The Use of Plasmapheresis in a Severe Case of Amiodarone-Induced Thyrotoxicosis.

Amiodarone-induced thyrotoxicosis (AIT) can be difficult to treat since amiodarone's long half-life leads to a persistent effect on thyroid function. We present a case of a 74-year-old male with severe AIT who presented with altered mentation and ultimately required intubation and intensive care for management of thyroid storm. Standard medical therapy for treatment of thyroid storm was initiated immediately, but the patient remained unresponsive with worsening biochemical parameters with increasing total T3 levels and sustained elevated levels of free T4 after 5 days of medical management. Due to the lack of a clinical and biochemical response to conventional medical therapy, the patient was started on plasmapheresis and underwent a total of 7 cycles of plasmapheresis over a period of 10 days. He significantly improved with plasmapheresis and was successfully bridged to a total thyroidectomy, which was completed without complications.

Ultrasensitive Multi-Species Detection of CRISPR-Cas9 by a Portable Centrifugal Microfluidic Platform.

The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has enabled the targeted editing of genomes from diverse organisms, but the permanent and inheritable nature of genome modification also poses immense risks. The potential for accidental exposure, malicious use, or undesirable persistence of Cas9 therapeutics and off-target genome effects highlight the need for detection assays. Here we report a centrifugal microfluidic platform for the measurement of both Cas9 protein levels and nuclease activity. Because Cas9 from many bacterial species have been adapted for biotechnology applications, we developed the capability to detect Cas9 from the widely-used S. pyogenes, as well as S. aureus, N. meningitides, and S. thermophilus using commercially-available antibodies. Further, we show that the phage-derived anti-CRISPR protein AcrIIC1, which binds to Cas9 from several species, can be used as a capture reagent to broaden the species range of detection. As genome modification generally requires Cas9 nuclease activity, a fluorescence-based sedimentation nuclease assay was also incorporated to allow the sensitive and simultaneous measurement of both Cas9 protein and activity in a single biological sample.

Integrated LAMP and immunoassay platform for diarrheal disease detection.

The challenges of diagnosing infectious disease, especially in the developing world, and the shortcomings of available instrumentation have exposed the need for portable, easy-to-use diagnostic tools capable of detecting the wide range of causative microbes while operating in low resource settings. We present a centrifugal microfluidic platform that combines ultrasensitive immunoassay and isothermal amplification-based screening for the orthogonal detection of both protein and nucleic acid targets at the point-of-care. A disposable disc with automatic aliquoting inlets is paired with a non-contact heating system and precise rotary control system to yield an easy-to-use, field-deployable platform with versatile screening capabilities. The detection of three enterotoxins (cholera toxin, Staphylococcal enterotoxin B, and Shiga-like toxin 1) and three enteric bacteria (C. jejuni, E. coli, and S. typhimurium) were performed independently and shown to be highly sensitive (limit of detection = 1.35-5.50 ng/mL for immunoassays and 1-30 cells for isothermal amplification), highly exclusive in the presence of non-specific targets, and capable of handling a complex sample matrix like stool. The full panel of toxins and bacteria were reliably detected simultaneously on a single disc at clinically relevant sample concentrations in less than an hour. The ability of our technology to detect multiple analyte types in parallel at the point-of-care can serve a variety of needs, from routine patient care to outbreak triage, in a variety of settings to reduce disease impact and expedite effective treatment.

Isoproterenol Induced Insulin Resistance Leading to Diabetic Ketoacidosis in Type 1 Diabetes Mellitus.

Isoproterenol is known to cause insulin resistance and is often used to treat bradyarrhythmias from atrioventricular block. We report a case of isoproterenol induced diabetic ketoacidosis in a 77-year-old female patient treated with isoproterenol for atrioventricular block prior to insertion of permanent pacemaker. Diabetic ketoacidosis (DKA) developed within hours of starting an isoproterenol drip, and there were no other precipitating factors at that time. DKA resolved quickly after discontinuing isoproterenol and starting insulin drip. DKA is a common complication of diabetes mellitus, with about 140,000 hospital admissions for DKA in 2009. While the rate of DKA has increased by nearly 50% between 1988 and 2009, the rate of mortality has decreased. There are many causes of diabetic ketoacidosis, such as medication noncompliance, infection, pancreatitis, stroke, myocardial infarction, and many others. Isoproterenol may lead to diabetic ketoacidosis by increasing insulin resistance.

