Anchor, Spacer, and Ligand-Modified Engineered Exosomes for Trackable Targeted Therapy.

Exosomes have been widely demonstrated as an effective anticancer therapeutic moiety. However, their clinical translation has been limited by the requirement of prohibitively high therapeutic doses due to their lack of specificity in delivery and, consequently, short systemic half-life. To overcome these challenges, we engineered a platform for modifying exosomes with an active targeting modality composed of membrane Anchor (BODIPY)-Spacer (PEG)-targeting Ligands (cyclic RGD peptide) (ASL). Herein, we show that the intramembrane incorporation of a trackable, targeting system renders ASL exosomes (AExs) a modular platform. AExs significantly overcome challenges associated with exosome modification, including potential damage for functionalization, or destabilizing interactions between dyes and drugs. ASL-modification not only enhanced stability in imparting active targeting but also introduced a built-in bioimaging modality. Our studies show that AExs target B16F10 melanoma tumor sites by the specific interaction of cyclic RGD and integrin. Doxorubicin encapsulated AExs (dAExs) significantly inhibited the growth of melanoma in vitro and in vivo. Thus, we conclude that ASL-modification allows exosomes to be transformed into a novel therapeutic vehicle uniquely integrating in vivo tracking and robust targeting with drug delivery. We anticipate that the therapeutic, targeting, and diagnostic modularity provided by ASL will potentiate translational applications of exosome-based vehicles beyond anticancer therapy.

Poor Appetite and Dietary Intake in Community-Dwelling Older Adults.

BACKGROUND/OBJECTIVES: Poor appetite in older adults leads to sub-optimal food intake and increases the risk of undernutrition. The impact of poor appetite on food intake in older adults is unknown. The aim of this study was to examine the differences in food intake among older community-dwelling adults with different reported appetite levels. DESIGN: Cross-sectional analysis of data from a longitudinal prospective study. SETTING: Health, aging, and body composition study performed in the USA. PARTICIPANTS: 2,597 community-dwelling adults aged 70-79. MEASUREMENTS: A semi-quantitative, interviewer-administered, 108-item food frequency questionnaire designed to estimate dietary intake. Poor appetite was defined as the report of a moderate, poor, or very poor appetite in the past month and was compared with good or very good appetite. RESULTS: The mean age of the study sample was 74.5 ± 2.8 years; 48.2% were men, 37.7% were black, and 21.8% reported a poor appetite. After adjustment for total energy intake and potential confounders (including biting/chewing problems), participants with a poor appetite had a significantly lower consumption of protein and dietary fiber, solid foods, protein rich foods, whole grains, fruits, and vegetables, but a higher consumption of dairy foods, fats, oils, sweets, and sodas compared to participants with very good appetite. In addition, they were less likely to report consumption of significant larger portion sizes. CONCLUSION: Older adults reporting a poor appetite showed a different dietary intake pattern compared to those with (very) good appetite. Better understanding of the specific dietary intake pattern related to a poor appetite in older adults can be used for nutrition interventions to enhance food intake, diet variety, and diet quality.

Lysosomally cleavable peptide-containing polymersomes modified with anti-EGFR antibody for systemic cancer chemotherapy.

Polymersomes (Ps) based on a biodegradable and biocompatible block copolymer of methoxy poly(ethylene glycol) (mPEG) and poly(D,L-lactide) (PDLLA) in which apeptide sequence, Gly-Phe-Leu-Gly-Phe (GFLGF), was introduced in between the two blocks(mPEG-pep-PDLLA) were developed. The peptide linker is cleavable by the lysosomal enzymecathepsin B (Cath B). Ps containing the peptide linker (Ps(pep)) with an average diameter of about 124 nm were prepared by injecting a THF solution of the block copolymer into DI water. The Ps had a membrane thickness of about 15 nm as determined by transmission electron microscopy (TEM). In order to investigate the enzymatic degradation of the Ps (pep), dynamic light scattering (DLS) measurements of Ps(pep) dispersions with different concentrations of Cath B at pH 5.5 and 7.4 were performed as a function of time. A gradual decrease in kilo counts per second (Kcps) of the Ps (pep) over 7 d was observed after incubation of the Ps (pep) dispersions with 5 units/ml of Cath B at pH 5.5 at 37 °C. The size distribution became also bimodal, indicating that aggregation and precipitation of Ps (pep) occurred by disintegration of the Ps (pep) as a result of cleavage of the peptide. The rate of disintegration of the Ps (pep) was depending on the concentration of Cath Band the pH. No changes by DLS were seen when the dispersions were incubated with the enzyme at pH 7.4. Acridine orange (AO) was encapsulated in Ps (pep)as a model drug and rapid release of AO triggered by Cath B degradation of Ps (pep) was observed at pH 5.5. Anti-epidermal growth factor receptor (anti-EGFR) antibody (abEGFR) was immobilized on the surface of Ps(pep)in order to enhance the cellular uptake of Ps (pep). Fluorescein isothiocyanate labeled dextran (40,000 g/mol) (FD40) was incorporated in the Ps (pep) for the cell study and Ps either without peptide or antibody or without both peptide and antibody were used as negative controls. After 3 d exposure to SKBR3 cells, abEGFR-conjugated Ps (pep) (abEGFR-Ps (pep)) were directly bound to the membrane of the cells and were endocytosed more rapidly as compared to Ps (pep)without abEGFR. Intracellular release of FD40 from Ps (pep) was observed, suggesting that the peptide linker in Ps (pep) was cleaved in the lysosomal compartments of the cells leading to Ps (pep) membrane disruption. An Alexa Fluor(®) 488 labeled fragment of anti-mouse IgG (F(ab')(2)A) was also coupled to Ps (pep). Specific binding of the Ps (pep) coupled IgG (F(ab')(2)A) onto SKBR3 cells treated with primary mouse antibody was observed, whereas no binding was found with SKBR3 cells treated with goat antibody.

