Porphyrin-Loaded TyroSpheres for the Intracellular Delivery of Drugs and Photoinduced Oxidant Species.

In order to understand the intracellular delivery of drugs and to improve the cell killing efficiency of photosensitizers (PSs) used in photodynamic therapy (PDT), we prepared TyroSphere nanoparticles, which are triblock polymer [poly(ethylene glycol)-block-oligo(desaminotyrosyltyrosine octyl ester suberate)-block-poly(ethylene glycol)] aggregates, loaded with amphiphilic porphyrins with either positive (CisDiMPyP) or negative (TPPS2a) charges. Their physicochemical and photochemical properties were investigated, as well as the efficiency and mechanism of PDT death in a cervical cancer cell line (HeLa). The photophysical properties of both PSs were improved when loaded in the nanocarrier, with a decrease in aggregation as well as an increase in the yield of singlet oxygen generation. The physical and chemical stability of TyroSphere nanoparticles allows them to enter cells and to promote the slow intracellular delivery of part of the PSs. Confocal steady-state and lifetime-resolved fluorescence imaging microscopy data showed that the released PSs are free to target their natural intracellular targets, which are mitochondria and lysosomes for CisDiMPyP and TPPS2a, respectively. The photodynamic efficiency of cell killing was increased considerably compared with the free PSs (∼3×), but the mechanism of cell death was the same as that of the free PSs, which are acute necro-apoptosis for CisDiMPyP and autophagy malfunction for TPPS2a, reflecting the specific damage in mitochondria and lysosomes, respectively. We are confident that TyroSpheres provide a novel and efficient platform to administrate PDT photosensitizers, as well as other drugs with intracellular targets.

Carvedilol Combined With Ivabradine Improves Left Ventricular Diastolic Dysfunction, Clinical Progression, and Survival in Cirrhosis.

BACKGROUND: Left ventricular diastolic dysfunction (LVDD) refers to impaired cardiac diastolic relaxation and may be improved by targeted heart rate reduction (THR). The authors evaluated whether a combination of carvedilol and ivabradine, an If channel blocker that reduces heart rate without affecting blood pressure, could improve LVDD and outcomes in cirrhosis. PATIENTS AND METHODS: THR was defined as heart rate reduction to 55 to 65 beats per minute. Of 260 patients with cirrhosis, 189 (72%) with LVDD were randomized to THR [group (Gr.)A; n=94; carvedilol±ivabradine)] or standard care (Gr.B; n=95; no ß-blockers) and followed for 12 months. RESULTS: In Gr.A, THR was achieved at 4 weeks in 88 (93%) patients (responders, R): 48 (61.5%) with carvedilol alone and 40 (86.9%) of 46 patients with additional ivabradine. In Gr.A, LVDD reversed in 16 (20.5%) and improved from grade 2 to 1 in 34 (35.4%)], whereas in Gr.B, it progressed from grade 1 to 2 in 10 (10.5%) patients. At 12 months, 21 (11.1%) patients died, 6 (14%) in Gr.A and 15 (18%) in Gr.B (P=0.240), but no mortality was seen in those who had persistent THR at 1 year (n=78; P=0.000). In multivariate analysis, model for end-stage liver disease [hazard ratio (HR), 1.52; 95% confidence interval (CI), 1.22-2.75; P=0.034] and E-wave transmitral/early diastolic mitral annular velocity (HR, 1.28; 95% CI, 1.23-2.42; P=0.048) predicted 1-year mortality. Nonresponders had an increased mortality risk (HR, 1.3; 95% CI, 1.2-1.8; P=0.046) independent of age, gender, and baseline model for end-stage liver disease. Levels of norepinephrine, N terminal brain natriuretic peptide, plasma renin activity, and aldosterone were reduced (P<0.01) in responders. More patients in Gr.B developed acute kidney injury (odds ratio, 4.2; 95% CI, 2.8-10.5; P=0.027) and encephalopathy (odds ratio, 6.6; 95% CI, 1.9-9.7; P=0.040). CONCLUSIONS: Ivabradine combined with carvedilol improves LVDD, achieves THR more often and reduces risk of encephalopathy, acute kidney injury with improved survival in patients with cirrhosis.

Left Ventricular Diastolic Dysfunction is Associated with Renal Dysfunction, Poor Survival and Low Health Related Quality of Life in Cirrhosis.

