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Lactobacillus fermentum is commonly found in food products, and some strains are known to have beneficial effects on human health. However, our previous research indicated that L. fermentum AGR1487 decreases in vitro intestinal barrier integrity. The hypothesis was that cell surface structures of AGR1487 are responsible for the observed in vitro effect. AGR1487 was compared to another human oral L. fermentum strain, AGR1485, which does not cause the same effect. The examination of phenotypic traits associated with the composition of cell surface structures showed that compared to AGR1485, AGR1487 had a smaller genome, utilized different sugars, and had greater tolerance to acid and bile. The effect of the two strains on intestinal barrier integrity was determined using two independent measures of paracellular permeability of the intestinal epithelial Caco-2 cell line. The transepithelial electrical resistance (TEER) assay specifically measures ion permeability, whereas the mannitol flux assay measures the passage of uncharged molecules. Both live and UV-inactivated AGR1487 decreased TEER across Caco-2 cells implicating the cell surfaces structures in the effect. However, only live AGR1487, and not UV-inactivated AGR1487, increased the rate of passage of mannitol, implying that a secreted component(s) is responsible for this effect. These differences in barrier integrity results are likely due to the TEER and mannitol flux assays measuring different characteristics of the epithelial barrier, and therefore imply that there are multiple mechanisms involved in the effect of AGR1487 on barrier integrity.

The conditional removal of floxed RFP from HEK293 genome by Cumate-induction. (PDF 412 kb)

Coding and amino-acid sequences of Cre recombinase. (PDF 40 kb)

Features of AAV-based Cre-delivery vectors. (PDF 367 kb)

Background: There are few data on echocardiographic indexes incorporating peak mitral inflow velocity (E), left atrial volume index (LAVi), and pulmonary artery pressure (PAP) for estimation of left ventricular (LV) filling pressure in patients with preserved LV ejection fraction (EF ≥ 50%). Methods: Patients underwent echocardiography ≤20 minutes of cardiac catheterization. Echocardiographic variables were compared to invasively measured LV end-diastolic pressure (LVEDP). Results: Of the 122 patients, 67 (55%) were women, the mean age was 55 ± 9 years, the mean left ventricular ejection fraction (LVEF) was 61 ± 6%, 107 (88%) were hypertensive, and 79 (65%) had significant coronary artery disease at catheterization. E/Ea correlated with LVEDP (R = 0.68, P 12 had 75% sensitivity and 78% specificity for LVEDP ≥ 20 mmHg (area under curve (AUC) = 0.79, P 30 (sensitivity = 72%, specificity = 80%, AUC = 0.84, P 57 (sensitivity = 73% and specificity = 81%, AUC = 0.82, P 90 cm/sec had 96% positive, predictive value for LVEDP ≥ 20 mmHg. (E + LAVi)/2 added incrementally to E/Ea when E/Ea was in the gray zone. Conclusions: New, simple echocardiographic equations, (E + LAVi)/2 and (PAP + LAVi)/2, have comparable accuracy to E/Ea for LVEDP estimation in patients with cardiac disease and preserved LVEF, and (E + LAVi)/2 added incrementally to E/Ea alone when E/Ea was in the gray zone. Peak E velocity alone had high negative and positive predictive value for elevated LVEDP in this population. These simple echocardiographic variables could be used—in isolation or with E/Ea—in patients with cardiac disease and preserved LVEF for the diagnosis of diastolic heart failure. Echocardiography 2010;27:946-953)

