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Given the significant impact of biofilms on human health and material corrosion, research in this field urgently needs more accessible techniques to facilitate the testing of new control agents and general understanding of biofilm biology. Microtiter plates offer a convenient format for standardized evaluations, including high-throughput assays of alternative treatments and molecular modulators. This study introduces a novel Biofilm Analysis Software (BAS) for quantifying biofilms from microtiter plate images. We focused on early biofilm growth stages and compared BAS quantification to common techniques: direct turbidity measurement, intrinsic fluorescence detection linked to pyoverdine production, and standard crystal violet staining which enables image analysis and optical density measurement. We also assessed their sensitivity for detecting subtle growth effects caused by cyclic AMP and gentamicin. Our results show that BAS image analysis is at least as sensitive as the standard method of spectrophotometrically quantifying the crystal violet retained by biofilms. Furthermore, we demonstrated that bacteria adhered after short incubations (from 10 min to 4 h), isolated from planktonic populations by a simple rinse, can be monitored until their growth is detectable by intrinsic fluorescence, BAS analysis, or resolubilized crystal violet. These procedures are widely accessible for many laboratories, including those with limited resources, as they do not require a spectrophotometer or other specialized equipment.

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BACKGROUND: Developing disposable paper-based devices has positively impacted analytical science, particularly in developing countries. Some benefits of those devices include their versatility, affordability, environmentally friendly, and the possibility of being integrated with portable electrochemical or colorimetric detectors. Paper-based analytical devices (PADs) comprising circular zones and microfluidic networks have been successfully employed in the analytical chemistry reign. However, the combination of the stencil-printing method and alternative binder has not been satisfactorily explored for fabricating colorimetric paper devices. RESULTS: We developed PADs exploring the stencil printing approach and glass varnish as the hydrophobic chemical agent. As a proof-of-concept, the colorimetric assay of salivary α-amylase (sAA) was performed in saliva samples. Through the scanning electron microscopy measurements, it was possible to indicate satisfactory definitions between native fibers and barrier, and that the measured values for the channel width revealed suitable fidelity (R2 = 0.99) with the nominal widths (ranging from 400 to 5000 µm). The proposed hydrophobic barrier exhibited excellent chemical resistance. The analytical applicability for detecting sAA revealed linear behavior in the range from 2 to 12 U mL-1 (R2 = 0.99), limit of detection of 0.75 U mL-1, reproducibility (RSD ≤2.4%), recovery experiments ranged from 89 to 108% and AGREE response (0.86). In addition, the colorimetric analysis of sAA in four different saliva samples demonstrated levels ranging from 202 to 2080 U mL-1, which enabled monitoring the absence and presence of periodontitis. SIGNIFICANCE: This report has presented the first use of a self-adhesive mask and glass varnish for creating circular zones and microfluidic architectures on paper without using thermic or UV curing treatments. Also, the proposed analytical methodology for detecting sAA exhibited suitable ecological impact considering the AGREE tool. We believe the proposed fabrication of paper devices emerges as a novel, simple, high-fidelity microfluidic channel and portable analytical approach for colorimetric sensing.

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PURPOSE: Arterial spin labeling (ASL) is a widely used contrast-free MRI method for assessing cerebral blood flow (CBF). Despite the generally adopted ASL acquisition guidelines, there is still wide variability in ASL analysis. We explored this variability through the ISMRM-OSIPI ASL-MRI Challenge, aiming to establish best practices for more reproducible ASL analysis. METHODS: Eight teams analyzed the challenge data, which included a high-resolution T1-weighted anatomical image and 10 pseudo-continuous ASL datasets simulated using a digital reference object to generate ground-truth CBF values in normal and pathological states. We compared the accuracy of CBF quantification from each team's analysis to the ground truth across all voxels and within predefined brain regions. Reproducibility of CBF across analysis pipelines was assessed using the intra-class correlation coefficient (ICC), limits of agreement (LOA), and replicability of generating similar CBF estimates from different processing approaches. RESULTS: Absolute errors in CBF estimates compared to ground-truth synthetic data ranged from 18.36 to 48.12 mL/100 g/min. Realistic motion incorporated into three datasets produced the largest absolute error and variability between teams, with the least agreement (ICC and LOA) with ground-truth results. Fifty percent of the submissions were replicated, and one produced three times larger CBF errors (46.59 mL/100 g/min) compared to submitted results. CONCLUSIONS: Variability in CBF measurements, influenced by differences in image processing, especially to compensate for motion, highlights the significance of standardizing ASL analysis workflows. We provide a recommendation for ASL processing based on top-performing approaches as a step toward ASL standardization.

