SARS-CoV-2 RNAemia Predicts Clinical Deterioration and Extrapulmonary Complications from COVID-19.

BACKGROUND: The determinants of coronavirus disease 2019 (COVID-19) disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterized relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNAemia and disease severity, clinical deterioration, and specific EPCs. METHODS: We used quantitative and digital polymerase chain reaction (qPCR and dPCR) to quantify SARS-CoV-2 RNA from plasma in 191 patients presenting to the emergency department with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterized the role of RNAemia in predicting clinical severity and EPCs using elastic net regression. RESULTS: Of SARS-CoV-2-positive patients, 23.0% (44 of 191) had viral RNA detected in plasma by dPCR, compared with 1.4% (2 of 147) by qPCR. Most patients with serial measurements had undetectable RNAemia within 10 days of symptom onset, reached maximum clinical severity within 16 days, and symptom resolution within 33 days. Initially RNAemic patients were more likely to manifest severe disease (odds ratio, 6.72 [95% confidence interval, 2.45-19.79]), worsening of disease severity (2.43 [1.07-5.38]), and EPCs (2.81 [1.26-6.36]). RNA loads were correlated with maximum severity (r = 0.47 [95% confidence interval, .20-.67]). CONCLUSIONS: dPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Because many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate.

Infected Ruptured Parathyroid Cyst Presenting with Hypercalcemia and Airway Obstruction: A Case Report.

Parathyroid cysts (PC) are infrequently encountered and characterize less than 1% of all head and neck masses. When present, PCs may present as a palpable neck mass and lead to hypercalcemia and rarely respiratory depression. Furthermore, the diagnostics of PCs is difficult as they can masquerade as a thyroid or mediastinal mass given their proximity. PCs are theorized to be a progression of parathyroid adenomas and often routine surgical excision is sufficient for cure. To our knowledge, there is no documented report of a patient with an infected parathyroid cyst that led to severe dyspnea. This case describes our experience of a patient with an infected parathyroid cyst presenting as hypercalcemia and airway obstruction.

A map of evidence using transcranial direct current stimulation (tDCS) to improve cognition in adults with traumatic brain injury (TBI).

Introduction: Cognition impairments often occur after a traumatic brain injury and occur at higher rates in military members. Cognitive symptoms impair daily function, including balance and life quality, years after the TBI. Current treatments to regain cognitive function after TBI, including medications and cognitive rehabilitation, have shown limited effectiveness. Transcranial direct current stimulation (tDCS) is a low-cost, non-invasive brain stimulation intervention that improves cognitive function in healthy adults and people with neuropsychologic diagnoses beyond current interventions. Despite the available evidence of the effectiveness of tDCS in improving cognition generally, only two small TBI trials have been conducted based on the most recent systematic review of tDCS effectiveness for cognition following neurological impairment. We found no tDCS studies that addressed TBI-related balance impairments. Methods: A scoping review using a peer-reviewed search of eight databases was completed in July 2022. Two assessors completed a multi-step review and completed data extraction on included studies using a priori items recommended in tDCS and TBI research guidelines. Results: A total of 399 results were reviewed for inclusion and 12 met the criteria and had data extracted from them by two assessors using Google Forms. Consensus on combined data results included a third assessor when needed. No studies using tDCS for cognition-related balance were found. Discussion: Guidelines and technology measures increase the identification of brain differences that alter tDCS effects on cognition. People with mild-severe and acute-chronic TBI tolerated and benefited from tDCS. TBI-related cognition is understudied, and systematic research that incorporates recommended data elements is needed to advance tDCS interventions to improve cognition after TBI weeks to years after injury.

Microfluidic array for three-dimensional perfusion culture of human mammary epithelial cells.

