Cyclic diarylheptanoids from Myrica javanica: isolation, transformation, and bioactivity evaluation.

Three cyclic diarylheptanoids, myricanol (1), myricanone (2), and porson (3), were isolated from Myrica javanica (Myricaceae). As a major component, myricanol (1) was obtained from dry powdered bark and twigs (up to 1.6%). Transformation of myricanol (1) afforded 5-prenylmyricanol (4) and 5-benzylmyricanol (5) in 84.5% and 65% yields, respectively. The bioactivities of the isolated cyclic diarylheptanoids and their derivatives were investigated to determine their cytotoxicity and DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activities. The cytotoxicity assay against murine leukaemia P-388 cells demonstrated that compounds 4 and 5 showed an almost two-fold increase in the activity of their parent molecule (1), with an IC50 value of 12 µM. Furthermore, the free radical scavenging assay showed that myricanol (1) had the highest radical scavenging activity, revealing the importance of the free phenolic group (IC50 39.3 µM).

In vitro evaluation of the selective cytotoxicity and genotoxicity of three synthetic ortho-nitrobenzyl derivatives in human cancer cell lines, with and without metabolic activation.

Although the presence of nitro groups in chemicals can be recognized as structural alerts for mutagenicity and carcinogenicity, nitroaromatic compounds have attracted considerable interest as a class of agents that can serve as source of potential new anticancer agents. In the present study, the in vitro cytotoxicity, genotoxicity, and mutagenicity of three synthetic ortho-nitrobenzyl derivatives (named ON-1, ON-2 and ON-3) were evaluated by employing human breast and ovarian cancer cell lines. A series of biological assays was carried out with and without metabolic activation. Complementarily, computational predictions of the pharmacokinetic properties and druglikeness of the compounds were performed in the Swiss ADME platform. The MTT assay showed that the compounds selectively affected selectively the cell viability of cancer cells in comparison with a nontumoral cell line. Additionally, the metabolic activation enhanced cytotoxicity, and the compounds affected cell survival, as demonstrated by the clonogenic assay. The comet assay, the cytokinesis-block micronucleus assay, and the immunofluorescence of the γ-H2AX foci formation assay have that the compounds caused chromosomal damage to the cancer cells, with and without metabolic activation. The results obtained in the present study showed that the compounds assessed were genotoxic and mutagenic, inducing double-strand breaks in the DNA structure. The high selectivity indices observed for the compounds ON-2 and ON-3, especially after metabolic activation with the S9 fraction, must be highlighted. These experimental biological results, as well as the theoretical properties predicted for the compounds have shown that they are promising anticancer candidates to be exploited in additional studies.

Intracranial tuberculomas diagnosed with Xpert MTB/RIF Ultra assay of formalin-fixed paraffin-embedded brain tissues and treated with an optimized antituberculosis regimen: A case report.

Diagnosis of intracranial tuberculoma remains a challenge due to its rarity, non-specific clinical presentation, and radiological findings. Herein, we describe a case of intracranial tuberculomas in a male diabetic patient who presented headache and vomiting on admission. Neuroimaging findings indicated multiple ring contrast-enhanced lesions with extensive perilesional edema. However, a cerebrospinal fluid (CSF) examination was normal. When a biopsy of brain lesions was performed, pathological characteristics of tuberculosis were absent and acid-fast staining was negative. A tuberculosis diagnosis was subsequently obtained from an Xpert MTB/RIF Ultra assay of formalin-fixed paraffin-embedded brain tissue. The patient was treated with an optimized anti-tuberculosis regimen which included high-dose intravenous administration of rifampicin and isoniazid, and oral administration of linezolid. The patient recovered well and exhibited marked clinical improvement. This case report demonstrates that when CSF analysis does not indicate the presence of intracranial tuberculomas, analysis of formalin-fixed paraffin-embedded brain tissue specimens with the Xpert MTB/RIF Ultra assay may be able to confirm a diagnosis. Furthermore, a high dose of rifampicin and isoniazid plus linezolid may improve patient outcome.

Behavioural changes in the city: The common black garden ant defends aphids more aggressively in urban environments.

Urbanisation alters biodiversity patterns and threatens to disrupt mutualistic interactions. Aside from pollination, however, little is known about how mutualisms change in cities. Our study aimed to assess how urbanisation affects the protective mutualism between ants and aphids, investigating potential behavioural changes in mutualistic ants and their implications for aphids in urban environments. To do so, we studied the protective mutualism between the pink tansy aphid (Metopeurum fuscoviride) and the black garden ant (Lasius niger) along an urbanisation gradient in Berlin, Germany. In nine locations along this gradient, we measured aphid colony dynamics and proxies for parasitism, quantified the investment of ants in tending aphids and conducted behavioural assays to test the aggressiveness of ant responses to a simulated attack on the aphids. We found that aphid colonies flourished and were equally tended by ants across the urbanisation gradient, with a consistent positive density dependence between aphid and ant numbers. However, ants from more urbanised sites responded more aggressively to the simulated attack. Our findings suggest that this protective mutualism is not only maintained in the city, but that ants might even rely more on it and defend it more aggressively, as other food resources may become scarce and more unpredictable with urbanisation. We thereby provide unique insights into this type of mutualism in the city, further diversifying the growing body of work on mutualisms across urbanisation gradients.

Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus.

In recent years, porcine diarrhea-associated viruses have caused significant economic losses globally. These viruses present similar clinical symptoms, such as watery diarrhea, dehydration, and vomiting. Co-infections with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are common. For the rapid and on-site preliminary diagnosis on the pig farms, this study aimed to develop a colloidal gold immunochromatography assay (GICA) strip for the detection of PEDV and TGEV simultaneously. The GICA kit showed that there was no cross-reactivity with the other five common porcine viruses. With visual observation, the lower limits were approximately 104 TCID50/mL and 104 TCID50/mL for PEDV and TGEV, respectively. The GICA strip could be stored at 4°C or 25°C for 12 months without affecting its efficacy. To validate the GICA strip, 121 clinical samples were tested. The positive rates of PEDV and TGEV were 42.9 and 9.9%, respectively, and the co-infection rate of the two viruses was 5.8% based on the duplex GICA strip. Thus, the established GICA strip is a rapid, specific, and stable tool for on-site preliminary diagnosis of PEDV- and TGEV-associated diarrhea.

Genistein transfersome-embedded topical delivery system for skin melanoma treatment: in vitro and ex vivo evaluations.

Skin melanoma is considered the most dangerous form of skin cancer due to its association with high risk of metastasis, high mortality rate and high resistance to different treatment options. Genistein is a natural isoflavonoid with known chemotherapeutic activity. Unfortunately, it has low bioavailability due to its poor aqueous solubility and excessive metabolism. In the current study, genistein was incorporated into transferosomal hydrogel to improve its bioavailability. The prepared transferosomal formulations were characterized regarding: particle size; polydispersity index; zeta potential; encapsulation efficiency; TEM; FTIR; DSC; XRD; in vitro drug release; viscosity; pH; ex vivo anti-tumor activity on 3D skin melanoma spheroids and 1-year stability study at different storage temperatures. The optimized formulation has high encapsulation efficiency with an excellent particle size that will facilitate its penetration through the skin. The transfersomes have a spherical shape with sustained drug release profile. The anti-tumor activity evaluation of genistein transfersome revealed that genistein is a potent chemotherapeutic agent with enhanced penetration ability through the melanoma spheroids when incorporated into transfersomes. Stability study results demonstrate the high physical and chemical stability of our formulations. All these outcomes provide evidence that our genistein transferosomal hydrogel is a promising treatment option for skin melanoma.

A colorimetric biosensor with infrared sterilization based on CuSe nanoparticles for the detection of E. coli O157:H7 in food samples.

It is critical to develop quick, accurate, and efficient sterilization for detecting Escherichia coli O157:H7 in order to prevent infections and outbreaks of foodborne illnesses. Herein, we established a colorimetric biosensor with sterilizing properties using copper selenide nanoparticles to detect E. coli O157:H7. The sample was mixed with magnetic nanoprobes and nanozyme probes to form a sandwich structure, and then the unbound nanozyme probes were collected by magnetic separation. Finally, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)-hydrogen peroxide (H2O2) reporting system was added for signal amplification. The change from colorless to green can be seen with the naked eye. Under the optimal conditions, the detection range of E. coli O157:H7 was 102-106 CFU/mL, and the detection limit was 0.35 × 102 CFU/mL. The total detection time was 80 minutes, which can be successfully applied to milk and mineral water. In addition, the colorimetric sensor can kill the target bacteria by irradiating it under a 980-nm laser for 5 minutes. In conclusion, this sensor is a promising tool for rapidly detecting foodborne pathogens and promptly eliminating bacteria. IMPORTANCE: Escherichia coli O157:H7 is a major threat to public health. At present, the detection methods for E. coli O157:H7 mainly include traditional bacterial culture, immunology (enzyme-linked immune-sorbent assay) and molecular biology techniques (polymerase chain reaction). These methods have the limitations of professional operation, waste of time and energy, and high cost. Therefore, we have developed a simple, fast, bactericidal colorimetric biosensor to detect E. coli. O157:H7. The entire process was completed in 80 minutes. The method has been successfully applied to milk and mineral water samples with satisfactory results, proving that the method is an effective method for real-time detection and inactivation of bacteria.

Designing and expression of novel recombinant fusion protein for efficient antigen screening of SARS-CoV-2.

Corona virus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), claimed millions globally. After the report of the first incidence of the virus, variants emerged with each posing a unique threat than its predecessors. Though many advanced diagnostic assays like real-time PCR are available for screening of SARS-CoV-2, their applications are being hindered because of accessibility and cost. With the advent of rapid assays for antigenic screening of SARS-CoV-2 made diagnostics far easy as the assays are rapid, cost-effective and can be used at point-of-care settings. In the present study, a fusion construct was made utilising highly immunogenic B cell epitopes from the three important structural proteins of SARS-CoV-2. The protein was expressed; purified capture mAbs generated and rapid antigen assay was developed. Eight hundred and forty nasopharyngeal swab samples were screened for the evaluation of the developed assay which showed 37.14% positivity, 96.51% and 100% sensitivity and specificity respectively. The assay developed was supposed to identify SARS-CoV-2 wild-type as well as variants of concern and variants of importance in real-time conditions.

[Cell membrane-penetrating capacity of hPP10-Cu, Zn-SOD fusion protein and its antioxidant and anti-inflammatory activity].

OBJECTIVE: To investigate the cell membrane-penetrating capacity of human cell-penetrating peptide hPP10 carrying human antioxidant protein Cu-Zn superoxide dismutase (Cu, Zn-SOD) and assess the antioxidant and anti-inflammatory activity of these fusion proteins. METHODS: The fusion protein hPP10-Cu, Zn-SOD was obtained by genetic engineering and identified by Western blotting. The membrane-penetrating ability of the fusion protein was evaluated by immunofluorescence assay, fluorescence colocalization assay and Western blotting, its SOD enzyme activity was detected using a commercial kit, and its effect on cell viability was assessed with MTT assay. In a HEK293 cell model of H2O2-induced oxidative stress, the effect of hPP10-Cu, Zn-SOD on cell apoptosis was analyzed with flow cytometry and RT-qPCR, and its antioxidant effect was assessed using reactive oxygen species (ROS) assay; its anti-inflammatory effect was evaluated in mouse model of TPA-induced ear inflammation by detecting expression of the inflammatory factors using RT-qPCR, Western blotting and immunohistochemistry. RESULTS: The fusion protein hPP10-Cu, Zn-SOD was successfully obtained. Immunofluorescence assay confirmed obvious membrane penetration of this fusion protein in HEK293 cells, localized both in the cell membrane and the cell nuclei after cell entry. hPP10-Cu, Zn-SOD at the concentration of 5 µmol/L exhibited strong antioxidant activity with minimal impact on cell viability at the concentration up to 10 µmol/L. The fusion protein obviously inhibited apoptosis and decreased intracellular ROS level in the oxidative stress cell model and significantly reduced mRNA and protein expression of the inflammatory factors in the mouse model of ear inflammation. CONCLUSION: The fusion protein hPP10-Cu, Zn-SOD capable of penetrating the cell membrane possesses strong antioxidant and anti-inflammatory activities with only minimal cytotoxicity, demonstrating the value of hPP10 as an efficient drug delivery vector and the potential of hPP10-Cu, Zn-SOD in the development of skincare products.

An efficient and high-throughput method for the evaluation of mitochondrial dysfunction in frozen brain samples after traumatic brain injury.

Mitochondrial function analysis is a well-established method used in preclinical and clinical investigations to assess pathophysiological changes in various disease states, including traumatic brain injury (TBI). Although there are multiple approaches to assess mitochondrial function, one common method involves respirometric assays utilizing either Clark-type oxygen electrodes or fluorescent-based Seahorse analysis (Agilent). However, these functional analysis methods are typically limited to the availability of freshly isolated tissue samples due to the compromise of the electron transport chain (ETC) upon storage, caused by freeze-thaw-mediated breakdown of mitochondrial membranes. In this study, we propose and refine a method for evaluating electron flux through the ETC, encompassing complexes I, II, and IV, in frozen homogenates or mitochondrial samples within a single well of a Seahorse plate. Initially, we demonstrate the impact of TBI on freshly isolated mitochondria using the conventional oxidative phosphorylation protocol (OxPP), followed by a comparison with ETC analysis conducted on frozen tissue samples within the context of a controlled cortical impact (CCI) model of TBI. Additionally, we explore the effects of mitochondrial isolation from fresh versus snap-frozen brain tissues and their storage at -80°C, assessing its impact on electron transport chain protocol (ETCP) activity. Our findings indicate that while both sets of samples were frozen at a single time point, mitochondria from snap-frozen tissues exhibited reduced injury effects compared to preparations from fresh tissues, which were either homogenized or isolated into mitochondria and subsequently frozen for later use. Thus, we demonstrate that the preparation of homogenates or isolated mitochondria can serve as an appropriate method for storing brain samples, allowing for later analysis of mitochondrial function, following TBI using ETCP.

Multisite studies for optimization of a highly efficient culture assay used for in vitro detection of residual undifferentiated human pluripotent stem cells intermingled in cell therapy products.

Introduction: MEASURE2 (Multisite Evaluation Study on Analytical Methods for Non-clinical Safety Assessment of HUman-derived REgenerative Medical Products 2) is a Japanese experimental public-private partnership initiative that aims to standardize testing methods for tumorigenicity evaluation of human pluripotent stem cell (hPSC)-derived cell therapy products (CTPs). MEASURE2 organized multisite studies to optimize the methodology of the highly efficient culture (HEC) assay, a sensitive culture-based in vitro assay for detecting residual undifferentiated hPSCs in CTPs. Methods: In these multisite studies, 1) the efficiency of colony formation by human induced pluripotent stem cells (hiPSCs) under two different culture conditions and 2) the sorting efficiency of microbeads conjugated to various anti-hPSC markers during hiPSC enrichment were evaluated using samples in which hiPSCs were spiked into hiPSC-derived mesenchymal stem cells. Results: The efficiency of colony formation was significantly higher under culture conditions with the combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) than with Y-27632, which is widely used for the survival of hPSCs. Between-laboratory variance was also smaller under the condition with CEPT than with Y-27632. The sorting efficiency of microbeads conjugated with the anti-Tra-1-60 antibody was sufficiently higher (>80%) than those of the other various microbeads investigated. Conclusions: Results of these multisite studies are expected to contribute to improvements in the sensitivity and robustness of the HEC assay, as well as to the future standardization of the tumorigenicity risk assessment of hPSC-derived CTPs.

Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia.

Background: The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor (LDLR) gene leading to familial hypercholesterolemia (FH) was chosen as a model. Methods: Hypercholesterolemic individuals (n = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY® technique. Results: Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, LDLR mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY®. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY®. Conclusion: The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the LDLR gene. This LFA can also be used to screen family members with E101K variant in the LDLR gene and is applicable for other SNP's detection.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 nanograms of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

A safe, cost-effective, and high-throughput SARS-CoV-2 antigen capture ELISA suitable for large-scale screening in low-resource settings.

Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening.

Comparative assessment of the diagnostic performances of particle-based multianalyte technology and commercial ELISA for antiphospholipid autoantibody testing.

BACKGROUND: Diagnosing Antiphospholipid Syndrome (APS) relies heavily on laboratory findings, particularly the detection of specific antibodies like lupus anticoagulant (LA), IgG and/or IgM anti-cardiolipin (aCL), and IgG and/or IgM anti-ß2 glycoprotein 1 (aB2GP1). Although ELISA is widely used in the US for this purpose, standardization between different assay methodologies remains challenging, leading to significant variability across laboratories. Particle-based multi-analyte technology (PMAT) offers a streamlined one-step detection for all six antiphospholipid (aPL) autoantibodies, covering aCL and aB2GP1 of IgA, IgG, and IgM isotypes. METHODS: In this study involving 224 subjects, including 34 clinically diagnosed with APS, alongside 160 non-APS patients and 30 healthy donors, PMAT's performance was evaluated against commercial ELISA in detecting aPL antibodies. RESULTS: At the manufacturer's suggested cutoff, PMAT exhibited sensitivity comparable to ELISA, albeit with a low to moderate decrease in specificity for certain antibodies. With anti-CL IgM alone, PMAT displayed a 17.7% decrease in sensitivity, accompanied by a corresponding 31.1% increase in specificity compared to ELISA. However, applying a stricter cutoff (88-90% specificity), IgA and IgM antibodies yielded 5.9-17.6% higher sensitivities with PMAT, and IgG antibodies displayed similar sensitivity. CONCLUSIONS: In this study cohort, PMAT demonstrated higher or comparable sensitivity to that of commercial ELISA for all six aPL antibodies at a specificity cutoff near 90%. Notably, PMAT demonstrated superior sensitivity and specificity overall in detecting IgA aCL and aB2GP1 antibodies. This study highlights the potential of automated PMAT for detecting aPL antibodies in APS evaluation.

High variability in assays of blood markers of collagen turnover in cardiovascular disease: Implications for research and clinical practice.

AIMS: Fibrosis is a common feature of many chronic diseases, including heart failure, which can have deleterious effects on cardiac structure and function that are associated with adverse outcomes. By-products of collagen synthesis and degradation, such as carboxy- and amino-terminal pro- or telo-peptides of collagen type I and III (PICP, PINP, PIIINP, and CITP) have been extensively investigated as markers of fibrosis. Although the majority of studies report on the reproducibility of their assay results, there is no a comparison of biomarker assays across studies. Therefore, we conducted a systematic review adhering to PRISMA guidelines. METHODS AND RESULTS: The search terms employed in Medline were: 'collagen AND cardiac' or 'collagen AND heart'. This query yielded a total of 1049 articles. Thereafter, specific search criteria were applied: (i) original English-language papers; (ii) human studies; (iii) in-vivo investigations; and (iv) blood/serum/plasma samples. Overall, 89 studies were identified (42 on PIIINP, 32 on PICP, 29 on CITP, and 17 on PINP). The range of reported values for PIIINP was between 0.06 to 11 800 µg/l; for PICP 0.006 to 1265 µg/l; for CITP 0.3 to 5450 µg/l; for PINP 0.15 to 80 µg/l. Extreme variations in values for fibrosis biomarkers were observed across studies, especially when different assays were used, but also with the same assays. CONCLUSIONS: Our findings show that it is challenging to ascertain normal ranges or compare studies for the measurement of fibrosis biomarkers. Given the potential implications for clinical practice and current lack of awareness of these issues, this subject warrants comprehensive acknowledgement and understanding.

A sensitive gold nanoflower-based lateral flow assay coupled with gold staining technique for the detection of SARS-CoV-2 antigen.

An enhanced lateral flow assay (LFA) is presented for rapid and highly sensitive detection of acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens with gold nanoflowers (Au NFs) as signaling markers and gold enhancement to amplify the signal intensities. First, the effect of the morphology of gold nanomaterials on the sensitivity of LFA detection was investigated. The results showed that Au NFs prepared by the seed growth method showed a 5-fold higher detection sensitivity than gold nanoparticles (Au NPs) of the same particle size, which may benefit from the higher extinction coefficient and larger specific surface area of Au NFs. Under the optimized experimental conditions, the Au NFs-based LFA exhibited a detection limit (LOD) of 25 pg mL-1 for N protein using 135 nm Au NFs as the signaling probes. The signal was further amplified by using a gold enhancement strategy, and the LOD for the detection of N protein achieved was 5 pg mL-1. The established LFA also exhibited good repeatability and stability and showed applicability in the diagnosis of SARS-CoV-2 infection.

Multi-analyte proteomic analysis identifies blood-based neuroinflammation, cerebrovascular and synaptic biomarkers in preclinical Alzheimer's disease.

