Microfluidics-based rapid measurement of nitrite in human blood plasma.

Nitric oxide (NO) is one of the vital gasotransmitters that takes part in many biological pathways such as infection, inflammation and ischemia, immune response, neurotransmission, and cardiovascular systems. Nitrite is one of the primary metabolites of NO and is considered to be a circulating storage pool for NO. Here, we report direct and rapid measurement of nitrite in human blood plasma using a fluorescence-based microfluidic method. The study revealed the factors that affect the endogenous concentration of nitrite in blood plasma, mainly the presence of blood cells, hemoglobin, and soluble proteins. We find that separation of blood plasma immediately after sample collection and subsequent dilution of plasma with buffer at a ratio of 1 : 4 eliminates the interference from cells and proteins, providing reliable measurements. The proposed method can measure plasma nitrite in the concentration range of 0-20 µM with a limit of detection of 60 nM and a sensitivity of 5.64 µM-1 within 10 min of sample collection. By spiking nitrite into plasma, a linear correlation between the nitrite concentration and FL intensity is obtained, which is utilized further to measure the endogenous concentration of nitrite present in the plasma of healthy volunteers and patients. The study revealed that the endogenous nitrite concentration in the blood plasma of healthy humans falls in the range of 0.4-1.2 µM. Furthermore, the study with blood samples obtained from patients showed that nitrite levels are inversely correlated with the total cholesterol and low-density lipoproteins levels, which is in good agreement with the literature.

Prevalence and Pattern of Self-medication with Alternative Medicine: Treatment-switch Analysis in Oral Submucous Fibrosis Patients.

AIM: The aim of this study was to investigate prevalence and pattern of self-medication with alternative medicine (ALM) among oral submucous fibrosis (OSF) patients with emphasis on treatment-switch analysis. METHODS: A total of 115 OSF patients were recruited and subjected to scientifically validated questionnaire. Two groups were identified: S-ALM: patients who have previously received modern medicinal treatment but switched to ALM; and DN-ALM: patients who started ALM from De Novo. RESULTS: A total of 37 (32.18%) patients reported use of the ALM at some point of time. Twenty-five (67.56%) switched to ALM, whereas 12 (32.43%) patients used ALM from De Novo. The pattern of switching to ALM in short period was significantly prevalent in patients with advanced stages (stage III and IV) as compared to early stages (stage I and II) of OSF. Clarified butter, glycerin, and honey were the most commonly used ALM in both the groups. The most common reasons for switching to ALM were the cost of treatment (32.00%), longer duration of medicinal treatment (24.00%) and ineffectiveness of the medications (24.00%). CONCLUSIONS: ALM usage is common in OSMF patients with significant percentage of patient switched from modern medicine to ALM. Therapeutic efficacy of ALM in OSF needs exploration in future.

Direct and rapid measurement of hydrogen peroxide in human blood using a microfluidic device.

The levels of hydrogen peroxide ([Formula: see text]) in human blood is of great relevance as it has emerged as an important signalling molecule in a variety of disease states. Fast and reliable measurement of [Formula: see text] levels in the blood, however, continues to remain a challenge. Herein we report an automated method employing a microfluidic device for direct and rapid measurement of [Formula: see text] in human blood based on laser-induced fluorescence measurement. Our study delineates the critical factors that affect measurement accuracy-we found blood cells and soluble proteins significantly alter the native [Formula: see text] levels in the time interval between sample withdrawal and detection. We show that separation of blood cells and subsequent dilution of the plasma with a buffer at a ratio of 1:6 inhibits the above effect, leading to reliable measurements. We demonstrate rapid measurement of [Formula: see text] in plasma in the concentration range of 0-49 µM, offering a limit of detection of 0.05 µM, a sensitivity of 0.60 µM-1, and detection time of 15 min; the device is amenable to the real-time measurement of [Formula: see text] in the patient's blood. Using the linear correlation obtained with known quantities of [Formula: see text], the endogenous [Formula: see text] concentration in the blood of healthy individuals is found to be in the range of 0.8-6 µM. The availability of this device at the point of care will have relevance in understanding the role of [Formula: see text] in health and disease.

An optomicrofluidic device for the detection and isolation of drop-encapsulated target cells in single-cell format.

Single-cell analysis has emerged as a powerful method for genomics, transcriptomics, proteomics, and metabolomics characterisation at the individual cell level. Here, we demonstrate a technique for the detection and selective isolation of target cells encapsulated in microdroplets in single-cell format. A sample containing a mixed population of cells with fluorescently labelled target cells can be focused using a sheath fluid to direct cells in single file toward a droplet junction, wherein the cells are encapsulated inside droplets. The droplets containing the cells migrate toward the centre of the channel owing to non-inertial lift force. The cells present in the droplets are studied and characterised based on forward scatter (FSC), side scatter (SSC), and fluorescence (FL) signals. The FL signals from the target cells can be used to activate a selective isolation module based on electro-coalescence, using suitable electronics and a program to sort droplets containing the target cells in single-cell format from droplets containing background cells. We demonstrated the detection and isolation of target cells (cancer cells: HeLa and DU145) from mixed populations of cells, peripheral blood mononuclear cells (PBMC) + cervical cancer cells (HeLa) and PBMC + human prostate cancer cells (DU145), at a concentration range of 104-106 ml-1 at 300 cells per s. The performance of the device is characterised in terms of sorting efficiency (>97%), enrichment (>1800×), purity (>98%), and recovery (>95%). The sorted target cells were found to be viable (>95% viability) and showed good proliferation when cultured, showing the potential of the proposed sorting technique for downstream analysis.

Rapid measurement of hydrogen sulphide in human blood plasma using a microfluidic method.

Hydrogen sulfide (H2S) is emerging as an important gasotransmitter in both physiological and pathological states. Rapid measurement of H2S remains a challenge. We report a microfluidic method for rapid measurement of sulphide in blood plasma using Dansyl-Azide, a fluorescence (FL) based probe. We have measured known quantities of externally added (exogenous) H2S to both buffer and human blood plasma. Surprisingly, a decrease in FL intensity with increase in exogenous sulphide concentration in plasma was observed which is attributed to the interaction between the proteins and sulphide present in plasma underpinning our observation. The effects of mixing and incubation time, pH, and dilution of plasma on the FL intensity is studied which revealed that the FL assay required a mixing time of 2 min, incubation time of 5 min, a pH of 7.1 and performing the test within 10 min of sampling; these together constitute the optimal parameters at room temperature. A linear correlation (with R2 ≥ 0.95) and an excellent match was obtained when a comparison was done between the proposed microfluidic and conventional spectrofluorometric methods for known concentrations of H2S (range 0-100 µM). We have measured the baseline level of endogenous H2S in healthy volunteers which was found to lie in the range of 70 µM - 125 µM. The proposed microfluidic device with DNS-Az probe enables rapid and accurate estimation of a key gasotransmitter H2S in plasma in conditions closely mimicking real time clinical setting. The availability of this device as at the point of care, will help in understanding the role of H2S in health and disease.

Development and validation of oral health-related quality of life measure in oral submucous fibrosis.

OBJECTIVE: The aim of this study was to develop and assess the validity and reliability of disease-specific oral health-related quality of life (OHRQoL) instrument for oral submucous fibrosis (OSF). MATERIALS AND METHODS: Items for the OHRQoL-OSF were generated from personal interviews and focus group discussions, the existing questionnaires, reviews of literature and inputs from expert's panel. Item reduction was performed by clinical impact method followed by pretesting of the developed questionnaire. The validity and reliability of the instrument were then examined. RESULTS: Forty-five items were generated from qualitative data and item pooling from various sources. After item reduction, 17 items were finalized with four domain-structure having Eigenvalues greater than 1. OHRQoL-OSF was shown to be valid in distinguishing patients with varying degrees of OSF severity. For the concurrent validity, the observed impact of OSF based on OHRQoL-OSF significantly correlated with Oral Health Impact Profile and global self-ratings of oral health and overall well-being. OHRQoL-OSF and all its domains demonstrated good internal consistency reliability with Cronbach's alpha ˃0.7 and excellent test-retest reliability (ICC = 0.96). CONCLUSION: The first disease-specific OHRQoL-OSF instrument appeared to be highly reliable and valid measure for assessing impact of OSF on life quality.

