Combining Bioorthogonal Chemistry with Fluorescent Silica Nanoparticles for the Ultrasensitive Detection of the HIV-1 p24 Antigen.

Early detection and viral concentration monitoring of human immunodeficiency virus in resource-poor settings are important to control disease spread and reduce mortality. Nucleic acid amplification tests are expensive for low-resource settings. Lateral flow antibody tests are not sensitive if testing is performed within 7-10 days, and these tests are not quantitative. We describe a signal enhancement technique based on fluorescent silica nanoparticles and bioorthogonal chemistries for the femtomolar detection of the HIV-1 p24 antigen. We developed a magnetic bead-based assay, wherein we used fluorescent-dye-encapsulated silica nanoparticles as reporters. The number of reporters was increased by using bioorthogonal chemistry to provide signal enhancement. The limit and range of detection of the sandwich immunoassay using alternating multiple layers for p24 in human serum were found to be 46 fg/mL (1.84 fM) and 46 fg/mL to 10 ng/mL, respectively. This simple assay was 217-fold higher in sensitivity compared to that of commercial enzyme-linked immunoassays (limit of detection of 10 pg/mL).

A two-generational reproductive study to assess the effects of Juglans regia on reproductive developments in the male and female rats.

Environmental pollutants and lifestyle severely threaten human and animal health, leading to disturbances of various functions, including infertility. So, exploring a safe treatment that could effectively reverse infertility remains a challenge. The current study was intended to explore the fertility-enhancing effect of Juglans Regia oil in two successive generations of rats; F0 and F1. J. Regia oil was initially tested for in vitro antioxidant assay via ROS and DPPH, followed by in vivo toxicity testing. In the fertility assessment, eighteen pairs of male and female rats (n=36, 1:1, F0 generation) were divided into three groups and dosed with 1 mL/kg and 2 mL/kg daily of J. Regia oil and saline, respectively, up to pre-cohabitation, cohabitation, gestation and lactation periods. The reproductive performance, including body weight, live birth index, fertility index, and litter size, was assessed. Hormonal and antioxidant markers of F1 generations were assessed with the histopathological evaluation of male and female organs. The oil of J. Regia showed great antioxidant potential (P < 0.05) in DPPH (1,1-diphenyl-2-picrylhydrazyl) and ROS (Reactive Oxygen Species) methods (P<0.05). The continued exposure of the F0 and F1 generations to J. Regia oil did not affect body weight, fertility index, litter size, and survival index. We have found pronounced fertility outcomes in both genders of F0 and F1 generations with J. Regia 2 mL/kg/day in comparison to the control. Results showed that J. Regia significantly increased (P < 0.05) luteinizing hormone (LH), plasma testosterone, follicular stimulating hormone (FSH), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities in both generations. Histology of both generations reveals improved spermatogenesis and folliculogenesis with enhanced architecture. Altogether, the present results suggest that J. Regia improved fertility in both male and female rats by improving hormonal activities and oxidative stress.

An evaluation of a one-day pain science education event in a high school setting targeting pain related beliefs, knowledge, and behavioural intentions.

BACKGROUND: Persistent pain is a common condition affecting one in four UK adults. Public understanding of pain is limited. Delivering pain education within schools may improve public understanding in the longer term. OBJECTIVE: To evaluate the impact of a one-day Pain Science Education (PSE) event on sixth form/high school students' pain beliefs, knowledge and behavioural intention. METHODS: Exploratory, single-site, mixed-methods, single-arm study involving secondary school students ≥16 years old attending a one-day PSE event. Outcome measures included the Pain Beliefs Questionnaire (PBQ), Concepts of Pain Inventory (COPI-ADULT), a vignette to assess pain behaviours; and thematic analysis of semi-structured interviews. RESULTS: Ninety (mean age 16.5 years, 74% female) of the 114 attendees, agreed to participate in the evaluation. PBQ scores improved on the Organic beliefs subscale [mean difference -5.9 (95% CI -6.8, -5.0), P < 0.01] and Psychosocial Beliefs subscale [1.6 (1.0, 2.2) P < 0.01]. The COPI-Adult revealed an improvement [7.1 (6.0-8.1) points, P < 0.01] between baseline and post intervention. Pain behavioural intentions improved post education for work, exercise, and bed rest related activities (p < 0.05). Thematic analysis of interviews (n = 3) identified increased awareness of chronic pain and its underpinning biology, beliefs that pain education should be widely available, and that pain management should be holistic. CONCLUSIONS: A one-day PSE public health event can improve pain beliefs, knowledge and behavioural intentions in high school students and increase openness to holistic management. Future controlled studies are needed to confirm these results and investigate potential long-term impacts.