There are a paucity of data comparing spectral and color tissue Doppler (TD) with non-Doppler, speckle-based myocardial velocities, and it is unknown how early transmitral diastolic velocity/mitral annular velocity (E/Ea) calculated using speckle velocities compares with TD-derived E/Ea.We measured systolic (Sa), Ea, and late diastolic (Aa) myocardial velocities using these 3 methods and compared calculated E/Ea with invasively measured left ventricular (LV) hemodynamics. Consecutive patients referred for cardiac catheterization were imaged, and LV pre-A contraction pressure was measured by retrograde aortic cardiac catheterization.Fifty patients (22 women, 44%) were studied with a mean age of 54 +/- 10 years and a mean LV ejection fraction of 48% +/- 19%. Speckle and color TD Sa, Ea, and Aa were significantly lower than spectral TD velocities, resulting in higher E/Ea values compared with spectral TD E/Ea. Spectral TD E/Ea (R = 0.65, P.001), color TD E/Ea (R = 0.69, P.001), and speckle E/Ea (R = 0.76, P.001) all correlated with LV pre-A pressure. Different cutoff values were needed for spectral TD, color TD, and speckle E/Ea to predict LV pre-A pressureor= 15 mm Hg; spectral E/Ea14 (area under the curve [AUC] = 0.87, P.001, sensitivity = 83%, specificity = 77%), color E/Ea16 (AUC = 0.89, P.0001, sensitivity = 79%, specificity = 81%), and speckle E/Ea18 (AUC = 0.87, P.0001, sensitivity = 88%, specificity = 74%; P = not significant for comparisons between the groups).Spectral TD, color TD, and speckle imaging measure different velocities, and consequently different cutoff values are needed for E/Ea to predict invasively measured LV filling pressure.

The effect of hydrolyzed casein (HC) on the expression of three mucin genes (Muc2, Muc3, and Muc4) in the rat intestine was investigated using quantitative real-time polymerase chain reaction. After a 10 day acclimatization period, rats received for 8 days the test diets containing either HC or a synthetic amino acid (SAA) mixture as the sole source of nitrogen or a protein-free (PF) diet (n = 12 per treatment). The addition of HC or the SAA mixture to the diet significantly improved average daily gain, average daily food intake, and gain:feed ratio as compared with the PF diet. Terminal ileal endogenous N flow was significantly higher for the HC-fed rats in comparison with either the SAA or the PF rats (p < or = 0.001). HC supported a significant increase of Muc3 mRNA (277 and 229% of that for diets PF and SAA, respectively; p < or = 0.05) in the small intestinal tissue and Muc4 mRNA (325 and 265% of that for diets PF and SAA, respectively; p < or = 0.05) in the colon. In conclusion, HC enhances ileal endogenous N flow and up-regulates in vivo the expression of some individual mucin genes.

Tissue Doppler indexes of left ventricular (LV) filling pressure are prone to angulation errors and tethering and are less reliable in patients with preserved LV ejection fraction and indeterminate early peak transmitral diastolic flow (E)/mitral early diastolic velocity (Ea) (8E/Ea15). Two-dimensional echocardiographic global longitudinal diastolic strain (Ds) and strain rate (DSr) were measured during peak mitral filling, and combined with E, derived new noninvasive indexes of LV filling pressure (E/Ds and E/10DSr). These indexes were compared with simultaneously invasively measured LV preatrial (pre-A) contraction pressure and E/Ea. Fifty patients were studied. Mean age was 55.9 +/- 9.9 years, 22 (43%) were women, and mean LV ejection fraction was 49.3 +/- 18.0%. Ds (R = 0.48, p0.001) and DSr (R = 0.43, p = 0.002) correlated with invasively measured -dP/dt. Correlations between E/Ds and E/10DSr with LV pre-A pressure were R = 0.81 (p0.001) and R = 0.80 (p0.001) compared with R = 0.63 (p0.001) between E/Ea and LV pre-A pressure, respectively. E/Dsor =8 had higher sensitivity and specificity (95% and 94%, respectively; area under the curve = 0.96, p0.0001) than E/Eaor =15 (sensitivity 81%, specificity 75%; area under the curve = 0.85, p0.0001) for the prediction of LV pre-A pressureor =15 mm Hg (p = 0.01 for comparison). In patients with LV ejection fractionor =50% and 8E/Ea15, E/Ds and E/DSr were more accurate than E/Ea for determination of LV pre-A pressure. In conclusion, a novel ratio 2-dimensional echocardiographic diastolic strain ratio (E/Ds) was a better predictor of LV filling pressure than E/Ea. In patients with LV ejection fractionor =50% or indeterminate E/Ea, both E/Ds and E/10DSr (a ratio based on global DSR) were better predictors of LV filling pressure than E/Ea.