Phenotypic and genotypic drug resistance profile of Salmonella serovars isolated from poultry farm and processing units located in and around Mumbai city, India.

BACKGROUND AND AIM: The extensive use of antimicrobials in poultry has led to an increase in bacterial multidrug resistance, and the emergence of multidrug-resistant nontyphoidal Salmonella is a global problem. This study was performed to detect antibiotic-resistant Salmonella serovars in poultry farming and processing environment. MATERIALS AND METHODS: A total of 956 various samples, comprising 432 farm origin, 324 poultry processing stage wise and environmental, and 154 product processing stages and environmental samples, were collected from poultry farms and processing units located in and around Mumbai city. Of a total of 71 recovered isolates, 42 randomly selected Salmonella isolates were subjected for antibiotic susceptibility testing by disk diffusion method and serotyping. A total of 31 serotypically confirmed isolates were characterized for the presence of tetA, tetB, bla TEM, and CTX-M gene. RESULTS: Higher resistance was recorded against Doxycycline (100%), followed by Oxytetracycline (97.62%), Neomycin (88.10%), Erythromycin (83.33%), Tetracycline (78.57%), and Ceftizoxime (35.71%). Resistance from 0.00 to 26.19 percent was found to antimicrobials, namely Norfloxacin (26.19%), Ampicillin (21.43%), Azithromycin (21.43%), Ciprofloxacin (19.05%), Colistin (4.76%), Streptomycin (16.67%), Cefotaxime (14.19%), Enrofloxacin (14.29%), Amoxyclav (14.29%), Gentamicin (7.14%), Chloramphenicol (4.76%), Amikacin (4.76%), and Ceftazidime (0.0%). Results demonstrate that the Salmonella Virchow dominated and all serotypes were found to carry Tetracycline resistance gene tetA, 5 isolates were found to be positive for blaTEM , whereas none of the isolates were carrying tetB and CTX-M gene. CONCLUSION: This study revealed that there is a significant rise of Tetracycline resistance with the presence of tetA gene in Salmonella spp. which indicates selective pressure for adopting resistance against tetracycline group of antibiotics.

Dual hit lipopolysaccharide & oleic acid combination induced rat model of acute lung injury/acute respiratory distress syndrome.

BACKGROUND & OBJECTIVES: Despite advances in therapy and overall medical care, acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) management remains a problem. Hence the objective of this study was to develop a rat model that mimics human ALI/ARDS. METHODS: Four groups of Wistar rats, 48 per group were treated with (i) intratracheal (IT) lipopolysaccharide (LPS) (5 mg/kg) dissolved in normal saline (NS), (ii) intravenous (iv) oleic acid (OA) (250 µl/kg) suspension in bovine serum albumin (BSA), (iii) dual hit: IT LPS (2 mg/kg) dissolved in NS and iv OA (100 µl/kg) and (iv) control group: IT NS and iv BSA. From each group at set periods of time various investigations like chest x-rays, respiratory rate (RR), tidal volume (TV), total cell count, differential cell count, total protein count and cytokine levels in bronchoalveolar lavage fluid (BALF), lung wet/dry weight ratio and histopathological examination were done. RESULTS: It was noted that the respiratory rate, and tumour necrosis factor-α (TNF-α) levels were significantly higher at 4 h in the dual hit group as compared to LPS, OA and control groups. Interleukin-6 (IL-6) levels were significantly higher in the dual hit group as compared to LPS at 8 and 24 h, OA at 8 h and control (at all time intervals) group. IL-1ß levels were significantly higher in LPS and dual hit groups at all time intervals, but not in OA and control groups. The injury induced in dual hit group was earlier and more sustained as compared to LPS and OA alone. INTERPRETATION & CONCLUSIONS: The lung pathology and changes in respiration functions produced by the dual hit model were closer to the diagnostic criteria of ALI/ARDS in terms of clinical manifestations and pulmonary injury and the injury persisted longer as compared to LPS and OA single hit model. Therefore, the ARDS model produced by the dual hit method was closer to the diagnostic criteria of ARDS in terms of clinical manifestations and pulmonary injury.

Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p < 0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, premalignant cells.

Formulation and Evaluation of Long Circulating Liposomal Amphotericin B: A Scinti-kinetic Study using Tc in BALB/C Mice.

In the present study, we formulated long circulating liposomes for amphotericin B and characterized them. The formulation was optimized using 2(3) factorial designs. Pegylated liposomal formulation showed favorable results with reference to particle size (247.33±9.60 nm), percent entrapment efficiency (94.55±3.34%). TEM studies revealed that the liposomes were essentially spherical, hollow, and appeared like powder puff structures. From DSC study it was concluded that the pegylated formulation containing Amp B showed better stability and membrane integrity of the formulation. During the stability studies the formulation was found to be stable. When subjected to gamma scintigraphy kinetic tracer studies the formulation showed longer residence time in the blood in BALB/C mice.

Cell surface as a fractal: normal and cancerous cervical cells demonstrate different fractal behavior of surface adhesion maps at the nanoscale.

Here we show that the surface of human cervical epithelial cells demonstrates substantially different fractal behavior when the cell becomes cancerous. Analyzing the adhesion maps of individual cervical cells, which were obtained using the atomic force microscopy operating in the HarmoniX mode, we found that cancerous cells demonstrate simple fractal behavior, whereas normal cells can only be approximated at best as multifractal. Tested on ~300 cells collected from 12 humans, the fractal dimensionality of cancerous cells is found to be unambiguously higher than that for normal cells.

Atomic force microscopy characterization of corneocytes: effect of moisturizer on their topology, rigidity, and friction.

BACKGROUND/PURPOSE: Atomic force microscopy (AFM) is a novel technique for skin characterization. OBJECTIVES: To develop AFM tests for characterization of the outermost epidermis layer, corneocytes. As an example, the effect of moisturizer on the corneocyte properties is studied. METHODS AND MATERIALS: Topology, rigidity, and friction (between individual corneocytes and AFM probe) of the top layer of corneocytes were measured by means of Veeco DM3100 AFM. Quench moisturizing cream was applied daily on the forearm of five volunteers for a period of 9 days. The skin flakes were collected before and after the treatment using Cuderm tape strips. No additional treatment of flakes was performed before the measurements. RESULTS: A protocol for the AFM study of corneocytes is developed. After the treatment, we observed overall smoothening of the corneocyte surface, an increase of friction, and a decrease of rigidity (the Young modulus). CONCLUSION: AFM can be used as a very sensitive tool for early detection of changes in corneocytes.

A novel in vitro stripping method to study geometry of corneocytes with fluorescent microscopy: example of aging skin.

BACKGROUND/PURPOSE: To develop modification of stripping method allowing high-resolution fluorescent visualization of corneocytes of human skin in vitro. To validate the method, the measured corneocyte areas on skin flakes are collected from individuals of different ages. MATERIALS AND METHODS: Two complimentary fluorescent dyes were used sequentially. First the adhesive layer of the stripping tape was stained with a cationic dye (rhodamine 640). This tape was used to collect skin flakes. Then both the tape and collected flakes were stained with anionic dye (fluorescein). The fluorescence of the adhesive tape exposed to the second staining is substantially decreased due to the mutual quenching of the dyes. Thirteen healthy, 6-86-year-old males participated to validate the method. The measurements were done on backhand and forearm. RESULTS: The method allows high-resolution imaging of corneocytes by means of fluorescent microscopy. Both absolute areas and the dependence of corneocyte areas on the individual age are in good agreement with the data reported previously. CONCLUSION: The developed method is fast and easy. It requires minimum interaction with the individual and allows using a broad variety of fluorescent dyes that may be potentially unsafe but beneficial for imaging. It can be used on any part of human or animal body.