Palladium encapsulated mesoporous silica nanoparticles for the rapid detection of analytes.

We designed a simple, inexpensive, and user-friendly assay using mesoporous silica nanoparticles to detect analytes. Highly stable and uniform palladium nanoparticles covered with mesoporous silica (Pd@mSiO2) were generated and characterized extensively using physical methods. Human Serum Albumin (HSA) protein or ssDNA specific to the HIV gag region was capped onto the Pd@mSiO2 electrostatically. This "cap" prevented the Pd(0) inside the mesoporous silica nanoparticles from catalyzing the conversion of non-fluorescent molecules to fluorescent molecules. In the presence of target anti-HSA antibodies or complementary sequence (HIV gag), HSA protein or DNA cap dissociated from the surface of Pd@mSiO2-NH2 through the specific antigen-antibody reaction or DNA hybridization, allowing Pd(0) to convert the non-fluorescent molecules to fluorescent molecules. The limit and range of detection of anti-HSA antibodies were 3.8 nM and 3.8 nM to 133.3 nM, respectively. The limit and range of detection of HIV gag were 1.6 nM and 1.6 nM to 15 nM, respectively. This simple, inexpensive, "add sample and measure" diagnostic assay could potentially be incorporated into point of care diagnostics for low-resource settings.

Change in surface characteristics and permeability of human enamel after subjecting to radiation therapy.

Background: Radiation-related caries is a complex destructive lesion leading to uncompromising damage of enamel and dentin in patients suffering from head and neck cancer managed with radiotherapy. Aim: The purpose of this study was to evaluate the changes in the permeability of enamel and to assess the morphological and chemical changes of teeth surface subjected to 6 MV photon beam irradiation. Materials and Methods: For this in vitro study, coronal portion of 20 premolars were sectioned mesiodistally into halves and then grouped into two. Samples in group 1 (control) were not subjected to cycles of irradiation and those in group 2 (experimental) were subjected to a cumulative uniform radiation dose of 70 Gray fractioned in 35 fractions with 6 MV photons. The silver nitrate penetration method was used to assess the change in permeability of enamel. The variations in surface topography and mineral content were assessed using scanning electron microscopy with energy dispersive X-ray analysis. Dye penetration scores of surface texture changes were compared between the two groups utilizing the Chi-square test. The change in the elemental levels between enamel surfaces of the two groups was compared using an independent t-test. Results: The application of 6 MV photon radiation did not change enamel permeability and surface topography. However, a noteworthy reduction in the carbon content (P = 0.002) was observed in teeth subjected to irradiation. Conclusions: Though radiation exposure did not alter the enamel permeability and surface topography, it had caused significant chemical compositional changes. Carbon content was significantly reduced in irradiated enamel samples.

Inflammatory bowel disease biomarkers.

Inflammatory bowel disease (IBD) is characterized as chronic inflammation in the gastrointestinal tract, which includes two main subtypes, Crohn's disease and ulcerative colitis. Endoscopy combined with biopsy is the most effective way to establish IBD diagnosis and disease management. Imaging techniques have also been developed to monitor IBD. Although effective, the methods are expensive and invasive, which leads to pain and discomfort. Alternative noninvasive biomarkers are being explored as tools for IBD prognosis and disease management. This review focuses on novel biomarkers that have emerged in recent years. These serological biomarkers and microRNAs could potentially be used for disease management in IBD, thereby decreasing patient discomfort and morbidity.

Toward Point-of-Care Diagnostics to Monitor MMP-9 and TNF-α Levels in Inflammatory Bowel Disease.

