Alternative Assay Research Articles

Fructosamine as an Indicator forGlycemic Control inthe Veteran Community.

Glycemic monitoring via Hemoglobin A1(HbA1c) proves to be inaccurate when a patient is diagnosed with a hemoglobinopathy/erythrocyte disorder. Moreover, any acute changes of glycemic intake within 6 weeks of blood sampling have been noted to impart a greater effect on HbA1c than the remaining days of the supposed overall 3-to-4-month average of glycemic control. Fructosamine, an alternative to HbA1c, allows physicians to analyze glycemic control in the presence of an underlying hemoglobinopathy/erythrocyte disorder. Fructosamine analyzes glycemic control over a two-to-three week period, compared to a 3-to-4-month period with HbA1c. Studies indicate that fructosamine is significantly correlated with HbA1c in various population types. Given the increased likelihood of hemaglobinopathies/erythrocyte disorders in the veteran population accompanied by the the necessity for tighter glycemic evaluation, fructosamine offers an assay for physician when analyzing glycemic control in veterans. The aim of our study was to evaluate the correlation of HbA1c and fructosamine in the veteran community. We hypothesize that fructosamine is significantly correlated with HbA1c in the veteran community and therefore can be used as an alternative assay in the Veterans Affairs. Following institutional review board review and exemption, a retrospective chart review was conducted at the Northport Veterans Affairs between the dates of December 2007 and December 2018. Inclusion criteria consisted of veterans above the age of 18 years with a HbA1c and fructosamine assay drawn from the same blood sample. Exclusion criteria consisted of veterans with an underlying hemoglobinopathy, erythrocyte disorder, or serum protein altering pathology. Linear regression analysis was conducted between HbA1c and fructosamine values. Linear regression equation, correlation coefficient, and coefficient of variance were reported. A paired t-test was conducted between assay HbA1c and predicted HbA1c from the determined regression equation. A total of 156 veterans were included in this study. A correlation coefficient and coefficient of variance of 0.75 and 0.56 were calculated from regression analysis (P < 0.05). A regression equation of the following was determined: HbA1c{%} = 0.0154*Fructosamine (mmol/L)+ 3.121. Paired t-test of assay HbA1c (M = 8.17, standard deviation = 1.64) and predicted HbA1c (M = 8.21, standard deviation = 1.53) returned without significance for difference of paired values. Given that both correlation coefficient and coefficient of variance were considered significantly strong and the paired t-test indicated no significant difference between assay HbA1c and predicted HbA1c, fructosamine can be considered as an alternative assessment of glycemic control compared to HbA1c in the veteran community. We advise physicians at the Veterans Affairs to use fructosamine as an assessment of glycemic control when a veteran is diagnosed with an underlying hemoglobinopathy/erythrocyte disorder or requires stringent analysis of glycemic control. Yet, at this point of time, fructosamine does not have the necessary data to indicate complete replacement of HbA1c with fructosamine.

Development of a loop-mediated isothermal amplification method for the rapid detection of Clostridium botulinum serotypes E and F.

Botulinum neurotoxin serotypes E and F (BoNT/E and BoNT/F) produced by the bacteria Clostridium botulinum (C. botulinum) found in a wide variety of foods cause poisoning in humans with high mortality rates. Mouse bioassays (MBAs), the gold standard method for BoNT detection, have a low detection limit; however, require experienced personnel and take a long time to obtain results. Therefore, it has been gradually replaced by nucleic acid amplification tests (NAATs) with primers targeting species-specific genes. In this study, for each serotype E and F, six LAMP primers were designed based on multiple sequence alignments of the conserved regions of the bont/E and bont/F genes collected from 180 serotype E strains and 23 serotype F strains published in NCBI. In silico PCR amplification with the outer primer pairs showed successful amplification of the target fragments. To validate the LAMP method, we constructed two synthetic plasmids containing the target sequences extended approximately 10-50bp to both ends. The specificity of the primers was further evaluated using six different Clostridium species and eight strains belonging to other common food poisoning-related bacterial species. Employing the synthetic plasmids, the optimal temperatures and limits of detection (LODs) were determined for bont/E (63°C, LOD ≤ 101 copies/reaction) and bont/F (65°C, LOD ≤ 102 copies/reaction) within 30min. In addition, the LAMP primer set for BoNT/F was redesigned with degenerate nucleotides that improved the coverage from 15 to 45%. For future directions, applications of the established method, especially with the degenerate primers, could be used as an alternative assay for the rapid and sensitive detection of C. botulinum.

Perforation of the host cell plasma membrane during Toxoplasma gondii invasion requires rhoptry exocytosis.

Toxoplasma gondii is an obligate intracellular parasite, and the delivery of effector proteins from the parasite into the host cell during invasion is critical for invasion itself and for parasite virulence. The effector proteins are released from specialized apical secretory organelles known as rhoptries. While much has been learned recently about the structure and composition of the rhoptry exocytic machinery and the function of individual rhoptry effector proteins that are exocytosed, virtually nothing is known about how the released proteins are translocated across the host cell plasma membrane. Previous electrophysiology experiments reported an unanticipated observation that invasion by T. gondii is preceded by a transient increase in host cell plasma membrane conductance. Here, we confirm this electrophysiological observation and propose that the conductance transient represents a parasite-induced perforation in the host cell plasma membrane through which rhoptry proteins are delivered. As a first step towards testing this hypothesis, and to provide higher throughput than patch clamp electrophysiology, we developed an alternative assay to detect the perforation. This assay utilizes high-speed, multi-wavelength fluorescence imaging to enable simultaneous visualization of host cell perforation and parasite invasion. Using this assay, we interrogated a panel of mutant parasites conditionally depleted of key invasion-related proteins. Parasites lacking signaling proteins involved in triggering rhoptry secretion ( e.g. , CLAMP) or components of the rhoptry exocytic machinery ( e.g. , Nd9, RASP2) are defective in their ability to induce the perforation. These data are consistent with a model in which the perforating agents that disrupt host cell membrane integrity during invasion - and may thereby provide the conduit for delivery of rhoptry effector proteins - are stored within the rhoptries themselves and released upon contact with the host cell.

Clinical and experimental evidence for differences between cardiac troponin I and T in acute and chronic cardiovascular conditions

Abstract Background Clinical and scientific practice operate under the assumption that systemic levels of cardiac troponin I (cTnI) and troponin T (cTnT) are indicative of the same underlying pathophysiological mechanisms and can be utilized interchangeably. Purpose To assess cTnI and cTnT levels and their ratio for differences in various cardiovascular conditions. Methods Individuals from three prospective clinical studies with a centrally adjudicated diagnosis were categorized into three groups: no known cardiac disease, chronic cardiac disease, and acute cardiac disease. Circulating cTnI and cTnT concentrations were measured using high-sensitivity assays (hs-cTnI-Architect, hs-cTnT-Elecsys) and the cTnI/cTnT ratio was calculated. Findings were validated using a second hs-cTnI assay (hs-cTnI-Clarity) and externally validated in independent cohorts. The cTnI/cTnT ratio was also assessed in five different non-lethal and lethal experimental models of cardiomyocyte injury. Results Of the 9704 included individuals, 9% had no known cardiac disease, 69% chronic cardiac disease, and 22% acute cardiac disease. No known cardiac disease and chronic cardiac disease were associated with a disproportional increase in cTnT compared to cTnI (cTnI/cTnT ratio, median 0.50, IQR 0.38-0.69 and 0.53, IQR 0.37-0.80). In contrast, cTnI concentrations were increased compared to cTnT in patients with acute cardiac disease, resulting in a cTnI/cTnT ratio of 1.62 (IQR 0.77-4.31). Findings were consistent using an alternative cTnI assay and in external validation cohorts (n=2776) where the cTnI/cTnT ratio was lower in those without cardiac disease (0.33 (IQR 0.25-0.50), 0.64 (IQR 0.40-1.05)) and with chronic cardiac disease (0.50 (IQR 0.32-0.75), 0.60 (IQR 0.38-1.00)), compared to those with acute cardiac disease (2.22 (IQR 1.08-5.27), 2.28 (IQR 1.01-5.67)). Non-lethal cardiomyocyte injury models showed a predominant release of cTnT with cTnI/cTnT ratios &amp;lt;0.5, and lethal injury models a predominant release of cTnI with cTnI/cTnT ratios &amp;gt;1. Conclusion Differences in cTnI and cTnT levels were observed in individuals with chronic and acute cardiac conditions, leading to distinct cTnI/cTnT ratios in both clinical and experimental settings. As a result, the interchangeability of cTnI and cTnT in present-day clinical and scientific settings needs to be reevaluated.Central illustration

Concordance Between Platelet Expression Assay (PEA) and Serotonin Release Assay (SRA) in Heparin-Induced Thrombocytopenia: Implications for Diagnostic Accuracy and Laboratory Test Utilization

Abstract Introduction The Serotonin Release Assay (SRA) serves as the gold standard functional assay for heparin-induced thrombocytopenia (HIT) diagnosis at our institution. The present turnaround time (TAT) for SRA results ranges from 4-5 days, occasionally extending beyond 8 days, given outsourcing to a reference laboratory. Recently, an alternative functional assay, the Platelet Expression Assay (PEA), has been introduced. The PEA detects platelet activation through p-selectin expression by flow cytometry, with a reported sensitivity of 100% and specificity of 95% for HIT. The PEA is also a send-out test, and thus has similar TAT and cost profiles as the SRA, but with potential to transition into an in-house assay in the future. Herein, we sought to determine the concordance of the PEA and SRA for the detection and diagnosis of HIT within our patient population. Methods A retrospective review was conducted on all cases in which a HIT ELISA (screening immunoassay) was performed by the Special Coagulation Laboratory during a one-year period. A 4Ts score was calculated if not already documented by chart review. In a subset analysis, specimens with a positive screening ELISA were concurrently tested by both SRA and PEA to compare performance. Results Of the 1341 cases, a 4Ts score was calculated and found to be less than 4 for 30-50% of cases. A total of 490 cases were sent out for SRA, of which 49 (10%) returned positive results. In a subset analysis of 20 samples from 14 patients, all with positive ELISAs, results revealed concordance between the SRA and PEA for all but one sample (19/20, 95%). Specifically, three successive samples from one patient had [negative, negative, positive] results for SRA, and [negative, positive, positive] results for PEA, respectively. This discordant result suggests a potential difference in sensitivity between the two assays. Further subset analysis focused on an additional 13 samples from 10 patients, all with SRA-positive results. Only 8 samples were concordant (8/13, 62%). One case showed a negative PEA and presumed false positive SRA, given the clinical history, while 4 samples showed likely true positive SRA results, given strength of ELISA results and 4Ts score, but were negative by PEA. Conclusions The lack of adherence to the 4Ts score algorithm to drive laboratory test utilization resulted in unnecessary ELISA and functional assay testing, potentially resulting in increased costs and false positive results. This result underscores the need for vigilance in assessing and refining diagnostic strategies. Additionally, our discordant findings in the subset analysis underscore the importance of evaluating the concordance and discordance between SRA and PEA results, providing insights into the diagnostic accuracy and potential limitations of each assay in identifying HIT.

8307 Molecularly Targeted Therapy in Oncogene-Driven Human Thyroid Cancer Cell Models: Assay Selection and Response Assessment

Abstract Disclosure: A.T. Yang: None. T.W. Laetsch: Consulting Fee; Self; Cellectis, Deciphera, GentiBio, Jazz Pharmaceuticals, Jumo Health, MassiveBio, Novartis Pharmaceuticals, Pyramid Biosciences, Y-mAbs Therapeutics, Advanced Microbubbles, AI Therapeutics, Bayer, Inc.. A.T. Franco: None. Background: The most common types of pediatric thyroid cancer (TC) demonstrate kinase-activating genomic alterations that are clinically inhibitor responsive – however, optimal integration of molecular therapy into practice remains uncertain. Human TC cell models can facilitate evaluation of combination therapies. However, prior studies vary from using traditional cell counts to newer reaction-based assays; we assessed whether a linear relationship between cell count and alternative assays exist for human TC cell models. Methods: Standardized curves for Cell TiterGlo (CTG; measures total ATP using luciferase-based luminescence) and NucBlue (DAPI; binds to DNA with nuclear fluorescence) were generated using TC cells seeded at escalating concentrations (5k to 300k cells/well). A linear equation was fitted to the CTG data and used to estimate cell count. DAPI counts were obtained with EVOS automation. Human TC cell lines TPC1 (RET fusion) / CUTC5 (BRAF V600E) were seeded at low (1.5k/2k) and high (3k/4k) cells per well, exposed for 3 days to 0-256 nM selpercatinib (RET inhibitor) or 0-192 nM dabrafenib (BRAF inhibitor), respectively, and response was measured using CTG and DAPI. All were measured in triplicate. Results: Standardized curves of CTG and DAPI demonstrated a 9-23% difference between known and measured cell counts. CTG was accurate within 20k-40k cells/well; DAPI showed linearity within 10k-80k cells/well. TC cells exhibited different IC50 depending on the assay and seeding concentration. Prior studies for selpercatinib and dabrafenib reported in vitro IC50 of 0.4-67 nM and 0.5-6 nM, respectively. In this study, IC50 for CTG/DAPI were closer at higher cell counts: TPC1 was 6.1 / 8.2 nM respectively; CUTC5 was 10.6 / 5.1 nM. Wells seeded at lower counts demonstrated larger inter-assay difference: IC50 for TPC1 was 10.1 / 5.9 nM respectively; CUTC5 was 38.4 / 5.9 nM. Conclusion: IC50 values varied by cell seeding concentrations and assay used, with a two to six fold difference at lower seeding and loss of linearity between luminescence measurements and cell count. This variability complicates evaluating the concordance among data from different laboratories, and in vitro and clinical response. Alternative assays intended to assess viability of inhibitor-exposed human TC cell models may require additional evaluation to ensure measured values demonstrate the expected relationship to the intended measure (ie cell number) and ultimately clinical outcome. Presentation: 6/3/2024

Dog complement system is less effective against Leishmania infantum than human complement

Dogs are important reservoir hosts for Leishmania infantum, the causative agent of visceral leishmaniasis. The complement system, as part of the innate immune defense, is responsible for initiating the fight against pathogens that may invade an organism. A failure of the complement to combat L. infantum may explain, at least in part, why a mammal species is more or less susceptible to visceral leishmaniasis. The objective of this study was to compare the effectiveness of human and dog complement systems against L. infantum parasites. The results showed that dog serum was less effective than human serum at killing promastigote and amastigote-like forms. We also compared the efficiency of human and canine sera in classic and alternative hemolytic assays, as well as the serum efficiency of non-infected and Leishmania-infected dogs. Serum from dogs was less hemolytic than human serum in both pathways tested, but the efficiency of serum from infected dogs was higher than that of non-infected dogs. When testing C3b deposition assays on parasite surfaces, serum from infected dogs was more effective against amastigote-like forms than serum from non-infected dogs. However, both types of serum proved equally effective on promastigotes, while serum from infected dogs was more effective on amastigote-like forms. Considering the efficiency of the complement system, our results indicate that dogs are more susceptible to visceral leishmaniasis than humans are.

Hirocidins, Cytotoxic Metabolites from Streptomyces hiroshimensis, Induce Mitochondrion-Mediated Apoptosis.

Recent advances in whole genome sequencing have revealed an immense microbial potential for the production of therapeutic small molecules, even from well-known producers. To access this potential, we subjected prominent antimicrobial producers to alternative antiproliferative assays using persistent cancer cell lines. Described herein is our discovery of hirocidins, novel secondary metabolites from Streptomyces hiroshimensis with antiproliferative activities against colon and persistent breast cancer cells. Hirocidin A is an unusual nine-membered carbocyclic maleimide and hirocidins B and C are relatives with an unprecedented, bridged azamacrocyclic backbone. Mode of action studies show that hirocidins trigger mitochondrion-dependent apoptosis by inducing expression of the key apoptotic effector caspase-9. The discovery of new cytotoxins contributes to scaffold diversification in anticancer drug discovery and the reported modes of action and concise total synthetic route for variant A set the stage for unraveling specific targets and biochemical interactions of the hirocidins.

A single-layer spot assay for easy, fast, and high-throughput quantitation of phages against multidrug-resistant Gram-negative pathogens.

Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.

