Sample Processing Research Articles

Epidemiological analysis of blood donors from college students in Hefei city.

College students represent a unique demographic group as they are adults no longer under direct parental care, yet often lack the institutional health support available to more established members of society, which can lead to their health needs being neglected, despite their substantial contributions to blood donation. The objective of this study is to shed light on the health status of college students in Hefei, with a specific focus on transfusion-transmitted diseases. Based on the detailed data analysis, the implementation of some constructive strategies will play a good warning role in improving clinical blood safety and promoting better health monitoring of this population in the future. The study aims to achieve two main objectives: On the one hand, to deepen our understanding of college students' participation in blood donation activities. On the other hand, to propose effective strategies for increasing youth involvement. Additionally, such research aims to afford recommendations for ensuring the safety of clinical blood supplies. Although the strategies and suggestions presented are speculative, they are grounded in the logical analysis of data and relevant theoretical frameworks. Therefore, these recommendations serve as a basis for potential future testing and implementation. The evaluation process for blood samples at the Hefei Blood Bank involves thorough serological testing for hepatitis B and C, HIV, and Treponema pallidum using both Enzyme-Linked Immunosorbent Assay (ELISA) and Nucleic Acid Testing (NAT). HIV reactive samples were further confirmed using Western Blot techniques at an external specialized laboratory. To ensure accuracy, each sample was tested with reagents from two different manufacturers. Moreover, any initially reactive sample was retested with the same reagent to eliminate potential false positives. In cases of conflicting results, alternative reagent kits are utilized for further confirmatory tests. Only samples that consistently show reactive results in consecutive tests are considered positive confirmation samples. Upon analysis of the data set, it was found that out of the total 663,105 donors, 177,089 (26.7%) were identified as college students, with the remaining 486,016 categorized as noncollege students. Our study demonstrated a notably lower prevalence of blood-transmitted infections among college students (0.48%) in contrast to the wider donor population (1.23%), underscoring their potential as safe blood donors. To uphold this level of safety and protect public health, it is crucial to increase disease prevention awareness and advocate for responsible donation practices within this specific demographic. Strengthening confidentiality protocols and expanding education on safe sexual behaviors in university settings are vital actions to cultivate an environment that promotes informed and secure blood donation. The overall group had prevalence rates of 0.44% for hepatitis B, 0.15% for hepatitis C, 0.02% for HIV, and 0.42% for Treponema pallidum. When focusing on the student cohort, the prevalence rates were 0.17% for hepatitis B, 0.04% for hepatitis C, 0.02% for HIV, and 0.23% for Treponema pallidum. Transmissibility through blood transfusion All donors (%) Students (%) Non-students (%) Any infection 1.23 0.48 1.51 HbsAg 0.44 0.17 0.54 Anti-HCV 0.15 0.04 0.19 HIV Ag/Ab 0.02 0.02 0.02 Anti-TP 0.42 0.23 0.49 Individuals with two or more infectious agents occur more than once in all categories. While it leads to numerical over-representation, such overlap is minimal and statistically insignificant.

Green endoscopy, one step towards a sustainable future: a review of the literature.

Rapid climate change or climate crisis is one of the most serious emergencies of the twenty-first century, accounting for highly impactful and irreversible changes worldwide. Climate crisis can also affect the epidemiology and disease burden of gastrointestinal (GI) diseases as they hold a connection with environmental factors and nutrition. GI endoscopy is a highly intensive procedure with a significant contribution to greenhouse gas (GHG) emissions. Moreover, endoscopy is the third highest generator of waste in healthcare facilities with significant contributions to carbon footprint. Main sources of direct carbon emission in endoscopy are the use of high-powered consumption devices (e.g. computers, anesthesia machines, wash machines for reprocessing, scope processors and lighting), and waste production derived mainly from the use of disposable devices. Indirect sources of emissions are those derived from the heating and cooling of facilities, processing of histological samples, transportation of patients and materials etc. Consequently, sustainable endoscopy and climate change have been the focus of discussions between endoscopy providers and professional societies with the aim of taking action to reduce environmental impact. The term "green endoscopy" refers to the practice of gastroenterology that aims to raise awareness, assess, and reduce endoscopy´s environmental impact. Nevertheless, while awareness has been growing, guidance on practical interventions to reduce carbon footprint of GI endoscopy are lacking. This review aims to summarize current data regarding the impact of endoscopy on GHG emissions and possible strategies to mitigate this phenomenon. Further, we aim to promote the evolution of a more sustainable "green endoscopy".

