Computational Identification and 3D Morphological Characterization of Renal Glomeruli in Optically Cleared Murine Kidneys

The aim of this study was to establish an accessible methodology for the objective identification and 3D morphological characterization of renal glomeruli in mice. 3D imaging of the renal cortex was performed by light sheet microscopy on iDISCO+ optical cleared kidneys of six C57BL/6J mice after labelling of the capillary endothelium by lectin injection. 3D images were processed with the open source software ImageJ, and statistical analysis done with GraphPad Prism. Non-visual delimitation of the external surface of the glomeruli was ensured by greyscale-based thresholding, the value of which was determined from the statistical analysis of the voxel frequency distribution. Exclusion of false-positive identification was done by successive volume- and shape-based segmentation. Renal glomeruli were characterized by their number, surface area, volume, and compactness. Average data were expressed as mean ± SD. The number of glomeruli was equal to 283 ± 35 per mm3 of renal tissue, representing 1.78 ± 0.49% of the tissue volume. The surface area, volume and compactness were equal to 20,830 ± 6200 µm², 62,280 ± 14,000 µm3 and 0.068 ± 0.026, respectively. The proposed standardized methodology allows the identification of the renal glomeruli and their 3D morphological characterization, and is easily accessible for biologists.

Citrobacter braakii Yield False-Positive Identification as Salmonella, a Note of Caution

Background: Globally, Salmonella enterica is one of the leading causes of foodborne illness in humans. Food of animal origin is obligatorily tested for the presence of this pathogen. Unfortunately, in meat and meat products, this is often hampered by the presence of background microbiota, which may present as false-positive Salmonella. Methods: For the identification of Salmonella spp. from meat samples of beef, pork, and poultry, the authorized detection method is PN-EN ISO 6579-1:2017-04 with the White–Kauffmann–Le Minor scheme, two biochemical tests: API 20E and VITEK II, and a real-time PCR-based technique. Results: Out of 42 presumptive strains of Salmonella, 83.3% Salmonella enterica spp. enterica, 14.3% Citrobacter braakii, and 12.4% Proteus mirabilis were detected from 180 meat samples. Conclusions: Presumptive strains of Salmonella should be identified based on genotypic properties such as DNA-based methods. The aim of this study was the isolation and identification of Salmonella spp. from miscellaneous meat sorts: beef, pork, and poultry.

Association Between Elevated Depressive Symptoms and Cognitive Function Moderated by APOE4 Status: Framingham Offspring Study

Background: Depression and Apolipoprotein E4 (APOE4) are associated with decreased cognitive function and differences in brain structure. Objective: This study investigated whether APOE4 status moderates the association between elevated depressive symptoms, cognitive function, and brain structure. Methods: Stroke- and dementia-free participants (n = 1,968) underwent neuropsychological evaluation, brain MRI, and depression screening. Linear and logistic regression was used to examine all associations. Secondary analyses were performed using interaction terms to assess effect modification by APOE4 status. Results: Elevated depressive symptoms were associated with lower cognitive performance in several domains. In stratified analyses, elevated depressive symptoms were associated with poorer visual short- and long-term memory performance for APOE4 + participants. Elevated depressive symptoms were not associated with any brain structure in this study sample. Conclusion: Elevated depressive symptoms impact cognitive function in non-demented individuals. Having the APOE4 allele may exacerbate the deleterious effects of elevated depressive symptoms on visual memory performance. Screening for elevated depressive symptoms in both research studies and clinical practice may be warranted to avoid false positive identification of neurodegeneration, particularly among those who are APOE4 + .