Secondary angle closure caused by air migrating behind the pupil in descemet stripping endothelial keratoplasty.

PURPOSE: To report secondary angle closure caused by air migrating behind the pupil in the context of intraocular pressure (IOP) elevation in the early postoperative period after Descemet stripping endothelial keratoplasty (DSEK). METHODS: A retrospective case series was conducted on 100 consecutive DSEK cases from 90 patients undergoing DSEK because of corneal disease from Fuchs corneal dystrophy, pseudophakic bullous keratopathy, aphakic bullous keratopathy, and iridocorneal endothelial syndrome. Preoperative and postoperative slit-lamp examinations and IOP measurements were ascertained for all 100 eyes. Main outcome measures included preoperative and postoperative IOP. RESULTS: Thirteen of 100 eyes developed an IOP rise of greater than 30 mm Hg on the first postoperative day. Six of these 13 patients developed angle closure from air migrating posterior to the iris and causing iridocorneal adhesions. One of these 13 patients developed pupillary block from air anterior to iris. Six of 13 patients developed increased IOP without pupillary block or iridocorneal adhesions and had a history of preexisting primary or secondary glaucoma. CONCLUSIONS: A secondary angle closure associated with DSEK is reported with air migrating behind the iris, resulting in extensive iridocorneal adhesions. An acute increase in IOP after DSEK can also be induced by air anterior to the iris causing pupillary block. IOP spikes are much more common in the first few postoperative days after DSEK. Medical treatment can occasionally resolve air posterior to the iris, but if iridocorneal adhesions are extensive and persistent, air removal and angle reformation may be necessary.

Evaluation of novel histone deacetylase inhibitors as therapeutic agents for colorectal adenocarcinomas compared to established regimens with the histoculture drug response assay.

BACKGROUND AND AIMS: This study was to evaluate the efficacy of histone deacetylase (HDAC) inhibitors in colorectal cancer together with other established regimens. MATERIALS AND METHODS: Chemosensitivities of 114 colorectal cancer patients to established regimens (fluorouracil (5-FU with leucovorin (FL), capecitabine, FL with irinotecan (FLIRI), and FL with oxaliplatin (FLOX)) as well as five hydroxamic acid derivatives (suberoylanilide hydroxamic acid, PXD101, and three novel candidates of CG-1, CG-2, and CG-3) were comparatively evaluated using the histoculture drug response assay. RESULTS: The chemosensitivity with established regimens was between 34.2% and 52.6%, when the cutoff value of the inhibition ratio was set at 30%, and between 54.5% and 84.1% with HDAC inhibitors. All HDAC inhibitors displayed synergistic effects in combination with established regimens of FLOX and FLIRI (P < or = 0.0001-0.002). Advanced T- and N-category tumors and patients with synchronous adenoma displayed higher chemosensitivity to CG-3, CG-2, and CG-1, respectively, on a multivariate analysis (P = 0.023, 0.044, and 0.045, respectively). Tumors with mismatch repair defects were closely correlated with chemosensitivities to combined regimens of PDX101 with FLOX and FLIRI (P = 0.044 and 0.048, respectively). CONCLUSIONS: Our findings firstly demonstrated the chemo-responsiveness of colorectal cancers to HDAC inhibitors with therapeutic efficacy comparable to the established regimens. Additionally, tumor growth and heredity were significantly associated with specific regimens, supporting their possible role as chemosensitive predictors.

Efficacy and safety of epirubicin and etoposide combination chemotherapy in advanced hepatocellular carcinoma: a retrospective analysis.

BACKGROUND AND AIM: Systemic treatments of advanced hepatocellular carcinoma (AHCC) have offered marginal clinical benefits. Recently, Italian investigators reported that etoposide and epirubicin combination (EE) chemotherapy was highly active against AHCC, with a response rate of 39% and a median overall survival (OS) of 10 months. We report our efficacy and safety results of EE in clinical practice. METHODS: Between December 1999 and October 2005, 35 patients with AHCC and fitting the preset eligibility criteria were treated with EE. Twenty-eight patients (80%) had liver disease associated with hepatitis B virus (HBV) and 26 (74%) had a prior history of transarterial chemoembolization (TACE) using cisplatin. The EE chemotherapy consisted of epirubicin 40 mg/m(2) on day 1 and etoposide 120 mg/m(2) on days 1, 3 and 5 every 4 weeks. RESULTS: A total of 102 chemotherapy cycles were administered, with a median of two cycles per patient (range one to eight cycles). Two patients had a partial response and nine had stable disease, with a tumor control rate of 32% (95% CI 17-48). The median progression-free survival (PFS) was 2.1 months (95% CI 1.8-2.4) and the median OS was 6.4 months (95% CI 4.4-8.5). There was a tendency toward improved PFS in patients seronegative for HBsAg and peritoneal seeding (P = 0.06 and P = 0.054, respectively). Overall survival was significantly better in patients without HBsAg and Cancer Liver Italian Program (CLIP) score 0-1 (P = 0.024 and P = 0.033, respectively). The main toxicities were hematological events, including grade 3/4 neutropenia in 29% and febrile neutropenia in 11% of patients. CONCLUSION: Treatment with EE showed minimal antitumor activity with acceptable toxicity in HBV-associated AHCC, especially in patients pretreated with TACE.

Effects of weight control during the menopausal transition on bone mineral density.

BACKGROUND: Studies of weight loss and changes in bone mineral density (BMD) have primarily been short-term trials in obese subjects. OBJECTIVE: We examined the effects of a 5-yr intervention designed to prevent menopausal weight gain or promote modest weight loss on BMD in premenopausal women participating in the Women's Healthy Lifestyle Project. DESIGN: We enrolled 373 premenopausal women (age 44-50 yr) and randomly assigned them to either lifestyle intervention (175 women, low-fat dietary modification, weight loss, and physical activity intervention) or control group (198 women). BMD and body weight were measured at baseline, annual follow-up visits (18, 30, 42, and 54 months), and two postintervention follow-ups (66 and 78 months). BMD was measured by dual x-ray absorptiometry. RESULTS: Over the 54 months of intervention, women in the intervention group lost 0.4 kg, whereas control women gained 2.6 kg (P = 0.011). The intervention group experienced significantly greater hip bone loss (-0.20%/yr) than the control group (-0.03%/yr). During the postintervention, differences in rates of bone loss disappeared. When considering both menopausal status and use of hormone therapy (HT), the annualized BMD changes were lower in women reporting HT use; nevertheless, among women on HT, those who lost more than 3% body weight experienced greater total hip BMD loss (-0.25%/yr) compared with those who gained weight (-0.02%/yr) (P = 0.025). CONCLUSIONS: Women randomized to a lifestyle intervention aimed at preventing menopausal weight gain or promoting modest weight loss experienced greater rates of hip bone loss than control women.

Diagnostic performances of double-phase tc-99m MIBI scintimammography in patients with indeterminate ultrasound findings: visual and quantitative analyses.

The aim of the study was to compare the diagnostic performances of visual and quantitative analyses of double-phase Tc-99m methoxyisobutylisonitrile (MIBI) scintimammography (SMM) in patients with ultrasonographically indeterminate findings. SMM (early: 10min; delayed: 3h) was performed on 78 patients (malignant 66, benign 12). For visual analysis, the five-scoring method was used. For quantitative analysis, ratios of early and delayed lesions to non-lesion (L/Ns) were calculated. When a visual grade of over 3 was used as the cut-off value for the detection of primary breast cancer, the sensitivity and specificity were 86.4%/ and 100%, respectively. The area under the curve (AUC) was 0.972. The optimal L/N ratios were 1.22 for early and 1.1 for delayed images. When early L/N 1.22 was used as the cut-off point, the sensitivity and specificity of SMM were 92.4% and 91.7'%, respectively. The AUC was 0.952. When delayed L/N 1.1 was used as the cut-off value, the sensitivity and specificity were 78.8% and 91.7%, respectively. The AUC was 0.863. Visual interpretation and early L/N were superior to delayed L/N for the detection of breast cancer. This study suggests the possible diagnostic role of visual and quantitative analyses of double-phase SMM for differentiating malignant breast lesions in patients with ultrasonographi-cally indeterminate findings.