Women Emancipation in Indonesia: A Psychological desire to choose the field of physics education at university level

The gender bias that places women as incapable people ultimately reduces their psychological drive to engage with the field of science, especially physics. This is in stark contrast to the prevailing attitudes at universities in Indonesia. This then became the basis for the aim of this research to explore whether women's interest in studying physics education was based on psychological factors in themselves by comparing them with men. The research was conducted using quantitative survey methods, and a sampling process using random sampling was carried out on the population of a university in Indonesia. The sample was divided into two groups based on gender: 77 women and 23 men. The data collection instrument was developed using the grounded theory of planned behavior (TPB) and social cognitive career theory (SCCT), which identified four main psychological factors: identity, interest, self-concept, and self-efficacy. The results of the descriptive and inferential data analysis, conducted using one-way MANOVA, indicate that the psychological factors of identity, interest, the self-concept, and self-efficacy for women are significantly higher than those for men. Furthermore, the Pearson analysis demonstrated a significantly strong positive correlation between the psychological factors tested

Comparison of DNA damage in fresh and frozen blood samples: implications for the comet assay in human biomonitoring studies.

The use of the comet assay in large biomonitoring studies may present logistical and technical challenges because of the processing of numerous samples. Proper sample preservation becomes imperative to prevent spurious DNA breakage. Previous research has shown the feasibility of conducting the comet assay on frozen blood samples, highlighting the potential of freezing at - 80°C in preserving DNA integrity. Nonetheless, this approach presents challenges, including potential DNA damage during freezing and thawing, variability in processing, and the need for standardized protocols. Our objective was to evaluate whether there are comparable results in DNA migration assessed by the comet assay between fresh and frozen blood samples on a larger scale (N = 373). In our findings, elevated DNA migration was evident in frozen samples relative to fresh ones. Additionally, smoking, alcohol consumption, and season were linked to increased DNA damage levels in whole blood cells. Based on our results and available literature, conducting the comet assay on frozen blood samples emerges as a practical and efficient approach for biomonitoring and epidemiological research. This method enables the assessment of DNA damage in large populations over time, with samples, if properly cryopreserved, that may be used for years, possibly even decades. These observations hold significant implications for large-scale human biomonitoring and long-term epidemiological studies, particularly when samples are collected during fieldwork or obtained from biobanks. Continued method optimization and validation efforts are essential to enhance the utility of this approach in environmental and occupational health studies, emphasizing caution when comparing data obtained between fresh and frozen blood samples.

The Effect of Residence Time during the Hydrothermal Carbonization Process of Sewage Sludge on the Properties of Hydrochar

The optimal process conditions concerning the hydrothermal carbonization of digested sewage sludge are crucial to the economically effective technology needed to produce a solid product, hydrochar, for energy purposes. Accordingly, different residence times, 0.5 h, 1 h and 2 h, were investigated in order to understand the effect of residence time on the process. Furthermore, the physical and chemical properties of hydrochar were investigated and compared to the raw material. For these reasons, analyses describing fuel properties were performed, including ultimate and proximate analyses, HHV, and TGA analysis. The latter method was employed to study the combustion process of solid samples. In addition, the oxide content of different elements within the ash of solid samples was determined using the XRF method to calculate indices related to operational problems during the combustion process. The results confirmed that time did not matter significantly and the physical and chemical properties of hydrochar were very similar to each other. However, the contact angle for 2 h of residence time confirmed that a longer processing time resulted in a more hydrophobic character of hydrochar and enabled more effective dewaterability of hydrothermal slurry. It was also noted that the hydrothermal carbonization process affected the sewage sludge in a positive way. In brief, the results confirmed that the hydrochar was a brittle, moderately hydrophilic, solid carbon-containing product that provided a different combustion performance than the raw sewage sludge.

Investigation of an Optimal Sampling Resolution to Support Soil Management Decisions for Urban Plots.

The main objective of the current study was to use seven lots in Hartford, CT that are planned for community reuse to determine the optimal sampling density that allows for the detection of hotspots of lead pollution while limiting the labor of the sampling process. The sampling density was investigated using soil Pb measured by in situ X-ray Fluorescence as the indicator to evaluate soil health, with a new threshold of 200-mg/kg proposed by the USEPA in January of 2024. Even though this study takes place in an urban setting, where the new USEPA policy requires the use of a 100-mg/kg threshold for Pb due to the fact that there are other identifiable sources of the contaminant, only the 200-mg/kg threshold is discussed because it is evident from the analysis that compliance of a 100 mg/kg threshold in urban plots is highly unlikely (five out of seven sites would require complete site excavation prior to reuse). Using the inverse distance weighted geospatial interpolation of in situ pXRF determined lead measurements, grid sampling resolutions of 3-m, 4-m, 5-m, 6-m, 8-m, 10-m, and 12-m were compared. Ultimately, the case study finds that the largest grid resolution that can be implemented for soil screening to maintain hotspots of pollution to properly inform soil management decisions is a 6-m grid, or a density of approximately 1/36-m2.