BACKGROUND: The presence of left ventricular diastolic dysfunction (LVDD) in patients with cirrhosis leads to a restriction of activities and a poor health related quality of life (HRQoL), which should be taken into consideration when treating them for liver and cardiac complications. AIMS: The prevalence, complications, predictors of HRQoL and survival in cirrhotic patients with LVDD were studied. METHODS: We report a prospective cohort study of 145 consecutive cirrhotic patients with LVDD who were evaluated for cardiac functional status at enrollment and followed up for hepatic complications, cardiac events, outcome and HRQoL using the Minnesota Living With Heart Failure Questionnaire (MLHFQ) over a period of 2 years. RESULTS: In total, 145 (mean age 61 years, 59% male) patients were included. Seventeen patients died with 10.5%, 22.5% and 40% mortality rates in patients with Grades 1, 2 and 3 LVDD respectively over 24 months. The parameters that were significant for predicting mortality on bivariate analysis were MELD, MELDNa, hepatic venous pressure gradient, MLHFQ, and left ventricular (LV) diastolic function (e' and E/e' ratio), but only MELD, MELDNa and E/e' remained significant on multivariate analysis. The E/e' ratio (8.7 ± 3.3 in survivors vs. 9.1 ± 2.3 in non-survivors) predicted outcome. On univariate analysis, the predictors of poor HRQoL were the Child-Pugh score ≥9.8 (OR 2.6; 95% confidence intervals (CI) 2.3-9.1, P = 0.041), MELD score ≥ 15.7 (OR 2.48; 95% CI 1.4-3.9, P = 0.029), refractory ascites (OR 1.9; 95% CI 1.1-6.1, P = 0.050), and E/e' ratio ≥7.6 (OR 1.9; 95% CI 1.8-7.1, P = 0.036) The presence of Class II/III (P = 0.046) symptoms of heart failure and MLHFQ≥ 45 (P = 0.042) were predictors of mortality at 24 months. CONCLUSION: The grade of LVDD correlates with liver function, clinical events, risk of renal dysfunction and HRQoL. Evaluation of novel therapies which target symptomatic improvement in LVDD, should be done with suitable outcome measures, including HRQoL assessment.

Layer-by-layer assembled gold nanoshells for the intracellular delivery of miR-34a.

INTRODUCTION: MicroRNAs (miRNAs) are short noncoding RNAs whose ability to regulate the expression of multiple genes makes them potentially exciting tools to treat disease. Unfortunately, miRNAs cannot passively enter cells due to their hydrophilicity and negative charge. Here, we report the development of layer-by-layer assembled nanoshells (LbL-NS) as vehicles for efficient intracellular miRNA delivery. Specifically, we developed LbL-NS to deliver the tumor suppressor miR-34a into triple-negative breast cancer (TNBC) cells, and demonstrate that these constructs can safely and effectively regulate the expression of SIRT1 and Bcl-2, two known targets of miR-34a, to decrease cell proliferation. METHODS: LbL-NS were made by coating negatively charged nanoshells with alternating layers of positive poly-L-lysine (PLL) and negative miRNA, with the outer layer consisting of PLL to facilitate cellular entry and protect the miRNA. Electron microscopy, spectrophotometry, dynamic light scattering, and miRNA release studies were used to characterize LbL-NS. The particles' ability to enter MDA-MB-231 TNBC cells, inhibit SIRT1 and Bcl-2 expression, and thereby reduce cell proliferation was examined by confocal microscopy, Western blotting, and EdU assays, respectively. RESULTS: Each successive coating reversed the nanoparticles' charge and increased their hydrodynamic diameter, resulting in a final diameter of 208±4 nm and a zeta potential of 53±5 mV. The LbL-NS released ~30% of their miR-34a cargo over 5 days in 1X PBS. Excitingly, LbL-NS carrying miR-34a suppressed SIRT1 and Bcl-2 by 46±3% and 35±3%, respectively, and decreased cell proliferation by 33%. LbL-NS carrying scrambled miRNA did not yield these effects. CONCLUSION: LbL-NS can efficiently deliver miR-34a to TNBC cells to suppress cancer cell growth, warranting their further investigation as tools for miRNA replacement therapy.

Spherical Nucleic Acid Architecture Can Improve the Efficacy of Polycation-Mediated siRNA Delivery.

