Eagle's Minimum Essential Medium Research Articles

Effect of total lens epithelial cell destruction on intraocular lens fixation in the human capsular bag

To evaluate the effect of complete destruction of lens epithelial cells (LECs) in the capsular bag on intraocular lens (IOL) stability. School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. Comparative evaluation. An in vitro organ culture model using the bag-zonule-ciliary body complex isolated from fellow human donor eyes was prepared. A capsulorhexis and fiber extraction were performed, and an Acrysof IOL was implanted. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining it in 6 mL Eagle minimum essential medium supplemented with 5% v/v fetal calf serum and 10 ng/mL transforming growth factor-β2 for 3 weeks or more. One bag of each pair was treated with 1 μM thapsigargin to destroy all LECs. Observations of LEC growth were captured by phase-contrast microscopy, IOL stability by video microscopy, and endpoint analysis through scanning electron microscopy and immunocytochemistry. The LECs in control capsular bags migrated centrally, closing the bag and fixating the IOL between the anterior and posterior capsules, as seen clinically. These events were not observed in the thapsigargin-treated group. After a period of controlled orbital movement, the IOL in the control group stabilized quicker than in the treated bags. There was no IOL rotation in the bag; however, the IOLs in the treated group rocked with axial movement. The LECs appeared to aid stabilization of current IOL designs in the capsular bag. The results have clinical implications for IOL design and for strategies to prevent posterior capsule opacification. No author has a financial or proprietary interest in any material or method mentioned.

High‐strength poly(para‐phenylene) as an orthopedic biomaterial

Poly(para-phenylene) (PPP) exhibits exceptional mechanical strength, stiffness, toughness, and chemical inertness, although it is not currently used in any biomedical applications. The purpose of this study is to serve as a preliminary investigation into the potential of PPP as a biomaterial in orthopedic load-bearing applications. Nuclear magnetic resonance (NMR) analysis confirmed a polymer structure composed of an aromatic backbone and side groups. Tensile PPP specimens along with samples from several other polymers often used for orthopedic applications were elongated to failure after being soaked in phosphate buffered saline (PBS) for 1 h, 1 day, 1 week, 2 weeks, 1 month, and more than 1 year. Results showed that PBS absorption of the PPP plateaued at 1 week at values of ∼0.7 wt % and remained within one standard deviation when soaked for over 1 year. PBS absorption did not affect elastic modulus (5.0 GPa), yield strength (141 MPa), fracture strength (120 MPa) and strain-to-failure (17%) more than one standard deviation. Zero-to-tension fatigue testing established an endurance limit of approximately 35 MPa, which was relatively insensitive to frequency (1-10 Hz). Eagle's minimum essential medium (MEM) elution assay with fibroblasts confirmed that the PPP was noncytotoxic. Relative to other polymers used for load-bearing biomedical applications, PPP displays promising mechanical properties that remain stable in aqueous solution. Lastly, prototype PPP and polyetheretherketone (PEEK) bone plates were manufactured and tested, with the PPP plate showing a 38% higher maximum tensile load before failure.

Dulbecco's modified eagle's medium and minimum essential medium--which one is more preferred for establishment of Caco-2 cell monolayer model used in evaluation of drug absorption?

The Caco-2 cell monolayer model is widely used in drug absorption studies. Dulbecco's Modified Eagle's Medium (DMEM) and Minimum Essential Medium (MEM) have been used alternatively in the development of this model, although they are different in composition which may affect the differentiation and junction formation of the Caco-2 cell monolayer. Two Caco-2 cell monolayers cultured in both media were compared herein in order to underlay the standardization of this model. These two monolayers were comparatively evaluated regarding reliability and stability by morphology, transepithelial electrical resistance (TEER), alkaline phosphatase (AKPase) activity and transport experiments. Although the results showed that characteristic microvilli were present at the apical side of both monolayers, the dynamic change of TEER of the monolayer cultured in DMEM was more stable than that cultured in MEM, and AKPase activity of the former was stronger than that of the latter. Furthermore, the quantity of atenolol, a key indicator usually used for assessment of this model, across the monolayer cultured in MEM was significantly more than that cultured in DMEM. Therefore, the Caco-2 monolayer cultured in DMEM was more reliable and stable than that cultured in MEM, and thus the former was preferred for drug absorption investigation in vitro.

Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro.

AimTo establish an organotypic in vitro model of limb bud development to verify whether epigenetic drug and teratogen 5-azacytidine (5azaC) has an effect on limb buds independent of its effects on the placenta.MethodsFischer strain rat fore- and hindlimb buds were microsurgically isolated from 13 days old embryos and cultivated in vitro for two weeks at the air-liquid interface in Eagle's minimum essential medium (MEM) with 50% rat serum. 30 µmol of 5azaC was added to the fresh medium. Overall growth was measured by an ocular micrometer. Routine histology, immunohistochemical detection of the proliferating cell nuclear antigen (PCNA), and stereological quantification of PCNA expression were performed.ResultsAt four time points, significantly lower overall growth was detected for fore- and hindlimb bud explants cultivated with 5azaC in comparison to controls. After the culture period, numerical density of the PCNA signal for both types of limb buds was lower than for controls (P < 0.001). Limb buds were initially covered by immature epithelium and contained mesenchyme, myotubes, single hemangioblasts, hemangioblast aggregates, blood islands, and capillaries. Regardless of the treatment, cartilage and epidermis differentiated, but cells and structures typical for vasculogenesis disappeared.ConclusionOur findings, obtained outside of the maternal organism, stress the importance of compromised cell proliferation for 5azaC impact on limb buds. This investigation points to the necessity to establish alternatives to in vivo research on animals using teratogenic agents.

Primary cell cultures from sea urchin ovaries: a new experimental tool

In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.

Angiostrongylus cantonensis (Nematode: Metastrongiloidea): In Vitro Cultivation of Infective Third-Stage Larvae to Fourth-Stage Larvae

The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.

Preexposure to PM2.5 exacerbates acute viral myocarditis associated with Th17 cell

It is increasingly recognized that exposure to ambient fine particles (PM(2.5)) is a risk factor for the development of cardiovascular events. This study was to explore the link between PM(2.5) exposure and viral myocarditis in the functional mechanism of Th17 cells. Male BALB/c mice were administered an intratracheal (i.t.) instillation of 10 mg/kg b.w. PM(2.5) particles. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100 μl of coxsackievirus B3 (CVB3) diluted in Eagle's minimal essential medium (EMEM). Seven days after the treatment, pulmonary and cardiac tissues were examined. The results showed that preexposure to PM(2.5) increased the cardiac and pulmonary injuries and viral replication in the heart of CVB3-infected mice along with an increase in CD4(+) IL-17(+) cells in the spleen and heart. The mRNA expressions of interleukin-17A (IL-17A), perforin, transforming growth factor-β (TGF-β) and RORγt were up-regulated in PM(2.5)-pretreated mice than that in the virus-treated mice. Additionally, compared to virus-treated mice, the cardiac protein expressions of IL-17A and matrix metalloproteinases-2 (MMP-2) were increased, but interferon-γ (IFN-γ) and metalloproteinases-1 (TIMP-1) were decreased in PM(2.5)-pretreated mice. Interestingly, PM(2.5) caused IFN-γ decreased, whereas CVB3 caused a dramatic increase in IFN-γ. Subsequently, preexposure to PM(2.5) induced a slight increase of IFN-γ in the sera of CVB3-infected mice. These results demonstrated that PM(2.5) exposure exacerbated virus-induced myocarditis possibly through the increase in Th17-mediated viral replication, perforin response and imbalance of MMP-2/TIMP-1. These findings provided supportive evidence for the epidemiological research that ambient particles could increase the occurrence and development of cardiovascular diseases.

Vaccination with the recombinant major outer membrane protein elicits antibodies to the constant domains and induces cross-serovar protection against intranasal challenge with Chlamydia trachomatis.