Schematic representation of the packaging plasmid, expressing GAG, POL, TAT and ENV. Panel A shows the configuration of the expression vector for wild-type pol gene, whereas panel B shows the site-directed mutant, resulting in the change of D116A (denoted by the red star). (PDF 590 kb)

It is unknown whether right ventricular (RV) tissue Doppler (TD) predicts outcome in patients with left ventricular (LV) heart failure (HF) independently of contemporary echocardiographic Doppler variables of LV diastolic function. Comprehensive echocardiographic Doppler examination was performed before discharge in 107 patients hospitalized with LV HF. The primary end point was cardiac death or rehospitalization for HF. Follow-up was complete for 100 of 107 patients a mean of 527 days after hospital discharge. There were no significant differences in baseline clinical variables (mean age 58 ± 12 years, 46% women, 77% hypertensive, 48% diabetic, 41% current smokers, and 23% known coronary artery disease) in prediction of the primary end point. Compared with patients without an event, patients with an event had a larger left atrial volume index (42 ± 16 vs 33 ± 13 ml/m 2 , p = 0.001), lower LV ejection fraction (35 ± 19% vs 46 ± 22%, p = 0.01), higher mitral peak early diastolic flow velocity/TD early diastolic velocity (19 ± 7 vs 14 ± 7, p = 0.001), lower RV fractional area change (39 ± 11% vs 43 ± 10%, p = 0.04), and lower RV TD systolic velocity (8 ± 2 vs 10 ± 3 cm/s, p = 0.005). On Cox proportional hazards multivariate analysis, left atrial volume index (p = 0.01), mitral peak early diastolic flow velocity/TD early diastolic velocity (p = 0.03), and RV TD systolic velocity (p = 0.04) were independent predictors of outcome. Even when contemporary echocardiographic Doppler measures of LV diastolic function are considered, RV TD systolic velocity is an independent predictor of cardiac death or rehospitalization for HF in patients hospitalized with HF and appears to be superior to conventional 2-dimensional parameters of RV function.

The authors utilized rapid right atrial pacing and handgrip exercise to provoke myocardial ischemia in 20 participants (age >65 years) who, for reasons of disability, were not candidates for exercise and pharmacologic stress testing. Echocardiographic left ventricular ejection fraction and left ventricular wall motions were obtained during pacing at baseline and at maximal pacing rates and were compared with coronary angiography. Using the failure of left ventricular ejection fraction to increase with pacing as an indicator of myocardial ischemia, the test yielded a sensitivity of 75%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 71%. When a pacing-induced decrease of wall-motion index was taken as an ischemia indicator, the sensitivity was 63%, specificity 100%, positive predictive value 100%, and negative predictive value 80%. Rapid atrial pacing echocardiography is a safe test. It may be considered in a select group of elderly patients as an alternative to exercise or pharmacologic tests before resorting to coronary angiography.

Lactobacillusspecies can exert health promoting effects in the gastrointestinal tract (GIT) through many mechanisms, which include pathogen inhibition, maintenance of microbial balance, immunomodulation, and enhancement of the epithelial barrier function. Different species of the genusLactobacilluscan evoke different responses in the host, and not all strains of the same species can be considered beneficial. Strain variations may be related to diversity of the cell surface architecture of lactobacilli and the bacteria's ability to express certain surface components or secrete specific compounds in response to the host environment. Lactobacilli are known to modify their surface structures in response to stress factors such as bile and low pH, and these adaptations may help their survival in the face of harsh environmental conditions encountered in the GIT. In recent years, multiple cell surface-associated molecules have been implicated in the adherence of lactobacilli to the GIT lining, immunomodulation, and protective effects on intestinal epithelial barrier function. Identification of the relevant bacterial ligands and their host receptors is imperative for a better understanding of the mechanisms through which lactobacilli exert their beneficial effects on human health.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction. There are three main components necessary for efficient lentiviral packaging. 1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance. An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at: http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf. This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

