An antibody-based molecular switch for continuous small-molecule biosensing.

We present a generalizable approach for designing biosensors that can continuously detect small-molecule biomarkers in real time and without sample preparation. This is achieved by converting existing antibodies into target-responsive "antibody-switches" that enable continuous optical biosensing. To engineer these switches, antibodies are linked to a molecular competitor through a DNA scaffold, such that competitive target binding induces scaffold switching and fluorescent signaling of changing target concentrations. As a demonstration, we designed antibody-switches that achieve rapid, sample preparation-free sensing of digoxigenin and cortisol in undiluted plasma. We showed that, by substituting the molecular competitor, we can further modulate the sensitivity of our cortisol switch to achieve detection at concentrations spanning 3.3 nanomolar to 3.3 millimolar. Last, we integrated this switch with a fiber optic sensor to achieve continuous sensing of cortisol in a buffer and blood with <5-min time resolution. We believe that this modular sensor design can enable continuous biosensor development for many biomarkers.

Diagnostic use of lung ultrasound compared to chest radiograph for suspected pneumonia in a resource-limited setting.

BACKGROUND: Lung ultrasound is an effective tool for diagnosing pneumonia in developed countries. Diagnostic accuracy in resource-limited countries where pneumonia is the leading cause of death is unknown. The objective of this study was to evaluate the sensitivity of bedside lung ultrasound compared to chest X-ray for pneumonia in adults presenting for emergency care in a low-income country. METHODS: Patients presenting to the emergency department with suspected pneumonia were evaluated with bedside lung ultrasound, single posterioranterior chest radiograph, and computed tomography (CT). Using CT as the gold standard, the sensitivity of lung ultrasound was compared to chest X-ray for the diagnosis of pneumonia using McNemar's test for paired samples. Diagnostic characteristics for each test were calculated. RESULTS: Of 62 patients included in the study, 44 (71%) were diagnosed with pneumonia by CT. Lung ultrasound demonstrated a sensitivity of 91% compared to chest X-ray which had a sensitivity of 73% (p = 0.01). Specificity of lung ultrasound and chest X-ray were 61 and 50% respectively. CONCLUSIONS: Bedside lung ultrasound demonstrated better sensitivity than chest X-ray for the diagnosis of pneumonia in Nepal. TRIAL REGISTRATION: ClinicalTrials.gov, registration number NCT02949141 . Registered 31 October 2016.

Characterization of a novel modification of a CHO-produced mAb: Evidence for the presence of tyrosine sulfation.

Herein we describe the investigation of a Chinese hamster ovary (CHO)-expressed human mAb molecule found partially modified by a +80 Da adduct. This mass difference, suggestive of a single sulfation or phosphorylation addition, was observed by mass analysis of the intact and reduced molecule by mass spectrometry (MS). The modification was located on tyrosine 31 (Y31) of the light chain in the complementarity-determining region 1 by liquid chromatography (LC)-MS peptide mapping and electron transfer dissociation fragmentation. The complete loss of the 80 Da modification moiety during collision induced dissociation fragmentation suggested this modification could not be a tyrosine phosphorylation. Treatment of the mAb with alkaline phosphatase confirmed our hypothesis. Western blot experiment using anti-tyrosine sulfation antibody and LC retention time correlation with corresponding synthetic sulfated peptides further confirmed the identification of tyrosine sulfation on the light chain. The unique sequence motif with neighboring acidic amino acids and local secondary structure might play a role to make Y31 a substrate residue for sulfation. This type of modification, to our knowledge, has not been previously reported for CHO-produced human IgG antibodies.

Rapid detection and quantification of apolipoprotein L1 genetic variants and total levels in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Human genetics studies in African Americans have shown a strong correlation between polymorphisms in the ApoL1 gene and chronic kidney disease (CKD). To gain further insight into the etiology of ApoL1-associated kidney diseases, the determination of circulating levels of both wild type as well as ApoL1 variants could be of significant use. To date, antibodies that discriminate between all three ApoL1 variant forms (wild type, G1 and G2) are not available. We aimed to develop a rapid method for detecting and quantifying ApoL1 variants and total levels in plasma. METHODS: Ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) in multiple-reaction monitoring acquisition mode was used to quantify ApoL1. RESULTS: We demonstrated that it is feasible to detect and quantify ApoL1 variants (wild type, G1 and G2), and total ApoL1 concentrations in plasma. ApoL1 genotypes determined by LC/MS agreed perfectly with the traditional method DNA sequencing for 74 human subjects. The method exhibited at least three orders of linearity with a lower limit of quantification of 10 nM. Moreover, the method can readily be multiplexed for the quantification of a panel of protein markers in a single sample. CONCLUSIONS: The method reported herein obviates the need to perform DNA genotyping of ApoL1 variants, which is of significant value in cases where stored samples are unsuitable for DNA analysis. More importantly, the method could potentially be of use in the early identification of individuals at risk of developing CKD, and for the stratification of patients for treatment with future ApoL1-modifying therapies.