To determine the ability of the major outer membrane protein (MOMP) to elicit cross-serovar protection, groups of mice were immunized by the intramuscular (i.m.) and subcutaneous (s.c.) routes with recombinant MOMP (rMOMP) from Chlamydia trachomatis serovars D (UW-3/Cx), E (Bour), or F (IC-Cal-3) or Chlamydia muridarum strain Nigg II using CpG-1826 and Montanide ISA 720 VG as adjuvants. Negative-control groups were immunized i.m. and s.c. with Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) or i.n. with Eagle's minimal essential medium (MEM-0). Following vaccination, the mice developed antibodies not only against the homologous serovar but also against heterologous serovars. The rMOMP-vaccinated animals also mounted cell-mediated immune responses as assessed by a lymphoproliferative assay. Four weeks after the last immunization, mice were challenged i.n. with 10(4) inclusion-forming units (IFU) of C. muridarum. The mice were weighed for 10 days and euthanized, and the number of IFU in their lungs was determined. At 10 days postinfection (p.i.), mice immunized with the rMOMP of C. muridarum or C. trachomatis D, E, or F had lost 4%, 6%, 8%, and 8% of their initial body weight, respectively, significantly different from the negative-control groups (Ng-rPorB, 13%; MEM-0, 19%; P < 0.05). The median number of IFU recovered from the lungs of mice immunized with C. muridarum rMOMP was 0.13 × 10(6). The median number of IFU recovered from mice immunized with rMOMP from serovars D, E, and F were 0.38 × 10(6), 7.56 × 10(6), and 11.94 × 10(6) IFU, respectively. All the rMOMP-immunized animals had significantly less IFU than the Ng-rPorB (40 × 10(6))- or MEM-0 (70 × 10(6))-immunized mice (P < 0.05). In conclusion, vaccination with rMOMP can elicit protection against homologous and heterologous Chlamydia serovars.

Thrombin-cleaved Fragments of Osteopontin Are Overexpressed in Malignant Glial Tumors and Provide a Molecular Niche with Survival Advantage

Osteopontin (OPN), which is highly expressed in malignant glioblastoma (GBM), possesses inflammatory activity modulated by proteolytic cleavage by thrombin and plasma carboxypeptidase B2 (CPB2) at a highly conserved cleavage site. Full-length OPN (OPN-FL) was elevated in cerebrospinal fluid (CSF) samples from all cancer patients compared with noncancer patients. However, thrombin-cleaved OPN (OPN-R) and thrombin/CPB2-double-cleaved OPN (OPN-L) levels were markedly increased in GBM and non-GBM gliomas compared with systemic cancer and noncancer patients. Cleaved OPN constituted ∼23 and ∼31% of the total OPN in the GBM and non-GBM CSF samples, respectively. OPN-R was also elevated in GBM tissues. Thrombin-antithrombin levels were highly correlated with cleaved OPN, but not OPN-FL, suggesting that the cleaved OPN fragments resulted from increased thrombin and CPB2 in this extracellular compartment. Levels of VEGF and CCL4 were increased in CSF of GBM and correlated with the levels of cleaved OPN. GBM cell lines were more adherent to OPN-R and OPN-L than OPN-FL. Adhesion to OPN altered gene expression, in particular genes involved with cellular processes, cell cycle regulation, death, and inflammation. OPN and its cleaved forms promoted motility of U-87 MG cells and conferred resistance to apoptosis. Although functional mutation of the RGD motif in OPN largely abolished these functions, OPN(RAA)-R regained significant cell binding and signaling function, suggesting that the SVVYGLR motif in OPN-R may substitute for the RGD motif if the latter becomes inaccessible. OPN cleavage contributes to GBM development by allowing more cells to bind in niches where they acquire anti-apoptotic properties.