Investigation of an Optimal Sampling Resolution to Support Soil Management Decisions for Urban Plots.

The main objective of the current study was to use seven lots in Hartford, CT that are planned for community reuse to determine the optimal sampling density that allows for the detection of hotspots of lead pollution while limiting the labor of the sampling process. The sampling density was investigated using soil Pb measured by in situ X-ray Fluorescence as the indicator to evaluate soil health, with a new threshold of 200-mg/kg proposed by the USEPA in January of 2024. Even though this study takes place in an urban setting, where the new USEPA policy requires the use of a 100-mg/kg threshold for Pb due to the fact that there are other identifiable sources of the contaminant, only the 200-mg/kg threshold is discussed because it is evident from the analysis that compliance of a 100 mg/kg threshold in urban plots is highly unlikely (five out of seven sites would require complete site excavation prior to reuse). Using the inverse distance weighted geospatial interpolation of in situ pXRF determined lead measurements, grid sampling resolutions of 3-m, 4-m, 5-m, 6-m, 8-m, 10-m, and 12-m were compared. Ultimately, the case study finds that the largest grid resolution that can be implemented for soil screening to maintain hotspots of pollution to properly inform soil management decisions is a 6-m grid, or a density of approximately 1/36-m2.

The moderating role of self-efficacy in the relationship between occupational stress and mental health issues among nurses

Occupational stress is a complex concept resulting from interaction among personnel, work environment, and cultural contexts. It can cause mental health issues, including clinical mental disorders, as well as emotional challenges like depression, anxiety, cognitive difficulties, and feeling sad. As a vulnerable group, healthcare professionals, especially nurses, experience a high rate of occupational stress. Therefore, we aimed to study the relationship between occupational stress, mental health problems, and self-efficacy among the nursing population. A cross-sectional study was conducted among 365 nurses selected through a two-stage sampling process from tertiary hospitals in Qazvin, Iran, between July and September 2022. Study variables included occupational stress measured by the persian version of the health and safety executive management standards revised indicator tool (MS-RIT), the general health questionnaire containing 28 questions measuring psychological distress in four primary areas of somatic and anxiety symptoms, sleep disorders, social dysfunction, and depression [GHQ–28], and general self-efficacy [GSE–10]. The mentioned variables and some of the nurses’ demographic characteristics, including age, gender, education, and work experience, were analyzed using moderated multiple regression, descriptive statistics, and the Pearson correlation coefficient.The Pearson correlation analysis revealed a statistically significant association between self-efficacy and occupational stress (r = 0.62, P = 0.000) and self-efficacy and mental health (r = 0.67, P = 0.01). Regression analysis demonstrated that occupational stress accounted for 42% of the variation in mental health (R2 = 0.42, P < 0.01). The results also showed that self-efficacy moderates the relationship between occupational stress and mental health, with a significant effect (P < 0.01). The elevated prevalence of occupational stress and the concerning state of mental health among nurses highlight the need for the implementation of intervention programs, including stress prevention strategies at the workplace through organizing better working shifts, as well as increasing nurses’ self-efficacy and their effective participation in work-related tasks aiming to enhance working conditions for nurses.

Sampling of plasma plume from atmosphere into vacuum for reliable Langmuir probe diagnostics

Langmuir probes cannot be used to diagnose cold atmospheric plasma jet, because their presence in the high electric field after-glow region modifies the plasma parameters that they are intended to measure. Here, we propose a system to sample the plasma plume from ambient conditions into a low-pressure region, where probe analysis can be accomplished. The effect of such a sampling process on the number density and velocity of the gas has been studied through simulations and using analytical equations. Simulation results regarding the effect of chamber and orifice dimensions on these parameters, have been presented. Based on this study an experimental chamber was fabricated and Langmuir probe analysis of the sampled plasma was done. Continuum flowing plasma theory was applied and the plasma density and electron temperature were estimated to be 1.8 × 1020m−3 and 4.7 eV respectively for the operating condition of 3 W plasma power at 12 kHz.

Analysis of size effect and its influencing factors of brittle red sandstone with different heights

We carried out uniaxial compression tests on brittle red sandstone with different heights. The test results show that the uniaxial compressive strength of rock sample increases first and then tends to be stable with the increase of the size, which is approximately stable between 75 and 81 MPa. Both elastic energy and dissipated energy increase with the increase of rock sample size. In order to further analyze the mechanism behind these phenomena, we combined advanced numerical simulation and theoretical analysis to explain these phenomena, and systematically analyzed the end face effect as one of the key factors affecting the uniaxial compression characteristics of brittle red sandstone for the first time. Small sized rock samples are very sensitive to end effect. The middle of the large sized rock samples is in a uniform compression state, and the effect of end effect is weakend. When there are rigid pads at both ends of the rock sample, there is an obvious elastic vertebral body during the loading process of the rock sample. The bearing capacity of rock samples with rigid pads is greater than that of rock samples without rigid pads, and the energy released during instantaneous failure of rock samples without rigid pads is greater than that of rock samples with rigid pads. The findings of this paper make a valuable contribution to establishing optimal study sample sizes and advancing the utilization of laboratory test mechanics parameters in engineering applications.