There are few data on echocardiographic indexes incorporating peak mitral inflow velocity (E), left atrial volume index (LAVi), and pulmonary artery pressure (PAP) for estimation of left ventricular (LV) filling pressure in patients with preserved LV ejection fraction (EF ≥ 50%).Patients underwent echocardiography ≤20 minutes of cardiac catheterization. Echocardiographic variables were compared to invasively measured LV end-diastolic pressure (LVEDP).Of the 122 patients, 67 (55%) were women, the mean age was 55 ± 9 years, the mean left ventricular ejection fraction (LVEF) was 61 ± 6%, 107 (88%) were hypertensive, and 79 (65%) had significant coronary artery disease at catheterization. E/Ea correlated with LVEDP (R = 0.68, P0.0001), compared to PAP (R = 0.53, P0.001), peak E velocity (R = 0.48, P0.001), and LAVi (R = 0.48, P0.001). E/Ea12 had 75% sensitivity and 78% specificity for LVEDP ≥ 20 mmHg (area under curve (AUC) = 0.79, P0.0001), compared with (PAP + LAVi)/230 (sensitivity = 72%, specificity = 80%, AUC = 0.84, P0.001) and (E + LAVi)/257 (sensitivity = 73% and specificity = 81%, AUC = 0.82, P0.001) (P = NS). E60 cm/sec had 94% negative, and E90 cm/sec had 96% positive, predictive value for LVEDP ≥ 20 mmHg. (E + LAVi)/2 added incrementally to E/Ea when E/Ea was in the gray zone.New, simple echocardiographic equations, (E + LAVi)/2 and (PAP + LAVi)/2, have comparable accuracy to E/Ea for LVEDP estimation in patients with cardiac disease and preserved LVEF, and (E + LAVi)/2 added incrementally to E/Ea alone when E/Ea was in the gray zone. Peak E velocity alone had high negative and positive predictive value for elevated LVEDP in this population. These simple echocardiographic variables could be used-in isolation or with E/Ea-in patients with cardiac disease and preserved LVEF for the diagnosis of diastolic heart failure.

There are few data on adding left atrial volume index (LAVi) or pulmonary artery systolic pressure (PAP) to the ratio of early mitral inflow to mitral annular velocity (E/e') for the estimation of left ventricular (LV) filling pressure in patients with preserved LV ejection fractions (LVEFs) (50%).Patients underwent echocardiography within 20 minutes of cardiac catheterization. Echocardiographic variables were compared with invasively measured LV preatrial contraction pressure (pre-A).Of the 122 patients studied (mean age, 55 +/- 9 years; mean LVEF, 61 +/- 6%), 67 (55%) were women, 108 (88%) had hypertension, and 79 (65%) had significant coronary artery disease at catheterization. E/e' was significantly correlated with pre-A (R = 0.63, P.0001) compared with LAVi (R = 0.49, P.001) and PAP (R = 0.48, P.001). E/e'13 had sensitivity of 70% and specificity of 93% (area under the curve [AUC], 0.82; P.0001), LAVi31 mL/m2 had sensitivity of 78% and specificity of 76% (AUC, 0.80, P.001), and PAP28 mm Hg had sensitivity of 80% and specificity of 64% for pre-A15 mm Hg (AUC, 0.77, P.001). Adding LAVi31 mL/m2 for E/e' = 8 to 13 significantly increased the accuracy of E/e'13 alone (sensitivity, 87%; specificity, 88%; AUC, 0.89; P = .01 for comparison). However, adding PAP28 mm Hg for E/e' = 8 to 13 did not significantly increase the accuracy of E/e'13 alone (AUC, 0.82; sensitivity, 82%; specificity, 72%; P = NS for comparison).In patients with preserved LVEFs, adding LAVi31 mL/m2 to E/e' (when E/e' was in the gray zone, but not when E/e' was13) significantly increased the accuracy of E/e' alone for the estimation of LV filling pressure. These data support the notion of using several, rather than any single, Doppler echocardiographic parameter for the accurate assessment of LV diastolic function.

