possible role of glucose-dependent-insulinotropic-polypeptide (GIP) and glucagon-like-peptide-1 (GLP-1) in reproductive function by examining receptor distribution and the effects of global GIPR and GLP-1R deletion on estrous cycling and reproductive outcomes in mice. GIPR and GLP-1R gene expression were readily detected by PCR in female reproductive tissues including pituitary, ovaries and uterine horn. Protein expression was confirmed with histological visualisation of incretin receptors using GIPR-Cre and GLP1R-Cre mice in which the incretin receptor expressing cells were fluorescently tagged. Functional studies revealed that female GIPR^−/− and GLP-1R^−/− null mice exhibited significantly (p < 0.05 and p < 0.01) deranged estrous cycling compared to wild-type controls, indicative of reduced fertility. Furthermore, only 50% and 16% of female GIPR^−/− and GLP-1R^−/− mice, respectively produced litters with wild-type males across three breeding cycles. Consistent with a physiological role of incretin receptors in pregnancy outcome, litter size was significantly (p < 0.001–p < 0.05) decreased in GIPR^−/− and GLP-1R^−/− mice. Treatment with oral metformin (300 mg/kg body-weight), an agent used clinically for treatment of PCOS, for a further two breeding periods showed no amelioration of pregnancy outcome except that litter size in the GIPR^−/− group was approximately 2 times greater in the second breeding cycle. These data highlight the significance of incretin receptors in modulation of female reproductive function which may provide future targets for pharmacological intervention in reproductive disorders. Full article

Cancer patients have a greater risk of thrombosis than individuals without cancer. Conversely, thrombosis is a diagnostic predictor of cancer, but the mechanisms by which thrombosis promotes tumor propagation are incompletely understood. Our previous studies showed that hypoxia-inducible factors (HIF) 1α and HIF2α are stabilized in myeloid cells of murine thrombi. We also previously showed that pulmonary thrombosis increases the levels of HIF1α and HIF2α in murine lungs, enhances the levels of tumorigenic factors in the circulation, and promotes pulmonary tumorigenesis. In this study, we aimed to investigate the regulation of thrombosis-induced tumorigenesis by myeloid cell-specific HIFs (i.e., HIF1 and HIF2 in neutrophils and macrophages). Our in vitro studies showed that multiple tumorigenic factors are upregulated in the secretome of hypoxic versus normoxic neutrophils and macrophages, which promotes lung cancer cell proliferation and migration in a myeloid-HIF-dependent manner. Next, we used a mouse model of pulmonary microvascular occlusion to study the impact of pulmonary thrombosis and myeloid HIFs on lung tumorigenesis. Experiments on mice lacking either HIF1α or HIF2α in myeloid cells demonstrated that loss of either factor eliminates the advantage given to pulmonary tumor formation by thrombotic insult. The myeloid HIF-dependent and tumorigenic impact of pulmonary thrombosis on tumor burden may be partly driven by paracrine thymidine phosphorylase (TP), given that TP levels were increased by hypoxia in neutrophil and macrophage supernates, and that plasma TP levels were positively correlated with multiple measures of tumor progression in wild type mice but not myeloid cell-specific HIF1α or HIF2α knockout mice. These data together demonstrate the importance of thrombotic insult in a model of pulmonary tumorigenesis and the essential role of myeloid HIFs in mediating tumorigenic success. Full article

P2X7 is an extracellular adenosine 5′-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca²⁺ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca²⁺ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y₁, P2Y₂ and P2Y₄. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted. Full article

A major barrier to the diagnosis and effective treatment of solid-tumor cancers is the difficulty in detection and visualization of tumor margins in primary and metastatic disease. The use of fluorescence can augment the surgeon’s ability to detect cancer and aid in its resection. Several cancer types express carcinoembryonic antigen (CEA) including colorectal, pancreatic and gastric cancer. Antibodies to CEA have been developed and tagged with near-infrared fluorescent dyes. This review article surveyed the use of CEA antibodies conjugated to fluorescent probes for in vivo studies since 1990. PubMed and Google Scholar databases were queried, and 900 titles and abstracts were screened. Fifty-nine entries were identified as possibly meeting inclusion/exclusion criteria and were reviewed in full. Forty articles were included in the review and their citations were screened for additional entries. A total of 44 articles were included in the final review. The use of fluorescent anti-CEA antibodies has been shown to improve detection and resection of tumors in both murine models and clinically. The cumulative results indicate that fluorescent-conjugated anti-CEA antibodies have important potential to improve cancer diagnosis and surgery. In an emerging technology, anti-CEA fluorescent antibodies have also been successfully used for photoimmunotherapy treatment for cancer. Full article