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The cooking process is fundamental for bean consumption and to increase the bioavailability of its nutritional components. The study aimed to determine the effect of cooking on bean seed coat through morphological analyses with different microscopy techniques and image analyses. The chemical composition and physical properties of raw black bean (RBB) and cooked black bean (CBB) seeds were determined. The surface and cross-sectional samples were studied by Optical microscopy (OM), environmental scanning electron microscopy (ESEM), atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). The composition of samples showed significant differences after the cooking process. OM images and gray level co-occurrence matrix algorithm (GLCM) analysis indicated that cuticle-deposited minerals significantly influence texture parameters. Seed coat surface ESEM images showed cluster cracking. Texture fractal dimension and lacunarity parameters were effective in quantitatively assessing cracks on CBB. AFM results showed arithmetic average roughness (Ra) (121.67 nm) and quadratic average roughness (Rq) (149.94 nm). The cross-sectional ESEM images showed a decrease in seed coat thickness. The CLSM results showed an increased availability of lipids along the different multilayer tissues in CBB. The results generated from this research work offer a valuable potential to carry out a strict control of bean seed cooking at industrial level, since the structural changes and biochemical components (cell wall, lipids and protein bodies) that occur in the different tissues of the seed are able to migrate from the inside to the outside through the cracks generated in the multilayer structure that are evidenced by the microscopic techniques used.

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Bee pollen is considered an excellent dietary supplement with functional characteristics, and it has been employed in food and cosmetics formulations and in biomedical applications. Therefore, understanding its chemical composition, particularly crude protein contents, is essential to ensure its quality and industrial application. For the quantification of crude protein in bee pollen, this study explored the potential of combining digital image analysis and Random Forest algorithm for the development of a rapid, cost-effective, and environmentally friendly analytical methodology. Digital images of bee pollen samples (n = 244) were captured using a smartphone camera with controlled lighting. RGB channels intensities and color histograms were extracted using open source softwares. Crude protein contents were determined using the Kjeldahl method (reference) and in combination with RGB channels and color histograms data from digital images, they were used to generate a predictive model through the application of the Random Forest algorithm. The developed model exhibited good performance and predictive capability for crude protein analysis in bee pollen (R2 = 80.93 %; RMSE = 1.49 %; MAE = 1.26 %). Thus, the developed analytical methodology can be considered environmentally friendly according to the AGREE metric, making it an excellent alternative to conventional analysis methods. It avoids the use of toxic reagents and solvents, demonstrates energy efficiency, utilizes low-cost instrumentation, and it is robust and precise. These characteristics indicate its potential for easy implementation in routine analysis of crude protein in bee pollen samples in quality control laboratories.

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A 30-year-old woman had 5 days of visual hallucinations, nystagmus, memory impairment and mutism. On examination, she was disorientated with reduced attention span, gaze-evoked nystagmus, paratonia and abnormal frontal reflexes. Cerebrospinal fluid (CSF) showed 80 cells, protein 0.41 g/L and glucose 3.2 mmol/L (plasma glucose 5.0 mmol/L). MR scan of the brain showed involvement of limbic and extra-limbic regions and brainstem. Commercial cell-based assays were negative, but tissue-based assays showed neuropil staining, and cell-based assays for anti-metabotropic glutamate receptor 5 (mGluR5) antibodies were positive in serum and CSF. Six months later, she was diagnosed with Hodgkin's lymphoma. This case emphasises the broader clinical spectrum of anti-mGluR5 encephalitis, challenging its initial characterisation as Ophelia syndrome. It underscores the significance of interpreting commercial cell-based assays and advocates for tissue-based assay testing followed by cell-based assay testing in serum and CSF for diagnosing rare autoimmune encephalitis.

