An open-source FACS automation system for high-throughput cell biology.

Recent advances in gene editing are enabling the engineering of cells with an unprecedented level of scale. To capitalize on this opportunity, new methods are needed to accelerate the different steps required to manufacture and handle engineered cells. Here, we describe the development of an integrated software and hardware platform to automate Fluorescence-Activated Cell Sorting (FACS), a central step for the selection of cells displaying desired molecular attributes. Sorting large numbers of samples is laborious, and, to date, no automated system exists to sequentially manage FACS samples, likely owing to the need to tailor sorting conditions ("gating") to each individual sample. Our platform is built around a commercial instrument and integrates the handling and transfer of samples to and from the instrument, autonomous control of the instrument's software, and the algorithmic generation of sorting gates, resulting in walkaway functionality. Automation eliminates operator errors, standardizes gating conditions by eliminating operator-to-operator variations, and reduces hands-on labor by 93%. Moreover, our strategy for automating the operation of a commercial instrument control software in the absence of an Application Program Interface (API) exemplifies a universal solution for other instruments that lack an API. Our software and hardware designs are fully open-source and include step-by-step build documentation to contribute to a growing open ecosystem of tools for high-throughput cell biology.

A handheld luminometer with sub-attomole limit of detection for distributed applications in global health.

Luminescence is ubiquitous in biology research and medicine. Conceptually simple, the detection of luminescence nonetheless faces technical challenges because relevant signals can exhibit exceptionally low radiant power densities. Although low light detection is well-established in centralized laboratory settings, the cost, size, and environmental requirements of high-performance benchtop luminometers are not compatible with geographically-distributed global health studies or resource-constrained settings. Here we present the design and application of a ~$700 US handheld, battery-powered luminometer with performance on par with high-end benchtop instruments. By pairing robust and inexpensive Silicon Photomultiplier (SiPM) sensors with a low-profile shutter system, our design compensates for sensor non-idealities and thermal drift, achieving a limit of detection of 1.6E-19 moles of firefly luciferase. Using these devices, we performed two pilot cross-sectional serology studies to assess sars-cov-2 antibody levels: a cohort in the United States, as well as a field study in Bangladesh. Results from both studies were consistent with previous work and demonstrate the device's suitability for distributed applications in global health.

The Evolving Treatment Landscape of Merkel Cell Carcinoma.

OPINION STATEMENT: Merkel cell carcinoma (MCC) has a high risk of recurrence and requires unique treatment relative to other skin cancers. The patient population is generally older, with comorbidities. Multidisciplinary and personalized care is therefore paramount, based on patient preferences regarding risks and benefits. Positron emission tomography and computed tomography (PET-CT) is the most sensitive staging modality and reveals clinically occult disease in ~ 16% of patients. Discovery of occult disease spread markedly alters management. Newly diagnosed, localized disease is often managed with sentinel lymph node biopsy (SLNB), local excision, primary wound closure, and post-operative radiation therapy (PORT). In contrast, metastatic disease is usually treated systemically with an immune checkpoint inhibitor (ICI). However, one or more of these approaches may not be indicated. Criteria for such exceptions and alternative approaches will be discussed. Because MCC recurs in 40% of patients and early detection/treatment of advanced disease is advantageous, close surveillance is recommended. Given that over 90% of initial recurrences arise within 3 years, surveillance frequency can be rapidly decreased after this high-risk period. Patient-specific assessment of risk is important because recurrence risk varies widely (15 to > 80%: Merkelcell.org/recur) depending on baseline patient characteristics and time since treatment. Blood-based surveillance tests are now available (Merkel cell polyomavirus (MCPyV) antibodies and circulating tumor DNA (ctDNA)) with excellent sensitivity that can spare patients from contrast dye, radioactivity, and travel to a cancer imaging facility. If recurrent disease is locoregional, management with surgery and/or RT is typically indicated. ICIs are now the first line for systemic/advanced MCC, with objective response rates (ORRs) exceeding 50%. Cytotoxic chemotherapy is sometimes used for debulking disease or in patients who cannot tolerate ICI. ICI-refractory disease is the major problem faced by this field. Fortunately, numerous promising therapies are on the horizon to address this clinical need.

Magnetic bead-based separation of pneumococcal serotypes.

The separation of pneumococcal serotypes from a complex polymicrobial mixture may be required for different applications. For instance, a minority strain could be present at a low frequency in a clinical sample, making it difficult to identify and isolate by traditional culture-based methods. We therefore developed an assay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. Using qPCR and colony counting methods, we first show that serotypes (12F, 23F, 3, 14, 19A, and 15A) present at ∼0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. We demonstrate two applications for this method: extraction of known pneumococcal serotypes from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. This method may have further laboratory or clinical applications when the selection of specific serotypes is required.