The ability to culture cells in three dimensional extracellular matrix (3D ECM) has proven to be an important tool for laboratory biology. Here, we demonstrate a microfluidic perfusion array on a 96-well plate format capable of long term 3D ECM culture within biomimetic microchambers. The array consists of 32 independent flow units, each with a 4 µl open-top culture chamber, and 350 µl inlet and outlet wells. Perfusion is generated using gravity and surface tension forces, allowing the array to be operated without any external pumps. MCF-10A mammary epithelial cells cultured in Matrigel in the microfluidic array exhibit acinus morphology over 9 days consistent with previous literature. We further demonstrated the application of the microfluidic array for in vitro anti-cancer drug screening.

Post-discharge interventions to enhance coping skills for survivors of stroke and their caregivers: a scoping review protocol.

OBJECTIVE: This scoping review aims to map the use of post-discharge interventions to enhance coping skills for adult stroke survivors and their informal caregivers. INTRODUCTION: Evidence shows that when stroke survivors and their informal caregivers are provided with post-discharge coping training, their outcomes improve. There is also evidence that healthcare providers currently focus on medication compliance and deliver little to no instruction on post-discharge coping interventions. As a result, more than 40% of stroke survivors and informal caregivers report a lack of awareness and access to community-based coping training once discharged from the hospital setting. INCLUSION CRITERIA: This scoping review will map the delivery modes, intervention types, locations, leadership and settings of current coping interventions for community-dwelling adults 18 years or older living with the effects of stroke and their informal caregivers. This review will be restricted to studies published in English that include interventions that are not reimbursed as part of United States healthcare coverage. METHODS: The search will include CINAHL, Cochrane Library, Embase, JBI Evidence Synthesis, MEDLINE, PEDro, PLoS, PsycINFO, and Web of Science. In addition, smaller health-related databases, stroke and rehabilitation journals, gray literature and stakeholder organization websites will be searched. A three-step search strategy will be utilized, including use of citation software to manage search results and de-duplication, abstract review and full-text review by two reviewers. Details of included studies will then be imported into information management software. The resulting map will be displayed in tabular form along with a narrative summary.

Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument.

Digital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.

SARS-CoV-2 RNAaemia predicts clinical deterioration and extrapulmonary complications from COVID-19.

Background: The determinants of COVID-19 disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterise the relationships between SARS-CoV-2 RNAaemia and disease severity, clinical deterioration, and specific EPCs. Methods: We used quantitative (qPCR) and digital (dPCR) PCR to quantify SARS-CoV-2 RNA from nasopharyngeal swabs and plasma in 191 patients presenting to the Emergency Department (ED) with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterised the role of RNAaemia in predicting clinical severity and EPCs using elastic net regression. Findings: 23·0% (44/191) of SARS-CoV-2 positive patients had viral RNA detected in plasma by dPCR, compared to 1·4% (2/147) by qPCR. Most patients with serial measurements had undetectable RNAaemia 10 days after onset of symptoms, but took 16 days to reach maximum severity, and 33 days for symptoms to resolve. Initially RNAaemic patients were more likely to manifest severe disease (OR 6·72 [95% CI, 2·45 - 19·79]), worsening of disease severity (OR 2·43 [95% CI, 1·07 - 5·38]), and EPCs (OR 2·81 [95% CI, 1·26 - 6·36]). RNA load correlated with maximum severity (r = 0·47 [95% CI, 0·20 - 0·67]). Interpretation: dPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAaemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Since many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAaemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate.

Dynamic cell culture: a microfluidic function generator for live cell microscopy.

We present a microfluidic system for time-lapsed, live cell microscopy with the ability to control solution exchange via a dynamic flow controller. The application specific microfluidic plates are designed to maintain adherent and non-adherent cell types for multiple days with continuous medium perfusion. Upstream channels with flow controlled via custom software allow the delivery of unique exposure profiles to the cultured cells, such as square waves, step functions, ramps, etc.

Precision cancer monitoring using a novel, fully integrated, microfluidic array partitioning digital PCR platform.

A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform's utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient's response to therapy and show the patient's achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring.