We have investigated the association of matrix metallopeptidase 9 (MMP-9) and tumor necrosis factor α (TNF-α) levels with colitis severity using an established IL10-/- mouse model, which reflects the severity of inflammation in humans with inflammatory bowel disease (IBD). We found that MMP-9 and TNF-α correlated with colitis severity. In parallel, we developed assays to detect fecal MMP-9 and serum TNF-α using "cap and release" mesoporous silica nanoparticles (MSNs). MMP-9 peptide substrates as "caps" were attached to dye-loaded MSNs. The introduction of MMP-9 resulted in substrate cleavage and subsequent dye release, which was rapidly detected using a fluorometer. For TNF-α, an anti-TNF antibody was used as the "cap". The introduction of TNF-α antigen leads to the release of the dyes because the antigen binds more strongly to the antibody cap. The MSN-based assays can detect MMP-9 and TNF-α effectively, although signal amplification is required to meet clinical sensitivity.

ASSURED-SQVM diagnostics for COVID-19: addressing the why, when, where, who, what and how of testing.

Introduction: SARS-CoV-2, the new coronavirus that originated in 2019, continues to impact every aspect of society in a profound manner. Testing will remain an important tool to mitigate the effects of this pandemic as early and accurate diagnosis can lead to appropriate countermeasures to reduce mortality and morbidity. However, testing isn't a simple yes/no answer as the target and host are complex, the virus is a moving target, there is a plethora of tests that identify different parts of the virus and have their own limits and range of detection, and when prevalence is low, false positives and negatives can be very high.Areas covered: This article covers all the major questions related to COVID-19 diagnostics, the why, when, where, who, what and how of testing, the different types of tests, interpretation of results and the ideal ASSURED-SQVM diagnostic. A comprehensive literature review using all the publicly available databases and government websites and reports was performed.Expert opinion: Diagnostics that meet the 'ASSURED-SQVM' (Affordable, Selective and Sensitive, User-friendly, Rapid and Robust, Equipment-free, Deliverable to end-users and additionally, allows for Self-testing, Quantifiable, detects if pathogens are Viable and can detect Multiple pathogens) would make a major impact in our fight against the current pandemic. While a significant majority of researchers focus on developing novel diagnostics that are highly selective and sensitive, it is the opinion of these authors that other aspects of the ASSURED-SQVM principles also be considered early in the development process for widespread use.

Point-of-Care Monitoring of Colitis Using Intestinal Alkaline Phosphatase in Inflammatory Bowel Disease.

Intestinal Alkaline Phosphatase (IAP) was investigated as a potential biomarker to monitor colitis in a mouse model of Inflammatory Bowel Disease (IBD). We developed a Point-Of-Care (POC) assay to detect IAP with a glucose meter in 15 min. We synthesized a paracetamol-bearing compound specifically cleaved by IAP to release paracetamol, which can be detected with a personal glucometer. Interleukin 10 deficient (IL 10-/-) mouse model samples were used to compare the IAP level in mice with mild or severe colitis. The results showed that fecal IAP level was significantly lower in each mouse sample with severe colitis than with mild colitis. Mice treated with anti-Tumor Necrosis Factor-alpha (anti-TNF-α) to decrease inflammation exhibited a much higher level of IAP than those without treatment (IAP levels from anti-TNF-α treated vs nontreated = 2.80 U vs 0.11 U, P < 0.0001). Taken together, IAP can be considered as a potential biomarker to monitor colitis, and a rapid, user-friendly POC glucometer-based assay can be potentially used to monitor colitis levels and inflammation flareups in IBD.

Rapid, user-friendly, and inexpensive detection of azidothymidine.

Strict adherence to highly active antiretroviral therapy (HAART) is very important to improve the quality of life for HIV-positive patients to reduce new infections and determine treatment success. Azidothymidine (AZT) is an antiretroviral drug commonly used in HAART treatment. In this research, an "add, mix, and measure" assay was developed to detect AZT within minutes. Three different probes designed to release fluorophores when samples containing AZT are added were synthesized and characterized. The limit of detection to AZT in simulated urine samples was determined to be 4 µM in 5 min for one of the probes. This simple and rapid point-of-care test could potentially be used by clinicians and health care workers to monitor the presence of AZT in low resource settings.

Dynamics of cluster-forming hub-filament systems: The case of the high-mass star-forming complex Monoceros R2.