Management and Prognosis of Cardiac Metastatic Merkel Cell Carcinoma: A Case-Control Study and Literature Review.

Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin cancer, has a high rate (20%) of distant metastasis. Within a prospective registry of 582 patients with metastatic MCC (mMCC) diagnosed between 2003-2021, we identified 9 (1.5%) patients who developed cardiac metastatic MCC (mMCC). We compared overall survival (OS) between patients with cardiac and non-cardiac metastases in a matched case-control study. Cardiac metastasis was a late event (median 925 days from initial MCC diagnosis). The right heart was predominantly involved (8 of 9; 89%). Among 7 patients treated with immunotherapy, 6 achieved a complete or partial response of the cardiac lesion. Among these 6 responders, 5 received concurrent cardiac radiotherapy (median 20 Gray) with immunotherapy; 4 of 5 did not have local disease progression or recurrence in the treated cardiac lesion. One-year OS was 44%, which was not significantly different from non-cardiac mMCC patients (45%, p = 0.96). Though it occurs relatively late in the disease course, cardiac mMCC responded to immunotherapy and/or radiotherapy and was not associated with worse prognosis compared to mMCC at other anatomic sites. These results are timely as cardiac mMCC may be increasingly encountered in the era of immunotherapy as patients with metastatic MCC live longer.

Advances in Trace Element Supplementation for Parenteral Nutrition.

Parenteral nutrition (PN) provides support for patients lacking sufficient intestinal absorption of nutrients. Historically, the need for trace element (TE) supplementation was poorly appreciated, and multi-TE products were not initially subjected to rigorous oversight by the United States Food and Drug Administration (FDA). Subsequently, the American Society for Parenteral and Enteral Nutrition (ASPEN) issued dosage recommendations for PN, which are updated periodically. The FDA has implemented review and approval processes to ensure access to safer and more effective TE products. The development of a multi-TE product meeting ASPEN recommendations and FDA requirements is the result of a partnership between the FDA, industry, and clinicians with expertise in PN. This article examines the rationale for the development of TRALEMENT® (Trace Elements Injection 4*) and the FDA's rigorous requirements leading to its review and approval. This combination product contains copper, manganese, selenium, and zinc and is indicated for use in adults and pediatric patients weighing ≥10 kg. Comprehensive management of PN therapy requires consideration of many factors when prescribing, reviewing, preparing, and administering PN, as well as monitoring the nutritional status of patients receiving PN. Understanding patients' TE requirements and incorporating them into PN is an important part of contemporary PN therapy.

Has the Sun Protection Campaign in Australia Reduced the Need for Pterygium Surgery Nationally?

BACKGROUND: The Slip! Slop! Slap! Sunsmart safety campaign was an Australian initiative implemented in the 1980s. To assess this campaign's effect on pterygium, we examined the rate of pterygium surgery across Australia and described the prevalence and associations of pterygium in Perth, Australia's sunniest capital city. METHODS: The rate of pterygium surgery was examined using Australian Medicare data. A cross-sectional analysis of the Generation 1 (Gen1) cohort of the Raine Study was performed to investigate the prevalence of pterygium in Perth. We investigated the association between pterygium and conjunctival ultraviolet autofluorescence (CUVAF) area, an objective biomarker of sun exposure, and demographics and health variables derived from a detailed questionnaire. RESULTS: Between 1994 and 2017, the rate of Medicare funded pterygium surgery in Western Australia fell 11%, well below the national average decline of 47%. Of the 1049 Gen1 Raine Study participants, 994 (571 females; mean age 56.7 years, range = 40.9-81.7) were included in the analysis. The lifetime prevalence of pterygium was 8.4% (n = 83). A higher prevalence of pterygium was associated with outdoor occupation (p-trend = 0.007), male sex (p-trend 0.01) and increasing CUVAF area (p-value <0.001). CONCLUSIONS: The effect of Australia's Slip! Slop! Slap! Sunsmart safety campaign on pterygium been mixed. Since 1994, the rate of private pterygium surgery has declined significantly in all Australian states except Western Australia. Perth, Western Australia, has the highest pterygium prevalence of any mainland-Australian cohort. Higher CUVAF area, male sex, and outdoor occupation were associated with an increased risk of pterygium.

Repurposing blue laser autofluorescence to measure ocular sun exposure.