Rapid, Portable, Multiplexed Detection of Bacterial Pathogens Directly from Clinical Sample Matrices.

Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. This platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated in diarrheal and enteric diseases in less than 20 min.

A Reversibly Sealed, Easy Access, Modular (SEAM) Microfluidic Architecture to Establish In Vitro Tissue Interfaces.

Microfluidic barrier tissue models have emerged as advanced in vitro tools to explore interactions with external stimuli such as drug candidates, pathogens, or toxins. However, the procedures required to establish and maintain these systems can be challenging to implement for end users, particularly those without significant in-house engineering expertise. Here we present a module-based approach that provides an easy-to-use workflow to establish, maintain, and analyze microscale tissue constructs. Our approach begins with a removable culture insert that is magnetically coupled, decoupled, and transferred between standalone, prefabricated microfluidic modules for simplified cell seeding, culture, and downstream analysis. The modular approach allows several options for perfusion including standard syringe pumps or integration with a self-contained gravity-fed module for simple cell maintenance. As proof of concept, we establish a culture of primary human microvascular endothelial cells (HMVEC) and report combined surface protein imaging and gene expression after controlled apical stimulation with the bacterial endotoxin lipopolysaccharide (LPS). We also demonstrate the feasibility of incorporating hydrated biomaterial interfaces into the microfluidic architecture by integrating an ultra-thin (< 1 µm), self-assembled hyaluronic acid/peptide amphiphile culture membrane with brain-specific Young's modulus (~ 1kPa). To highlight the importance of including biomimetic interfaces into microscale models we report multi-tiered readouts from primary rat cortical cells cultured on the self-assembled membrane and compare a panel of mRNA targets with primary brain tissue signatures. We anticipate that the modular approach and simplified operational workflows presented here will enable a wide range of research groups to incorporate microfluidic barrier tissue models into their work.

Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

Centrifugal sedimentation immunoassays for multiplexed detection of enteric bacteria in ground water.

Waterborne pathogens pose significant threat to the global population and early detection plays an important role both in making drinking water safe, as well as in diagnostics and treatment of water-borne diseases. We present an innovative centrifugal sedimentation immunoassay platform for detection of bacterial pathogens in water. Our approach is based on binding of pathogens to antibody-functionalized capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk. Beads at the distal end of the disk are imaged to quantify the fluorescence and determine the bacterial concentration. Our platform is fast (20 min), can detect as few as ∼10 bacteria with minimal sample preparation, and can detect multiple pathogens simultaneously. The platform was used to detect a panel of enteric bacteria (Escherichia coli, Salmonella typhimurium, Shigella, Listeria, and Campylobacter) spiked in tap and ground water samples.

Centrifugal microfluidic platform for ultrasensitive detection of botulinum toxin.

We present an innovative centrifugal microfluidic immunoassay platform (SpinDx) to address the urgent biodefense and public health need for ultrasensitive point-of-care/incident detection of botulinum toxin. The simple, sample-to-answer centrifugal microfluidic immunoassay approach is based on binding of toxins to antibody-laden capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk and quantification by laser-induced fluorescence. A blind, head-to-head comparison study of SpinDx versus the gold-standard mouse bioassay demonstrates 100-fold improvement in sensitivity (limit of detection = 0.09 pg/mL), while achieving total sample-to-answer time of <30 min with 2-µL required volume of the unprocessed sample. We further demonstrate quantification of botulinum toxin in both exogeneous (human blood and serum spiked with toxins) and endogeneous (serum from mice intoxicated via oral, intranasal, and intravenous routes) samples. SpinDx can analyze, without any sample preparation, multiple sample types including whole blood, serum, and food. It is readily expandable to additional analytes as the assay reagents (i.e., the capture beads and detection antibodies) are disconnected from the disk architecture and the reader, facilitating rapid development of new assays. SpinDx can also serve as a general-purpose immunoassay platform applicable to diagnosis of other conditions and diseases.

