A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.     

PCSK9 Plasma Concentrations Are Independent of GFR and Do Not Predict Cardiovascular Events in Patients with Decreased GFR

Background

Impaired renal function causes dyslipidemia that contributes to elevated cardiovascular risk in patients with chronic kidney disease (CKD). The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator of the LDL receptor and plasma cholesterol concentrations. Its relationship to kidney function and cardiovascular events in patients with reduced glomerular filtration rate (GFR) has not been explored.

Methods

Lipid parameters including PCSK9 were measured in two independent cohorts. CARE FOR HOMe (Cardiovascular and Renal Outcome in CKD 2–4 Patients—The Forth Homburg evaluation) enrolled 443 patients with reduced GFR (between 90 and 15 ml/min/1.73 m²) referred for nephrological care that were prospectively followed for the occurrence of a composite cardiovascular endpoint. As a replication cohort, PCSK9 was quantitated in 1450 patients with GFR between 90 and 15 ml/min/1.73 m² enrolled in the Ludwigshafen Risk and Cardiovascular Health Study (LURIC) that were prospectively followed for cardiovascular deaths.

Results

PCSK9 concentrations did not correlate with baseline GFR (CARE FOR HOMe: r = -0.034; p = 0.479; LURIC: r = -0.017; p = 0.512). 91 patients in CARE FOR HOMe and 335 patients in LURIC reached an endpoint during a median follow-up of 3.0 [1.8–4.1] years and 10.0 [7.3–10.6] years, respectively. Kaplan-Meier analyses showed that PCSK9 concentrations did not predict cardiovascular events in either cohort [CARE FOR HOMe (p = 0.622); LURIC (p = 0.729)]. Sensitivity analyses according to statin intake yielded similar results.

Conclusion

In two well characterized independent cohort studies, PCSK9 plasma levels did not correlate with kidney function. Furthermore, PCSK9 plasma concentrations were not associated with cardiovascular events in patients with reduced renal function.     

Effects of soil warming and nitrogen foliar applications on bud burst of black spruce

Key message

In mature black spruce, bud burst process is anticipated by soil warming, while delayed by foliar applications of nitrogen; however, the effects depend on growth conditions at the site.

Abstract

The observation of phenological events can be used as biological indicator of environmental changes, especially from the perspective of climate change. In boreal forests, the onset of the bud burst is a key factor in the length of the growing season. With current climate change, the major factors limiting the growth of boreal trees (i.e., temperature and nitrogen availability) are changing and studies on mature trees are limited. The aim of this study was to investigate the effects of soil warming and increased nitrogen (N) deposition on bud burst of mature black spruce [Picea mariana (Mill.) BSP]. From 2008 onwards, an experimental manipulation of these environmental growth conditions was conducted in two stands (BER and SIM) at different altitudes in the boreal forest of Quebec, Canada. An increase in soil temperature (H treatment) and a canopy application of artificial rain enriched with nitrogen (N treatment) were performed. Observations of bud phenology were made during May–July 2012 and 2013. In BER, H treatment caused an anticipation (estimated as 1–3 days); while N treatment, a delay (estimated as 1–2 days but only in 2012) in bud burst. No treatments effect was significant in SIM. It has been demonstrated that soil temperature and N availability can play an important role in affecting bud burst in black spruce but the effects of these environmental factors on growth are closely linked with site conditions.

     

Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern,phagosome maturation,and escape to the cytosol

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication‐permissive compartment termed the Mycobacterium‐containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid‐borne genes. Fluorescence microscopy of M. marinum‐infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol‐3‐phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V‐ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.     

Desulfoferrodoxin of Clostridium acetobutylicum functions as a superoxide reductase

Desulfoferrodoxin (cac2450) of Clostridium acetobutylicum was purified after overexpression in E. coli. In an in vitro assay the enzyme exhibited superoxide reductase activity with rubredoxin (cac2778) of C. acetobutylicum as the proximal electron donor. Rubredoxin was reduced by ferredoxin:NADP(+) reductase from spinach and NADPH. The superoxide anions, generated from dissolved oxygen using Xanthine and Xanthine oxidase, were reduced to hydrogen peroxide. Thus, we assume that desulfoferrodoxin is the key factor in the superoxide reductase dependent part of an alternative pathway for detoxification of reactive oxygen species in this obligate anaerobic bacterium.     

DNA sequences from Arabidopsis, which encode protein kinases and function as upstream regulators of Snf1 in yeast

Sucrose nonfermenting-1 (Snf1)-related protein kinase-1 (SnRK1) of plants is a global regulator of carbon metabolism through the modulation of enzyme activity and gene expression. It is structurally and functionally related to the yeast protein kinase, Snf1, and to mammalian AMP-activated protein kinase. Two DNA sequences from Arabidopsis thaliana, previously known only by their data base accession numbers of NM_ 125448.3 (protein ID NP_200863) and NM_114393.3 (protein ID NP_566876) each functionally complemented a Saccharomyces cerevisiae elm1 sak1 tos3 triple mutant. This indicates that the Arabidopsis proteins are able to substitute for one of the missing yeast upstream kinases, which are required for activity of Snf1. Both plant proteins were shown to phosphorylate a peptide with the amino acid sequence of the phosphorylation site in the T-loop of SnRK1 and by inference SnRK1 in Arabidopsis. The proteins encoded by NM_125448.3 and NM_114393.3 have been named AtSnAK1 and AtSnAK2 (Arabidopsis thaliana SnRK1-activating kinase), respectively. We believe this is the first time that upstream activators of SnRK1 have been described in any plant species.     

Proteomics demonstration that normal breast epithelial cells can induce apoptosis of breast cancer cells through insulin-like growth factor-binding protein-3 and maspin

Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.     

Glutamine regulates the human epithelial intestinal HCT-8 cell proteome under apoptotic conditions

Glutamine plays a key role in the metabolism of rapidly dividing cells, including enterocytes and lymphocytes, which may contribute to its beneficial clinical effects. Gut mucosal homeostasis is achieved through a balance between cell proliferation and apoptosis. In T cells, glutamine up-regulates antiapoptotic proteins and down-regulates proapoptotic proteins. In gut mucosa, glutamine prevents apoptosis in rat epithelial cell lines, whereas glutamine starvation induces apoptosis through caspase activation. Finally glutamine specifically prevents tumor necrosis factor-alpha-related apoptosis in the human intestinal cell line HT-29. Comparative functional proteomics enables the characterization of each differentially expressed protein in intestinal cells in response to modifications of nutritional environment. The influence of glutamine on intestinal proteome expression in apoptotic conditions has not been studied and evaluated. This comparative proteomics study was performed in the human epithelial intestinal cell line HCT-8 under experimental apoptotic conditions to investigate the influence of glutamine on protein expression during apoptosis. The pharmaconutritional effects of glutamine were determined under 2 mm (physiological concentration) and 10 mm (pharmaconutritional concentration) conditions. About 1,800 protein spots were revealed in both conditions. Comparative assessments indicated that 28 proteins were differentially expressed significantly (i.e. at least 2-fold modulated and Student's t test with p 相似文献