Clinical translation of small interfering RNA (siRNA) nanocarriers is hindered by limited knowledge regarding the parameters that regulate interactions between nanocarriers and biological systems. To address this, we investigated the influence of polycation-based nanocarrier architecture on intracellular siRNA delivery. We compared the cellular interactions of two polycation-based siRNA carriers that have similar size and surface charge but different siRNA orientation: (1) polyethylenimine-coated spherical nucleic acids (PEI-SNAs), in which polyethylenimine is wrapped around a spherical nucleic acid core containing radially oriented siRNA and (2) randomly assembled polyethylenimine-siRNA polyplexes that lack controlled architecture. We found that PEI-SNAs undergo enhanced and more rapid cellular uptake than polyplexes, suggesting a prominent role for architecture in cellular uptake. Confocal microscopy studies demonstrated that while PEI-SNAs and polyplexes exhibit similar intracellular stability, PEI-SNAs undergo decreased accumulation within lysosomes, identifying another advantage conferred by their architecture. Indeed, these advantageous cellular interactions enhanced the gene silencing potency of PEI-SNAs by 10-fold relative to polyplexes. Finally, cytocompatibility studies showed that PEI-SNAs exhibit decreased toxicity per PEI content relative to polyplexes, allowing the use of more polycation. Our studies provide critical insight into design considerations for engineering siRNA carriers and warrant future investigation of how nanocarrier architecture influences cellular-, organ-, and organism-level interactions.

Is Antifungal Resistance a Cause for Treatment Failure in Dermatophytosis: A Study Focused on Tinea Corporis and Cruris from a Tertiary Centre?

BACKGROUND: Dermatophytoses are one of the most common skin diseases that have been largely simple to treat. However, in recent years, these infections have become recalcitrant to treatment which can possibly be due to antifungal resistance. AIM: To analyze the resistance pattern of patients with recalcitrant dermatophytoses. MATERIALS AND METHODS: A cross-sectional evaluation was undertaken of 40 consecutive patients with recalcitrant tinea corporis/cruris/both who had taken systemic antifungal treatment and did not respond completely to therapy or had recurrent lesion within 1 month of stopping the therapy. Terbinafine, fluconazole, itraconazole, ketoconazole, amphotericin B, and voriconazole were the antifungals tested using broth microdilution assay for antifungal susceptibility testing of dermatophytes, and MIC50, 90 values were recorded. RESULTS: KOH mount was positive in 18 (45%) patients, culture was positive in 28 (70%) patients. Trichophyton mentagrophytes (35%) and T. rubrum (27.5%) were the predominant isolates. Overall, activity of terbinafine and itraconazole were significantly higher than the other drugs tested. For terbinafine, both T. mentagrophytes and T. rubrum were inhibited at MIC90 of 0.125 µg/ml. Itraconazole-inhibited T. mentagrophytes and T. rubrum at MIC90 of 0.0625 and 0.25 µg/ml, respectively. All isolates had reduced susceptibility to fluconazole. CONCLUSION: While MIC seen were higher than western data, in-vitro resistance (>1 µg/ml) to antifungals was not seen and probably may not be a cause of treatment failure. Possibly, treatment failure lies in the intricate host fungal interaction and virulence of species which help it to evade host immune response.

Clinico-mycological analysis and antifungal resistance pattern in human immunodeficiency virus-associated candidiasis: An Indian perspective.

OBJECTIVES: Candidiasis is a common human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome-associated opportunistic mycoses. The present study ascertained the species spectrum of Candida strains recovered from different clinical samples from symptomatic HIV-positive individuals and determined the antifungal susceptibility profile of the isolates. MATERIALS AND METHODS: A variety of specimens were collected from 234 symptomatic HIV seropositive individuals depending on their clinical manifestations and subjected to direct microscopic examination. Blood samples were inoculated in biphasic blood culture medium and all other specimens on Sabouraud dextrose agar with chloramphenicol and incubated at 35°C-37°C. Species identification of the recovered Candida isolates was attempted on the basis of germ tube production, micromorphology on corn meal agar, color and morphology on HiCrome Candida Differential agar, and carbohydrate fermentation and assimilation tests. Susceptibility testing of the isolates was performed employing the VITEK 2 system. RESULTS: A total of 167 Candida isolates were obtained; Candida albicans (136), Candida tropicalis (13), Candida krusei (8), Candida parapsilosis (5), Candida glabrata (4), and Candida kefyr (1). Fluconazole resistance was more frequent among nonalbicans species, and significantly higher 5-fluorocytosine resistance compared to C. albicans was also observed. Eight Candida strains (six C. krusei, one C. kefyr, and one C. albicans) were multidrug resistant. CONCLUSION: Although C. albicans continues to be the leading etiological agent of candidiasis, the incidence of nonalbicans species among HIV-positive Indian individuals is rising. Antifungal resistance was higher among nonalbicans Candida species. Another issue of therapeutic concern is the possible emergence of multidrug-resistant Candida strains among these patients.