A microfluidic system for dynamic yeast cell imaging.

The investigation of cellular processes and gene regulatory networks within living cells requires the development of improved technology for dynamic, single cell imaging. Here, we demonstrate a microfluidic system capable of mechanical trapping of yeast cells with continuous flow and flow switching capability during time-lapse high magnification fluorescence imaging. The novel functionality of the system was validated by observing the response of pheromone-induced expression of GFP in Saccharomyces cerevisiae.

Microfluidic tissue model for live cell screening.

We have developed a microfluidic platform modeled after the physiologic microcirculation for multiplexed tissue-like culture and high-throughput analysis. Each microfabricated culture unit consisted of three functional components: a 50 microm wide cell culture pocket, an artificial endothelial barrier with 2 microm pores, and a nutrient transport channel. This configuration enabled a high density of cancer cells to be maintained for over 1 week in a solid tumor-like morphology when fed with continuous flow. The microfluidic chip contained 16 parallel units for "flow cell" based experiments where live cells were exposed to a soluble factor and analyzed via fluorescence microscopy or flow-through biochemistry. Each fluidically independent tissue unit contained approximately 500 cells fed with a continuous flow of 10 nL/min. As a demonstration, the toxicity profile of the anti-cancer drug paclitaxel was collected on HeLa cells cultured in the microfluidic format and compared with a 384-well dish for up to 5 days of continuous drug exposure.

Open-access microfluidic patch-clamp array with raised lateral cell trapping sites.

A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.

A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array.

We present a high aspect ratio microfluidic device for culturing cells inside an array of microchambers with continuous perfusion of medium. The device was designed to provide a potential tool for cost-effective and automated cell culture. The single unit of the array consists of a circular microfluidic chamber 40 microm in height surrounded by multiple narrow perfusion channels 2 microm in height. The high aspect ratio (approximately 20) between the microchamber and the perfusion channels offers advantages such as localization of the cells inside the microchamber as well as creating a uniform microenvironment for cell growth. Finite element methods were used to simulate flow profile and mass transfer of the device. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C and was grown to confluency. The microfluidic cell culture array could potentially offer an affordable platform for a wide range of applications in high throughput cell-based screening, bioinformatics, synthetic biology, quantitative cell biology, and systems biology.

On-chip cell lysis by local hydroxide generation.

We present a novel method for on-chip cell lysis based on local hydroxide electro-generation. Hydroxide ions porate the cell membrane, leading to cell lysis. After lysis occurs, hydrogen ions, also generated on chip, react with excess hydroxide, creating a neutral pH lysate and eliminating the need for a wash step. Three different cell types are shown to be effectively lysed by this method: red blood cells, HeLa (human tumor line) and Chinese Hamster Ovary (CHO) cell lines. The release of cytoplasmic molecules from HeLa and CHO cells is demonstrated by monitoring the escape of a membrane impermeant dye from the cytoplasm. In the vicinity of the cathode, the hydroxide concentration is predicted by finite element simulations and shown to fit the lysis rates at different distances from the generating cathode. For flow-through experiments, a second device integrating a mechanical filter with hydroxide generation is fabricated and tested. The purpose of the filter is to trap whole cells and only allow lysate to pass through. The flow rate dependence of hydroxide concentration at the lysis filter is modeled and lysis efficiency is experimentally determined to be proportional to the hydroxide concentration for flow rates from 15 to 30 microl min(-1).

Immunizations, immunology, and autism.

Public fears of rising rates of children being diagnosed with autistic spectrum disorders has led to a fear that immunizations, specifically the measles-mumps-varicella vaccine (MMR), may trigger autism. This article reviews theories of immunization as a risk factor for autism, including thimerosal exposure. We also review theories of autoimmunity as a predisposing genetic risk in autistic patients. We summarize from multiple population-based studies and extensive review committee reports that neither immunization nor thimerosal exposure has been conclusively linked to autism. Current treatments for autoimmunity in autism are reviewed and summarized as being only anecdotally effective, with no controlled studies to conclusively determine effectiveness. The goal of this article is to allow child neurologists to effectively counsel parents of autistic patients about vaccination risks and treatment options in presumed cases of autoimmune dysfunction.

