Micro-Electrical Impedance Spectroscopy and Identification of Patient-Derived, Dissociated Tumor Cells.

Fine needle aspirate sampling of tumors requires acquisition of sufficient cells to complete a diagnosis. Aspirates through such fine needles are typically composed of small cell clusters in suspension, making them readily amenable to microfluidic analysis. Here we show a microfluidic device with integrated electrodes capable of interrogating and identifying cellular components in a patient-derived sample of dissociated tumor cells using micro-electrical impedance spectroscopy ( µ EIS). We show that the µ EIS system can distinguish dissociated tumor cells in a sample consisting of red blood cell (RBCs) and peripheral blood mononucleated cells (PBMCs). Our µ EIS system can also distinguish dissociated tumor cells from normal cells and we show results for five major cancer types, specifically, lung, thyroid, breast, ovarian, and kidney cancer. Moreover, our µ EIS system can make these distinctions in a label-free manner, thereby opening the possibility of integration into standard clinical workflows at the point of care.

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.

Inertio-elastic focusing of bioparticles in microchannels at high throughput.

Controlled manipulation of particles from very large volumes of fluid at high throughput is critical for many biomedical, environmental and industrial applications. One promising approach is to use microfluidic technologies that rely on fluid inertia or elasticity to drive lateral migration of particles to stable equilibrium positions in a microchannel. Here, we report on a hydrodynamic approach that enables deterministic focusing of beads, mammalian cells and anisotropic hydrogel particles in a microchannel at extremely high flow rates. We show that on addition of micromolar concentrations of hyaluronic acid, the resulting fluid viscoelasticity can be used to control the focal position of particles at Reynolds numbers up to Re≈10,000 with corresponding flow rates and particle velocities up to 50 ml min(-1) and 130 m s(-1). This study explores a previously unattained regime of inertio-elastic fluid flow and demonstrates bioparticle focusing at flow rates that are the highest yet achieved.

Cellular bias on the microscale: probing the effects of digital microfluidic actuation on mammalian cell health, fitness and phenotype.

The potential benefits of using new technologies such as microfluidics for life science applications are exciting, but it is critical to understand and document potential biases imposed by these technologies on the observed results. Here, we report the first study of genome-level effects on cells manipulated by digital microfluidics. These effects were evaluated using a broad suite of tools: cell-based stress sensors for heat shock activation, single-cell COMET assays to probe changes in DNA integrity, and DNA microarrays and qPCR to evaluate changes in genetic expression. The results lead to two key observations. First, most DMF operating conditions tested, including those that are commonly used in the literature, result in negligible cell-stress or genome-level effects. Second, for DMF devices operated at high driving frequency (18 kHz) and with large driving electrodes (10 mm × 10 mm), there are significant damage to DNA integrity and differential genomic regulation. We hypothesize that these effects are caused by droplet heating. We recommend that for DMF applications involving mammalian cells that driving frequencies be kept low (≤ 10 kHz) and electrode sizes be kept small (≤ 5 mm) to avoid detrimental effects.

Mitochondrial localization and the persistent migration of epithelial cancer cells.

During cancer cell invasion, faster moving cancer cells play a dominant role by invading further and metastasizing earlier. Despite the importance of these outlier cells, the source of heterogeneity in their migratory behavior remains poorly understood. Here, we show that anterior localization of mitochondria, in between the nucleus and the leading edge of migrating epithelial cancer cells, correlates with faster migration velocities and increased directional persistence. The asymmetry of mitochondrial localization along the axis of migration is absent during spontaneous cell migration on two-dimensional surfaces and only occurs in the presence of chemical attractant cues or in conditions of mechanical confinement. Moreover, perturbing the asymmetric distribution of mitochondria within migrating cells by interfering with mitochondrial fusion (opa-1) or fission (drp-1) proteins, significantly reduces the number of cells with anterior localization of mitochondria and significantly decreases the velocity and directional persistence of the fastest moving cells. We also observed similar changes after perturbing the linkage between mitochondria and microtubules by the knockdown of mitochondrial rhoGTPase-1 (miro-1). Taken together, the changes in migration velocity and directional persistence in cells with anterior-localized mitochondria could account for an order of magnitude differences in invasive abilities between cells from otherwise homogenous cell populations.

Patellar eversion does not adversely affect quadriceps recovery following total knee arthroplasty.

Although avoiding patellar eversion during a total knee arthroplasty (TKA) has theoretical benefit in quadriceps recovery, there has been paucity of supportive objective clinical results. We prospectively designed the study whether TKA without patellar eversion has better quadriceps recovery in an objective, dynamometer study. Seventy-two knees undergoing TKA with midvastus approach were randomized into two groups according to patellar eversion or not. Clinical data and objective quadriceps recovery using a dynamometer were investigated preoperatively and postoperative at 6weeks, 3months, 6months and 1year. There were no statistical differences between two groups throughout the follow-up periods in recovery of quadriceps force or power and clinical data. Choosing to evert patella during TKA using midvastus approach would not adversely affect postoperative quadriceps recovery.

Cell-based sensors for quantifying the physiological impact of microsystems.

Microsystems are increasingly used in the manipulation, patterning and sorting of cells. Critical to the widespread adoption of these new technologies is development of an understanding of their impact on cellular physiology. Here we show the integration of a cell-based sensor, a microfabricated electrical screening platform, and quantitative imaging to enable the first large-scale physiological screens of the impact of microsystems on cells. To perform physiological screening, we developed a cell-based sensor that reports on stress-mediated transcription (via Heat Shock Factor 1 induced expression of GFP). This cell-based sensor was quantitatively characterized using automated imaging. The integration of this quantitative physiological sensor with a microfabricated system enabled the execution of multiplexed screens across electric field strength, frequency, and application duration. Voltage sweeps indicate increasing physiological stress with increasing voltage due to Joule heating, while frequency sweeps indicate increased stress at lower frequencies (<500 kHz) compared with higher frequencies (>1 MHz) due to generation of reactive species at lower frequencies. Combined voltage and frequency sweeps enable the generation of complex maps of physiological state.

Electroactive hydrodynamic weirs for microparticle manipulation and patterning.

We present a platform for parallelized manipulations of individual polarizable micron-scale particles (i.e., microparticles) that combines negative dielectrophoretic forcing with the passive capture of hydrodynamic weir-based trapping. Our work enables manipulations using ejection- andor exclusion-based methods. In ejection operations, we unload targeted weirs by displacing microparticles from their capture faces via electrode activation. In exclusion-based operations, we prevent weir loading by activating selected on-chip electrodes before introducing microparticles into the system. Our work describes the device's passive loading dynamics and demonstrates enhanced functionalities by forming a variety of particle patterns.

Plastic masters-rigid templates for soft lithography.

We demonstrate a simple process for the fabrication of rigid plastic master molds for soft lithography directly from (poly)dimethysiloxane devices. Plastics masters (PMs) provide a cost-effective alternative to silicon-based masters and can be easily replicated without the need for cleanroom facilities. We have successfully demonstrated the use of plastics micromolding to generate both single and dual-layer plastic structures, and have characterized the fidelity of the molding process. Using the PM fabrication technique, world-to-chip connections can be integrated directly into the master enabling devices with robust, well-aligned fluidic ports directly after molding. PMs provide an easy technique for the fabrication of microfluidic devices and a simple route for the scaling-up of fabrication of robust masters for soft lithography.

5,6-Diarylanthranilo-1,3-dinitriles as a new class of antihyperglycemic agents.

Various functionalized mono- and diarylanthranilo-1,3-dinitriles were synthesized and evaluated for their in vitro antihyperglycemic activity against the PTP-1B, glucose-6-phosphatase, glycogen phosphorylase and alpha-glucosidase enzymes. Among various screened compounds, 5,6-diaryl substituted anthranilo-1,3-dinitriles 3a, 3b, and 3d showed good inhibitory activity against PTP-1B with IC(50) values of 58-72 microM. Three of the test compounds showed significant (25-37%) lowering of plasma glucose level at 24h in sucrose-challenged streptozotocin-induced diabetic Sprague-Dawley rat model.