A diagnostic LAMP assay for the destructive grapevine insect pest, phylloxera (Daktulosphaira vitifoliae)

Grape phylloxera (Daktulosphaira vitifoliae) is a destructive insect pest of grapevines that is highly invasive worldwide, despite strict biosecurity containment measures in place at farm and regional levels. Current phylloxera identification by visual inspection and laboratory-based molecular methods is time-consuming and costly. More rapid and cost-effective methods for identification of this pest would benefit industry, growers, and biosecurity services. Loop mediated isothermal amplification (LAMP) is a new portable technology available for rapid and accurate in-field molecular diagnostics. This study outlines the development of a new LAMP assay to enable the identification of phylloxera specimens. New LAMP primers were developed to specifically amplify phylloxera mitochondrial DNA (5′-COI), which we have shown is effective as a DNA barcode for identification of phylloxera, using LAMP technology. Positive LAMP reactions, containing phylloxera DNA, amplified in less than twelve minutes with an anneal derivative temperature of approximately 79 °C to 80 °C compared to a newly designed synthetic DNA (gBlock) fragment which had an anneal derivative temperature of 82 °C. No LAMP amplification was detected in any of the non-target species tested, i.e. no false-positive identification resulted for these species. We also successfully optimised a non-destructive DNA extraction procedure, HotSHOT “HS6”, for use in the field on phylloxera adults, nymphs and eggs, to retain physical specimens. DNA extracted using this method was also suitable for species and genotype molecular identification methods, such as DNA barcoding, qPCR and microsatellite genotyping. The new LAMP assay provides a novel visual molecular tool for accurate diagnostics of phylloxera in the laboratory and field.

Mass Spectrometry: A Rosetta Stone to Learn How Fungi Interact and Talk

Fungi are a highly diverse group of heterotrophic organisms that play an important role in diverse ecological interactions, many of which are chemically mediated. Fungi have a very versatile metabolism, which allows them to synthesize a large number of still little-known chemical compounds, such as soluble compounds that are secreted into the medium and volatile compounds that are chemical mediators over short and long distances. Mass spectrometry (MS) is currently playing a dominant role in mycological studies, mainly due to its inherent sensitivity and rapid identification capabilities of different metabolites. Furthermore, MS has also been used as a reliable and accurate tool for fungi identification (i.e., biotyping). Here, we introduce the readers about fungal specialized metabolites, their role in ecological interactions and provide an overview on the MS-based techniques used in fungal studies. We particularly present the importance of sampling techniques, strategies to reduce false-positive identification and new MS-based analytical strategies that can be used in mycological studies, further expanding the use of MS in broader applications. Therefore, we foresee a bright future for mass spectrometry-based research in the field of mycology.

One-month Progress of COVID-19 Cases in East Kalimantan, Indonesia

BACKGROUND: East Kalimantan, Indonesia, will play a significant geopolitical role as the province has been selected as the location of the future capital city of Indonesia. As a buffer zone to the capital city, there is urgent attention on the preparedness of the cities and regencies in East Kalimantan to respond to emergent infectious disease events such as coronavirus disease-19 (COVID-19). AIM: The aim of the present study was to descriptively convey information about COVID-19 cases in East Kalimantan during the period of March 18, 2020–April 18, 2020, in terms of the isolation, testing, and tracing mechanisms used. METHODS: The initial distribution of COVID-19 was identified in 5 of 10 districts and is now present in almost all districts except for one very remote regency. RESULTS: The tracing performance of the fast response teams in East Kalimantan during this period was considered satisfactory with a mean of 0.7% of people under observation testing positive for severe acute respiratory syndrome coronavirus 2, and a mean of 14.4% patients under investigations testing positive. The use of rapid tests since March 30, 2020 has improved the detection ability, with confirmed positive cases as a percentage of confirmed negatives increasing from 20.2% to 31.8%. The use of the COVID-19 rapid test was cross-checked with a dengue rapid test to prevent false-positive identification. Confirmed clusters were announced to the public, urging people to respond and report. CONCLUSION: The 1-month progress of COVID-19 cases in East Kalimantan showed a total case fatality rate (CFR) of 1.85%, a closed CFR of 8.3% and a closed case recovery rate of 91.7%.

Cost-Effective Spectrum Utilization for Futuristic Cognitive Radio based Services

The proliferation of various mobile users in the context of advanced wireless technologies, spectrum scarcity arises as a crucial problem. The notion of cognitive radio (CR) principle offers cost-effective spectrum reusability with intelligent mode of transmission model. It basically enables a radio-driven technology to utilize dynamic spectrum accessing to meet the quality-of-services (QoS) requirements by satisfying enormous communication and connectivity demands. To alleviate the spectrum scarcity problem the study proposes a novel analytical solution of spectrum allocation considering a simplified evolutionary learning model. The prime target of the formulated concept is to detects the spectrum holes effectively for reusability and the possibility of identification has be maximized for best possible spectrum allocation to the radios. And on the other hand also the occurrence of false positive identification has to be minimized. The outcome obtained after performing the simulation shows promising aspects in the context of effective spectrum allocation for higher priority user with better throughput performance.

Eumelanin and pheomelanin pigmentation in mollusc shells may be less common than expected: insights from mass spectrometry

Background The geometric patterns that adorn the shells of many phylogenetically disparate molluscan species are comprised of pigments that span the visible spectrum. Although early chemical studies implicated melanin as a commonly employed pigment, surprisingly little evidence generated with more recent and sensitive techniques exists to support these observations. Results Here we present the first mass spectrometric investigations for the presence of eumelanin and pheomelanin in 13 different molluscan species from three conchiferan classes: Bivalvia, Cephalopoda and Gastropoda. In the bivalve Mytilus edulis we demonstrate that eumelanin mainly occurs in the outermost, non-mineralised and highly pigmented layer of the shell (often referred to as the periostracum). We also identified eumelanin in the shells of the cephalopod Nautilus pompilius and the marine gastropods Clanculus pharaonius and Steromphala adriatica. In the terrestrial gastropod Cepaea nemoralis we verify the presence of pheomelanin in a mollusc shell for the first time. Surprisingly, in a large number of brown/black coloured shells we did not find any evidence for either type of melanin. Conclusions We recommend methods such as high-performance liquid chromatography with mass spectrometric detection for the analysis of complex biological samples to avoid potential false-positive identification of melanin. Our results imply that many molluscan species employ as yet unidentified pigments to pattern their shells. This has implications for our understanding of how molluscs evolved the ability to pigment and pattern their shells, and for the identification of the molecular mechanisms that regulate these processes.

Managing Risks Associated with Establishing the Metabolome of Dietary Phytochemicals (P06-010-19)

Objectives Establishing the metabolome of dietary phytochemicals is complicated by the influence of the microbiome. Due to large numbers of human, microbial and hybrid human-microbial metabolites, false-positive identification via mass spectrometry (MS) is probable. Users must be aware of the influence of matrix and instrumental background environment upon the mass spectrum, which produce spectral features arising from fragmentation, gas-phase artifacts, molecular rearrangement, quasi-molecular ion or radical formation. Hydroxylated cyclic and polycyclic structures such as polyphenols are prone to multiple gas-phase artifacts, including water elimination (loss −17), hydrogen elimination (−1), radical fragmentation (loss −15, −14), Retro-Diels-Alder reactions (C-ring electron rearrangement; + or −2), or combinations thereof. In-source fragmentation is often observed for phase II conjugates, such as sulfate, glucuronide and glycine. Finally, polyphenol metabolites such as valerolactones, benzoic, phenylpropanoic and phenylacetic acids, are also products of MS fragmentation. As there are few reference standards available for confirmation or optimization, false-positive identification is likely. The objective of the present study was to highlight limitations with MS to ensure researchers make appropriate assumptions from their spectral data. Methods A quantitative metabolomics database comprising optimized spectral signals for fragmentation profiling of over 400 poly/phenols and metabolites was established using a UHPLC-coupled electrospray triple quadrupole-linear ion trap mass spectrometer (SCIEX QTRAP 6500+). Methods were established and utilized to interrogate over 3000 biospecimens derived from studies feeding various polyphenol-rich diets. Results Scanning for numerous metabolites reported in the literature using single transition monitoring, in both neat and extracted human and animal tissue matrices consistently identify peaks which were either artifacts, isomers, fragments or background noise, as confirmed relative to authentic reference standards. Conclusions Without ample MS experience, method development, validation and data interrogation, falsely identified metabolites will continue to occur and undoubtedly hinder future discovery. Funding Sources NIFA-USDA Hatch 1011757.