Predictors of Depression and Anxiety among Medical Students.

BACKGROUND: Medical undergraduates are more prone to emotional distress in comparison to the general population and non-medical undergraduates. This study aimed to identify the prevalence and factors associated with depression and anxiety symptoms among undergraduate medical students. METHODS: A cross-sectional study was conducted among 204 medical students in a medical institute in Kathmandu. Depression, Anxiety and Stress Scale-42 was used to identify the prevalence of depression and anxiety symptoms. The data were analyzed using multivariable logistic regression models. RESULTS: The prevalence of depression and anxiety symptoms was 30.9% and 38.7% respectively. Depression symptoms were more likely to be prevalent among fourth and fifth-year students, with an adjusted odds ratio (aOR 1.96, 95% CI: 1.03-3.75) compared to second and third-year students, and those who failed in the last academic examination (aOR 2.55, 95% CI: 1.28-5.09). Anxiety symptoms were more prevalent among male students (aOR 2.11, 95% CI: 1.04-4.27), those who were from the relatively less advantaged ethnic group (aOR 2.08, 95% CI: 1.04-4.16) and those who stayed outside the dormitory (aOR 2.90, 95% CI: 1.46-5.78). CONCLUSIONS: The prevalence of depression and anxiety symptoms among medical students was high. Psychological support is needed to ensure the mental well-being of medical students.

Preparation and optimisation of anionic liposomes for delivery of small peptides and cDNA to human corneal epithelial cells.

Drug delivery to corneal epithelial cells is challenging due to the intrinsic mechanisms that protect the eye. Here, we report a novel liposomal formulation to encapsulate and deliver a short sequence peptide into human corneal epithelial cells (hTCEpi). Using a mixture of Phosphatidylcholine/Caproylamine/Dioleoylphosphatidylethanolamine (PC/CAP/DOPE), we encapsulated a fluorescent peptide, resulting in anionic liposomes with an average size of 138.8 ± 34 nm and a charge of -18.2 ± 1.3 mV. After 2 h incubation with the peptide-encapsulated liposomes, 66% of corneal epithelial (hTCEpi) cells internalised the FITC-labelled peptide, demonstrating the ability of this formulation to effectively deliver peptide to hTCEpi cells. Additionally, lipoplexes (liposomes complexed with plasmid DNA) were also able to transfect hTCEpi cells, albeit at a modest level (8% of the cells). Here, we describe this novel anionic liposomal formulation intended to enhance the delivery of small cargo molecules in situ.

Targeting Acidity in Pancreatic Adenocarcinoma: Multispectral Optoacoustic Tomography Detects pH-Low Insertion Peptide Probes In Vivo.

BACKGROUND: pH-low insertion peptides (pHLIP) can serve as a targeting moiety that enables pH-sensitive probes to detect solid tumors. Using these probes in conjunction with multispectral optoacoustic tomography (MSOT) is a promising approach to improve imaging for pancreatic cancer. METHODS: A pH-sensitive pHLIP (V7) was conjugated to 750 NIR fluorescent dye and evaluated as a targeted probe for pancreatic adenocarcinoma. The pH-insensitive K7 pHLIP served as an untargeted control. Probe binding was assessed in vitro at pH 7.4, 6.8, and 6.6 using human pancreatic cell lines S2VP10 and S2013. Using MSOT, semiquantitative probe accumulation was then assessed in vivo with a murine orthotopic pancreatic adenocarcinoma model. RESULTS: In vitro, the V7-750 probe demonstrated significantly higher fluorescence at pH 6.6 compared with pH 7.4 (S2VP10, P = 0.0119; S2013, P = 0.0160), whereas no difference was observed with the K7-750 control (S2VP10, P = 0.8783; S2013, P = 0.921). In the in vivo S2VP10 model, V7-750 probe resulted in 782.5 MSOT a.u. signal compared with 5.3 MSOT a.u. in K7-750 control in tumor (P = 0.0001). Similarly, V7-750 probe signal was 578.3 MSOT a.u. in the S2013 model compared with K7-750 signal at 5.1 MSOT a.u. (P = 0.0005). There was minimal off-target accumulation of the V7-750 probe within the liver or kidney, and probe distribution was confirmed with ex vivo imaging. CONCLUSIONS: Compared with pH-insensitive controls, V7-750 pH-sensitive probe specifically targets pancreatic adenocarcinoma and has minimal off-target accumulation. The noninvasive detection of pH-targeted probes by means of MSOT represents a promising modality to improve the detection and monitoring of pancreatic cancer.

Orthotopic pancreatic tumors detected by optoacoustic tomography using Syndecan-1.