Electrically addressable vesicles: tools for dielectrophoresis metrology.

Dielectrophoresis (DEP) has emerged as an important tool for the manipulation of bioparticles ranging from the submicron to the tens of microns in size. Here we show the use of phospholipid vesicle electroformation techniques to develop a new class of test particles with specifically engineered electrical propserties to enable identifiable dielectrophoretic responses in microfabricated systems. These electrically addressable vesicles (EAVs) enable the creation of electrically distinct populations of test particles for DEP. EAVs offer control of both their inner aqueous core and outer membrane properties; by encapsulating solutions of different electrolyte strength inside the vesicle and by incorporating functionalized phospholipids containing poly(ethylene glycol) (PEG) brushes attached to their hydrophilic headgroup in the vesicle membrane, we demonstrate control of the vesicles' electrical polarizabilities. This combined with the ability to encode information about the properties of the vesicle in its fluorescence signature forms the first steps toward the development of EAV populations as metrology tools for any DEP-based microsystem.

A photopatternable silicone for biological applications.

We show the application of a commercially available photopatternable silicone (PPS) that combines the advantageous features of both PDMS and SU-8 to address a critical bioMEMS materials deficiency. Using PPS, we demonstrate the ability to pattern free-standing mechanically isolated elastomeric structures on a silicon substrate: a feat that is challenging to accomplish using soft lithography-based fabrication. PPS readily integrates with many cell-based bioMEMS since it exhibits low autofluorescence and cells easily attach and proliferate on PPS-coated substrates. Because of its inherent photopatternable properties, PPS is compatible with standard microfabrication processes and easily aligns to complex featured substrates on a wafer scale. By leveraging PPS' unique properties, we demonstrate the design of a simple dielectrophoresis-based bioMEMS device for patterning mammalian cells. The key material properties and integration capabilities explored in this work should present new avenues for exploring silicone microstructures for the design and implementation of increasingly complex bioMEMS architectures.

Infantile tibia vara: treatment of Langenskiold stage IV.

An eight year old girl presented with a progressively increasing deformity of the left proximal tibia since last 2 years. She had no history of trauma, fever and swelling of left knee. There were no obvious signs of rickets/muscular dystrophy. She had 25 degrees of tibia vara clinically with lateral thrust and a prominent fibular head. The radiograph of left knee revealed tibia vara with medial beaking and a significant depression of the medial tibial epiphysis and metaphysis. A computed tomography (CT) scan revealed significant depression of the medial tibial epiphysis but no bony bar in the physis or fusion of the medical tibial epiphysis. There was a posterior slope in addition to the medial one. She was treated with elevation of the medial tibial hemiplateau with subtuberosity valgus derotation dome osteotomy. She also underwent a lateral proximal tibial hemiphysiodesis (temporary stapling). A prophylactic subcutaneons anterolateral compartment fasciotomy was also performed. All osteotomies united in 2 months. All deformities were corrected and she regained a knee range of 0 to 130 degrees. At final followup (4 years), there was no recurrence of varus deformity, knee was stable, with 1cm of leg length discrepancy. In Langenskiold stage IV tibia vara, elevation of medial tibial plateau, a subtuberosity valgus derotation osteotomy and a concomitant lateral hemiephiphysiodesis has given good results.

Transfusion medicine in India: a survey of current practice.