Age-Related Differences in Signaling Efficiency of Human Lens Cells Underpin Differential Wound Healing Response Rates following Cataract Surgery

Cataract surgery is blighted by posterior capsule opacification (PCO), which is more severe and frequent in the young than the elderly (>60 years). Our aim was to understand the biological basis for these age-related differences in PCO/wound healing rates. Human capsular bags were prepared by cataract surgery on donor lenses (young [<40 years] and elderly [>60 years] groups) and maintained in serum-free Eagle's minimum essential medium. Cell growth was determined using the MTS assay. Fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) levels were determined using ELISA. Protein synthesis rates were elucidated by 35S-methionine incorporation. U0126, SB203580, and SP600125 were used to disrupt ERK-, p38-, and JNK-mediated signaling, respectively. Level of total and phospho-ERK, -c-jun, -P38, and -JNK plus cytokines were detected using a BIOPLEX array system. Following a 2-day culture period, significant decreases in IL-1β and IL-6, and increases in IL-10, IL-12, IL-13, and VEGF in the >60 years group were observed compared with their younger counterparts. Capsular bags (cells and capsule) from aged donors contained greater than or equal levels of HGF and FGF than younger counterparts and had greater rates of protein synthesis. Inhibition of ERK, p38, and JNK signaling significantly suppressed cell coverage on the posterior capsule. pERK, p-c-jun, p-p38, and pJNK were consistently lower in aged cell populations; total signaling protein expression was unaffected by age. Serum stimulation increased pERK, p-c-jun, and pJNK levels in cells of all ages; p-p38 was significantly increased in the >60 years group only. Ligand availability to cells is not a limiting factor as we age, but the ability to convert this resource into signaling activity is. We therefore propose that overall signaling efficiency is reduced as a function of age, which consequently limits wound-healing response rates after injury.

Transplantation of Adipose Tissue Mesenchymal Stem Cells in Experimental Chronic Chagasic Cardiopathy

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major cause of heart failure in Latin America. Tissue therapy has been investigated as a possible therapeutic option for patients with cardiovascular disease. This study evaluated the effects of therapy with mesenchymal stem cells in an experimental model of chronic Chagasic cardiomyopathy. C57BL/6 mice were infected with 1000 trypomastigotes from the Colombian strain of T. cruzi and, after six months of infection, were treated with mesenchymal human stem cells from adipose tissue (STAT) or with Dulbecco/Vogt modified Eagle's minimal essential medium - DMEM (control). The treated group received two intraperitoneal injections of STAT (1x10(6) cells/dose), with a month interval between the two doses. Before and after the first and second months of treatment, the chagasic and normal control animals underwent cardiopulmonary exercise testing and electrocardiography. All animals were sacrificed under anesthesia after two months of treatment for histopathological analysis of the heart. No improvement was observed in arrhythmias and cardiovascular function in the group of animals treated with STAT; however, sections of mice hearts in this group revealed a significant reduction in the number of inflammatory cells (p<0.0001) and areas of fibrosis (p<0.01) in comparison with chagasic animals treated with DMEM. Thus, it is concluded that administration of intraperitoneal STAT can reduce inflammation and fibrosis in the heart of mice chronically infected with T. cruzi; however, there were no effects on the cardiac function two months after transplantation.

Cytotoxicity of alginate for orthodontic use

OBJECTIVE: To evaluate the cytotoxicity of three different alginate impression materials for orthodontic use. METHODS: Three different brands of alginate were divided into three groups, namely, Group JCO (Jeltrate Chromatic Ortho), OP (Orthoprint) and CO (Cavex Orthotrace). Three control groups were also included: Group C+ (positive control), consisting of detergent Tween 80; Group C- (negative control), consisting of PBS, and Group CC (cell control), consisting of cells not exposed to any material. After manipulating the materials according to the respective manufacturer instructions, samples were made with the use of silicon rings. Then the samples were immersed in Eagle's minimum essential medium (MEM) for 2 minutes. The supernatants were then removed and brought into direct contact with L929 fibroblasts. After exposure to the medium, the cells were incubated for 24 hours. Then 100 µl of 0.01% neutral red dye were added. The cells were incubated again for 3 hours so that the dye could be absorbed. After this 3-hour period, the cells were fixed to perform the viable cell count, using a spectrophotometer (BioTek, Winooski, Vermont, USA) at a wavelength of 492 nm. RESULTS: Statistical differences were found when Groups CC and C- were compared with the other experimental groups. Group JCO had the highest cytotoxicity, followed by Groups OP and CO. CONCLUSION: Based on the results obtained in this work, it was concluded that all alginate impression materials are potentially cytotoxic.