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BACKGROUND: Spinal cord damage is a feature of many spinocerebellar ataxias (SCAs), but well-powered in vivo studies are lacking and links with disease severity and progression remain unclear. Here we characterise cervical spinal cord morphometric abnormalities in SCA1, SCA2, SCA3 and SCA6 using a large multisite MRI dataset. METHODS: Upper spinal cord (vertebrae C1-C4) cross-sectional area (CSA) and eccentricity (flattening) were assessed using MRI data from nine sites within the ENIGMA-Ataxia consortium, including 364 people with ataxic SCA, 56 individuals with preataxic SCA and 394 nonataxic controls. Correlations and subgroup analyses within the SCA cohorts were undertaken based on disease duration and ataxia severity. RESULTS: Individuals in the ataxic stage of SCA1, SCA2 and SCA3, relative to non-ataxic controls, had significantly reduced CSA and increased eccentricity at all examined levels. CSA showed large effect sizes (d>2.0) and correlated with ataxia severity (r<-0.43) and disease duration (r<-0.21). Eccentricity correlated only with ataxia severity in SCA2 (r=0.28). No significant spinal cord differences were evident in SCA6. In preataxic individuals, CSA was significantly reduced in SCA2 (d=1.6) and SCA3 (d=1.7), and the SCA2 group also showed increased eccentricity (d=1.1) relative to nonataxic controls. Subgroup analyses confirmed that CSA and eccentricity are abnormal in early disease stages in SCA1, SCA2 and SCA3. CSA declined with disease progression in all, whereas eccentricity progressed only in SCA2. CONCLUSIONS: Spinal cord abnormalities are an early and progressive feature of SCA1, SCA2 and SCA3, but not SCA6, which can be captured using quantitative MRI.

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Tomato fruit is susceptible to chilling injury (CI) during its postharvest handling at low temperature. The symptoms caused by this physiological disorder have been commonly evaluated by visual inspection at a macro-observation scale on fruit surface; however, the structure at deeper scales is also affected by CI. This work aimed to propose a descriptive model of the CI development in tomato tissue under the micro-scale, micro-nano-scale and nano-scale approaches using fractal analysis. For that, quality and fractal parameters were determined. In this sense, light microscopy, Environmental Scanning Electron Microscopy (ESEM) and Atomic Force Microscopy (AFM) were applied to analyse micro-, micro-nano- and nano-scales, respectively. Results showed that the morphology of tomato tissue at the micro-scale level was properly described by the multifractal behaviour. Also, generalised fractal dimension (Dq=0) and texture fractal dimension (FD) of CI-damaged pericarp and cuticle were higher (1.659, 1.601 and 1.746, respectively) in comparison to non-chilled samples (1.606, 1.578 and 1.644, respectively); however, FD was unsuitable to detect morphological changes at the nano-scale. On the other hand, lacunarity represented an appropriate fractal parameter to detect CI symptoms at the nano-scale due to differences observed between damaged and regular ripe tissue (0.044 and 0.025, respectively). The proposed multi-scale approach could improve the understanding of CI as a complex disorder to the development of novel techniques to avoid this postharvest issue at different observation scales.

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Around the world, citrus production and quality are threatened by diseases caused by fungi, bacteria, and viruses. Citrus growers are currently demanding technological solutions to reduce the economic losses caused by citrus diseases. In this context, image analysis techniques have been widely used to detect citrus diseases, extracting discriminant features from an input image to distinguish between healthy and abnormal cases. The dataset presented in this article is helpful for training, validating, and comparing citrus abnormality detection algorithms. The data collection comprises 953 color images taken from the orange leaves of Citrus sinensis (L.) Osbeck species. There are 12 nutritional deficiencies and diseases supporting the development of automatic detection methods that can reduce economic losses in citrus production.

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This article presents a dataset of thermographic images of terrain with antipersonnel mines to identify the presence or absence of these artifacts using machine learning and artificial vision techniques. The dataset has 2700 thermographic images acquired at different heights, using a Zenmuse XT infrared camera (7-13 µm), embedded in the DJI Matrice 100 drone. The data acquisition experiment consists of capturing aerial infrared images of a terrain where elements with characteristics similar to antipersonnel mines type legbreaker were buried. The mines were planted in the ground between 0 cm and 10 cm deep and were spread over an area of 10 m x 10 m. The drone used a flight protocol that set the trajectory, the time of the flight, the acquisition height, and the image sampling frequency. This dataset was used in "Detection of "legbreaker" antipersonnel landmines by analysis of aerial thermographic images of the soil" [7].