The new Indian National Blood Policy intends to improve the provision of easily accessible safe blood and blood components, available according to need. There is a requirement for information on patterns of blood transfusion in India, to help understand the potential for changes in transfusion practice and the organization of blood services, and to help ensure optimal use of this valuable and limited human resource. This study involved a questionnaire survey of blood transfusion practice with reference to Indian National AIDS Control Organisation clinical guidelines at selected blood centres in four study areas (Delhi, Bangalore, Nasik District and Imphal). Information on 1062 transfusion episodes was analysed. Adult recipients accounted for 87% of transfusions, and amongst the age group of 25-34, 73% of transfusions were to women. Anaemia was listed as a reason for 60% of transfusions, surgery for 42%, acute haemorrhage for 26% and pregnancy for 16%. Seventy-four per cent of adult transfusions were inappropriate when assessed against criteria derived from government transfusion guidelines. Possible common proximate causes for inappropriate transfusions include unnecessary transfusion for iron-deficiency anaemia and transfusion as a first choice for volume replacement. Options to promote good transfusion practice in India should be appraised.

Intervention to promote appropriate blood use in India.

The decision to transfuse should be guided by information on the risks and benefits of transfusion. Safer alternatives should be considered. Properly screened blood or components from a reputable source should be used. In this study, a simple, self-educating transfusion request form was developed, and its effects on transfusion practice were assessed, using a cluster-randomized trial. Transfusions at six study hospitals and six control hospitals at four locations in India (Delhi, Bangalore, Nasik and Imphal) were monitored over a 4-month pre-intervention period and a 5-month post-intervention period. During the trial, 56 171 units were transfused to in-patients at the participating hospitals. Among the six intervention hospitals, there was some evidence of a nonsignificant post-intervention reduction in all the three main outcome measures: number of transfusion requests per admission (P = 0.09), number of units transfused per admission (P = 0.11) and number of crossmatches per admission (P = 0.06). No such changes were seen at control sites over the same period. Simple interventions to promote good clinical practice can have an effect, but may be better placed within longer term, broad-based strategies that are able to consider some of the background factors. Lack of clinical training, the hospital environment and fragmented blood bank services influence the way blood is used in India. It is thought that the intervention was ultimately unsuccessful because these factors remained as detrimental influences. A focus on education, policy and infrastructure in line with the new National Blood Policy will be important in coming years.

Brucellosis in children of Dhofar Region, Oman.

OBJECTIVE: To study the epidemiological and clinical pattern of brucellosis in children of Dhofar and to ascertain the efficacy of a pre-determined antibiotic regimen to treat the disease. METHODS: The study was hospital based and was carried out prospectively for 3 years. All cases diagnosed to have brucellosis on clinical and serological basis were entered into the study. The epidemiological background and clinical presentations were analyzed and the clinical response to a combination of oral rifampicin and co-trimoxazole was evaluated. RESULTS: Three hundred and seventy five cases of brucellosis were eligible for the study. Ingestion of raw milk and its products were responsible for causation of the disease in 63% of cases. Eighty three per cent had direct contact with animals mainly cattle. A minority of 4.5% denied ingestion of raw milk or coming into direct contact with animals. Fever was the most common presenting feature at 91%. We identified 2 distinct groups of presentation: Seventy per cent of those who presented with arthritis belonged to the older age group (7.34 years, standard deviation 2.64). They did not have a systemic illness. The younger age group presented with severe systemic illness associated with severe leucopenia and thrombocytopenia. The clinical response to the combination of rifampicin and co-trimoxazole was satisfactory in 90% of patients and 98% of brucella species isolated from the blood of patients were sensitive to both antibiotics used. CONCLUSION: Ingestion of infected milk and contact with infected animals are the main causes of human brucellosis, although aerial transmission from contaminated environmental soil could not be excluded. The main clinical presentation of brucellosis in children is fever but the skeletal manifestations of the disease are significant. The hematological manifestations of the disease in endemic areas deserve special attention. The combination of oral rifampicin and co-trimoxazole for 6 weeks is adequate to treat most cases of brucellosis in children.

Trends of HIV infection in the blood donors of Delhi.

