Single-step genome engineering in the bee gut symbiont Snodgrassella alvi.

Honey bees are economically relevant pollinators experiencing population declines due to a number of threats. As in humans, the health of bees is influenced by their microbiome. The bacterium Snodgrassella alvi is a key member of the bee gut microbiome and has a role in excluding pathogens. Despite this importance, there are not currently any easy-to-use methods for modifying the S. alvi chromosome to study its genetics. To solve this problem, we developed a one-step procedure that uses electroporation and homologous recombination, which we term SnODIFY (Snodgrassella-specific One-step gene Deletion or Insertion to alter FunctionalitY). We used SnODIFY to create seven single-gene knockout mutants and recovered mutants for all constructs tested. Nearly all transformants had the designed genome modifications, indicating that SnODIFY is highly accurate. Mutant phenotypes were validated through knockout of Type 4 pilus genes, which led to reduced biofilm formation. We also used SnODIFY to insert heterologous sequences into the genome by integrating fluorescent protein-coding genes. Finally, we confirmed that genome modification is dependent on S. alvi's endogenous RecA protein. Because it does not require expression of exogenous recombination machinery, SnODIFY is a straightforward, accurate, and lightweight method for genome editing in S. alvi. This workflow can be used to study the functions of S. alvi genes and to engineer this symbiont for applications including protection of honey bee health.

Reduced urinary angiotensinogen excretion in preeclampsia.

OBJECTIVE: This study evaluated urinary angiotensinogen in preeclampsia. METHODS: Normal pregnant (n = 57) and preeclamptic patients (n = 31); Normal pregnant (n = 10) and preeclamptic rats (n = 10) were studied. Urinary angiotensinogen and plasma angiotensin II were assayed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Urinary angiotensinogen in preeclampsia patients (2.0 ± 1.1 ng/mg creatinine) was suppressed (*p < 0.05) compared to normal pregnant (2.7 ± 1.5 ng/mg creatinine). Plasma angiotensin II in preeclampsia patients (preeclampsia: 36.2 ± 7; normal pregnant: 48.1 ± 5 fmol/mL) was lower. The similar result was observed in preeclampsia rat model. CONCLUSIONS: The reduced urinary excretion of angiotensinogen was both in human preeclampsia patients and rat model of preeclampsia.

Cartiotonic steroids affect monolayer permeability in lymphatic endothelial cells.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 µM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of ß-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure ß-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1 µM L-NAME attenuated (p < 0.05) the MPLEC. The ß-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.

Differential Effects of In Vitro Treatment with Cinobufotalin on Three Types of Ovarian Cancer Cells.

BACKGROUND/AIM: Cinobufotalin (CINO), a cardiotonic steroid, has been used as an anticancer agent. This study assessed the cell-specific effect of CINO on SK-OV-3, CRL-1978 and CRL-11731 ovarian cancer cells which differ in terms of their respective karyotypes. MATERIALS AND METHODS: Cell cultures were treated with CINO (0.1, 1, 5 and 10 µM) for 24, 48, and 72 h. Cell proliferation, migration, and invasion were measured using CellTiter, Cytoselect, and FluoroBlock assays, respectively. Expression of proliferating cell nuclear antigen (PCNA) was evaluated by western blot analysis. Cell viability was determined by fluorescence-activated cell sorting. Immunofluorescence was performed using Annexin-V staining and fluorescein isothiocyanate (FITC). Mitochondrial membrane potential (MMP) was measured using MitoTracker™ Red. RESULTS: CINO at 0.5 µM inhibited SK-OV-3, CRL-1978, and CRL-11731 proliferation, migration, and invasion. Each cell type differed in response to CINO doses for PCNA, Annexin-V expression and MMP. CONCLUSION: The antineoplastic property of CINO is consistent, but its mode of action varies among cell lines.

Differential Mechanism of Action of 3,4',7-O-trimethylquercetin in Three Types of Ovarian Cancer Cells.

BACKGROUND/AIM: 3,4',7-O-trimethylquercetin (34'7TMQ), a derivative of quercetin, inhibited ovarian cancer cell migration and invasion without affecting proliferation. In this study, the apoptotic effect of 34'7TMQ on three cancer cell lines (CRL-1978, CRL-11731, SK-OV-3) was evaluated. MATERIALS AND METHODS: Expression of pro-apoptotic proteins such as Bax/Bcl-2 ratio, p38 MAP kinase, and caspase-9 were measured by western blot analysis. Annexin-V staining was performed to visualize apoptotic signaling. RESULTS: Caspase-9 was up-regulated in all three cell lines. Bax/Bcl-2 ratio was up-regulated in CRL-1978 and SK-OV-3 but down-regulated in CRL-11731. The p38 MAPK was down-regulated in CRL-1978, up-regulated in SK-OV-3, and had differential expression in CRL-11731. Annexin V staining indicated that 34'7TMQ at 6.25 µM induced apoptotic signaling in the CRL-1978 ovarian cancer cell line. CONCLUSION: 34'7TMQ induced apoptosis in three types of cancer cell lines but it appears to have a different mechanism of action in each cell line.