Dual-Functional Peroxidase-Copper Phosphate Hybrid Nanoflowers for Sensitive Detection of Biological Thiols

An effective strategy to detect biological thiols (biothiols), including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), holds significant incentive since they play vital roles in many cellular processes and are closely related to many diseases. Here, we demonstrated that hybrid nanoflowers composed of crystalline copper phosphate and horseradish peroxidase (HRP) served as a functional unit exhibiting dual catalytic activities of biothiol oxidase and HRP, yielding a cascade reaction system for a sensitive one-pot fluorescent detection of biothiols. The nanoflowers were synthesized through the anisotropic growth of copper phosphate petals coordinated with the amine/amide moieties of HRP, by simply incubating HRP and copper(II) sulfate for three days at room temperature. Copper phosphates within the nanoflowers oxidized target biothiols to generate H2O2, which activated the entrapped HRP to oxidize the employed Amplex UltraRed substrate to produce intense fluorescence. Using this strategy, biothiols were selectively and sensitively detected by monitoring the respective fluorescence intensity. This nanoflower-based strategy was also successfully employed for reliable quantification of biothiols present in human serum, demonstrating its great potential for clinical diagnostics.

Olfactory Sensory Neuron: Immunohistochemical Markers, Technique of Flat-Mounted Mucosa and Cell Count

A classification of olfactory sensory neuron (OSN) markers, a simple and robust technique of immunostaining on flat-mounted olfactory epithelium (OE) and a reliable quantification of the density of mature and immature OSNs are three crucial tools to study the pathophysiology of olfactory dysfunction. Using the rat model, we first categorized the main OSN markers by immunohistochemistry (IHC) on cross sections of OE. The OSN markers were divided into 3 groups: mature OSNs (OMP), immature OSNs (Tuj-1, DCX, OLIG2) and both (N-cadherin, LHX2, PGP9.5). The subcellular localization of each marker was also described. Tuj-1, OMP, DCX, PGP9.5 and N-cadherin were selected for immunostaining on flat-mounted OE because of their staining of OSN dendrites. We were able to successfully label OE with all the 5 markers using a simple technique for flat-mounted OE. In addition, this technique allowed the first mapping of the OE directly on the ethmoid turbinates. Finally, we quantified the mature (OMP+, Tuj-1-), immature (OMP-, Tuj-1+), transitory (OMP+, Tuj-1+) and total OSN density which were respectively 42080 ± 11820, 49384 ± 7134, 14448 ± 5865 and 105912 ± 13899 per mm2 (mean ± SD). The transitory population was quantified for the first time.

Application of Parallel Reaction Monitoring in 15N labeled Samples for Quantification

Accurate relative quantification is critical in proteomic studies. The incorporation of stable isotope 15N to plant-expressed proteins in vivo is a powerful tool for accurate quantification with a major advantage of reducing preparative and analytical variabilities. However, 15N labeling quantification has several challenges. Less identifications are often observed in the heavy labeled samples because of incomplete labeling, resulting in missing values in reciprocal labeling experiments. Inaccurate quantification can happen when there is contamination from co-eluting peptides or chemical noise in the MS1 survey scan. These drawbacks in quantification can be more pronounced in less abundant but biologically interesting proteins, which often have very few identified peptides. Here we demonstrate the application of parallel reaction monitoring (PRM) to 15N labeled samples on a high resolution, high mass accuracy Orbitrap mass spectrometer to achieve reliable quantification even of low abundance proteins in samples.

Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04–6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.

Advances in Scanning Microwave Impedance Microscopy

The never-ending scaling and increase in complexity of integrated circuits requires continual development of tools to understand and control the process of fabrication. However, scaling alone has not been enough to achieve improvements in performance. Material properties, dopant levels and other non-dimensional properties are now critical in increasing device performance beyond dimensional scaling. In the study presented here we report on advancements made in the measurement of electrical properties of materials using scanning Microwave Impedance Microscopy (sMIM). These advancements address the needs for certain electrical property measurements in semiconductors and have room for future growth. The latest incarnation of sMIM, the ScanWave Pro, achieves a sensitivity of less than 0.1 aF in capacitance and can measure minute changes in dielectric constant (k-value) and dopant levels in films. Additionally, we studied the response of these measurements across a range of materials and dopants used in semiconductor manufacturing and report here the results. For dielectric films and dopant levels, the response of the sMIM signal is log-linear in each case thus enabling easy and reliable quantification of measurements. The repeatability of the measurements was also studied and was found to be well within the requirements for process monitoring. This in turn, has enabled reliable quantification of results for this wide range of material properties. Up until now, sMIM measurements were qualitative and that limited their value. We implemented quantification of sMIM results and report on the approach and real-device results. Lastly, we explore additional potential applications and future developments.