Atomic force microscopy detects differences in the surface brush of normal and cancerous cells.

The atomic force microscope is broadly used to study the morphology of cells, but it can also probe the mechanics of cells. It is now known that cancerous cells may have different mechanical properties to those of normal cells, but the reasons for these differences are poorly understood. Here, we report quantitatively the differences between normal and cancerous human cervical epithelial cells by considering the brush layer on the cell surface. These brush layers, which consist mainly of microvilli, microridges and cilia, are important for interactions with the environment. Deformation force curves obtained from cells in vitro were processed according to the 'brush on soft cell model'. We found that normal cells have brushes of one length, whereas cancerous cells have mostly two brush lengths of significantly different densities. The observed differences suggest that brush layers should be taken into account when characterizing the cell surface by mechanical means.

Dose-dependent response of central dopaminergic systems to buspirone in mice.

Buspirone, a partial agonist of 5-hydroxytryptaminelA autoreceptors, preferentially blocks the presynaptic rather than the postsynaptic D2 dopamine (DA) receptors. Behavioural effects of a wide dose range of buspirone were therefore studied in mice. Buspirone at 0.625 to 5 mg/kg ip induced stereotyped cage climbing behaviour which was antagonized by pretreatment with haloperidol, alpha-methyl-p-tyrosine and small doses of apomorphine. Buspirone at 10, 20 and 40 mg/kg ip induced catalepsy and antagonized oral stereotypies induced by high doses of apomorphine and methamphetamine and apomorphine-induced cage climbing behaviour. The findings indicate that buspirone at 0.625 to 5 mg/kg selectively blocks the presynaptic mesolimbic D2 DA autoreceptors and releases DA which stimulates the postsynaptic mesolimbic D2 and D1 DA receptors and induces cage climbing behaviour. Buspirone, at 10, 20 and 40 mg/kg blocks the postsynaptic striatal and mesolimbic D2 and D1 DA receptors. Pretreatment with 1-tryptophan, dexfenfluramine and fluoxetine antagonized buspirone induced cage climbing behaviour and potentiated buspirone induced catalepsy. Pretreatment with trazodone, mianserin and p-chlorophenylalanine potentiated buspirone induced cage climbing behaviour and antagonized buspirone induced catalepsy. The results indicate that drugs which influence the activity of central serotonergic systems modulate the intensity of buspirone induced cage climbing behaviour and catalepsy.

Silica nanoparticles to polish tooth surfaces for caries prevention.

Although silica particles have been used for tooth polishing, polishing with nanosized particles has not been reported. Here we hypothesize that such polishing may protect tooth surfaces against the damage caused by cariogenic bacteria, because the bacteria can be easily removed from such polished surfaces. This was tested on human teeth ex vivo. The roughness of the polished surfaces was measured with atomic force microscopy (AFM). A considerably lower nanometer-scale roughness was obtained when silica nanoparticles were used to polish the tooth surfaces, as compared with conventional polishing pastes. Bacterial attachment to the dental surfaces was studied for Streptococcus mutans, the most abundant cariogenic bacteria. We demonstrated that it is easier to remove bacteria from areas polished with silica nanoparticles. The results demonstrate the advantage of using silica nanoparticles as abrasives for tooth polishing.

The TAR model: use of therapeutic state transitions for quality assurance reporting in chronic disease management.

Chronic disease management represents one of the challenges for health informatics and demands the appropriate application of information technology for improved patient care. This paper presents an approach to quality assurance reporting wherein the recommendations of evidence-based clinical practice guidelines are considered in the context of empirical therapeutic state-transitions (in terms of changes in individual patient prescriptions over time). We apply a Transition-based Audit Report (TAR) model to antihypertensive prescribing and related data as stored in a New Zealand General Practice Management System database. The results provide a set of quality indicators and specific patient cohorts for potential practice quality improvement with strong linkage to the selected guidelines and observed practice patterns. We see the TAR model primarily as a tool to enable internal quality improvement efforts, but also to be of relevance for focusing pay-for-performance programs.