A universal flow cytometry assay for screening carbohydrate-active enzymes using glycan microspheres.

We describe a simple, multiplexed assay that integrates glycan synthesis, bioconjugation to microspheres, fluorescent chemical/biochemical detection and multiparameter flow cytometric analysis to screen activities of different families of carbohydrate-active enzymes.

Single cell microRNA analysis using microfluidic flow cytometry.

MicroRNAs (miRNAs) are non-coding small RNAs that have cell type and cell context-dependent expression and function. To study miRNAs at single-cell resolution, we have developed a novel microfluidic approach, where flow fluorescent in situ hybridization (flow-FISH) using locked-nucleic acid probes is combined with rolling circle amplification to detect the presence and localization of miRNA. Furthermore, our flow cytometry approach allows analysis of gene-products potentially targeted by miRNA together with the miRNA in the same cells. We demonstrate simultaneous measurement of miR155 and CD69 in 12-O-tetradecanoylphorbol 13-acetate (PMA) and Ionomycin stimulated Jurkat cells. The flow-FISH method can be completed in ∼10 h, utilizes only 170 nL of reagent per experimental condition, and is the first to directly detect miRNA in single cells using flow cytometry.

Insulin in the medical management of postprandial hypoglycemia in a patient with type 2 diabetes after gastric bypass surgery.

Objective. We evaluated a 47-year-old woman with a history of type 2 diabetes and severe obesity who developed postprandial hypoglycemia after undergoing Roux-en-Y gastric bypass surgery and losing 60% of her total body weight. We studied her insulin secretion and blood glucose dynamics and were able to tailor a therapeutic regimen involving insulin that eliminated episodes of hypoglycemia. Methods. We studied blood glucose levels during a prolonged fast, performed continuous glucose monitoring studies using a subcutaneous glucose sensor, and evaluated regional pancreatic insulin secretion using selective arterial calcium stimulation. Results. Continuous glucose monitoring revealed that the patient had early (1-2 hr) postprandial hyperglycemia followed by late (3-4 hr) postprandial hypoglycemia. Biochemical studies confirmed endogenous pancreatogenous insulin secretion as the cause of episodic hypoglycemia, but imaging studies and selective arterial calcium stimulation failed to localize an insulinoma. The patient was treated with preprandial doses of insulin aspart in order to attenuate the early postprandial hyperglycemia, and the late hypoglycemic episodes were avoided. Conclusion. We describe an interesting and novel nonsurgical approach to the prevention of postprandial hypoglycemia in a patient with noninsulinoma pancreatogenous hypoglycemia after gastric bypass.

Safe and simple emergency department discharge therapy for patients with type 2 diabetes mellitus and severe hyperglycemia.

OBJECTIVE: To investigate the safety and effectiveness of 2 simple discharge regimens for use in patients with type 2 diabetes mellitus (DM2) and severe hyperglycemia, who present to the emergency department (ED) and do not need to be admitted. METHODS: We conducted an 8-week, open-label, randomized controlled trial in 77 adult patients with DM2 and blood glucose levels of 300 to 700 mg/dL seen in a public hospital ED. Patients were randomly assigned to receive glipizide XL, 10 mg orally daily (G group), versus glipizide XL, 10 mg orally daily, plus insulin glargine, 10 U daily (G+G group). The primary outcome was to maintain safe fasting glucose and random glucose levels of <350 and <500 mg/dL up to 4 weeks and <300 and <400 mg/dL, respectively, thereafter and to have no return ED visits (responders). RESULTS: Baseline characteristics were similar between the 2 treatment groups. The primary outcome was achieved in 87% of patients in both treatment groups. The enrollment mean blood glucose values of 440 and 467 mg/dL in the G and G+G groups, respectively, declined by the end of week 1 to 298 and 289 mg/dL and by week 8 to 140 and 135 mg/dL, respectively. Homeostasis model assessment of beta-cell function and early insulin response improved 7-fold and 4-fold, respectively, in responders at the end of the 8-week study. CONCLUSION: Sulfonylurea with and without use of a small dose of insulin glargine rapidly improved blood glucose levels and beta-cell function in patients with DM2. Use of sulfonylurea alone once daily can be considered a safe discharge regimen for such patients and an effective bridge between ED intervention and subsequent follow-up.