Topical Delivery of Immunosuppression to Prolong Xenogeneic and Allogeneic Split-Thickness Skin Graft Survival.

Cadaveric skin allograft is the current standard of treatment for temporary coverage of large burn wounds. Porcine xenografts are viable alternatives but undergo α-1,3-galactose (Gal)-mediated hyperacute rejection and are lost by post-operative day (POD) 3 because of naturally occurring antibodies to Gal in primate recipients. Using baboons, we previously demonstrated that xenografts from GalT-KO swine (lacking Gal) provided wound coverage comparable with allografts with systemic immunosuppression. In this study, we investigate topical immunosuppression as an alternative to prolong xenograft survival. Full-thickness wounds in baboons were created and covered with xenogeneic and allogeneic split-thickness skin grafts (STSGs). Animals were treated with slow-release (TyroSphere-encapsulated) topical formulations (cyclosporine-A [CSA] or Tacrolimus) applied 1) directly to the STSGs only, or 2) additionally to the wound bed before STSG and 1). Topical CSA did not improve either xenograft or allograft survival (median: treated grafts = 12.5 days, control = 14 days; P = 0.27) with similar results when topical Tacrolimus was used. Pretreatment of wound beds resulted in a significant reduction of xenograft survival compared with controls (10 vs 14 days; P = 0.0002), with comparable results observed in allografts. This observation was associated with marked reduction of inflammation on histology with Tacrolimus and not CSA. Prolongation of allograft and xenograft survival after application to full-thickness wound beds was not achieved with the current formulation of topical immunosuppressants. Modulation of inflammation within the wound bed was effective with Tacrolimus pretreatment before STSG application and may serve as a treatment strategy in related fields.

The Relationship Between Uterine Artery two-dimensional Color Doppler Measurement and Pregnancy Outcome: A Prospective Observational Study.

BACKGROUND: The purpose of the study was to evaluate the role of uterine blood flow parameters measured by uterine artery two-dimensional (2D)-power color doppler (PCD) ultrasound in predicting fertility outcomes in women undergoing IVF-ET cycles. METHODS: In this prospective observational study, a total of 188 infertile women who underwent IVF-ET cycles were investigated. Uterine artery 2D-PD measurements were taken during early follicular phase and on day of trigger. Pulsatility Index (PI), Resistant Index (RI), Peak Systolic Velocity (PSV), and Systolic/Diastolic ratio (S/D) were measured. Statistical correlation was sought between the doppler parameters and fertility outcomes. RESULTS: The pregnancy rate was 40.43% (76/188). The women who conceived (n= 76) (Group A) were found to have mean age of 31.2±3.9 years whereas the non-pregnant group of women (n=112) (Group B) had mean age of 31.45±4.25 years. The mean PI measurements subsequently during early follicular phase and on the day of hCG trigger between group A and group B were comparable (2.09±1.15 versus 1.9±0.95; p=0.385 and 1.86±1.12 versus 2.03±1.0; p=0.192, respectively). No significant changes in the uterine artery PSV values and S/D values and RI were noted during the cycle. CONCLUSION: Uterine artery doppler evaluation in women undergoing IVF cycles was not predictive of the pregnancy outcomes.

Pulmonary aspergillosis as opportunistic mycoses in a cohort of human immunodeficiency virus-infected patients: Report from a tertiary care hospital in North India.

OBJECTIVE: The incidence of pulmonary aspergillosis in human immunodeficiency virus (HIV)-infected persons is rising. This study was designed to determine the prevalence of pulmonary aspergillosis in a cohort of HIV-positive patients (n = 71) presenting with lower respiratory tract infection at a tertiary care medical center in India. METHODS: Sputum samples were collected, and potassium hydroxide mount, cultural characteristics, and lactophenol cotton blue preparations were employed to aid in the identification of Aspergillus species. In addition, serum galactomannan antigen testing was also performed. RESULTS: Pulmonary aspergillosis was diagnosed in 7 patients, five of whom showed a positive antigenemia indicating invasive form of disease. The prevalence of pulmonary aspergillosis was highest in individuals 21-40 years of age (13.3%). The gender-wise prevalence of pulmonary aspergillosis was 18.7% and 7.7% in females and males, respectively. The common chest radiographic findings noted in patients with pulmonary aspergillosis included a normal chest radiograph in 3 (42.8%), infiltrates in 2 (28.6%), and pleural effusion in 2 (28.6%). The common Aspergillus species recovered from sputa of these patients were Aspergillus flavus (4; 57.1%); Aspergillus fumigatus (2; 28.6%), and Aspergillus niger (1; 14.3%). A predisposing lung condition in the form of pulmonary tuberculosis was identified in 2; Pneumocystis carinii pneumonia in 2 and a dual tubercular and P. carinii infection in one. The mean CD4 count of these patients was 155.86 ± 119.33 cells/µl (median = 117 cells/µl; range = 18-329 cells/µl). CONCLUSION: Our findings suggest that Aspergillus species be considered possible etiological agents in HIV-positive patients with pulmonary infection.