BACKGROUND: Advances in small animal imaging have improved the detection and monitoring of cancer in vivo; although with orthotopic models, precise localization of tumors remains a challenge. In this study, we evaluated multispectral optoacoustic tomography (MSOT) as an imaging modality to detect pancreatic adenocarcinoma in an orthotopic murine model. METHODS: In vitro binding of Syndecan-1 probe to the human pancreatic cancer cell line S2VP10 was evaluated on flow cytometry. For in vivo testing, S2VP10 cells were orthotopically implanted into the pancreas of severe combined immunodeficiency mice. At 7 d after implantation, the mice were intravenously injected with Syndecan-1 probe, and tumor uptake was evaluated with MSOT at multiple time points. Comparison was made with a free-dye control, indocyanine green (ICG). Probe uptake was verified ex vivo with fluorescent imaging. RESULTS: Syndecan-1 probe demonstrated partial binding to S2VP10 cells in vitro. In vivo, Syndecan-1 probe preferentially accumulated in the pancreas tumor (480 MSOT a.u.) compared with off-target organs, including the liver (67 MSOT a.u.) and kidney (96 MSOT a.u.). Syndecan-1 probe accumulation peaked at 6 h (480 MSOT a.u.), whereas the ICG control dye failed to demonstrate similar retention within the tumor bed (0.0003 MSOT a.u.). At peak accumulation, signal intensity was 480 MSOT a.u., resulting in several times greater signal in the tumor bed than in the kidney or liver. Ex vivo fluorescent imaging comparing tumor signal with that within off-target organs confirmed the in vivo results. CONCLUSIONS: MSOT demonstrates successful accumulation of Syndecan-1 probe within pancreatic tumors, and provides high-resolution images, which allow noninvasive, real-time comparison of signal within individual organs. Syndecan-1 probe preferentially accumulates within a pancreatic adenocarcinoma model, with minimal off-target effects.

Targeted noninvasive imaging of EGFR-expressing orthotopic pancreatic cancer using multispectral optoacoustic tomography.

Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe before MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a nonspecific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at 6 hours postinjection, demonstrating specific intratumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of the EGF receptor.

Microgel-encapsulated methylene blue for the treatment of breast cancer cells by photodynamic therapy.

PURPOSE: Photodynamic therapy (PDT) is gaining increasing recognition for breast cancer treatment because it offers local selectivity and reduced toxic side effects compared to radiotherapy and chemotherapy. In PDT, photosensitizer drugs are loaded in different nanomaterials and used in combination with light exposure. However, the most representative issue with PDT is the difficulty of nanomaterials to encapsulate anticancer drugs at high doses, which results in low efficacy of the PDT treatment. Here, we proposed the development of the poly(N-isopropylacrylamide) (PNIPAM) microgel for the encapsulation of methylene blue, an anticancer drug, for its use as breast cancer treatment in MCF-7 cell line. METHODS: We developed biocompatible microgels based on nonfunctionalized PNIPAM and its corresponding anionically functionalized PNIPAM and polyacrylic acid (PNIPAM-co-PAA) microgel. Methylene blue was used as the photosensitizer drug because of its ability to generate toxic reactive oxygen species upon exposure to light at 664 nm. Core PNIPAM and core/shell PNIPAM-co-PAA microgels were synthesized and characterized using ultraviolet-visible spectroscopy and dynamic light scattering. The effect of methylene blue was evaluated using the MCF-7 cell line. RESULTS: Loading of methylene blue in core PNIPAM microgel was higher than that in the core/shell PNIPAM-co-PAA microgel, indicating that electrostatic interactions did not play an important role in loading a cationic drug. This behavior is probably due to the skin layer inhibiting the high uptake of drugs in the PNIPAM-co-PAA microgel. Core PNIPAM microgel effectively retained the cationic drug (i.e., methylene blue) for several hours compared to core/shell PNIPAM-co-PAA and enhanced its photodynamic efficacy in vitro more than that of free methylene blue. CONCLUSION: Our results showed that the employment of core PNIPAM and core/shell PNIPAM-co-PAA microgels enhanced the encapsulation of methylene blue. Core PNIPAM microgel released the drug more slowly than did core/shell PNIPAM-co-PAA, and it effectively inhibited the growth of MCF-7 cells.

Pulsed hydrogen/deuterium exchange mass spectrometry for time-resolved membrane protein folding studies.

Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D(2) O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N-H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR.

Cationic liposome colloidal stability in the presence of guar derivatives suggests depletion interactions may be operative in artificial tears.