CONTEXT: High-mass stars and star clusters commonly form within hub-filament systems. Monoceros R2 (hereafter Mon R2), at a distance of 830 pc, harbors one of the closest such systems, making it an excellent target for case studies. AIMS: We investigate the morphology, stability and dynamical properties of the Mon R2 hub-filament system. METHODS: We employ observations of the 13CO and C18O 1→0 and 2→1 lines obtained with the IRAM-30m telescope. We also use H2 column density maps derived from Herschel dust emission observations. RESULTS: We identified the filamentary network in Mon R2 with the DisPerSE algorithm and characterized the individual filaments as either main (converging into the hub) or secondary (converging to a main filament) filaments. The main filaments have line masses of 30-100 M ⊙ pc-1 and show signs of fragmentation, while the secondary filaments have line masses of 12-60 M ⊙ pc-1 and show fragmentation only sporadically. In the context of Ostriker's hydrostatic filament model, the main filaments are thermally supercritical. If non-thermal motions are included, most of them are trans-critical. Most of the secondary filaments are roughly transcritical regardless of whether non-thermal motions are included or not. From the morphology and kinematics of the main filaments, we estimate a mass accretion rate of 10-4-10-3 M ⊙ yr-1 into the central hub. The secondary filaments accrete into the main filaments with a rate of 0.1-0.4×10-4 M ⊙ yr-1. The main filaments extend into the central hub. Their velocity gradients increase towards the hub, suggesting acceleration of the gas.We estimate that with the observed infall velocity, the mass-doubling time of the hub is ~ 2:5 Myr, ten times larger than the free-fall time, suggesting a dynamically old region. These timescales are comparable with the chemical age of the Hii region. Inside the hub, the main filaments show a ring- or a spiral-like morphology that exhibits rotation and infall motions. One possible explanation for the morphology is that gas is falling into the central cluster following a spiral-like pattern.

Fluorescent sialic derivatives for the specific detection of influenza viruses.

Early and accurate diagnosis of influenza viruses can decrease its harmful impact. Here, we have synthesized fluorescent sialic acid derivatives that are cleaved by influenza neuraminidases (NAs) and not by Streptococcus pneumoniae that also inhabits the human olfactory. We have also attempted to develop assays that could differentiate between influenza virus and S. pneumoniae by taking advantage of the structural differences between NAs from these pathogens.

Disruption of the Orion molecular core 1 by wind from the massive star θ1 Orionis C.

Massive stars inject mechanical and radiative energy into the surrounding environment, which stirs it up, heats the gas, produces cloud and intercloud phases in the interstellar medium, and disrupts molecular clouds (the birth sites of new stars1,2). Stellar winds, supernova explosions and ionization by ultraviolet photons control the lifetimes of molecular clouds3-7. Theoretical studies predict that momentum injection by radiation should dominate that by stellar winds8, but this has been difficult to assess observationally. Velocity-resolved large-scale images in the fine-structure line of ionized carbon ([C II]) provide an observational diagnostic for the radiative energy input and the dynamics of the interstellar medium around massive stars. Here we report observations of a one-square-degree region (about 7 parsecs in diameter) of Orion molecular core 1-the region nearest to Earth that exhibits massive-star formation-at a resolution of 16 arcseconds (0.03 parsecs) in the [C II] line at 1.9 terahertz (158 micrometres). The results reveal that the stellar wind originating from the massive star θ1 Orionis C has swept up the surrounding material to create a 'bubble' roughly four parsecs in diameter with a 2,600-solar-mass shell, which is expanding at 13 kilometres per second. This finding demonstrates that the mechanical energy from the stellar wind is converted very efficiently into kinetic energy of the shell and causes more disruption of the Orion molecular core 1 than do photo-ionization and evaporation or future supernova explosions.

Detection of Enzymes, Viruses, and Bacteria Using Glucose Meters.

We have developed innovative assays that can detect enzymes rapidly. Paracetamol- or catechol-bearing compounds, when exposed to their respective enzymes, released paracetamol or catechol, which can be detected using a standard glucose meter. This approach was used to detect a number of diverse analytes that include enzymes such as ß-galactosidase and α-mannosidase and pathogens such as influenza viruses, Streptococcus pneumoniae, and E. coli rapidly. The limit of detection for all analytes was extremely low and clinically relevant for influenza viruses. We also demonstrate that glucose oxidase or glucose dehydrogenase is not required because the paracetamol gets oxidized directly on the electrode surface. This indicates that test strips without glucose oxidase or dehydrogenase can be used, and we can detect analytes in the presence of high levels of background glucose. We demonstrate this unique nature of the assay to detect paracetamol in simulated urine and sheep blood without background interference of intrinsic glucose, indicating that glucose meters can be used to detect nonglucose analytes without background glucose interference.