Cell type-specific extracellular matrix guided the differentiation of human mesenchymal stem cells in 3D polymeric scaffolds.

The tissue microenvironment has profound effects on tissue-specific regeneration. The 3-dimensional extracellular matrix (ECM) niche influences the linage-specific differentiation of stem cells in tissue. To understand how ECM guides tissue-specific regeneration, we established a series of 3D composite scaffolds containing ECMs derived from different primary cells isolated from a single animal species and assessed their impact on the differentiation of human mesenchymal stem cells (hMSCs). Synthetic microfiber scaffolds (fiber mats) were fabricated by electrospinning tyrosine-derived polycarbonates (pDTEC). The bovine primary fibroblasts, chondrocytes and osteoblasts cultured on the fiber mats produced and assembled their ECMs, infiltrating the pores of the fibrous scaffold. The composite scaffolds were decellularized to remove cellular components, preserve ECM and minimally affect polymer integrity. Characterization of the ECMs derived from different primary cells in the composite scaffolds showed overlapping but distinct compositions. The chondrogenic and osteogenic differentiation of hMSCs on the different composite scaffolds were compared. Our results showed that ECM derived from chondrocytes cultured in synthetic fiber mats promoted the chondrogenic differentiation of hMSC in the presence or absence of soluble inducing factors. ECM derived from co-culture of osteoblasts and chondrocytes promoted osteogenic differentiation in hMSCs better than ECM derived from chondrocytes. This study demonstrated that decellularized ECMs derived from different cell types formed within synthetic fiber scaffolds guide the tissue-specific differentiation of hMSCs. These composite scaffolds may be developed into models to study the mechanisms of ECM-induced tissue regeneration.

Development of hybrid scaffolds with natural extracellular matrix deposited within synthetic polymeric fibers.

A major challenge of tissue engineering is to generate materials that combine bioactivity with stability in a form that captures the robust nature of native tissues. Here we describe a procedure to fabricate a novel hybrid extracellular matrix (ECM)-synthetic scaffold biomaterial by cell-mediated deposition of ECM within an electrospun fiber mat. Synthetic polymer fiber mats were fabricated using poly(desamino tyrosyl-tyrosine carbonate) (PDTEC) co-spun with poly(ethylene glycol) (PEG) used as a sacrificial polymer. PEG removal increased the overall mat porosity and produced a mat with a layered structure that could be peeled into separate sheets of about 50 µm in thickness. Individual layers had pore sizes and wettability that facilitated cell infiltration over the depth of the scaffold. Confocal microscopy showed the formation of a highly interpenetrated network of cells, fibronectin fibrils, and synthetic fibers mimicking a complex ECM as observed within tissues. Decellularization did not perturb the structure of the matrix or the fiber mat. The resulting hybrid ECM-scaffold promoted cell adhesion and spreading and stimulated new ECM assembly by stem cells and tumor cells. These results identify a new technique for fabricating highly porous synthetic fibrous scaffolds and an approach to supplement them with natural biomimetic cues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2162-2170, 2017.

Fungal Opportunistic Pneumonias in HIV/AIDS Patients: An Indian Tertiary Care Experience.

INTRODUCTION: Opportunistic pneumonias are a major cause of mortality and morbidity in Human Immunodeficiency Virus (HIV) reactive patients. Despite the significant role that fungi play in causation of this opportunistic mycoses, very few Indian studies have attempted to investigate the burden and aetiological spectrum of HIV/AIDS-associated fungal pneumonias. AIM: To document the prevalence of fungal aetiology in HIV/AIDS-related opportunistic pneumonias in an Indian setting; and to elucidate the various fungal opportunists responsible for the same. MATERIALS AND METHODS: The present study was a prospective, cross-sectional analysis conducted at Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi from October 2008 to September 2011. Expectorated sputa were collected from 71 HIV reactive patients with a clinical diagnosis of pneumonia and subjected to direct microscopic examination employing Gram stain, 10% KOH wet mount and India ink preparation. In addition, direct immunofluorescence of sputum samples was performed for detection of cysts and trophozoites of Pneumocystis carinii. Also, each sputum sample was inoculated in duplicate onto Sabouraud Dextrose Agar (SDA) for culture. A blood sample was drawn from each patient and a battery of serological tests was performed, including Cryptococcal Antigen Latex Agglutination System (CALASTM) for detection of cryptococcal capsular polysaccharide antigen; Platelia™ Aspergillus EIA for detection of Aspergillus galactomannan antigen; SERION ELISA antigenCandida for detection of Candida antigen and Histoplasma DxSelect™ for detecting antibodies to Histoplasma species. Descriptive statistics were employed to depict results as proportions and figures. Further, arithmetic mean and standard deviation were calculated for central tendencies and median for non-normal/skewed distributions. RESULTS: A definite fungal aetiology was established in 25 (35.2%) of 71 HIV reactive patients with pneumonic involvement. Of these, sputa of 21 patients yielded single fungal isolates, while mixed fungal isolates were reported in four patients. Pneumocystis carinii was the predominant fungal pathogen isolated in our study and was reported in 14 (19.7%) patients. Pulmonary aspergillosis was reported in 7 (9.9%) patients, with Aspergillus flavus (4), Aspergillus fumigatus (2) and Aspergillus niger (1) being the commonly recovered Aspergillus species. Candida pneumonia was documented in 6 (8.5%) patients and the Candida species isolated included Candida albicans in four, Candida glabrata in one and Candida tropicalis in one of these six patients respectively. Pulmonary cryptococcosis was diagnosed in 2 (2.8%) patients; a coexisting cryptococcal meningitis was documented in one of them. Furthermore, antibodies against Histoplasma species were detected in 21 (29.6%) cases suggesting its possible aetiological role. CONCLUSION: Fungal opportunistic pneumonias are common in HIV reactive patients in Indian setting and warrant a prompt and accurate diagnostic evaluation in the form of a combination of microbiological, serological and histopathological techniques, for an effective prophylactic and therapeutic management.

Optimization of Polymer-ECM Composite Scaffolds for Tissue Engineering: Effect of Cells and Culture Conditions on Polymeric Nanofiber Mats.

The design of composite tissue scaffolds containing an extracellular matrix (ECM) and synthetic polymer fibers is a new approach to create bioactive scaffolds that can enhance cell function. Currently, studies investigating the effects of ECM-deposition and decellularization on polymer degradation are still lacking, as are data on optimizing the stability of the ECM-containing composite scaffolds during prolonged cell culture. In this study, we develop fibrous scaffolds using three polymer compositions, representing slow (E0000), medium (E0500), and fast (E1000) degrading materials, to investigate the stability, degradation, and mechanics of the scaffolds during ECM deposition and decellularization, and during the complete cellularization-decell-recell cycle. We report data on percent molecular weight (% Mw) retention of polymeric fiber mats, changes in scaffold stiffness, ECM deposition, and the presence of fibronectin after decellularization. We concluded that the fast degrading E1000 (Mw retention ≤ 50% after 28 days) was not sufficiently stable to allow scaffold handling after 28 days in culture, while the slow degradation of E0000 (Mw retention ≥ 80% in 28 days) did not allow deposited ECM to replace the polymer support. The scaffolds made from medium degrading E0500 (Mw retention about 60% at 28 days) allowed the gradual replacement of the polymer network with cell-derived ECM while maintaining the polymer network support. Thus, polymers with an intermediate rate of degradation, maintaining good scaffold handling properties after 28 days in culture, seem best suited for creating ECM-polymer composite scaffolds.

Identification and Antifungal Susceptibility Testing of Candida Species: A Comparison of Vitek-2 System with Conventional and Molecular Methods.