Adrenocorticotropic hormone stimulation test during high-dose glucocorticoid therapy.

OBJECTIVE: To define the effective time frame of adequate serum cortisol response to the short standard 250-microg adrenocorticotropic hormone stimulation test (ACTH-ST) after initiation of high-dose glucocorticoid therapy in order to assist in the diagnosis of adrenal insufficiency. METHODS: We performed an ACTH-ST at 4 different times in hospitalized patients, who had a documented intact hypothalamic-pituitary-adrenal axis and were receiving high-dose dexamethasone therapy for nonendocrine diseases, to determine the time until the serum cortisol response is compromised. The ACTH-ST was performed at the following time intervals after initiation of dexamethasone therapy - 24, 48, 72, and 96 hours. The outcome measures were cortisol levels measured at 0, 30, and 60 minutes after administration of 250 microg of cosyntropin. RESULTS: Of the overall group of 11 study patients, all those (n = 8) tested within the first 72 hours after initiation of dexamethasone therapy had an adequate response (serum cortisol level at 60 minutes, >or=18 microg/dL). Two of the 3 patients tested at 96 hours after initiation of dexamethasone therapy had a suppressed cortisol response at 60 minutes. At 30 minutes, all cortisol levels except 1 were inadequate (<18 micro/dL). CONCLUSION: This study suggests indirectly that the ACTH-ST may be used for the diagnosis of suspected adrenal insufficiency within the first 3 days after initiation of empiric glucocorticoid therapy. An inadequate response of serum cortisol at 60 minutes to the ACTH-ST may be indicative of adrenal insufficiency in this setting. Additional studies with more patients in each therapy time point are needed to confirm these initial results.

Bioactive nanofibers instruct cells to proliferate and differentiate during enamel regeneration.

During tooth development, ectoderm-derived ameloblast cells create enamel by synthesizing a complex protein mixture serving to control cell to matrix interactions and the habit of hydroxyapatite crystallites. Using an in vitro cell and organ culture system, we studied the effect of artificial bioactive nanostructures on ameloblasts with the long-term goal of developing cell-based strategies for tooth regeneration. We used branched peptide amphiphile molecules containing the peptide motif Arg-Gly-Asp, or "RGD" (abbreviated BRGD-PA), known to self-assemble in physiologic environments into nanofibers that display on their surfaces high densities of this biological signal. Ameloblast-like cells (line LS8) and primary enamel organ epithelial (EOE) cells were cultured within PA hydrogels, and the PA was injected into the enamel organ epithelia of mouse embryonic incisors. The expression of amelogenin, ameloblastin, integrin alpha 5, and integrin alpha 6 was detected by quantitative real-time PCR and immunodetection techniques. We performed cell proliferation assay using BrdU labeling and a biomineralization assay using Alizarin red S staining with quantitative Ca(2+) measurements. In the cell culture model, ameloblast-like cells (LS8) and primary EOE cells responded to the BRGD-PA nanostructures with enhanced proliferation and greater amelogenin, ameloblastin, and integrin expression levels. At the site of injection of the BRGD-PA in the organ culture model, we observed EOE cell proliferation with differentiation into ameloblasts as evidenced by their expression of enamel specific proteins. Ultrastructural analysis showed the nanofibers within the forming extracellular matrix, in contact with the EOE cells engaged in enamel formation and regeneration. This study shows that BRGD-PA nanofibers present with enamel proteins participate in integrin-mediated cell binding to the matrix with delivery of instructive signals for enamel formation.

Covalent functionalization of NiTi surfaces with bioactive peptide amphiphile nanofibers.

Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel-titanium (NiTi) shape memory alloy in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO(2) layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO(2) with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM techniques. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers.