Specific Assay Research Articles

Cinnamaldehyde Alleviates Alveolar Epithelial Cell Injury in ALI by Inhibiting the CaMKII Pathway.

Alveolar epithelial cell injury plays a key role in acute lung injury (ALI) and is a vital determinant of its severity. Here, we aimed to assess the protective effects of cinnamaldehyde (CA) on lipopolysaccharide (LPS)-induced A549 cells and elucidate the underlying mechanisms. A549 cells were stimulated with 1 μg/mL LPS for 24 h to establish an alveolar epithelial cell injury model and subsequently treated with CA or Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93. Flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and lactate dehydrogenase release assays were used to evaluate apoptosis, cell viability, and lactate dehydrogenase activity, respectively. Levels of inflammatory cytokines (interleukin-6, interleukin-1β, tumor necrosis tactor-α, and interferon-γ) and oxidative stress markers (reactive oxygen species, superoxide dismutase, catalase, and malondialdehyde) were determined using enzyme-linked immunosorbent assay and specific assay kits, respectively. Furthermore, levels of apoptosis-related proteins (cleaved caspase-3, Bcl-2-associated X, and Bcl-2) and CaMKII were assessed via western blotting. CA did not exhibit significant cytotoxicity in A549 cells. It dose-dependently improved the cell viability, suppressed apoptosis, decreased cleaved caspase-3 and Bcl-2-associated X levels, and increased Bcl-2 levels in LPS-treated A549 cells. It also inhibited inflammatory factor release and oxidative stress in LPS-induced A549 cells. Similar results were observed in the KN93- and CA-treated groups. Western blotting assay revealed that CA and KN93 inhibited CaMKII pathway activation, as indicated by the reduced p-CaMKII and p-phospholamban (PLN) levels and p-CaMKII/CaMKII and p-PLN/PLN ratios. Overall, CA alleviated alveolar epithelial cell injury by inhibiting the inflammatory response and oxidative stress and inducing cell apoptosis in LPS-induced A549 cells by regulating the CaMKII pathway, serving as a potential candidate for ALI prevention and treatment.

Mass mortality associated with carpione rhabdovirus in golden pompano (Trachinotus ovatus) in China: First report

From August to November 2023, a severe epizootic outbreak occurred in an offshore cultured golden pompano (Trachinotus ovatus) population in Zhanjiang City, Guangdong Province, China, resulting in a cumulative mortality rate of 65–82 %. The diseased fish showed clinical signs of lethargy, anorexia, whirling movements, and hemorrhage at the base of the fins and in the upper and lower jaws before mass mortality. A rhabdovirus strain, designated as CARPV2023, was successfully isolated using fathead minnow (FHM) cells. The cytopathic effects of the tissue filtrate from the diseased golden pompano on FHM and epithelioma papulosum cyprini (EPC) cell monolayers were characterized by rounded cells, grape-like cluster formation, detachment, and lysis. Histopathology revealed congestion, hemorrhage, and necrosis in the spleen, liver, trunk kidney, and intestinal tissues of naturally and experimentally infected golden pompanos. Electron microscopy showed that bullet-shaped viral particles, measuring 183.5–217.6 nm in length and 57.3–82.7 nm in width, budded from the cell membrane or aggregated around the infected FHM cells. The virus remained stable for 7 d at 4–33 °C and replicated optimally at 28 °C. Whole-genome sequencing and subsequent phylogenetic analysis further indicated that the carpione rhabdovirus (CARPV)2023 strain is a new carpione rhabdovirus, and is related to novirhabdoviruses, however, is thus far unclassified. A specific nested reverse transcription-polymerase chain reaction assay confirmed the presence of CARPV in the affected golden pompano. Significantly higher mortality was observed in challenged golden pompanos than that in the controls through intraperitoneal injection and immersion infection. Fluorescence in situ hybridization showed showed positive reactions for RNA in multiple tissues (liver, spleen, trunk kidney, brain, heart, gill, and muscle) from infected fish. The highest expression level was confirmed in the spleen through qPCR. This is the first study to report natural CARPV infections in marine fish.

Glutaminase 1 plays critical roles in myelodysplastic syndrome and acute myeloid leukemia cells.

Myelodysplastic syndrome (MDS) features bone marrow failure and a heightened risk of evolving into acute myeloid leukemia (AML), increasing with age and reducing overall survival. Given the unfavorable outcomes of MDS, alternative treatments are necessary. Glutamine, the most abundant amino acid in the blood, is metabolized first by the enzyme glutaminase (GLS). To investigate whether GLS is involved in the progression of MDS. The efficacy of GLS inhibitors (CB839 or IPN60090) and BCL2 inhibitor venetoclax was also examined. We employed GLS inhibitors (CB839, IPN60090) and the BCL2 inhibitor venetoclax, prepared as detailed. MDS and AML cell lines were cultured under standard and modified (hypoxic, glutamine-free) conditions. Viability, proliferation, and caspase activity were assessed with commercial kits. RT-PCR quantified gene expression post-shRNA transfection. Mitochondrial potential, ATP levels, proteasome activity, and metabolic functions were evaluated using specific assays. Statistical analyses (t-tests, ANOVA) validated the findings. The glutamine-free medium inhibited the growth of MDS cells. GLS1 expression was higher in AML cells than in normal control samples (GSE15061), whereas GLS2 expression was not. Treatment of MDS and AML cells for 72 h was inhibited in a dose-dependent manner by GLS inhibitors. Co-treatment with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax and GLS inhibitors increased potency. Cells transfected with GLS1 short hairpin RNA showed suppressed proliferation under hypoxic conditions and increased sensitivity to venetoclax. Targeting glutaminolysis and BCL2 inhibition enhances the therapeutic efficacy and has been proposed as a novel strategy for treating high-risk MDS and AML.

Salinity Tolerance Mechanism of Crithmum maritimum L.: Implications for Sustainable Agriculture in Saline Soils

Edible halophytes are attracting attention due to their potential for agriculture in saline and marginal areas. The salt tolerance mechanism was analyzed in Crithmum maritimum L., based on ionic, osmotic, and redox homeostasis strategies under salt stress. The methodology involved growing C. maritimum seeds in pots under controlled greenhouse conditions and exposing them to different NaCl concentrations (0, 100, 200, and 300 mM) for five months. High salinity levels decreased plant length and biomass, but the shoot-to-root length and biomass ratio increased significantly. Photosynthetic pigments (chlorophyll and carotenoids) were quantified using spectrophotometric analysis, while macro- and micro-nutrient contents were determined via the Kjeldahl method, flame photometry, and atomic absorption spectrophotometry. Osmolyte accumulation, including proline and glycine betaine, was analyzed using specific biochemical assays, and antioxidant enzyme activities (SOD, CAT, and POX) were measured to assess redox homeostasis. Photosynthetic pigments in C. maritimum leaves slightly increased at 100 mM NaCl, but significantly declined at 200 and 300 mM NaCl. A high Na content in the shoots indicated no restriction in mineral uptake in the roots. Nitrogen and phosphorus slightly decreased under high salinity. The cation content in the shoots varied: potassium decreased, while calcium and magnesium increased with salinity, although the Mg+2/Na+ and K+/Na+ ratios showed similar declining patterns. The micro-nutrients iron and manganese increased in the shoots, while copper remained unchanged. The content of osmolytes proline and glycine betaine significantly increased under the 200 and 300 mM NaCl treatments. Antioxidant enzyme activities (SOD, CAT, and POX) decreased at 100 and 200 mM NaCl, but were strongly induced at 300 mM NaCl. The total antiradical activity of the leaves increased with higher salinity levels. Our results indicated that the facultative halophyte characteristics of C. maritimum emerged after exposure to 200 mM NaCl. Increased calcium content may be a key factor in salinity tolerance. We concluded that C. maritimum employs strong osmotic adjustment and redox homeostasis mechanisms, making it a promising candidate for cultivation in saline environments.

Salmonella Detection in Food Using a HEK-hTLR5 Reporter Cell-Based Sensor.

The development of a rapid, sensitive, specific method for detecting foodborne pathogens is paramount for supplying safe food to enhance public health safety. Despite the significant improvement in pathogen detection methods, key issues are still associated with rapid methods, such as distinguishing living cells from dead, the pathogenic potential or health risk of the analyte at the time of consumption, the detection limit, and the sample-to-result. Mammalian cell-based assays analyze pathogens' interaction with host cells and are responsive only to live pathogens or active toxins. In this study, a human embryonic kidney (HEK293) cell line expressing Toll-Like Receptor 5 (TLR-5) and chromogenic reporter system (HEK dual hTLR5) was used for the detection of viable Salmonella in a 96-well tissue culture plate. This cell line responds to low concentrations of TLR5 agonist flagellin. Stimulation of TLR5 ligand activates nuclear factor-kB (NF-κB)-linked alkaline phosphatase (AP-1) signaling cascade inducing the production of secreted embryonic alkaline phosphatase (SEAP). With the addition of a ρ-nitrophenyl phosphate as a substrate, a colored end product representing a positive signal is quantified. The assay's specificity was validated with the top 20 Salmonella enterica serovars and 19 non-Salmonella spp. The performance of the assay was also validated with spiked food samples. The total detection time (sample-to-result), including shortened pre-enrichment (4 h) and selective enrichment (4 h) steps with artificially inoculated outbreak-implicated food samples (chicken, peanut kernel, peanut butter, black pepper, mayonnaise, and peach), was 15 h when inoculated at 1-100 CFU/25 g sample. These results show the potential of HEK-DualTM hTLR5 cell-based functional biosensors for the rapid screening of Salmonella.

Comparison of Roka Atlas® System Performance and Health Canada Reference Method for Listeria Detection From Plastic, Sealed Concrete, and Stainless-Steel Surface Samples.

There is a high demand in the food industry and in public health for rapid automated methods capable of high-volume sample processing. In an unpaired study, Roka Atlas® System performance was compared with Health Canada reference method MFHPB-30 for Listeria spp. (LSP Roka assay) and Listeria monocytogenes (LmG2 Roka Assay) detection on plastic (PL), sealed concrete (SC), and stainless-steel (SS) surfaces (45 samples each per candidate or reference method). Seeking shorter enrichment time for the candidate method, R2 medium pre-enrichment for 14, 16, and 24 h at 35°C was combined with the Roka assay. Listeria welshimeri, L. innocua, and L. monocytogenes were employed to individually inoculate each of the three surfaces, with two competing microorganisms within the 10-100-fold higher concentration range. False negative, false positive, sensitivity, and specificity were 0, 0, 100, and 100%, respectively, for the plastic, sealed concrete, and stainless-steel surfaces, regardless of inoculation level (high, low, and uninoculated) and enrichment time. Candidate method detected 10, 7, and 9 true positives, versus 10, 6, and 10 by the reference method in individually inoculated SS, PL, and SC, respectively. Probability of detection for all the three surfaces for the Roka Atlas System was comparable to the reference method in this unpaired study. The Roka Atlas System detected targets after as little as 14 h enrichment. Surface type did not negatively affect assay sensitivity or specificity. The Roka Atlas System was comparable to the reference method.

PSI-15 Validation of a rapid test to evaluate humoral immunity in Labrador Retrievers across multiple life stages

Abstract This study sought to validate the D2Dx rapid immunity test in canines and investigate the age-related changes in the humoral immune system of healthy large breed dogs. Blood plasma with EDTA anticoagulant was collected from healthy Labrador Retrievers (n = 80) housed at Four Rivers Kennel under IACUC protocol FRK-56. Age groups were defined as follows: puppy 0-1.5 yr, adults 1.5-7 yr, senior 7-10, and geriatric 10-14. Immunity biomarkers including c-reactive protein (CRP), interleukin 4 (IL-4), interferon gamma (IFN-γ), complement protein 3 (C3), and immunoglobulin G (IgG) were determined through canine specific enzyme-linked immunosorbent assay (ELISA) kits. The nanoparticle based D2Dx test, which evaluates the cumulative humoral immune response, was also performed. Data were analyzed using a one-way ANOVA between age groups, with a Tukey’s post hoc, and correlations between variables were evaluated through multivariate analysis. The D2Dx score was correlated with body weight, age, IgG, and C3 validating that the test was able to adequately measure the humoral immunity in canines (r ≥ 0.49). D2Dx score was significantly different between age groups (P < 0.01) with puppies having significantly reduced scores compared with all other groups (P < 0.01). C3 was significantly different between age groups (P < 0.01), with puppies significantly less than other life stages (P ≤ 0.03). The C3 of adults averaged significantly greater than seniors and trended greater than geriatric subjects (P ≤ 0.01, 0.07). IgG between ages was also significantly different, with puppies significantly less than other age groups (P < 0.01). There was no significant difference between age groups for IFN-γ, IL-4 ratio, IFN-γ, IL-4, or CRP (P ≥ 0.18). This study showed that the D2Dx Rapid Immunity Test is able to detect changes to humoral immunity in canines. Beyond evaluating the effectiveness of the D2Dx test, this study also provides important information about the under researched topic of canine humoral immunity development and senescence in a robust, healthy cohort of large breed dogs.

A highly sensitive and specific fluorescent probe for thrombin detection and high-throughput screening of thrombin inhibitors in complex matrices

Thrombin plays a critical role in hemostasis and hemolysis, and is a significant biomarker for blood-related diseases. Detection and inhibitors screening of thrombin are essential in medical research. In this study, we developed a fluorescent sensor based on the interaction between quantum dots (QDs) and fibrinogen (Fib) for thrombin detection and its inhibitors screening. Upon the presence of thrombin, the fibrinogen of soluble QDs-Fib were converted into insoluble fibrin precipitate, causing a change of fluorescence intensity in the supernatant. Under optimized conditions, our method exhibited an excellent linearity (R2 ≥0.99) over the range of 2∼100 U/L with a limit of detection (LOD) as low as 0.29 U/L. Moreover, we employed this method to screen for thrombin inhibitors using dabigatran as an exemplary direct thrombin inhibitor (DTI), even at concentrations as low as 1 nM. Finally, the established method was successfully used to screen thrombin inhibitors in 23 different extracts from Eupolyphaga sinensis walker. The method provided not only a sensitive, specific and high throughput assay for the detection of thrombin activity in biological samples, but also a reliable strategy for the screening of thrombin inhibitors in complex matrices.

Biochemical and structural insights of a recombinant AA16 LPMO from the marine and sponge-symbiont Peniophora sp

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidize polysaccharides, leading to their cleavage. LPMOs are classified into eight CAZy families (AA9-11, AA13-17), with the functionality of AA16 being poorly characterized. This study presents biochemical and structural data for an AA16 LPMO (PnAA16) from the marine sponge symbiont Peniophora sp. Phylogenetic analysis revealed that PnAA16 clusters separately from previously characterized AA16s. However, the structural modelling of PnAA16 showed the characteristic immunoglobulin-like fold of LPMOs, with a conserved his-brace motif coordinating a copper ion. The copper-bound PnAA16 showed greater thermal stability than its apo-form, highlighting copper's role in enzyme stability. Functionally, PnAA16 demonstrated oxidase activity, producing 5 μM H₂O₂ after 30 min, but showed 20 times lower peroxidase activity (0.27 U/g) compared to a fungal AA9. Specific activity assays indicated that PnAA16 acts only on cellohexaose, generating native celloligosaccharides (C3 to C5) and oxidized products with regioselective oxidation at C1 and C4 positions. Finally, PnAA16 boosted the activity of a cellulolytic cocktail for cellulose saccharification in the presence of ascorbic acid, hydrogen peroxide, or both. In conclusion, the present work provides insights into the AA16 family, expanding the understanding of their structural and functional relationships and biotechnological potential.

Novel antibody- and aptamer-based approaches for sensitive detection of mycotoxin fusaric acid in cereal

This study presents the first successful generation of polyclonal antibodies (pAbs) and oligonucleotide aptamers specifically targeting fusaric acid (FA). Utilizing these pAbs and aptamers, three highly sensitive and specific assays were developed for the detection of FA in cereals with limits of detection (LOD) ranging from 5 to 50 ng/g: an antibody-based enzyme-linked immunosorbent assay (ELISA), an aptamer-based enzyme-linked aptamer-sorbent assay (ELASA), and a hybrid enzyme-linked aptamer-antibody sandwich assay (ELAAA). The recovery rates of FA in spiked cereal samples ranged from 87 % to 112 % across all assays. Analysis of 15 cereal feed samples revealed FA contamination levels of 459 to 1743 ng/g (ELISA), 427 to 1960 ng/g (ELASA), and 381 to 1987 ng/g (ELAAA). These results were further validated by HPLC analysis, confirming high consistency within developed assays. Overall, the ELISA, ELASA, and ELAAA are promising tools for the rapid detection of FA, significantly contributing to food safety monitoring.

Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale.

The parasite species formerly known as Plasmodium ovale, Plasmodium ovalecurtisi (P. ovalecurtisi) and Plasmodium ovalewallikeri (P. ovalewallikeri), are endemic across multiple African countries. These species are thought to differ in clinical symptomatology and latency, but only a small number of existing diagnostic assays can detect and distinguish them. In this study, we sought to develop new assays for the detection and differentiation of P. ovalecurtisi and P. ovalewallikeri by leveraging recently published whole-genome sequences for both species. Repetitive sequence motifs were identified in available P. ovalecurtisi and P. ovalewallikeri genomes and used for assay development and validation. We evaluated the analytical sensitivity of the best-performing singleplex and duplex assays using synthetic plasmids. We then evaluated the specificity of the duplex assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), and validated its performance using 55 P. ovale samples and 40 non-ovale Plasmodium samples from the DRC. The best-performing P. ovalecurtisi and P. ovalewallikeri targets had 9 and 8 copies within the reference genomes, respectively. The P. ovalecurtisi assay had high sensitivity with a 95% confidence lower limit of detection (LOD) of 3.6 parasite genome equivalents/μl, while the P. ovalewallikeri assay had a 95% confidence LOD of 25.9 parasite genome equivalents/μl. A duplex assay targeting both species had 100% specificity and 95% confidence LOD of 4.2 and 41.2 parasite genome equivalents/μl for P. ovalecurtisi and P. ovalewallikeri, respectively. We identified promising multi-copy targets for molecular detection and differentiation of P. ovalecurtisi and P. ovalewallikeri and used them to develop real-time PCR assays. The best performing P. ovalecurtisi assay performed well in singleplex and duplex formats, while the P. ovalewallikeri assay did not reliably detect low-density infections in either format. These assays have potential use for high-throughput identification of P. ovalecurtisi, or for identification of higher density P. ovalecurtisi or P. ovalewallikeri infections that are amenable to downstream next-generation sequencing.

Analytical Validation of Esopredict, an Epigenetic Prognostic Assay for Patients with Barrett's Esophagus.

EsopredictTM is a prognostic assay that risk-stratifies Barrett's esophagus patients to predict future progression to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). Established based on foundational studies at Johns Hopkins University, a risk algorithm was developed and clinically validated in two independent studies (n = 320). EsopredictTM is currently offered as a clinical test under the Clinical Laboratory Improvement Amendments (CLIA) guidelines. Here we present the analytical validation by repeated testing of FFPE tissues (n = 26 patients), cell lines, and contrived DNA controls to determine assay performance regarding analytical sensitivity (as defined by the limit of detection (LOD)), analytical specificity (as defined by the limit of blank (LOB)), accuracy as determined from the average positive and negative agreement, repeatability, and reproducibility. The LOD for the assay at 1.5% DNA methylation was significantly higher than the LOB, as determined by an unmethylated DNA control (0% methylated DNA). Inter- and intra-assay average positive agreement (APA) were 88% and 94%, respectively, while average negative agreement (ANA) values were 90% and 94%, respectively. Average inter- and intra-assay precision were <9% and <5% coefficient of variation (CV), respectively. These results confirm that EsopredictTM is a highly reproducible, sensitive, and specific risk categorization assay for the prediction of progression to HGD or EAC within 5 years.

TAK-242 alleviates diabetic cardiomyopathy via inhibiting pyroptosis and TLR4/CaMKII/NLRP3 pathway.

Diabetic cardiomyopathy (DCM) is identified as a progressive disease that may lead to irreparable heart failure. Toll-like receptor (TLR) signaling is believed to be implicated in the pathogenesis of DCM. This study intended to explore the potential impact of Toll-like receptor 4 (TLR4) on DCM in vitro and in vivo. Streptozotocin and HG medium were utilized to induce diabetes in animal and cell models, respectively. Selective TLR4 inhibitor TAK-242 and calcium/calmodulin-dependent protein kinase-II (CaMKII) inhibitor KN-93 were employed to explore the involvement of TLR4/CaMKII in DCM. TLR4 expression was increased in DCM hearts, while inhibition of TLR4 activation by TAK-242 improved cardiac function, attenuated heart hypertrophy, and fibrosis, as well as reduced oxidative stress and proinflammatory cytokine levels in rats, which were confirmed by Doppler echocardiography, hematoxylin and eosin staining, and Masson Trichome staining and specific enzyme-linked immunosorbent assay kits. Besides, the expression of hypertrophy-related molecules and oxidative stress damage were also inhibited by TAK-242. Furthermore, TAK-242 treatment reduced CaMKII phosphorylation accompanied by decreased expression of NOD-like pyrin domain-containing protein 3, gasdermin D (GSDMD), The N-terminal domain of Gasdermin D (GSDMD-N), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and Caspase-1 both in vivo and in vitro. Similar positive impacts on HG-induced pyroptosis were also observed with KN-93 treatment, and this was achieved without affecting TLR4 expression. Collectively, our work suggested that TAK-242 demonstrated substantial benefits against DCM both in vivo and in vitro, potentially attributed to the suppression of the TLR4-mediated CaMKII/NLRP3 pathway activity.

The riverscape on a chip: High-throughput qPCR enables basin-wide fishery assessments

To achieve reliable detection of environmental DNA from multiple species, a method which combines the specificity of single-taxon assays with the broad taxonomic coverage of metabarcoding is needed. High-throughput quantitative PCR (HT-qPCR) has emerged as one such method that holds particular promise for ecological applications. Here, we present the development and validation of a HT-qPCR biochip (i.e., biochip) protocol for a suite of western North American fishes with high cultural, economic, and ecological significance. Coupling this biochip with systematically distributed eDNA samples collected by citizen scientists, we evaluated the distribution of multiple species throughout a large river basin in the U.S. Pacific Northwest. Patterns of detection using the biochip were comparable to those using single-taxon qPCR (ST-qPCR; mean concordance = 0.917), despite the biochip using 90% less DNA per taxon and reducing costs by an estimated 40%. Comparisons between biochip results and distributions derived from conventional methods demonstrate that this approach not only offers more reliable and high-resolution distribution data for these species but also achieves this more efficiently than traditional sampling methods.

Streamlined detection of Nipah virus antibodies using a split NanoLuc biosensor

ABSTRACT Nipah virus (NiV) is an emerging zoonotic RNA virus that can cause fatal respiratory and neurological diseases in animals and humans. Accurate NiV diagnostics and surveillance tools are crucial for the identification of acute and resolved infections and to improve our understanding of NiV transmission and circulation. Here, we have developed and validated a split NanoLuc luciferase NiV glycoprotein (G) biosensor for detecting antibodies in clinical and animal samples. This assay is performed by simply mixing reagents and measuring luminescence, which depends on the complementation of the split NanoLuc luciferase G biosensor following its binding to antibodies. This anti-NiV-G “mix-and-read” assay was validated using the WHO's first international standard for anti-NiV antibodies and more than 700 serum samples from the NiV-endemic country of Bangladesh. Anti-NiV antibodies from survivors persisted for at least 8 years according to both ⍺NiV-G mix-and-read and NiV neutralization assays. The ⍺NiV-G mix-and-read assay sensitivity (98.6%) and specificity (100%) were comparable to anti-NiV IgG ELISA performance but failed to detect anti-NiV antibodies in samples collected less than a week following the appearance of symptoms. Overall, the anti-NiV-G biosensor represents a simple, fast, and reliable tool that could support the expansion of NiV surveillance and retrospective outbreak investigations.

A nonadjuvanted HLA-restricted peptide vaccine induced both T and B cell immunity against SARS-CoV-2 spike protein

During COVID-19 pandemic, cases of postvaccination infections and restored SARS-CoV-2 virus have increased after full vaccination, which might be contributed to by immune surveillance escape or virus rebound. Here, artificial linear 9-mer human leucocyte antigen (HLA)-restricted UC peptides were designed based on the well-conserved S2 region of the SARS-CoV-2 spike protein regardless of rapid mutation and glycosylation hindrance. The UC peptides were characterized for its effect on immune molecules and cells by HLA-tetramer refolding assay for HLA-binding ability, by HLA-tetramer specific T cell assay for engaged cytotoxic T lymphocytes (CTLs) involvement, by HLA-dextramer T cell assay for B cell activation, by intracellular cytokine release assay for polarization of immune response, Th1 or Th2. The specific lysis activity assay of T cells was performed for direct activation of cytotoxic T lymphocytes by UC peptides. Mice were immunized for immunogenicity of UC peptides in vivo and immunized sera was assay for complement cytotoxicity assay. Results appeared that through the engagement of UC peptides and immune molecules, HLA-I and II, that CTLs elicited cytotoxic activity by recognizing SARS-CoV-2 spike-bearing cells and preferably secreting Th1 cytokines. The UC peptides also showed immunogenicity and generated a specific antibody in mice by both intramuscular injection and oral delivery without adjuvant formulation. In conclusion, a T-cell vaccine could provide long-lasting protection against SARS-CoV-2 either during reinfection or during SARS-CoV-2 rebound. Due to its ability to eradicate SARS-CoV-2 virus-infected cells, a COVID-19 T-cell vaccine might provide a solution to lower COVID-19 severity and long COVID-19.

SARS-COV-2 SEROSURVEY OF HEALTHY, PRIVATELY OWNED CATS PRESENTING TO A NEW YORK CITY ANIMAL HOSPITAL IN THE EARLY PHASE OF THE COVID-19 PANDEMIC (2020-2021).

SARS-CoV-2, the cause of the ongoing COVID-19 pandemic, not only infects humans but is also known to infect various species, including domestic and wild animals. While many species have been identified as susceptible to SARS-CoV-2, there are limited studies on the prevalence of SARS-CoV-2 in animals. Both domestic and non-domestic cats are now established to be susceptible to infection by SARS-CoV-2. While serious disease in cats may occur in some instances, the majority of infections appear to be subclinical. Differing prevalence data for SARS-CoV-2 infection of cats have been reported, and are highly context-dependent. Here, we report a retrospective serological survey of cats presented to an animal practice in New York City, located in close proximity to a large medical center that treated the first wave of COVID-19 patients in the U.S. in the Spring of 2020. We sampled 79, mostly indoor, cats between June 2020 to May 2021, the early part of which time the community was under a strict public health "lock-down". Using a highly sensitive and specific fluorescent bead-based multiplex assay, we found an overall prevalence of 13/79 (16%) serologically-positive animals for the study period; however, cats sampled in the Fall of 2020 had a confirmed positive prevalence of 44%. For SARS-CoV-2 seropositive cats, we performed viral neutralization test with live SARS-CoV-2 to additionally confirm presence of SARS-CoV-2 specific antibodies. Of the thirteen seropositive cats, 7/13 (54%) were also positive by virus neutralization, and two of seropositive cats had previously documented respiratory signs, with high neutralization titers of 1/1024 and 1/4096; overall however, there was no statistically significant association of SARS-CoV-2 seropositivity with respiratory signs, or with breed, sex or age of the animals. Follow up sampling of cats showed that positive serological titers were maintained over time. In comparison, we found an overall confirmed positive prevalence of 51% for feline coronavirus (FCoV), an endemic virus of cats, with 30% confirmed negative for FCoV. We demonstrate the impact of SARS-CoV-2 in a defined feline population during the first wave of SARS-CoV-2 infection of humans, and suggest that human-cat transmission was substantial in our study group. Our study provide a new context for SARS-CoV-2 transmission events across species.

A multiplexed real‐time PCR assay for simultaneous quantification of human immunodeficiency virus and Hepatitis B virus for low‐and‐middle‐ income countries

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16–74.3] for HBV and 82 % [78.09–85.90] for HIV and an overall specificity of 100 % [94.95–100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26–88.73] and >1000 IU/mL for HBV was 100 % [95.51–100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.

An aptamer-guided fluorescence polarisation platform for extracellular vesicle liquid biopsy.

The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.