BACKGROUND: Candida infection is a major cause of morbidity and mortality in immunocompromised patients; an accurate and early identification is a prerequisite need to be taken as an effective measure for the management of patients. The purpose of this study was to compare the conventional identification of Candida species with identification by Vitek-2 system and the antifungal susceptibility testing (AST) by broth microdilution method with Vitek-2 AST system. MATERIALS AND METHODS: A total of 172 Candida isolates were subjected for identification by the conventional methods, Vitek-2 system, restriction fragment length polymorphism, and random amplified polymorphic DNA analysis. AST was carried out as per the Clinical and Laboratory Standards Institute M27-A3 document and by Vitek-2 system. RESULTS: Candida albicans (82.51%) was the most common Candida species followed by Candida tropicalis (6.29%), Candida krusei (4.89%), Candida parapsilosis (3.49%), and Candida glabrata (2.79%). With Vitek-2 system, of the 172 isolates, 155 Candida isolates were correctly identified, 13 were misidentified, and four were with low discrimination. Whereas with conventional methods, 171 Candida isolates were correctly identified and only a single isolate of C. albicans was misidentified as C. tropicalis. The average measurement of agreement between the Vitek-2 system and conventional methods was >94%. Most of the isolates were susceptible to fluconazole (88.95%) and amphotericin B (97.67%). The measurement of agreement between the methods of AST was >94% for fluconazole and >99% for amphotericin B, which was statistically significant (P < 0.01). CONCLUSION: The study confirmed the importance and reliability of conventional and molecular methods, and the acceptable agreements suggest Vitek-2 system an alternative method for speciation and sensitivity testing of Candida species infections.

Off-Target Effect of Sildenafil on Postsurgical Erectile Dysfunction: Alternate Pathways and Localized Delivery System.

INTRODUCTION: There is no consensus on the best oral phosphodiesterase type 5 inhibitor (PDE5I) for patients undergoing penile rehabilitation after surgical nerve injury. AIM: To determine the mechanism of PDE5I on cultured neuronal cells and the effectiveness of local drug delivery using nanospheres (NSPs) to sites of nerve injury in a rat model of bilateral cavernous nerve injury (BCNI). METHODS: The effects of sildenafil, tadalafil, and vardenafil on cyclic adenosine monophosphate, cyclic guanosine monophosphate, and cell survival after exposure to hypoxia and H2O2 were measured in PC12, SH-SY5Y, and NTERA-2 (NT2) cell cultures. The effects of phosphodiesterase type 4 inhibitor (PDE4I) and PDE5I on neuronal cell survival were evaluated. Male rats underwent BCNI and were untreated (BCNI), immediately treated with application of empty NSPs (BCNI + NSP), NSPs containing sildenafil (Sild + NSP), or NSPs containing rolipram (Rol + NSP). MAIN OUTCOME MEASURES: Viability of neuronal cells was measured. Intracavernous pressure changes after cavernous nerve electrostimulation and expression of neurofilament, nitric oxide synthase, and actin in mid-shaft of penis were analyzed 14 days after injury. RESULTS: Sildenafil and rolipram significantly decreased cell death after exposure to H2O2 and hypoxia in PC12, SH-SY5Y, and NT2 cells. PC12 cells did not express PDE5 and knockdown of PDE4 significantly increased cell viability in PC12, SH-SY5Y, and NT2 cells exposed to hypoxia. The ratio of intracavernous pressure to mean arterial pressure and expression of penile neurofilament, nitric oxide synthase, and actin were significantly higher in the Sild + NSP and Rol + NSP groups than in the BCNI and BCNI + NSP groups. Limitations included analysis in only two PDE families using only a single dose. CONCLUSION: Sildenafil showed the most profound neuroprotective effect compared with tadalafil and vardenafil. Sildenafil- or rolipram-loaded NSP delivery to the site of nerve injury prevented erectile dysfunction and led to increased neurofilament, nitric oxide synthase, smooth muscle content in rat penile tissue after BCNI.

Spectrum of Opportunistic Fungal Infections in HIV/AIDS Patients in Tertiary Care Hospital in India.

HIV related opportunistic fungal infections (OFIs) continue to cause morbidity and mortality in HIV infected patients. The objective for this prospective study is to elucidate the prevalence and spectrum of common OFIs in HIV/AIDS patients in north India. Relevant clinical samples were collected from symptomatic HIV positive patients (n = 280) of all age groups and both sexes and subjected to direct microscopy and fungal culture. Identification as well as speciation of the fungal isolates was done as per the standard recommended methods. CD4+T cell counts were determined by flow cytometry using Fluorescent Activated Cell Sorter Count system. 215 fungal isolates were isolated with the isolation rate of 41.1%. Candida species (86.5%) were the commonest followed by Aspergillus (6.5%), Cryptococcus (3.3%), Penicillium (1.9%), and Alternaria and Rhodotorula spp. (0.9% each). Among Candida species, Candida albicans (75.8%) was the most prevalent species followed by C. tropicalis (9.7%), C. krusei (6.4%), C. glabrata (4.3%), C. parapsilosis (2.7%), and C. kefyr (1.1%). Study demonstrates that the oropharyngeal candidiasis is the commonest among different OFIs and would help to increase the awareness of clinicians in diagnosis and early treatment of these infections helping in the proper management of the patients especially in resource limited countries like ours.