Neurologic treatment strategies in autism: an overview of medical intervention strategies.

Child neurologists are likely to be caring for an increasing number of patients with autistic spectrum disorder (ASD). ASD may occur in as many as 1/100 to 1/200 births. It appears to be a multifactorial disease, with many phenotypes or subgroups. No simple treatment is currently approved for curing or managing core symptoms of autism. We rationally propose a symptom-based review of what treatments may offer relief to specific subtypes of clinical behaviors seen in autism. There is a lack of clinically based evidence on which to universally recommend a rational clinical algorithm for treatment; we suggest that rational pharmacotherapy may offer symptomatic relief to core areas of dysfunction in the autistic population. Future research into rational medical treatment options is desperately needed.

Microfluidic systems for live cell imaging.

Microfluidic systems provide many advantages for live cell imaging, including improved cell culture micro-environments, control of flows and dynamic exposure profiles, and compatibility with existing high resolution microscopes. Here, we will discuss our approach for design and engineering of microfluidic cell culture environments as well as interfacing with standard laboratory tools and protocols. We focus on an application specific design concept, whereby a shared fabrication process is used to deliver multiple products for different biological applications. As adoption of advanced in vitro models increases, we envision the use of microfluidic cell culture technology to become commonplace.

Microfluidic environment for high density hepatocyte culture.

We present a microfluidic bioreactor for culturing high-density arrays of hepatocytes in a tissue-like micro-architecture. The microfluidic environment mimicked physiological liver mass transport, enabling sustained culture of high density cells (>2,000 cells/mm(2)) without nutrient limitation for over 1 week. The key feature of this design was a microporous microfluidic barrier that formed a sieved-pocket to concentrate cells during loading. Nutrient depletion within the cell mass was avoided by maintaining a continuous flow of medium (10 microl/day) that diffused across the porous barrier. Human hepatoma cells (HepG2/C3A) remained viable and functional as demonstrated by fluorescent viability assays and secretion of albumin for the one-week culture period.

An artificial liver sinusoid with a microfluidic endothelial-like barrier for primary hepatocyte culture.

Primary hepatocytes represent a physiologically relevant model for drug toxicity screening. Here, we created a biologically inspired artificial liver sinusoid with a microfluidic endothelial-like barrier having mass transport properties similar to the liver acinus. This unit consisted of a cord of hepatocytes (50 x 30 x 500 microm) fed by diffusion of nutrients across the microfluidic endothelial-like barrier from a convective transport vessel (10 nL/min). This configuration sustained rat and human hepatocytes for 7 days without an extracellular matrix (ECM) coating. Experiments with the metabolism mediated liver toxicant diclofenac showed no hepatotoxicity after 4 h and an IC(50) of 334 +/- 41 microM after 24 h.

Microfluidic System for Automated Cell-based Assays.

Microfluidic cell culture is a promising technology for applications in the drug screening industry. Key benefits include improved biological function, higher quality cell-based data, reduced reagent consumption, and lower cost. In this work, we demonstrate how a microfluidic cell culture design was adapted to be compatible with the standard 96-well plate format. Key design features include the elimination of tubing and connectors, the ability to maintain long term continuous perfusion cell culture using a passive gravity driven pump, and direct analysis on the outlet wells of the microfluidic plate. A single microfluidic culture plate contained 8 independent flow units, each with 10(4) cells at a flow rate of 50 µl/day (6 minute residence time). The cytotoxicity of the anti-cancer drug etoposide was measured on HeLa cells cultured in this format, using a commercial lactate dehydrogenase (LDH) plate reader assay. The integration of microfluidic cell culture methods with commercial automation capabilities offers an exciting opportunity for improved cell-based screening.