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Leukodystrophies are a group of genetic diseases with diverse clinical features and prominent involvement of the central nervous system white matter. We describe a 27-year-old man who presented with a progressive neurological disease, and striking involvement of the brainstem and symmetrical white matter lesions on MR scanning. Having excluded several other causes of leukodystrophy, we confirmed Alexander disease when a genetic panel showed a probable pathogenic variant in GFAP: p.Leu359Pro. Clinicians should suspect Alexander disease in people with a progressive neurological motor decline who has pyramidal and bulbar signs and compatible neuroimaging.

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BACKGROUND: In tissues and organisms, the coordination of neighboring cells is essential to maintain their properties and functions. Therefore, knowing which cells are adjacent is crucial to understand biological processes that involve physical interactions among them, e.g. cell migration and proliferation. In addition, some signaling pathways, such as Notch or extrinsic apoptosis, are highly dependent on cell-cell communication. While this is straightforward to obtain from membrane images, nuclei labelling is much more ubiquitous for technical reasons. However, there are no automatic and robust methods to find neighboring cells based only on nuclear markers. RESULTS: In this work, we describe Nfinder, a method to assess the cell's local neighborhood from images with nuclei labeling. To achieve this goal, we approximate the cell-cell interaction graph by the Delaunay triangulation of nuclei centroids. Then, links are filtered by automatic thresholding in cell-cell distance (pairwise interaction) and the maximum angle that a pair of cells subtends with shared neighbors (non-pairwise interaction). We systematically characterized the detection performance by applying Nfinder to publicly available datasets from Drosophila melanogaster, Tribolium castaneum, Arabidopsis thaliana and C. elegans. In each case, the result of the algorithm was compared to a cell neighbor graph generated by manually annotating the original dataset. On average, our method detected 95% of true neighbors, with only 6% of false discoveries. Remarkably, our findings indicate that taking into account non-pairwise interactions might increase the Positive Predictive Value up to + 11.5%. CONCLUSION: Nfinder is the first robust and automatic method for estimating neighboring cells in 2D and 3D based only on nuclear markers and without any free parameters. Using this tool, we found that taking non-pairwise interactions into account improves the detection performance significantly. We believe that using our method might improve the effectiveness of other workflows to study cell-cell interactions from microscopy images. Finally, we also provide a reference implementation in Python and an easy-to-use napari plugin.

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Prebiotic effects have been attributed to agave fructans through bacterial and yeast fermentations, but there are few reports on their use as raw materials of a carbon source. Kefir milk is a fermented drink with lactic acid bacteria and yeast that coexist in a symbiotic association. During fermentation, these microorganisms mainly consume lactose and produce a polymeric matrix called kefiran, which is an exopolysaccharide composed mainly of water-soluble glucogalactan, suitable for the development of bio-degradable films. Using the biomass of microorganisms and proteins together can be a sustainable and innovative source of biopolymers. In this investigation, the effects of lactose-free milk as a culture medium and the addition of other carbon sources (dextrose, fructose, galactose, lactose, inulin and fructans) in concentrations of 2, 4 and 6% w/w, coupled with initial parameters such as temperature (20, 25 and 30 °C), % of starter inoculum (2, 5 and 10% w/w) was evaluated. The method of response surface analysis was performed to determine the optimum biomass production conditions at the start of the experiment. The response surface method showed that a 2% inoculum and a temperature of 25 °C were the best parameters for fermentation. The addition of 6% w/w agave fructans in the culture medium favored the growth of biomass (75.94%) with respect to the lactose-free culture medium. An increase in fat (3.76%), ash (5.57%) and protein (7.12%) content was observed when adding agave fructans. There was an important change in the diversity of microorganisms with an absence of lactose. These compounds have the potential to be used as a carbon source in a medium culture to increase kefir granule biomass. There was an important change in the diversity of microorganisms with an absence of lactose, where the applied image digital analysis led to the identification of the morphological changes in the kefir granules through modification of the profile of such microorganisms.

