Microwave assisted saponification (MAS) followed by on-line liquid chromatography (LC)-gas chromatography (GC) for high-throughput and high-sensitivity determination of mineral oil in different cereal-based foodstuffs

A high throughput, high-sensitivity procedure, involving simultaneous microwave-assisted extraction (MAS) and unsaponifiable extraction, followed by on-line liquid chromatography (LC)-gas chromatography (GC), has been optimised for rapid and efficient extraction and analytical determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) in cereal-based products of different composition. MAS has the advantage of eliminating fat before LC-GC analysis, allowing an increase in the amount of sample extract injected, and hence in sensitivity. The proposed method gave practically quantitative recoveries and good repeatability. Among the different cereal-based products analysed (dry semolina and egg pasta, bread, biscuits, and cakes), egg pasta packed in direct contact with recycled paperboard had on average the highest total MOSH level (15.9mgkg(-1)), followed by cakes (10.4mgkg(-1)) and bread (7.5mgkg(-1)). About 50% of the pasta and bread samples and 20% of the biscuits and cake samples had detectable MOAH amounts. The highest concentrations were found in an egg pasta in direct contact with recycled paperboard (3.6mgkg(-1)) and in a milk bread (3.6mgkg(-1))

Designer Leptin Receptor Antagonist Allo-aca Inhibits VEGF Effects in Ophthalmic Neoangiogenesis Models

Experimental and clinical data suggest that pro-angiogenic, pro-inflammatory and mitogenic cytokine leptin can be implicated in ocular neovascularization and other eye pathologies. At least in part, leptin action appears to be mediated through functional interplay with vascular endothelial growth factor (VEGF). VEGF is a potent regulator of neoangiogenesis and vascular leakage with a proven role in conditions such as proliferative diabetic retinopathy, age-related macular degeneration and diabetic macular edema. Accordingly, drugs targeting VEGF are becoming mainstream treatments for these diseases. The crosstalk between leptin and VEGF has been noted in different tissues, but its involvement in the development of eye pathologies is unclear. Leptin is coexpressed with VEGF during ocular neovascularization and can potentiate VEGF synthesis and angiogenic function. However, whether or not VEGF regulates leptin expression or signaling has never been studied. Consequently, we addressed this aspect of leptin/VEGF crosstalk in ocular models, focusing on therapeutic exploration of underlying mechanisms.Here we show, for the first time, that in retinal (RF/6A) and corneal (BCE) endothelial cells, VEGF (100 ng/mL, 24 h) stimulated leptin mRNA synthesis by 70 and 30%, respectively, and protein expression by 56 and 28%, respectively. In parallel, VEGF induced RF/6A and BCE cell growth by 33 and 20%, respectively. In addition, VEGF upregulated chemotaxis and chemokinesis in retinal cells by ~40%. VEGF-dependent proliferation and migration were significantly reduced in the presence of the leptin receptor antagonist, Allo-aca, at 100-250 nmol/L concentrations. Furthermore, Allo-aca suppressed VEGF-dependent long-term (24 h), but not acute (15 min) stimulation of the Akt and ERK1/2 signaling pathways. The efficacy of Allo-aca was validated in the rat laser-induced choroidal neovascularization model where the compound (5 μg/eye) significantly reduced pathological vascularization with the efficacy similar to that of a standard treatment (anti-VEGF antibody, 1 μg/eye).Cumulatively, our results suggest that chronic exposure to VEGF upregulates leptin expression and function. As leptin can in turn activate VEGF, the increased abundance of both cytokines could amplify pro-angiogenic and pro-inflammatory environement in the eye. Thus, combined therapies targeting ObR and VEGF should be considered in the treatment of ocular diseases

Posttranscriptional regulation of PARG mRNA by HuR facilitates DNA repair and resistance to PARP inhibitors

The majority of pancreatic ductal adenocarcinomas (PDAC) rely on the mRNA stability factor HuR (ELAV-L1) to drive cancer growth and progression. Here, we show that CRISPR-Cas9–mediated silencing of the HuR locus increases the relative sensitivity of PDAC cells to PARP inhibitors (PARPi). PDAC cells treated with PARPi stimulated translocation of HuR from the nucleus to the cytoplasm, specifically promoting stabilization of a new target, poly (ADP-ribose) glycohydrolase (PARG) mRNA, by binding a unique sequence embedded in its 30 untranslated region. HuR-dependent upregulation of PARG expression facilitated DNA repair via hydrolysis of polyADP-ribose on related repair proteins. Accordingly, strategies to inhibit HuR directly promoted DNA damage accumulation, inefficient PAR removal, and persistent PARP-1 residency on chromatin (PARP-1 trapping). Immunoprecipitation assays demonstrated that the PARP-1 protein binds and posttranslationally modifies HuR in PARPi-treated PDAC cells. In a mouse xenograft model of human PDAC, PARPi monotherapy combined with targeted silencing of HuR significantly reduced tumor growth compared with PARPi therapy alone. Our results highlight the HuR–PARG axis as an opportunity to enhance PARPi-based therapies. ©2017 AACR

Biomass and Productivity of Thalassia testudinum in Estuaries of the Florida Panhandle

Thalassia testudinum often dominates seagrass meadows of the Florida panhandle but few measurements of productivity, biomass, density, turnover or leaf area index in this region have been made. We targeted 5 estuaries located at similar latitudes, 30⁰ ± 0.3⁰N: Big Lagoon, Santa Rosa Sound, St. Andrew Bay, St. Joseph Bay, and St. George Sound. This study was one component of a collaborative partnership of state and local researchers examining factors preventing recovery in panhandle estuarine areas that had historically contained seagrass in the 1940s and 1950s. Measurements were made twice in 2016, once in June and then again in summer or fall, except in Santa Rosa Sound where measurements were made 3 times. In the estuaries sampled for the second time in July or August, aboveground productivity was greater than in June. St. Joseph Bay had the highest aboveground productivity (4.3 g/m2/d) and 1—sided leaf area index (4.2) while St. George Sound had the lowest values (0.41 g/m2/d and 1.0). Principal component analysis suggested that St. Andrew Bay, Big Lagoon and Santa Rosa Sound were the most similar, with higher values for shoot densities and leaf turnover and lower salinities and watershed:water ratios. St. Joseph Bay had high aboveground productivity and salinity, and low turbidity. St. George Sound had low aboveground productivity, high total suspended solids and the highest watershed:water ratio. These baseline productivity estimates will be useful to assess the success of restoration efforts targeting seagrasses in the Florida panhandle and evaluate impacts of climate change on seagrasses

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Synthesis of Glycosylated Tuftsins and Tuftsin-containing IGg Fragment Undecapeptide

Syntheses are described of two new tuftsin derivatives containing a 2-acetamido-2-deoxy-D-galactopyranosyl unit alpha- or beta-glycosidically linked to the threonine's hydroxy side chain function and of the glycosylated undecapeptide corresponding to the tuftsin region of the heavy chain of IgG (amino acid sequence 289-299). The glycosylated tuftsins were synthesized by the solution procedure. Fmoc-[Gal NAc(Ac)3-alpha]Thr-OH and Fmoc-[GalNAc(Ac)3-beta]Thr-OH were allowed to react with H-Lys(Z)-Pro-Arg(NO2)-OBzl by the mixed anhydride procedure and the resulting glycosylated tetrapeptides were fully deblocked by catalytic hydrogenation followed by treatment with potassium cyanide, purified by ion exchange chromatography and characterized by analytical HPLC, elemental and amino acid analyses, optical rotation, and proton NMR spectroscopy. Synthesis of the glycosylated undecapeptide was achieved by the continuous flow solid phase procedure on 4-hydroxymethylphenoxyacetyl-norleucyl derivatized Kieselguhr-supported resin. Fmoc-amino acid symmetrical anhydrides or pentafluorophenyl esters, in the presence of N-hydroxybenzotriazole, were used as the acylating agents. To mimic the native sequence of the tuftsin region at the Fc-domain of immunoglobulin G a 2-acetamido-2-deoxy-beta-D-glucopyranosyl unit was N-glycosidically linked to the amide side chain of Asn 297. The glycosylated asparagine residue was introduced as N2-fluorenylmethyloxycarbonyl-N4-(2-acetamido-3,4, 6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-asparagine pentafluorophenyl ester. After cleavage from the resin the glycopeptide was deprotected, purified by ion exchange chromatography, and characterized by analytical HPLC, amino acid analysis, high voltage electrophoresis, and proton NMR. The conformational features of the glyco-undecapeptide were determined by circular dichroism measurements both in water and in 98% trifluoroethanol. Results of biological assays will be published elsewhere

Exploring leptin antagonism in ophthalmic cell models.

BACKGROUND:Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions. METHODS AND RESULTS:The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca. CONCLUSIONS:Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models

Interaction of Aggregated Cationic Porphyrins with Human Serum Albumin

The interaction of an equilibrium mixture of monomeric and aggregated cationic trans-5,15-bis(N-methylpyridinium-4-yl)-10,15-bis-diphenylporphine (t-H2Pagg) chloride salt with human serum albumin (HSA) has been investigated through UV/Vis absorption, fluorescence emission, circular dichroism and resonant light scattering techniques. The spectroscopic evidence reveals that both the monomeric t-H2Pagg and its aggregates bind instantaneously to HSA, leading to the formation of a tight adduct in which the porphyrin is encapsulated within the protein scaffold (S430) and to clusters of aggregated porphyrins in electrostatic interaction with the charged biomolecules. These latter species eventually interconvert into the final S430 species following pseudo-first-order kinetics. Molecular docking simulations have been performed to get some insights into the nature of the final adduct. Analogously to hemin bound to HSA, the obtained model supports favorable interactions of the porphyrin in the same 1B subdomain of the protein. Hydrophobic and van der Waals energy terms are the main contributions to the calculated ΔGbind value of −117.24 kcal/mol