New approach methodologies to confirm developmental toxicity of pharmaceuticals based on weight of evidence

The aim of embryo-fetal developmental toxicity assessments for pharmaceuticals is to inform potential risk of adverse pregnancy outcome, which has traditionally relied on studies in pregnant animals. Recent updates to international safety guidelines (ICH S5R3) have incorporated information on how to use weight of evidence and alternative assays to reduce animal use while still informing risk of fetal harm. Uptake of these alternative approaches has been slow due to limitations in understanding how alternative assays translate to in vivo effects and then relevance to human exposure. To understand the predictivity of new approach methodologies for developmental toxicity (DevTox NAMs), we used two pharmaceutical examples (glasdegib and lorlatinib) to illustrate the value of DevTox NAMs to complement weight of evidence (WoE) assessments while considering the relationship of concentration-effect levels in NAMs to in vivo studies. The in vitro results generated in a battery of assays (mEST, rWEC, zebrafish, and human based stem cells) confirmed the WoE based on literature and further confirmed by preliminary embryo-fetal development data. The data generated for these two compounds supports integrating DevTox NAMs into the developmental toxicity assessment for advanced cancer indications.

Hirocidins, Cytotoxic Metabolites from Streptomyces hiroshimensis, Induce Mitochondrion‐Mediated Apoptosis

AbstractRecent advances in whole genome sequencing have revealed an immense microbial potential for the production of therapeutic small molecules, even from well‐known producers. To access this potential, we subjected prominent antimicrobial producers to alternative antiproliferative assays using persistent cancer cell lines. Described herein is our discovery of hirocidins, novel secondary metabolites from Streptomyces hiroshimensis with antiproliferative activities against colon and persistent breast cancer cells. Hirocidin A is an unusual nine‐membered carbocyclic maleimide and hirocidins B and C are relatives with an unprecedented, bridged azamacrocyclic backbone. Mode of action studies show that hirocidins trigger mitochondrion‐dependent apoptosis by inducing expression of the key apoptotic effector caspase‐9. The discovery of new cytotoxins contributes to scaffold diversification in anticancer drug discovery and the reported modes of action and concise total synthetic route for variant A set the stage for unraveling specific targets and biochemical interactions of the hirocidins.

In vitro Generated Megakaryocytes for the Detection of Human Platelet Antigen-Specific Alloantibodies

Plain Language SummaryThe identification of antibodies to human platelet antigens (HPA) is crucial. The standard test (MAIPA assay) is difficult because it requires fresh platelets from specific donors. This study investigated a new method, monoclonal antibody immobilization of megakaryocyte antigens (MAIMA), using in vitro generated megakaryocytes from CD34+ cells. The assay was tested against WHO reference reagents and patient samples with known anti-HPA antibodies. Results showed that MAIMA successfully detected anti-HPA-1a antibodies and correlated well with MAIPA. However, MAIMA had lower sensitivity for anti-HPA-3a and anti-HPA-5b antibodies. Notably, anti-HPA-5b antibodies were not detectable by MAIMA. In conclusion, in vitro generated megakaryocytes can effectively detect anti-HPA-1a antibodies. However, MAIMA may be less suitable for antibodies against other HPAs, such as HPA-5b. This study highlights the potential of MAIMA as an alternative assay and provides insight into its strengths and limitations in detecting specific anti-HPA antibodies.

Conventional PCR-based versus next-generation sequencing-based approach for T-cell receptor γ gene clonality assessment in mature T-cell lymphomas: A phase 3 diagnostic accuracy study.