水道水でつくったEagle's minimum essential mediumの培養細胞への傷害作用は同じ水道水でつくったDulbecoo's modified Eagle's medium培地で消失する

水道水でつくったEagle's minimum essential mediumの培養細胞への傷害作用は同じ水道水でつくったDulbecoo's modified Eagle's medium培地で消失する

Culturing with trehalose produces viable endothelial cells after cryopreservation

Dimethylsulfoxide, the most commonly employed cryoprotectant for cells, has well documented cytotoxic effects in patients. Among the compounds available that may provide protection to cells and tissues during preservation with less cytotoxicity is trehalose. Some animals, such as brine shrimp and tardigrades, accumulate trehalose during periods of extreme environmental stress. In this study, experiments were performed to evaluate the effects of culturing a bovine endothelial cell line (ATCC #CCL-209) in the presence of trehalose prior to preservation by freezing. A number of factors were shown to contribute to cell retention of metabolic activity and proliferative potential including cell culture time with trehalose and the solution conditions during cryopreservation. Using an optimized protocol consisting of 24h of cell culture with 0.2M trehalose followed by cryopreservation with 0.2–0.4M trehalose in sodium bicarbonate buffered Eagles minimum essential medium at pH 7.4 resulted in 87±4% post-preservation cell metabolic activity expressed as relative fluorescence based upon reduction of resazurin to resorufin. This new method provides an alternative preservation strategy to the more classical preservation methods employing dimethylsulfoxide available for cells and tissues.

Aberrant Stratifin Overexpression Is Regulated by Tumor-Associated CpG Demethylation in Lung Adenocarcinoma

We previously have shown the aberrant overexpression of stratifin (SFN, 14-3-3 ς) in lung adenocarcinoma. Although SFN is known to facilitate tumor cell proliferation, the mechanism that underlies its aberrant expression has remained unclear. SFN, the downstream target of p53, often has been reported to be hypermethylated and subsequently silenced in certain cancers; however, its hypomethylation-linked reactivation has not yet been validated. In this study, we investigated the DNA methylation status of the SFN promoter region using 8 lung cancer cell lines and 32 specimens of adenocarcinoma tissue. Real-time methylation-specific PCR analysis showed that although both normal lung tissue and adenocarcinoma in situ bore a completely methylated SFN promoter, the promoter region in almost all invasive adenocarcinomas was at least partially methylated. The expression of SFN and its level of methylation were correlated strongly. Furthermore, statistical analysis revealed that the level of methylation became reduced with progression of the pathologic stage, although no clear relationship between methylation level and p53 abnormality was found. These results suggest that methylation-related silencing of SFN occurs in both normal lung tissues and adenocarcinoma in situ, and that demethylation of the SFN promoter participates in the aberrant expression of SFN in invasive adenocarcinoma cells, independently of p53 alteration. This novel finding might be informative for clarifying the mechanism that underlies the progression of early lung adenocarcinoma.

A Fully Human In Vitro Capsular Bag Model to Permit Intraocular Lens Evaluation

To establish a fully human in vitro culture model with which to test the putative effects of intraocular lens (IOL) designs in preventing posterior capsule opacification (PCO) after cataract surgery. A sham cataract operation was performed to prepare human capsular bags from donor lenses. In one capsular bag of a donor pair, an intraocular lens (PMMA round-edge IOL or acrylic IOL) was implanted while the other capsular bag remained aphakic. Bags were transferred to a Petri dish and secured anterior-face down using entomological pins. Capsular bags were maintained in Eagle's minimum essential medium supplemented with 2% human serum and 10 ng/mL TGF-β to drive growth and matrix contraction. In the absence of an IOL, cells appeared within the central posterior capsule at 4.38 ± 0.26 days, whereas in the presence of a PMMA round-edge IOL or an acrylic IOL they appeared at 8 ± 0.41 days and 11 ± 0.7 days, respectively. Immunocytochemical analysis showed an accumulation of cells at the edge of the acrylic IOL and a less evident accumulation with the PMMA round-edge IOL. Moreover, matrix contraction was more prominent in the absence of an IOL but was still apparent, to a lesser degree, in the presence of a PMMA round-edge IOL. The acrylic IOL greatly suppressed matrix contraction. The authors have developed a fully human in vitro capsular bag system that relates well to clinical observations and permits the testing of novel intraocular lenses.