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Aromatic plants represent about 0.7% of all medicinal plants. The most common are peppermint (main active ingredient: menthol) and chamomile (main active ingredient: luteolin), which are usually consumed in "tea bags" to make infusions or herbal teas. In this study, menthol and luteolin encapsulates using different hydrocolloids were obtained to replace the conventional preparation of these beverages. Encapsulation was carried out by feeding an infusion of peppermint and chamomile (83% aqueous phase = 75% water - 8% herbs in equal parts, and 17% dissolved solids = wall material in 2:1 ratio) into a spray dryer (180 °C-4 mL/min). A factorial experimental design was used to evaluate the effect of wall material on morphology (circularity and Feret's diameter) and texture properties of the powders using image analysis. Four formulations using different hydrocolloids were evaluated: (F1) maltodextrin-sodium caseinate (10 wt%), (F2) maltodextrin-soy protein (10 wt%), (F3) maltodextrin-sodium caseinate (15 wt%), and (F4) maltodextrin-soy protein (15 wt%). The moisture, solubility, bulk density, and bioavailability of menthol in the capsules were determined. The results showed that F1 and F2 presented the best combination of powder properties: higher circularity (0.927 ± 0.012, 0.926 ± 0.011), lower moisture (2.69 ± 0.53, 2.71 ± 0.21), adequate solubility (97.73 ± 0.76, 98.01 ± 0.50), and best texture properties. Those suggest the potential of these powders not only as an easy-to-consume and ecofriendly instant aromatic beverage but also as a functional one.

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The textural changes during storage of two freeze-dried candies developed from blackcurrant fruits, unflavored yogurt, and different alternative sweeteners, one sweetened with honey/isomalt (HI) and another sweetened with isomalt/stevia (IS), were analyzed using three different methods (instrumental, sensory, and image analysis). Fresh candies were in the supercooled state and presented different structural and textural characteristics (HI: compact and homogeneous, and IS: porous and crunchy), with Fmax values of 139 ± 14 and 174 ± 16 N for HI and IS, respectively. After storage, the instrumental analysis showed approximately 60% average drop in Fmax and W values, in agreement with the decrease observed by sensory analysis in hardness, fracturability, and crispness. Image analysis showed an increase in parameters related to the homogeneity and the uniformity/smoothness for HI. Pearson's correlation coefficients analysis showed that there was a good correlation between the three techniques used, suggesting that the joint use of these methods could be performed for a better understanding of complex food texture.

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Facial nerve stimulation (FNS) is a potential complication which may affect the auditory performance of children with cochlear implants (CIs). We carried out an exploratory prospective observational study to investigate the effects of the electrical stimulation pattern on FNS reduction in young children with CI. Ten ears of seven prelingually deafened children with ages up to 6 years old who undergone a unilateral or bilateral CI surgery were included in this study. Electromyographic (EMG) action potentials from orbicularis oculi muscle were recorded using monopolar biphasic stimulation (ST1) and multi-mode monophasic stimulation with capacitive discharge (ST2). Presence of EMG responses, facial nerve stimulation thresholds (T-FNS) and EMG amplitudes were compared between ST1 and ST2. Intra-cochlear electrodes placement, cochlear-nerve and electrode-nerve distances were also estimated to investigate their effects on EMG responses. The use of ST2 significantly reduced the presence of intraoperative EMG responses compared to ST1. Higher stimulation levels were required to elicit FNS with ST2, with smaller amplitudes, compared to ST1. No and weak correlation was observed between cochlea-nerve and electrode-nerve distances and EMG responses, respectively. ST2 may reduce FNS in young children with CI. Differently from the electrical stimulation pattern, the cochlea-nerve and electrode-nerve distances seem to have limited effects on FNS in this population.

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We propose a new theory for enamel cupping lesions formation. At early stages, naturally formed cupping lesions showed increased porosity at two structural prismatic traits: the central cone extending into the enamel-dentine junction and the type-I Hunther-Schreger bands (HSB), suggesting them to be the main drivers for cupping lesion formation and development. In addition, these lesions were circumscribed by type-II HSBs, which present lower surface porosity and higher resistance to wear. This theory was verified in in vitro observations, where both the central cone and the type-I HSB of cuspal enamel showed higher susceptibility to wear, potentially elucidating the mechanisms involved on cupping lesion formation.

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