Comparison of gated blood pool SPECT and spiral multidetector computed tomography in the assessment of right ventricular functional parameters: validation with first-pass radionuclide angiography.

OBJECTIVE: The aim of this study was to compare gated blood pool single photon emission computed tomography (SPECT) (GBPS) and multidetector row computed tomography (MDCT) for the determination of right ventricular ejection fraction (RVEF) and right ventricular volumes (RVV) and to compare first-pass radionuclide angiography (FP-RNA) as the gold standard. METHODS: Twenty consecutive patients (11 men, 9 women) referred for MDCT for the evaluation of the presence of coronary artery disease underwent FP-RNA and GBPS. RESULTS: The mean right ventricular end-diastolic volume (EDV) calculated with GBPS revealed a statistically significant lower value than that of MDCT. The mean right ventricular end-systolic volume (ESV) calculated with GBPS was also lower than that of MDCT. A comparison of right ventricular EDV from GBPS and MDCT yielded a correlation coefficient of 0.5972. Right ventricular ESV between GBPS and MDCT showed a correlation coefficient of 0.5650. The mean RVEFs calculated with FP-RNA (39.8% +/- 4.0%), GBPS (43.7% +/-6.9%0), and MDCT (40.4% + 7.7%) showed no statistical differences (Kruskal-Wallis statistics 4.538, P = 0.1034). A comparison of RVEFs from FP-RNA and GBPS yielded a correlation coefficient of 0.7251; RVEFs between FP-RNA and MDCT showed a correlation coefficient of 0.6166 and between GBPS and MDCT showed a correlation coefficient of 0.6367. CONCLUSION: The RVEF, EDV, and ESV calculated by GBPS had good correlation with those obtained with MDCT. In addition, there were no statistical differences of RVEF calculated from FP-RNA, GBPS, and MDCT. However, with regard to RVV, EDV and ESV from GBPS revealed statistically significantly lower values than those of MDCT. Although reasonable correlations among these modalities were obtained, the agreement among these three modalities was not good enough for interchangeable use in the clinical setting. Also, these results should be confirmed in patients with cardiac diseases in future larger population-based studies.

Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells.

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.

Synthesis and structure--activity relationships of aroylpyrrole alkylamide bradykinin (B2) antagonists.

The synthesis and structure-activity relationships of a novel series of aroylpyrrole alkylamides as potent selective bradykinin B(2) receptor antagonists are described. Several members of this series display nanomolar affinity at the B(2) receptor and show activity in an animal model of antinociception.

Mass production of methane from food wastes with concomitant wastewater treatment.

We developed a process for production of methane at a pilot scale. This process consists of three stages. The first stage is a semianaerobic hydrolysis/acidogenic step in which organic wastes are converted to various sugars, amino acids, and volatile fatty acids (VFAs). Operation temperature and pH were 45 degrees C, and 5.0-5.5, respectively. Hydraulic retention time (HRT) was 2 d. To remove the putrid odor and to enhance the hydrolysis of organic wastes, a mixture of bacteria isolated from landfill soil was inoculated into the reactor. Total chemical oxygen demand (tCOD) and biological oxygen demand (BOD) were 36,000 mg/L and 40,000 mg/L, respectively. The second stage was an anaerobic acidogenic process, which can produce large amount of VFAs including acetate, propionate, butyrate, valerate, and caproate. Operation temperature and pH were 35 degrees C, and 5.0-5.5, respectively. HRT was 2 d. The third stage was a strictly anaerobic methane fermentation step producing methane and carbon dioxide from VFAs. The working volume of upflow anaerobic sludge blanket (UASB) type reactor was 1200 L, and operation temperature and pH were 41 degrees C, and 7.7-7.9, respectively. HRT was 12 d. Seventy two percent of methane at maximum was generated and the yield was 0.45-0.50 m3/kgVS of food wastes. Through the process, 88% of tCOD and 95% of BOD were removed. The wastewater was treated with the biological aerobic and anaerobic filters immobilized with heterotrophic and autotrophic nitrifying and denitrifying bacteria. Ninety percent of total nitrogen (T-N) was removed by this treatment. The residual T-N and total phosphorous (T-P) were removed by the algal periphyton treatment system. The final concentrations of nitrogen and phosphorous in the drain water were 53 and 7 mg/L, respectively.