There is controversy regarding the nature of systolic function in patients with elevated filling pressure and preserved left ventricular (LV) ejection fraction. In this study, tissue Doppler variables and 2-dimensional echocardiographic systolic strain (SS) and systolic strain rate (SSr) were measured in patients who underwent cardiac catheterization to determine correlations with invasively measured LV end-diastolic pressure (LVEDP), dP/dt, and LV mass. Forty patients were studied. Their mean age was 55.9+/-9.9 years, and their mean LV ejection fraction was 59.8+/-5.2%. Tissue Doppler systolic annular velocity (5.4+/-1.1 vs 6.4+/-1.0 cm/s, p=0.04), SS (13.4+/-3.7% vs 18.8+/-2.3%, p0.001), and SSr (0.73+/-0.17 vs 0.98+/-0.14 s(-1), p0.001) were significantly lower in patients with LVEDP20 mm Hg compared with those with LVEDP20 mm Hg. Tissue Doppler systolic velocity, SSr, and SS were correlated with LV mass (R=0.58, R=0.57, and R=0.52, respectively, all p values0.001) and with LVEDP (R=0.49, p=0.002; R=0.79, p0.001; and R=0.70, p0.001, respectively). However, dP/dt was not significantly different between patients with LVEDP20 mm Hg and those with LVEDP20 mm Hg (1,387+/-520 vs 1,495+/-594 mm Hg/s, respectively, p=0.55) and was not correlated with LV mass (R=0.18, p=0.25). The optimum cut-off values for LVEDP20 mm Hg were SSr0.85 s(-1) (area under the curve 0.88, p0.001, positive predictive value 89%, negative predictive value 86%) and SS16% (area under the curve 0.84, p=0.002, positive predictive value 88%, negative predictive value 79%). In conclusion, as opposed to invasively measured dP/dt, tissue Doppler systolic velocity and 2-dimensional echocardiographic SS and SSr are significantly depressed in patients with preserved LV ejection fraction and LVEDP20 mm Hg, suggesting that systolic abnormalities are present in at least some of these patients. These differences are likely because invasively measured dP/dt and these echocardiographic variables measure different systolic properties in patients with preserved LV ejection fraction.

Noninvasive left ventricular (LV) pressure estimation in obese patients has not been well described. Simultaneous B-type natriuretic peptide (BNP) and echocardiographic Doppler examinations were performed in patients with dyspnea undergoing cardiac catheterization. Patients were divided into body mass index (BMI)35 (markedly obese), 31 to 35 (obese), andor =30 kg/m2 (nonobese). BNP levels and mitral early diastolic/tissue Doppler annular velocity (E/Ea) were compared with invasively measured LV end-diastolic and pre-atrial (pre-A) pressures. Seventy-two patients were studied. Except for BMI, LV mass index, and LV diastolic dimension, there were no significant differences in baseline, echocardiographic Doppler, or hemodynamic characteristics among the groups. However, BNP was significantly lower in markedly obese compared with obese and nonobese patients (116 +/- 187 vs 241 +/- 674 and 277 +/- 352 pg/ml, respectively; p = 0.03). BNP did not correlate with LV pre-A pressure in markedly obese patients (R = 0.13, p = 0.47), whereas BNP significantly correlated with this variable in the obese (R = 0.64) and nonobese (R = 0.58) groups. Mitral E/Ea significantly correlated with LV pre-A and LV end-diastolic pressures in all BMI groups. In markedly obese patients with dyspnea, BNP did not correlate with invasively measured LV filling pressure, whereas this correlated in obese and nonobese patients. However, mitral E/Ea significantly correlated with LV filling pressures in all BMI groups. In conclusion, BNP is not recommended for LV filling pressure estimation in ambulatory patients with dyspnea with BMI35 kg/m2.

Objective. —To assess the potential availability and utility of fetal tissues obtained from spontaneous abortions and from ectopic pregnancies for human transplantation therapy. Design. —Tissue collection and analysis by personnel skilled in tissue banking. Setting. —Procurement programs in five tissue banks located in diverse geographical areas that are funded by the National Institutes of Health. Patients. —All women entering obstetric clinics during 1993 who consented to participate in the study. Interventions. —None. Main Outcome Measures. —Evaluation of the products of conception by standard developmental, histological, microbiological, and cytogenetic criteria. Results. —From 22235 obstetric admissions, 1250 spontaneously aborted embryos and 247 products of ectopic pregnancies were obtained. Of these, seven embryos (0.5%) were potentially useful for human transplantation therapy. Conclusion. —Fetal tissues from spontaneous abortions and from ectopic pregnancies are quite limited as feasible sources for human transplantation therapy. ( JAMA . 1995;273:66-68)

Background A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done. Results Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed. Conclusions An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells. Electronic supplementary material The online version of this article (doi:10.1186/s12575-016-0044-z) contains supplementary material, which is available to authorized users.