IMPORTANCE: Excessive ocular sun exposure is linked to various eye pathologies. Conjunctival ultraviolet autofluorescence (CUVAF) is a method of detecting sun-related conjunctival damage; however, the custom-built camera system required is not readily available. BACKGROUND: We investigated whether blue laser autofluorescence (BAF) on a commonly used confocal scanning laser ophthalmoscope (cSLO) can be utilized to measure CUVAF area. DESIGN: Cross-sectional evaluation of a diagnostic technology at a medical research institute. PARTICIPANTS: Sixty-four participants recruited from three on-going observational eye studies in Western Australia. METHODS: All participants had four images, two of each eye, captured using the CUVAF camera and BAF on the same day. Participants with pterygium or poor quality images were excluded from the analysis. Two graders measured CUVAF area in each image twice. CUVAF area measured by BAF was then compared to measurements determined with the conventional camera system. MAIN OUTCOME MEASURES: CUVAF area. RESULTS: After exclusions, 50 participants' images were analysed. Intra- and inter-observer repeatability were similar between the two systems. When comparing CUVAF area measured by BAF to the camera measurement, grader 1 had a mean difference of +1.00 mm2 , with 95% limits of agreement -5.75 to 7.77 mm2 . Grader 2 had a mean difference of +0.21mm2 , with 95% limits of agreement -7.22 to 7.64 mm2 . CONCLUSIONS AND RELEVANCE: BAF on a commercially available cSLO is a valid method for measuring CUVAF area. This finding provides broader opportunity for identifying, monitoring and educating patients with sun-exposure-related ocular conditions and for researching the ocular impacts of sun exposure.

Acoustic Methods for Pulmonary Diagnosis.

Recent developments in sensor technology and computational analysis methods enable new strategies to measure and interpret lung acoustic signals that originate internally, such as breathing or vocal sounds, or are externally introduced, such as in chest percussion or airway insonification. A better understanding of these sounds has resulted in a new instrumentation that allows for highly accurate as well as portable options for measurement in the hospital, in the clinic, and even at home. This review outlines the instrumentation for acoustic stimulation and measurement of the lungs. We first review the fundamentals of acoustic lung signals and the pathophysiology of the diseases that these signals are used to detect. Then, we focus on different methods of measuring and creating signals that have been used in recent research for pulmonary disease diagnosis. These new methods, combined with signal processing and modeling techniques, lead to a reduction in noise and allow improved feature extraction and signal classification. We conclude by presenting the results of human subject studies taking advantage of both the instrumentation and signal processing tools to accurately diagnose common lung diseases. This paper emphasizes the active areas of research within modern lung acoustics and encourages the standardization of future work in this field.

Past, Present, and Future Concepts of the Choroidal Scleral Interface Morphology on Optical Coherence Tomography.

The choroid is the most vascular tissue in the eye and it has been implicated in the pathophysiology of a variety of ocular diseases. A new era of research in the choroid began with the improved ability to visualize this layer and its inner and outer boundaries using spectral domain optical coherence tomography (OCT) with enhanced depth imaging and swept source OCT. The accuracy and precision of qualitative and quan-titative assessments of the choroidal layer support the potential use of OCT-derived choroidal parameters for diagnosis, monitoring of disease progression, planning surgical access, and evaluating treatment response. Although there is increasing interest in measuring choroidal thickness, there is currently no consensus nomenclature to classify choroidal layers and boundaries. Furthermore, the definition and description of the choroidal scleral interface is inconsistent in the literature, contributing to interstudy variation in choroidal thickness measurements. The purpose of this review is to provide an overview of the literature on the definition of choroidal layers and choroidal scleral boundary, review the discrepan-cies, and harmonize the terminology so that a consensus nomenclature can be proposed.

Quantification of sun-related changes in the eye in conjunctival ultraviolet autofluorescence images.

Quantification of sun-related changes in conjunctival ultraviolet autofluorescence (CUVAF) images is a subjective and tedious task, in which reproducibility of results is difficult. Thus, we have developed a semiautomatic method in MATLAB(®) to analyze CUVAF images retrospectively. The algorithm was validated on 200 images from 50 randomly selected participants from the Western Australian Pregnancy Cohort (Raine) study 20-year follow-up assessment, in which CUVAF area measurements were available from previous manual analysis. Algorithm performance was compared to manual measurements and yielded better than 95% correspondence in both intra- and interobserver agreement. Furthermore, the semiautomatic method significantly reduced analysis time by 50%.

Spectral-Domain Optical Coherence Tomography-Derived Characteristics of Bruch Membrane Opening in a Young Adult Australian Population.