The colloidal stability of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes was measured in the presence of guar, carboxymethyl guar, hydroxypropyl guar (HPG), and in mixtures of HPG with boric acid. Carboxymethyl guar induced DOTAP aggregation, presumably by bridging flocculation. The interaction with HPG-borate, an anionic polyelectrolyte with labile charge groups, depended on ionic strength. Without added salt, HPG-borate (pH 9.2) adsorbed on the liposomes and destabilized them. In contrast, in 0.1 M NaCl, HPG-borate did not adsorb onto the liposomes. HPG, HPG-borate, and guar induced depletion flocculation of the liposomes at high polymer concentration. Depletion flocculation may be an important mechanism when HPG is employed in artificial tear formulations.

Environmental management plan (EMP) for Melamchi water supply project, Nepal.

More than 1.5 million people live in the Kathmandu valley. The valley is facing an extreme shortage of water supply. At the same time the demand is escalating rapidly. To address this issue of scarcity of water, the government of Nepal has proposed a project of inter-basin transfer of water from Melamchi River located 40 km north-east of the Kathmandu valley. The project will cover two districts and three municipalities and will potentially have significant impacts on the environment. In accordance with the Environmental Protection Regulation of Nepal (1997), the Melamchi Water Supply Project (MWSP) has undergone an EIA during the feasibility study stage of the proposed project. The recommendations contained in the EIA were integrated into the project design for implementation in 2006. This paper summarizes the background of MWSP, the environmental concerns described in the EIA and the status of Environmental Management Plan (EMP) developed to address environmental compliance and other issues involving participation and support of the local people. This paper also provides some lessons to learn on the modalities of addressing the demands and grievances of the local people concerning environmental management.

Fabrication of nanoaggregates of a triple hydrophilic block copolymer by cetyltrimethylammonium chloride binding.

A triple hydrophilic block copolymer composed of poly(ethylene oxide), poly(sodium 2-acrylamido-2-methylpropanesulfonate), and poly(methacrylic acid) (PEO-PAMPS-PMAA) does not form a micelle by itself when it is dissolved in water. However, if the anionic PAMPS and/or PMAA blocks are electrically neutralized with a cationic surfactant, such as cetyltrimethylammonium chloride (CTAC), micelle-like nanoaggregates are obtained, where the core is formed by the insolubilized PAMPS and/or PMAA blocks. Formation of the nanoaggregates was confirmed by dynamic light scattering (DLS) measurements and scanning electron microscopy (SEM), while the binding of CTAC to PEO-PAMPS-PMAA was monitored by electrophoresis measurements. The aggregates were characterized by fluorescence spectroscopy as well as DLS and SEM. It was found that the nanoaggregates have a spherical structure, and the hydrodynamic diameter ranges from 125 to 193 nm depending on the concentrations of the PEO-PAMPS-PMAA and CTAC. The critical aggregate concentration is on the order of 10-4 g L-1 when the ionic blocks of PEO-PAMPS-PMAA are fully neutralized with CTAC.

Incorporation and release of cloxacillin sodium in micelles of poly(styrene-b-2-vinyl pyridine-b-ethylene oxide).

A novel drug carrier system was constructed from anionic drug cloxacillin sodium (CLX) and micelle of poly(styrene-b-2-vinyl pyridine-b-ethylene oxide) (PS-PVP-PEO) by incorporating the former into the micelle of the latter. The incorporation of CLX into the micelle of PS-PVP-PEO was confirmed by zeta-potential measurements, dynamic light scattering (DLS), and fluorescence spectroscopy. In the absence of the anionic drug, CLX, the zeta-potential of neat PS-PVP-PEO micelle was +13 mV under aqueous acidic condition due to the presence of positive charges in the PVP unit. The addition of CLX into the micelle of PS-PVP-PEO decreased the zeta-potential of the micelle smoothly and finally led to a minimum zeta-potential around 0 mV. This fact shows that the added CLX is effectively incorporated into the PS-PVP-PEO micelle by electrostatic attraction. In concomitant with the decrease in zeta-potential, a decrease in hydrodynamic diameter from 94 to 69 nm was observed on addition of CLX to the PS-PVP-PEO micellar solution. This fact also indicates the incorporation of CLX into the PS-PVP-PEO micelles because the binding of CLX to the PVP block of the micelles induces a conformational change from an extended to a shrunken form due to the cancellation of the repulsive force in the PVP blocks by CLX. Fluorescence quenching of pyrene by CLX gave additional evidence for the effective bindings of CLX to the PS-PVP-PEO micelles. Further, release of CLX from the nanoaggregates of CLX/PS-PVP-PEO was investigated in vitro. It was found that the release kinetics of the CLX is conformed to a model based on the consecutive chemical kinetics.