Background: Blood-based biomarkers are gaining grounds for Alzheimer's disease (AD) detection. However, two key obstacles need to be addressed: the lack of methods for multi-analyte assessments and the need for markers of neuroinflammation, vascular, and synaptic dysfunction. Here, we evaluated a novel multi-analyte biomarker platform, NULISAseq CNS disease panel, a multiplex NUcleic acid-linked Immuno-Sandwich Assay (NULISA) targeting ~120 analytes, including classical AD biomarkers and key proteins defining various disease hallmarks. Methods: The NULISAseq panel was applied to 176 plasma samples from the MYHAT-NI cohort of cognitively normal participants from an economically underserved region in Western Pennsylvania. Classical AD biomarkers, including p-tau181 p-tau217, p-tau231, GFAP, NEFL, Aß40, and Aß42, were also measured using Single Molecule Array (Simoa). Amyloid pathology, tau pathology, and neurodegeneration were evaluated with [11C] PiB PET, [18F]AV-1451 PET, and MRI, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA biomarkers and AD pathologies. Spearman correlations were used to compare NULISA and Simoa. Results: NULISA concurrently measured 116 plasma biomarkers with good technical performance, and good correlation with Simoa measures. Cross-sectionally, p-tau217 was the top hit to identify Aß pathology, with age, sex, and APOE genotype-adjusted AUC of 0.930 (95%CI: 0.878-0.983). Fourteen markers were significantly decreased in Aß-PET+ participants, including TIMP3, which regulates brain Aß production, the neurotrophic factor BDNF, the energy metabolism marker MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aß PET-dependent yearly increases in Aß-PET+ participants. Markers with tau PET-dependent longitudinal changes included the microglial activation marker CHIT1, the reactive astrogliosis marker CHI3L1, the synaptic protein NPTX1, and the cerebrovascular markers PGF, PDGFRB, and VEFGA; all previously linked to AD but only reliably measured in cerebrospinal fluid. SQSTM1, the autophagosome cargo protein, exhibited a significant association with neurodegeneration status after adjusting age, sex, and APOE ε4 genotype. Conclusions: Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD.

The Spectrum of Presumed Tubercular Uveitis in a Referral Eye Clinic in Qatar.

PURPOSE: To analyze the clinical spectrum of patients with presumed tubercular uveitis in a referral eye clinic in Qatar. METHODS: We retrospectively reviewed the clinical records of 50 patients (80 eyes) diagnosed with presumed ocular tuberculosis who presented to the uveitis clinic, department of Ophthalmology Hamad Medical Corporation, Qatar, from January 2014 till December 2019. RESULTS: Mean age at presentation was 34.5 ± 9.3 years. Forty one patients were males (82%) and 30 patients had bilateral involvement (60%). Forty eyes (50%) had posterior uveitis, 21 eyes (26.3%) intermediate uveitis, 11 eyes (13.7%) panuveitis, and 8 eyes (10%) anterior uveitis. Ocular findings included vitritis in 82.5% of eyes, retinal vasculitis in 46.3% (92% of which were occlusive in nature), multifocal choroiditis in 18.8%, serpiginous-like choroiditis in 11.3%, Most common complications at presentation or during follow-up included macular edema in 32 eyes (40%), preretinal or optic disc neovascularization in 29 eyes (36.3%) and vitreous hemorrhage in 17 eyes (21.3%). Anti-tubercular treatment was provided to 46 patients (92%). Systemic corticosteroids and corticosteroid sparing agents were associated, respectively, in 39 patients (78%) and 14 patients (28%). After 1 year of follow up, inflammation was controlled, with a significant improvement in visual acuity (p < 0.0001). CONCLUSIONS: In Qatar, tubercular uveitis has a broad spectrum of ocular features, with posterior and intermediate uveitis being the most common anatomic forms. Vitritis, multifocal choroiditis without or with a serpiginous-like pattern, and occlusive retinal vasculitis are the most common ocular findings. Main sight-threatening ocular complications are macular edema, posterior segment neovascularization, and vitreous hemorrhage.

Application of a Fluorescence Recovery-Based Polo-Like Kinase 1 Binding Assay to Polo-Like Kinase 2 and Polo-Like Kinase 3.

Assay systems for evaluating compound protein-binding affinities are essential for developing agonists and/or antagonists. Targeting individual members of a protein family can be extremely important and for this reason it is critical to have methods for evaluating selectivity. We have previously reported a fluorescence recovery assay that employs a fluorescein-labelled probe to determine IC50 values of ATP-competitive type 1 inhibitors of polo-like kinase 1 (Plk1). This probe is based on the potent Plk1 inhibitor BI2536 [fluorescein isothiocyanate (FITC)-polyethylene glycol (PEG)-lysine (Lys) (BI2536) 1]. Herein, we extend this approach to the highly homologous Plk2 and Plk3 members of this kinase family. Our results suggest that this assay system is suitable for evaluating binding affinities against Plk2 and Plk3 as well as Plk1. The new methodology represents the first example of evaluating N-terminal catalytic kinase domain (KD) affinities of Plk2 and Plk3. It represents a simple and cost-effective alternative to traditional kinase assays to explore the KD-binding compounds against Plk2 and Plk3 as well as Plk1.