Effects of dextromethorphan on dopamine dependent behaviours in rats.

Dextromethorphan, a noncompetitive blocker of N-methyl-D- aspartate (NMDA) type of glutamate receptor, at 7.5-75 mg/kg, ip did not induce oral stereotypies or catalepsy and did not antagonize apomorphine stereotypy in rats. These results indicate that dextromethorphan at 7.5-75 mg/kg does not stimulate or block postsynaptic striatal D2 and D1 dopamine (DA) receptors. Pretreatment with 15 and 30 mg/kg dextromethorphan potentiated dexamphetamine stereotypy and antagonised haloperidol catalepsy. Pretreatment with 45, 60 and 75 mg/kg dextromethorphan, which release 5-hydroxytryptamine (5-HT), however, antagonised dexamphetamine stereotypy and potentiated haloperidol catalepsy. Apomorphine stereotypy was not potentiated or antagonised by pretreatment with 7.5-75 mg/kg dextromethorphan. This respectively indicates that at 7.5-75 mg/kg dextromethorphan does not exert facilitatory or inhibitory effect at or beyond the postsynaptic striatal D2 and D1 DA receptors. The results are explained on the basis of dextromethorphan (15-75 mg/kg)-induced blockade of NMDA receptors in striatum and substantia nigra pars compacta. Dextromethorphan at 15 and 30 mg/kg, by blocking NMDA receptors, activates nigrostriatal dopaminergic neurons and thereby potentiates dexampetamine stereotypy and antagonizes haloperidol catalepsy. Dextromethorphan at 45, 60 and 75 mg/kg, by blocking NMDA receptors, releases 5-HT and through the released 5-HT exerts an inhibitory influence on the nigrostriatal dopaminergic neurons with resultant antagonism of dexampetamine stereotypy and potentiation of haloperidol catalepsy.

Effects of buspirone on dopamine dependent behaviours in rats.

Buspirone, a partial agonist of 5-hydroxytryptamine autoreceptors, selectively blocks presynaptic nigrostriatal D2 dopamine (DA) autoreceptors. At doses which antagonised action of apomorphine in biochemical presynaptic nigrostriatal D2 DA autoreceptor test systems buspirone neither induced catalepsy nor antagonised apomorphine-induced turning behaviour in rats indicating that at these doses buspirone does not block postsynaptic striatal D2 and D1 DA receptors. This study determines whether at high doses buspirone blocks postsynaptic striatal D2 and D1 DA receptors and provides behavioural evidence for selective blockade of presynaptic nigrostriatal D2 DA autoreceptors by smaller doses of buspirone. We investigated in rats whether buspirone induces catalepsy and effect of its pretreatment on DA agonist induced oral stereotypies and on cataleptic effect of haloperidol and small doses (0.05, 0.1 mg/kg, ip) of apomorphine. Buspirone at 1.25, 2.5, 5 mg/kg, ip neither induced catalepsy nor antagonised apomorphine stereotypy but did potentiate dexamphetamine stereotypy and antagonised cataleptic effect of haloperidol and small doses of apomorphine. Buspirone at 10, 20, 40 mg/kg, ip induced catalepsy and antagonised apomorphine and dexamphetamine stereotypies. Our results indicate that buspirone at 1.25, 2.5, 5 mg/kg blocks only presynaptic nigrostriatal D2 DA autoreceptors while at 10, 20, 40 mg/kg, it blocks postsynaptic striatal D2 and D1 DA receptors. Furthermore, buspirone at 1.25, 2.5, 5 mg/kg by selectively blocking presynaptic nigrostriatal D2 DA autoreceptors, increases synthesis of DA and makes more DA available for release by dexamphetamine and during haloperidol-induced compensatory 'feedback' increase of nigrostriatal DAergic neuronal activity and thus potentiates dexamphetamine stereotypy and antagonizes haloperidol catalepsy.