Clonality assessment is currently the major molecular analysis utilized to support the diagnosis of suspicious lymphoid malignancies. Clonal rearrangements of the V-J segments of T-cell receptor G chain locus (TCRγ or TRG) have been observed in almost all types of T neoplasms, such as T-cell-related non-Hodgkin lymphomas and leukemias. At present, the gold standard for clonality evaluation is multiplex polymerase chain reaction (PCR), plus subsequent capillary electrophoresis/heteroduplex analyses, and/or Sanger sequencing. This approach overcomes the problem with the conventional Southern blot hybridization and is more efficient, simple, fast, and reproducible. In the recent years, the new next-generation sequencing (NGS) technologies provided alternative techniques for the analysis of antigen receptors genes, which presented several advantages, such as increased efficiency, specificity (SP), sensitivity (ST), resolution, and objectivity of the results, leading to a better classification, stratification, and monitoring of lymphoid malignancies. Nonetheless, these technologies are still far from being the new gold standard since further studies are warranted to prove their utility. The present study aimed to assess the diagnostic accuracy of these two methods by comparing a commercial NGS-based assay for the evaluation of TRG locus with the gold standard PCR-based one, to fulfill the requirements of a phase 3 diagnostic accuracy study. We assessed the TRG gene rearrangements in 72 cases using the conventional and highly-validated PCR-based assay proposed by EuroClonality consortium, an alternative commercial PCR-based assay, namely, IdentiClone® TCR Gamma Gene Rearrangement Assay 2.0, and a commercial NGS-based assay, that is, Invivoscribe LymphoTrack® Dx MiSeq® (both by Invivoscribe Technologies Inc., San Diego, CA, USA), to determine the diagnostic accuracy of the latter, and compare them with reference diagnoses made based on observation of clinical manifestations, cytohistological, and immunohistochemical analyses. Statistical values were calculated using the Oxford CATmaker software package. Using standardized criteria of interpretation, the obtained results showed a diagnostic accuracy of 90.3% (correspondence in 65 out of 72 cases) of the test under investigation, with a ST of 86%, a SP of 95%, a positive predicting value of 94%, and a negative predicting value of 88%, demonstrating that it had high efficiency and reliability in detecting clonal TRG gene rearrangements in T-cell non-Hodgkin lymphomas. This diagnostic accuracy study yielded comparable results using a validated PCR-based approach and a new NGS-based one. Subsequent studies and cost-effectiveness evaluation are needed to put the NGS-based clonality assessment into routine diagnostic practice.

Structure-optimized iridium complex coordination-functionalized ZIF-8-based nanoemitters with HEPES as novel efficient and eco-friendly ECL system for multiplex assays of HSV and VZV

Both herpes simplex virus (HSV) and varicella-zoster virus (VZV) belong to herpesviruses family, and the cutaneous infections with HSV or VZV often present similar clinical symptoms, making it challenging in differentiation. Hence, simultaneous detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) holds great significance for protecting the public health safety. Herein, simultaneous detection of HSV and VZV was innovatively realized based on electrochemiluminescence (ECL) biosensor. Structure-optimized iridium(III) complex coordinated in ZIF-8 nanoemitters (Irbt-ZIF-8-NH2) were synthesized as anodic emitters. Notably, Irbt-ZIF-8-NH2 showed remarkable ECL performance with N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) as the novel efficient co-reactant over the traditional TPrA. The proposed biosensor in this work demonstrated outstanding performance within the linear range of 10 fM to 1 nM, with the detection limit of 1.5 fM for HSV and 2.2 fM for VZV. Importantly, it also exhibited satisfactory recoveries with RSDs in the analysis of viral nucleic acids samples extracted from HSV or VZV infected cells. This work inevitably provides a promising alternative assay for simultaneously detecting HSV and VZV, which would promote to the advancement of efficient and sensitive techniques to tackle significant global health concerns caused by herpesviruses outbreak in the future.

A conserved RNA switch for acetylcholine receptor clustering at neuromuscular junctions in chordates.

In mammals, neuromuscular synapses rely on clustering of acetylcholine receptors (AChRs) in the muscle plasma membrane, ensuring optimal stimulation by motor neuron-released acetylcholine neurotransmitter. This clustering depends on a complex pathway based on alternative splicing of Agrin mRNAs by the RNA-binding proteins Nova1/2. Neuron-specific expression of Nova1/2 ensures the inclusion of small "Z" exons in Agrin, resulting in a neural-specific form of this extracellular proteoglycan carrying a short peptide motif that is required for binding to Lrp4 receptors on the muscle side, which in turn stimulate AChR clustering. Here we show that this intricate pathway is remarkably conserved in Ciona robusta, a non-vertebrate chordate in the tunicate subphylum. We use in vivo tissue-specific CRISPR/Cas9-mediated mutagenesis and heterologous "mini-gene" alternative splicing assays in cultured mammalian cells to show that Ciona Nova is also necessary and sufficient for Agrin Z exon inclusion and downstream AChR clustering. We present evidence that, although the overall pathway is well conserved, there are some surprising differences in Nova structure-function between Ciona and mammals. We further show that, in Ciona motor neurons, the transcription factor Ebf is a key activator of Nova expression, thus ultimately linking this RNA switch to a conserved, motor neuron-specific transcriptional regulatory network.

Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling.

Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked β-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gαs:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.

Expanded Antigen-Specific Elimination Assay to Measure Human CD8+ T Cell Cytolytic Potential.

Durable cellular immunity against pathogens is dependent upon a coordinated recall response to antigen by memory CD8+ T cells, involving their proliferation and the generation of secondary cytotoxic effector cells. Conventional assays measuring ex vivo cytotoxicity fail to capture this secondary cytolytic potential, especially in settings where cells have not been recently exposed to their cognate antigen in vivo. Here we describe the expanded antigen-specific elimination assay (EASEA), a flow cytometric endpoint assay to measure the capacity of human CD8+ T cells to expand in vitro upon antigen re-exposure and generate secondary effector cells capable of selectively eliminating autologous antigen-pulsed target cells across a range of effector-to-target ratios. Unlike alternative assays, EASEA avoids the hazards of radioactive labeling and viral infection and can be used to study responses to individual or pooled antigens of interest. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Expanded antigen-specific elimination assay.

Integration of Image Pattern Recognition and Photon Sensor for Analyzing Cytokine Gene Expression Using πCode MicroDisc.

Current quantitative gene expression detection in genomic and transcriptomic research heavily relies on quantitative real-time PCR (qPCR). While existing multiplex gene detection techniques offer simultaneous analysis of multiple targets, we present an alternative assay capable of detecting gene expression simultaneously within a single well. This highly sensitive method utilizes πCode MicroDiscs, featuring unique identification patterns and fluorescent detection. Our study compared this multiplex πCode platform with a qPCR platform for profiling cytokine gene expression. The πCode MicroDisc assay successfully demonstrated the expression of polymerization markers for M1- and M2-like macrophages generated from THP-1-derived macrophages in a qualitative assay. Additionally, our findings suggest a pattern agreement between the πCode assay and the qPCR assay, indicating the potential of the πCode technology for comparative gene expression analysis. Regarding the inherent sensitivity and linearity, the developed πCode assay primarily provides qualitative gene expression to discriminate the polarization of macrophages. This remarkable capability presents substantial advantages for researchers, rendering the technology highly suitable for high-throughput applications in clinical diagnosis and disease monitoring.

Development of a human papillomavirus (HPV) multiplex immunoassay to profile HPV antibodies.

Neutralizing antibodies (NAbs) are considered the primary mechanism of vaccine-mediated protection against human papillomaviruses (HPV), the causative agent of cervical cancer. However, the minimum level of NAb needed for protection is currently unknown. The HPV pseudovirion-based neutralization assay (PBNA) is the gold standard method for assessing HPV antibody responses but is time-consuming and labor-intensive. With the development of higher valency HPV vaccines, alternative serological assays with the capacity for multiplexing would improve efficiency and output. Here we describe a multiplex bead-based immunoassay to characterize the antibody responses to the seven oncogenic HPV types (HPV16/18/31/33/45/52/58) contained in the current licensed nonavalent HPV vaccine. This assay can measure antibody isotypes and subclasses (total IgG, IgM, IgA1-2, IgG1-4), and can be adapted to measure other antibody features (e.g., Fc receptors) that contribute to vaccine immunity. When tested with serum samples from unvaccinated and vaccinated individuals, we found high concordance between HPV-specific IgG using this multiplex assay and NAbs measured with PBNA. Overall, this assay is high-throughput, sample-sparing, and time-saving, providing an alternative to existing assays for the measurement and characterization of HPV antibody responses.

Identification and analysis of immunoreactive proteins of Shigella flexneri in human sera and stool specimens.

The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of Shigella that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. The SAPs of clinical isolates of S. dysenteriae, S. boydii, Shigella flexneri, and S. sonnei were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain Shigella SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Immunoblot analysis revealed that sera from patients infected with S. flexneri recognized 31 proteins. These SAP antigens are recognized by the host humoral response during Shigella infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 S. flexneri proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in S. dysenteriae, S. boydii, and S. sonnei. This is the first report that demonstrates the presence of immunoreactive Shigella SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.