Multivalent oleanolic acid human serum albumin conjugate as nonglycosylated neomucin for influenza virus capture and entry inhibition.

We report the synthesis of multivalent oleanolic acid (OA) protein conjugates as nonglycosylated neomucin mimic for the capture and entry inhibition of influenza viruses. Oleanolic acid derivatives bearing an amine-terminated linker were synthesized by esterification of carboxylic acid and further grafted onto the human serum albumin (HSA) via diethyl squarate method. The binding of hemagglutinin (HA) on the virion surface to the synthetic neomucin was evaluated by hemagglutination inhibition assay. The influenza virus capture ability of the PEGylated OA-HSA conjugate was further investigated by Dynamic Light Scattering (DLS), virus capture assay and Isothermal Titration Calorimeter (ITC) techniques. The pronounced agglutination of viral particles, the high capture efficiency and affinity constant indicate that this neoprotein is comparable to natural glycosylated mucin, suggesting that this material could potentially be used as anti-infective barriers to prevent virus from invading host cells. The study also rationalizes the feasibility of antiviral drug development based on OA or other antiviral small molecules conjugated protein strategies.

Permanent tooth agenesis in individuals with non-syndromic Robin sequence: a systematic review and meta-analysis.

OBJECTIVES: To evaluate prevalence rates and patterns of permanent tooth agenesis in individuals with non-syndromic Robin sequence (ns-RS). MATERIAL AND METHODS: Two investigators independently carried out a literature search, using PubMed, Web of Science, Ovid, EMBASE and the Cochrane Library, identifying articles relating to permanent tooth agenesis, diagnosed using panoramic radiographs, in individuals with ns-RS. The outcomes desired were prevalence rates and patterns of permanent tooth agenesis. The final search was carried out in January 2017. RESULTS: Six articles were selected for inclusion, with a total of 448 individuals with ns-RS. Data available in each study varied which allowed conducting further specific meta-analyses only on sample sizes of 339 or 378 individuals from three or four of the six included articles. The overall estimated prevalence rate of permanent tooth agenesis was 42% (95% CI: 35%-48%), excluding third molars, with no sex predilection. In individuals with tooth agenesis, approximately 30% had one missing tooth while 40% had two missing teeth. Tooth agenesis was more commonly bilateral (Relative Risk 2) and found in the mandible (Relative Risk 1.6). The highest prevalence of permanent tooth agenesis was found for mandibular second premolars (26%) followed by maxillary second premolars (14%). The most common tooth agenesis patterns were the agenesis of both mandibular second premolars, followed by the agenesis of all second premolars. CONCLUSION: Individuals with ns-RS have high prevalence rates of permanent tooth agenesis. The most prevalent tooth agenesis patterns are bilateral symmetric tooth agenesis, most commonly agenesis of both mandibular second premolars.

Impact of automated ICA-based denoising of fMRI data in acute stroke patients.

Different strategies have been developed using Independent Component Analysis (ICA) to automatically de-noise fMRI data, either focusing on removing only certain components (e.g. motion-ICA-AROMA, Pruim et al., 2015a) or using more complex classifiers to remove multiple types of noise components (e.g. FIX, Salimi-Khorshidi et al., 2014 Griffanti et al., 2014). However, denoising data obtained in an acute setting might prove challenging: the presence of multiple noise sources may not allow focused strategies to clean the data enough and the heterogeneity in the data may be so great to critically undermine complex approaches. The purpose of this study was to explore what automated ICA based approach would better cope with these limitations when cleaning fMRI data obtained from acute stroke patients. The performance of a focused classifier (ICA-AROMA) and a complex classifier (FIX) approaches were compared using data obtained from twenty consecutive acute lacunar stroke patients using metrics determining RSN identification, RSN reproducibility, changes in the BOLD variance, differences in the estimation of functional connectivity and loss of temporal degrees of freedom. The use of generic-trained FIX resulted in misclassification of components and significant loss of signal (< 80%), and was not explored further. Both ICA-AROMA and patient-trained FIX based denoising approaches resulted in significantly improved RSN reproducibility (p < 0.001), localized reduction in BOLD variance consistent with noise removal, and significant changes in functional connectivity (p < 0.001). Patient-trained FIX resulted in higher RSN identifiability (p < 0.001) and wider changes both in the BOLD variance and in functional connectivity compared to ICA-AROMA. The success of ICA-AROMA suggests that by focusing on selected components the full automation can deliver meaningful data for analysis even in population with multiple sources of noise. However, the time invested to train FIX with appropriate patient data proved valuable, particularly in improving the signal-to-noise ratio.