Occurrence of Nanobacteria-Like Particles in Renal Stones of a Southern Brazilian Population

Purpose: Identifying the source of stone formation in recurrent stone formers has always been a big problem. Material and Methods: In this study kidney stones from 52 patients were submitted to direct examination by scanning electronic microscopy (SEM) after manual fracture and 27 calculi were cultured in Eagle’s Minimum Essential Medium (E-MEM) and Brain-Heart Infusion (BHI) for eight weeks at 37°C in 5% CO2 atmosphere. Twenty-seven powdered and demineralized stones were suspended in sterile PBS, filtered through 0.22 m-pore-size sterile filters Millex (Millipore, Massachusetts, USA) and submitted to DNA extraction (Quiagen-Brazil). Platinum PCR SuperMIX (GIBCO-BRL), primers (Invitrogen), and Ultra Pure Water (Advanced Biotechnologies, Columbia, MD) were used for PCR (Polymerase Chain Reaction), which was generally conducted for 30 or 35 cycles with annealing of primers for 40 sec at 55°C and extension for 1 min at 72°C. Results: In 36 out 52 (69%) kidney stones it was detected the presence of biofilm coating the mineral surface of the stone when examined by SEM, consisting of coccoid particles, isolated or clustered, with diameter of 500 nm or less. Eleven out 27 (41%) kidney stone cultures produced white-colored sediment on the bottom of the tubes after eight-week incubation, revealing tiny structures similar to those observed directly by SEM. These structures were similar in size and morphology to spherical apatite particles previously observed in human kidney stones and named as nanobacteria (NB). No PCR products were observed in the samples. Conclusion: We found a strong correlation between renal stones and calcifying nanoparticles (CNP) in this study and these results open a new insight on this area to explore the etiology of stone formation. Whether NB/CNP are truly microorganisms or self-propagating mineral compounds is still controversial and its contribution, if any, in apatite nucleation and crystal growth remains uncertain.

Lactobacillus rhamnosus GG Treatment Potentiates Intestinal Hypoxia-Inducible Factor, Promotes Intestinal Integrity and Ameliorates Alcohol-Induced Liver Injury

Gut-derived endotoxin is a critical factor in the development and progression of alcoholic liver disease (ALD). Probiotics can treat alcohol-induced liver injury associated with gut leakiness and endotoxemia in animal models, as well as in human ALD; however, the mechanism or mechanisms of their beneficial action are not well defined. We hypothesized that alcohol impairs the adaptive response-induced hypoxia-inducible factor (HIF) and that probiotic supplementation could attenuate this impairment, restoring barrier function in a mouse model of ALD by increasing HIF-responsive proteins (eg, intestinal trefoil factor) and reversing established ALD. C57BJ/6N mice were fed the Lieber DeCarli diet containing 5% alcohol for 8 weeks. Animals received Lactobacillus rhamnosus GG (LGG) supplementation in the last 2 weeks. LGG supplementation significantly reduced alcohol-induced endotoxemia and hepatic steatosis and improved liver function. LGG restored alcohol-induced reduction of HIF-2α and intestinal trefoil factor levels. In vitro studies using the Caco-2 cell culture model showed that the addition of LGG supernatant prevented alcohol-induced epithelial monolayer barrier dysfunction. Furthermore, gene silencing of HIF-1α/2α abolished the LGG effects, indicating that the protective effect of LGG is HIF-dependent. The present study provides a mechanistic insight for utilization of probiotics for the treatment of ALD, and suggests a critical role for intestinal hypoxia and decreased trefoil factor in the development of ALD.

Novel Role for Pendrin in Orchestrating Bicarbonate Secretion in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-expressing Airway Serous Cells

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.

β-Hairpin Peptide That Targets Vascular Endothelial Growth Factor (VEGF) Receptors

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1(D2), we designed a VEGF receptor-binding peptide reproducing the placental growth factor β-hairpin region Gln(87)-Val(100) that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1(D2)-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln(87)-Val(100). Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.