Self-Assembly and Critical Aggregation Concentration Measurements of ABA Triblock Copolymers with Varying B Block Types: Model Development, Prediction, and Validation.

The dissipative particle dynamics (DPD) simulation technique is a coarse-grained (CG) molecular dynamics-based approach that can effectively capture the hydrodynamics of complex systems while retaining essential information about the structural properties of the molecular species. An advantageous feature of DPD is that it utilizes soft repulsive interactions between the beads, which are CG representation of groups of atoms or molecules. In this study, we used the DPD simulation technique to study the aggregation characteristics of ABA triblock copolymers in aqueous medium. Pluronic polymers (PEG-PPO-PEG) were modeled as two segments of hydrophilic beads and one segment of hydrophobic beads. Tyrosine-derived PEG5K-b-oligo(desaminotyrosyl tyrosine octyl ester-suberate)-b-PEG5K (PEG5K-oligo(DTO-SA)-PEG5K) block copolymers possess alternate rigid and flexible components along the hydrophobic oligo(DTO-SA) chain, and were modeled as two segments of hydrophilic beads and one segment of hydrophobic, alternate soft and hard beads. The formation, structure, and morphology of the initial aggregation of the polymer molecules in aqueous medium were investigated by following the aggregation dynamics. The dimensions of the aggregates predicted by the computational approach were in good agreement with corresponding results from experiments, for the Pluronic and PEG5K-oligo(DTO-SA)-PEG5K block copolymers. In addition, DPD simulations were utilized to determine the critical aggregation concentration (CAC), which was compared with corresponding results from an experimental approach. For Pluronic polymers F68, F88, F108, and F127, the computational results agreed well with experimental measurements of the CAC measurements. For PEG5K-b-oligo(DTO-SA)-b-PEG5K block polymers, the complexity in polymer structure made it difficult to directly determine their CAC values via the CG scheme. Therefore, we determined CAC values of a series of triblock copolymers with 3-8 DTO-SA units using DPD simulations, and used these results to predict the CAC values of triblock copolymers with higher molecular weights by extrapolation. In parallel, a PEG5K-b-oligo(DTO-SA)-b-PEG5K block copolymer was synthesized, and the CAC value was determined experimentally using the pyrene method. The experimental CAC value agreed well with the CAC value predicted by simulation. These results validate our CG models, and demonstrate an avenue to simulate and predict aggregation characteristics of ABA amphiphilic triblock copolymers with complex structures.

Nanoparticles and nanofibers for topical drug delivery.

This review provides the first comprehensive overview of the use of both nanoparticles and nanofibers for topical drug delivery. Researchers have explored the use of nanotechnology, specifically nanoparticles and nanofibers, as drug delivery systems for topical and transdermal applications. This approach employs increased drug concentration in the carrier, in order to increase drug flux into and through the skin. Both nanoparticles and nanofibers can be used to deliver hydrophobic and hydrophilic drugs and are capable of controlled release for a prolonged period of time. The examples presented provide significant evidence that this area of research has - and will continue to have - a profound impact on both clinical outcomes and the development of new products.

The Clinical Outcome of Post Placental Copper-T-380A Insertion with Long Placental Forceps (Kelly's Forceps) After Normal Vaginal Delivery and Cesarean Section.

OBJECTIVES: To study the efficacy safety effect on menstrual cycles, expulsion, continuation, and failure rate of post placental Copper-T-380A after vaginal and cesarean birth in a tertiary center, over the period of 1 year. METHODS: A total of 150 women who opted for insertion of Copper-T-380A within 10 min of expulsion of placenta whether delivered vaginally or by cesarean section were enrolled into study. Women having past history of ectopic pregnancy or any genital tract infection or hemorrhagic disorders, uterine anomaly, chorioamnionitis, LPV > 18 h, unresolved PPH, Hb < 8 g% were excluded from the study. RESULTS: No incidence of perforation, PID, and failure of contraception was detected. Percentage satisfaction among users after 6 weeks-91.7 %, 3 months-92.9 %, and 6 months-95.6 %. CONCLUSION: Although there was high incidence of missing IUCD threads (probably owing to coiling of long threads), the actual expulsion rate was far lesser. Removal rate due to menorrhagia, pain in abdomen, and vaginal discharge was low, and 6 months continuation rate was considerably good.