Screening of HIV Infection was made mandatory for every unit of blood collected for transfusion in Delhi, India since 1989. Ten Zonal Blood Testing Centres have been identified which test all the blood collected for HIV by 29 blood blanks for the city. Reports from these testing centres have been analysed yearwise to find out the magnitude and trends of HIV infection in different groups of blood donors. Although initially there was no difference in HIV Sero-reactivity in different blood donors categories (between 1 & 2 per 1000 blood donors samples tested) but subsequently there is significant increase (5.24/1000 in 1992 & 7.48/1000 in 1993) in the HIV sero-reactivity in replacement donor category possibly because professional donors donate blood in the guise of being replacement donors. The fact which comes out clearly is that HIV infection is present in all sections of the population in Delhi and mandatory HIV Screening of all blood collected for transfusion is justified.

PIP: Since 1989, screening of all blood collected for transfusion for HIV has been mandatory in Delhi, India. Blood collected at Delhi's 29 blood banks is sent to 1 of 10 zonal blood testing centers. About 90% of blood donors are men 20-40 years of age. Only about 20% of donors are voluntary; 35% of blood is contributed by paid professional donors and the remaining 45% by replacement donors. The HIV seropositivity rates per 1000 samples screened among voluntary donors were 0.63, 0.45, 1.9, 3.03, and 3.87 in 1989, 1990, 1991, 1992, and 1993, respectively. Among replacement donors, these rates were 0.46, 0.50, 1.9, 5.24, and 7.48, respectively. Among professional donors, these rates were 1.50, 0.90, 1.3, 3.28, and 3.76, respectively. Notable is the significant increase in HIV infection in replacement donors. It is speculated that this trend represents a tendency for many professional donors to present as replacement donors to obtain payment directly from a patient's family rather than the blood bank. If voluntary blood donors are representative of the general male adult population, HIV prevalence appears to be steadily increasing in Delhi and continued blood screening is certainly warranted.

Distribution and trends of HIV infection in blood donors of four metropolitan cities.

Screening for HIV infection has been made mandatory for every unit of blood collected for transfusion in major cities of the country, having facilities for such screening. Results of HIV screening among blood donors received from the 4 Metropolitan cities of Delhi, Bombay, Calcutta and Madras have been analysed year wise from 1989 to 1991 to determine the magnitude and trends of HIV infection in different categories of blood donors and the seropositivity rates seems to be increasing over time. Universal coverage of HIV screening for donated blood has not been fully achieved and the justification and urgency for achieving complete coverage is highlighted.

PIP: In India, an analysis of monthly reports (January 1989-December 1991) received from 140 blood banks in India's 4 Zonal Blood Testing Centres in Delhi, Calcutta, Bombay, ad Madras compared the cities HIV infection rates and examined the progression of these rates among different categories of blood donors. Blood screening did not begin in Calcutta until 1990. In 1991, a considerable difference existed between the number of blood units collected and the units tested in Madras and Calcutta (a difference of 32.5% and 61.04%, respectively). The proportion tested was highest in Delhi (95.5%) followed by Bombay (87.3%). HIV seropositivity rates remained elevated in all cities among all blood donor groups, except Calcutta. In Bombay, these rates were 9.81/1000 samples screened in 1989, 7.36 in 1990, and 8.65 in 1991. In Delhi, they were 0.89 0.64, and 1.69, respectively. In Madras, they were 1.02, 3.2, and 6.21, respectively. The 1990 and 1991 rates in Calcutta were just 0.12 and 0.02, respectively. The highest HIV infection rates were greatest in professional blood donors in Bombay and Madras. Between 1989 and 1991, they fell in Bombay (42.02-12.34) and rose in Madras (2.81-18.47). Professional blood donors made up 16.82% of all donors in Madras, 10.56% in Bombay, 18.94% in Calcutta, and 33.8% in Delhi. Since 90% of voluntary blood donors were males, the 1991 HIV infection rate of voluntary blood donors indicated that HIV had established itself in males in Bombay (9.06) and Madras (4.03) and less so in Delhi (1.9). In Calcutta, the HIV infection rate among voluntary donors was 013 in 1990, while it was 0 in 1991. India has yet to completely achieve HIV screening. These findings confirm the necessity for universal HIV screening.