Highly specific and rapid glycan based amperometric detection of influenza viruses.

Rapid and precise detection of influenza viruses in a point of care setting is critical for applying appropriate countermeasures. Current methods such as nucleic acid or antibody based techniques are expensive or suffer from low sensitivity, respectively. We have developed an assay that uses glucose test strips and a handheld potentiostat to detect the influenza virus with high specificity. Influenza surface glycoprotein neuraminidase (NA), but not bacterial NA, cleaved galactose bearing substrates, 4,7di-OMe N-acetylneuraminic acid attached to the 3 or 6 position of galactose, to release galactose. In contrast, viral and bacterial NA cleaved the natural substrate, N-acetylneuraminic acid attached to the 3 or 6 position of galactose. The released galactose was detected amperometrically using a handheld potentiostat and dehydrogenase bearing glucose test strips. The specificity for influenza was confirmed using influenza strains and different respiratory pathogens that include Streptococcus pneumoniae and Haemophilus influenzae; bacteria do not cleave these molecules. The assay was also used to detect co-infections caused by influenza and bacterial NA. Viral drug susceptibility and testing with human clinical samples was successful in 15 minutes, indicating that this assay could be used to rapidly detect influenza viruses at primary care or resource poor settings using ubiquitous glucose meters.

Multivalent S-sialoside protein conjugates block influenza hemagglutinin and neuraminidase.

A new class of S-sialoside Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) conjugates were prepared to enhance the binding affinity to hemagglutinin (HA) and neuraminidase (NA). The valency of glycoconjugates was controlled by the reaction ratio of the S-sialoside monomer and protein. Hemagglutination inhibition assay showed that these synthetic glycoproteins have higher affinity to HA than the small clusters of sialosides with lower valency, due to multivalent effect and optimized three dimensional presentation of sialosides on the protein platform. The results of fluorescent NA inhibition assay showed that some of the conjugates have moderate NA inhibitory activity, in comparison to the monomer and low valent conjugates with weak or none inhibitory activity. These synthetic sialylated proteins were not cytotoxic with concentrations up to 100 µM, since the sialylation did not change the secondary structure of protein. This new kind of conjugates can be used as lead compounds for antiviral drug design and the construction of pseudo sialoside-protein conjugates library to investigate the carbohydrate-HA/NA recognition process and a platform for the influenza virus capturing.

Toward the Development of the Next Generation of a Rapid Diagnostic Test: Synthesis of Glycophosphatidylinositol (GPI) Analogues of Plasmodium falciparum and Immunological Characterization.

A large number of proteins in malaria parasites are anchored using glycophosphatidylinositols (GPIs) with lipid tails. These GPIs are structurally distinct from human GPIs. Plasmodium falciparum GPIs have been considered as potential vaccine candidates because these molecules are involved in inducing inflammatory responses in human hosts, and natural anti-GPI antibody responses have been shown to be associated with protection against severe disease. GPIs can also be considered as targets for rapid diagnostic tests. Because isolation of native GPIs in large quantities is challenging, development of synthetic GPI molecules can facilitate further exploration of GPI molecules for diagnostics. Here, we report synthesis and immunological characterization of a panel of malaria-specific GPI analogues. A total of three GPI analogues were chemically synthesized and conjugated to a carrier protein to immunize and generate antibodies in rabbits. The rabbit immune sera showed reactivity with synthetic GPIs and native GPIs extracted from P. falciparum parasite, as determined by Luminex and ELISA methods.