Assessment of Citrinin in Spices and Infant Cereals Using Immunoaffinity Column Clean-Up with HPLC-Fluorescence Detection

Historically, the analysis of citrinin has mainly been performed on cereals such as red yeast rice; however, in recent years, more complex and abnormal commodities such as spices and infant foods are becoming more widely assessed. The aim of this study was to develop and validate clean-up methods for spices and cereal-based infant foods using a citrinin immunoaffinity column before HPLC analysis with fluorescence detection. Each method developed was validated with a representative matrix, spiked at various citrinin concentrations, based around European Union (EU) regulations set for ochratoxin A (OTA), with recoveries >80% and % RSD <9% in all cases. The limit of detection (LOD) and the limit of quantification (LOQ) were established at 1 and 3 µg/kg for spices and 0.1 and 0.25 µg/kg for infant cereals, respectively. These methods were then tested across a variety of spices and infant food products to establish efficacy with high recoveries >75% and % RSD <5% across all matrices assessed. Therefore, these methods proved suitable for providing effective clean-up of spices and infant cereals, enabling reliable quantification of citrinin detected. Samples such as nutmeg and infant multigrain porridge had higher levels of citrinin contamination than anticipated, indicating that citrinin could be a concern for public health. This highlighted the need for close monitoring of citrinin contamination in these commodities, which may become regulated in the future.

Rapid and Accurate Approach for Honeybee Pollen Analysis Using ED-XRF and FTIR Spectroscopy

Since honeybee pollen is considered a “perfectly complete food” and is characterized by many beneficial properties (anti-inflammatory, antioxidant, anti-bacterial, etc.), it has begun to be used for therapeutic purposes. Consequently, there is a high need to develop methods for controlling its composition. A thorough bee pollen analysis can be very informative regarding its safety for consumption, the variability of its composition, its biogeographical origin, or harvest date. Therefore, in this study, two reliable and non-destructive spectroscopy methods, i.e., ED-XRF and ATR–FTIR, are proposed as a fast approach to characterize bee pollen. The collected samples were derived from apiaries located in west-central Poland. Additionally, some commercially available samples were analyzed. The applied methodology was optimized and combined with sophisticated chemometric tools. Data derived from IR analyses were also subjected to two-dimensional correlation spectroscopy. The developed ED-XRF method allowed the reliable quantification of eight macro- and micro-nutrients, while organic components were characterized by IR spectroscopy. Principal component analysis, cluster analysis, and obtained synchronous and asynchronous maps allowed the study of component changes occurring dependently on the date and location of harvest. The proposed approach proved to be an excellent tool to monitor the variability of the inorganic and organic content of bee pollen.

Standardized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

Patients with COVID-19 shed SARS-CoV-2 RNA in stool, sometimes well after their respiratory infection has cleared. This may be significant for patient health, epidemiology, and diagnosis. However, methods to preserve stool, and to extract and quantify viral RNA are not standardized. We test the performance of three preservative approaches at yielding detectable SARS-CoV-2 RNA: the OMNIgene-GUT kit, Zymo DNA/RNA shield kit, and the most commonly applied, storage without preservative. We test these in combination with three extraction kits: QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. We also test the utility of ddPCR and RT-qPCR for the reliable quantification of SARS-CoV-2 RNA from stool. We identify that the Zymo DNA/RNA preservative and the QiaAMP extraction kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR. Taken together, we recommend a comprehensive methodology for preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

A parameter-free statistical test for neuronal responsiveness

Neurophysiological studies depend on a reliable quantification of whether and when a neuron responds to stimulation. Simple methods to determine responsiveness require arbitrary parameter choices, such as binning size, while more advanced model-based methods require fitting and hyperparameter tuning. These parameter choices can change the results, which invites bad statistical practice and reduces the replicability. New recording techniques that yield increasingly large numbers of cells would benefit from a test for cell-inclusion that requires no manual curation. Here, we present the parameter-free ZETA-test, which outperforms t-tests, ANOVAs, and renewal-process-based methods by including more cells at a similar false-positive rate. We show that our procedure works across brain regions and recording techniques, including calcium imaging and Neuropixels data. Furthermore, in illustration of the method, we show in mouse visual cortex that 1) visuomotor-mismatch and spatial location are encoded by different neuronal subpopulations; and 2) optogenetic stimulation of VIP cells leads to early inhibition and subsequent disinhibition.

Microplastic concentrations, characteristics, and fluxes in water bodies of the Tollense catchment, Germany, with regard to different sampling systems

The widespread presence of microplastics in multiple environmental compartments has largely been demonstrated. Assessing the ecological risk that microplastics pose is, at the present stage, hindered due to methodical differences. Moreover, different methods hamper meaningful comparisons between studies and data on microplastics <300 μm is scarce. Therefore, we focused on microplastics >20 μm in freshwater and sampling-related aspects in this concern. Sampling was conducted between 2018 and 2020 in the Tollense catchment in northeastern Germany and was carried out by in situ pump filtration. Two different sampling systems (cutoff sizes 20 μm and 63 μm) were applied to filter water volumes of 0.075–1.836 m3. Retained particles were analyzed by a combination of Nile red staining and micro-Raman spectroscopy. Thereby, we found microplastic concentrations between 123 and 1728 particles m−3 using the 63-μm cut-off size and between 1357 and 2146 particles m−3 using the 20-μm cut-off size. Local hydrodynamics (discharge and flow velocity) and land cover are likely influencing the observed microplastic concentrations and fluxes. The variability between both sampling systems cannot fully be explained by the different mesh sizes used. We argue that differentiation between a theoretical cut-off size (finest mesh) and a factual cut-off size (reliable quantification) can help to understand sampling related differences between studies.