PURPOSE: To characterize and quantify Bruch membrane opening (BMO)-based optic nerve head (ONH) parameters in a large, young and healthy, predominantly white population. DESIGN: Cross-sectional study and reliability analysis. METHODS: The ONH of 1344 predominantly white subjects were imaged with spectral-domain optical coherence tomography (SD-OCT). A customized script, coded in Matlab, was used to manually segment and measure multiple BMO-based parameters of the ONH. Measurements were compared to those obtained with confocal scanning laser ophthalmoscopy (Heidelberg Retina Tomograph; HRT). Regression analysis was performed to assess the relationship between BMO parameters and other ocular and demographic variables. RESULTS: Mean BMO disc and neuroretinal rim (NRR) areas ranged from 0.94 to 4.06 mm(2) (mean 1.77 ± 0.38 mm(2)) and 0.94 to 3.99 mm(2) (mean 1.56 ± 0.31 mm(2)), respectively. When compared to the equivalent HRT measurements, SD-OCT-derived measures differed significantly for all comparable ONH parameters (P < .001). The limits of agreement computed from Bland-Altman plots comparing SD-OCT and HRT measurements showed suboptimal agreement between the techniques. Linear regression analysis demonstrated an effect of ethnicity, axial length, and refractive error on BMO-based parameters. CONCLUSIONS: We have quantified BMO-based parameters in a large cohort of young adults using SD-OCT. These data will be informative in constructing normative profiles for clinical and research purposes in glaucoma diagnosis and management.

A computer-assisted method for pathogenicity assessment and genetic reporting of variants stored in the Australian Inherited Retinal Disease Register.

The assignment of pathogenicity to variants suspected of causing an inherited retinal disease and the subsequent creation of molecular genetic reports sent to clinical geneticists and ophthalmologists has traditionally been time-consuming and subject to error and ambiguity. The purpose of this paper is to describe a computer-assisted method we have developed for (1) assessment of the predicted pathogenicity of genetic variants identified in patients diagnosed with an inherited retinal disease and (2) the incorporation of these results into the Australian Inherited Retinal Disease Register and DNA Bank's databases, for the production of molecular genetics reports. This method has significantly accelerated the assessment of variant pathogenicity prediction and subsequent patient report generation for the Australian Inherited Retinal Disease Register and DNA Bank, and has reduced the potential for human error. The principles described in this paper may be applied in any situation where genetic variants and patient information are stored in a well-organised database.

Semi-automated identification of cones in the human retina using circle Hough transform.

A large number of human retinal diseases are characterized by a progressive loss of cones, the photoreceptors critical for visual acuity and color perception. Adaptive Optics (AO) imaging presents a potential method to study these cells in vivo. However, AO imaging in ophthalmology is a relatively new phenomenon and quantitative analysis of these images remains difficult and tedious using manual methods. This paper illustrates a novel semi-automated quantitative technique enabling registration of AO images to macular landmarks, cone counting and its radius quantification at specified distances from the foveal center. The new cone counting approach employs the circle Hough transform (cHT) and is compared to automated counting methods, as well as arbitrated manual cone identification. We explore the impact of varying the circle detection parameter on the validity of cHT cone counting and discuss the potential role of using this algorithm in detecting both cones and rods separately.

Peripheral thyroid hormones and response to selective serotonin reuptake inhibitors.

OBJECTIVE: To examine the relation between baseline measurements of thyroid function and response to selective serotonin reuptake inhibitors (SSRIs) and to consider the effect of these antidepressants on thyroid hormone levels. METHODS: Nineteen subjects with major depression, but without a history of thyroid treatment or lithium treatment, were treated openly with either sertraline or fluoxetine in a university- affiliated tertiary care hospital. Hamilton Depression Rating Scale (Ham-D) scores were measured before and after treatment. Clinical Global Impressions (CGI) scores were measured at study end. Thyroid data, consisting of values for thyroid-stimulating hormone (TSH), triiodothyronine (T(3), measured by radioimmunoassay [RIA]), thyroxine (T(4), measured by RIA) and free T(4), were collected before and after treatment. Complete thyroid data were available for 17 subjects. Data were collected during 1997-1999. RESULTS: Baseline TSH correlated strongly with response to treatment as measured by change in Ham-D scores (r = 0.64, p = 0.003). Low TSH values correlated with greater improvement in depressive symptoms. Thyroid hormone levels decreased with treatment, but these decreases did not correlate with clinical improvement. CONCLUSION: Baseline thyroid function, as measured by serum TSH, may predict a patient's response to antidepressant treatment with SSRIs. Optimal thyroid function, beyond simply being within the normal laboratory values, may be necessary for an optimal response to antidepressants.