A high-throughput automated platform for the development of manufacturing cell lines for protein therapeutics.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics. The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest (1), followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis (2-4). Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup. This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

Repression of cardiac phospholamban gene expression is mediated by thyroid hormone receptor-{alpha}1 and involves targeted covalent histone modifications.

Phospholamban (PLB) is a critical regulator of Ca(2+) cycling in heart muscle cells, and its gene expression is markedly down-regulated by T(3). Nonetheless, little is known about the molecular mechanisms of T(3)-dependent gene silencing in cardiac muscle, and it remains unclear whether thyroid hormone receptors (TRs) directly bind at the PLB gene in vivo and facilitate transcriptional repression. To investigate the regulatory role of TRs in PLB transcription, we used a physiological murine heart muscle cell line (HL-1) that retains cardiac electrophysiological properties, expresses both TRalpha1 and TRbeta1 subtypes, and exhibits T(3)-dependent silencing of PLB expression. By performing RNA interference assays with HL-1 cells, we found that TRalpha1, but not TRbeta1, is essential for T(3)-dependent PLB gene repression. Interestingly, a PLB reporter gene containing only the core promoter sequences -156 to +64 displayed robust T(3)-dependent silencing in HL-1 cells, thus suggesting that transcriptional repression is facilitated by TRalpha1 via the PLB core promoter, a regulatory region highly conserved in mammals. Consistent with this notion, chromatin immunoprecipitation and in vitro binding assays show that TRalpha1 directly binds at the PLB core promoter region. Furthermore, addition of T(3) triggered alterations in covalent histone modifications at the PLB promoter that are associated with gene silencing, namely a pronounced decrease in both histone H3 acetylation and histone H3 lysine 4 methylation. Taken together, our data reveal that T(3)-dependent repression of PLB in cardiac myocytes is directly facilitated by TRalpha1 and involves the hormone-dependent recruitment of histone-modifying enzymes associated with transcriptional silencing.

The combination of electrically stimulated gracilis neoanal sphincter and continent colonic conduit: a step forward for total anorectal reconstruction?

PURPOSE: Patients undergoing total anorectal reconstruction for anorectal atresia or following abdominoperineal resection of the rectum do not fare as well after an electrically stimulated gracilis neoanal sphincter as patients with incontinence alone. This retrospective study reports the outcome for the combination of a continent colonic conduit or antegrade continence enema procedure with an electrically stimulated gracilis neoanal sphincter in patients with atresia or following an abdominoperineal resection of the rectum as part of total anorectal reconstruction to overcome combined incontinence and evacuatory dysfunction. METHODS: Between September 1994 and September 1999, 11 continent colonic conduits were fashioned in 11 patients with an electrically stimulated gracilis neoanal sphincter as part of total anorectal reconstruction for end-stage fecal incontinence. In addition, three patients had an antegrade continence enema procedure in situ, one of which was converted to a colonic conduit at a later stage. Five patients had a colonic conduit fashioned subsequent to an electrically stimulated gracilis neoanal sphincter, four had both procedures in a combined operation, and five had a conduit formed before an electrically stimulated gracilis neoanal sphincter (including the three with an antegrade continence enema procedure). RESULTS: Median follow-up was 53 (range, 7-98) months until July 2002 or until exit from this study group because of end stoma formation (n = 6). Seven patients (50 percent) had a successful outcome, defined as continent to solid and liquid stool. Overall, eight patients (57 percent) reported some degree of improvement in their bowel function and were successfully managed by this combination of procedures. An end stoma was formed in six patients (43 percent). CONCLUSIONS: The combination of antegrade irrigation via a colonic conduit or an antegrade continence enema procedure provides a successful outcome for some patients when incorporated into total anorectal reconstruction, provided that sepsis does not occur, thus avoiding permanent stoma formation. The combination of these procedures may represent an improvement in total anorectal reconstruction and warrants further clinical trial.