3D Reconstruction from a Single Sketch via View-dependent Depth Sampling.

Reconstructing a 3D shape based on a single sketch image is challenging due to the inherent sparsity and ambiguity present in sketches. Existing methods lose fine details when extracting features to predict 3D objects from sketches. Upon analyzing the 3D-to-2D projection process, we observe that the density map, characterizing the distribution of 2D point clouds, can serve as a proxy to facilitate the reconstruction process. In this work, we propose a novel sketch-based 3D reconstruction model named SketchSampler. It initiates the process by translating a sketch through an image translation network into a more informative 2D representation, which is then used to generate a density map. Subsequently, a two-stage probabilistic sampling process is employed to reconstruct a 3D point cloud: firstly, recovering the 2D points (i.e., the x and y coordinates) by sampling the density map; and secondly, predicting the depth (i.e., the z coordinate) by sampling the depth values along the ray determined by each 2D point. Additionally, we convert the reconstructed point cloud into a 3D mesh for wider applications. To reduce ambiguity, we incorporate hidden lines in sketches. Experimental results demonstrate that our proposed approach significantly outperforms other baseline methods.

Unsupervised domain adaptation with hard-sample dividing and processing strategy

In Unsupervised Domain Adaptation(UDA), the hard samples cause serious negative transfer to model performance. However, the existing UDA methods don’t fully pay attention to the fine-grained processing of hard samples in target and source domains. To address these problems, the UDA framework with Hard-sample Dividing and Processing Strategy (HDPS) is proposed in this paper. In HDPS, we define sample division criteria in target domain and source domain respectively and divide the samples into easy samples, hard samples with low confidence and hard samples with high confidence. We further design systematic processing strategy of the three types of samples. Specifically, we firstly define geometric metrics based on the class prototype to divide the hard samples and eliminate them in source domain. We further design two weight allocation strategies based on sample classification entropy in target domain. Finally, we build a teacher-student guidance mechanism to learn the discriminative features of hard samples. The HDPS framework can be easily loaded to other methods as a plug-in, can promote the diversity feature learning of samples, and significantly improve the discriminant performance of the model. Extensive experiments on six benchmark datasets verify that HDPS is effective and superior to most existing UDA methods.

Rapid Mass Spectrometry-Based Multiattribute Method for Glycation Analysis with Integrated Afucosylation Detection Capability.

The multiattribute method (MAM) has emerged as a powerful tool for simultaneously screening multiple product quality attributes of therapeutic antibodies. One such potential critical quality attribute (CQA) is glycation, a common modification that can impact the heterogeneity, functional activity, and immunogenicity of therapeutic antibodies. However, current methods for monitoring glycation levels in MAM are rare and not sufficiently rapid and accurate. In this study, an improved mass spectrometry (MS)-based MAM was developed to simultaneously monitor glycation and other quality attributes including afucosylation. The method was evaluated using two therapeutic antibodies with different glycosylation site numbers. Treatment with IdeS, Endo F2, and dithiothreitol generated three distinct subunits, and the glycation results obtained were similar to those treated with PNGase F, which is routinely used to release glycans; the sample processing time was greatly reduced while providing additional quality attribute information. The MS-based MAM was also employed to assess the glycation progression following forced glycation in various buffer solutions. A significant increase in oxidation was observed when forced glycation was conducted in an ammonium bicarbonate buffer solution, and a total of 23 potential glycation sites and 4 significantly oxidized sites were identified. Notably, we found that ammonium bicarbonate was found to specifically stimulate oxidation, while glycation had a synergistic effect on oxidation. These findings establish this study as a novel methodology for achieving a technologically advanced platform and concept that enhances the efficacy of product development and quality control, characterized by its broad-spectrum, rapid, and accurate nature.

P-077 The role of glycosylation in the male reproduction revealed by omics technologies

Abstract Study question What is the current status of art and future perspective of glycosylation in the male reproduction using omics-based technology? Summary answer The precise site-specific glycoproteome of fertile human semen was generally established while the alteration in the pathogenic processes needed to be further investigated. What is known already The entire lifespan of human sperm concomitant with the glycosylation, which have been considered to be profoundly implicated in the male reproduction. In the past, only a few glycan patterns or glycoproteins in the male reproductive system were characterized due to technical limitations. Traditional strategies based on either glycomics or deglycosylated proteomics also are far from sufficient for fully understanding glycosylation in male reproduction. The development of the precise site-specific glycosylation of human semen was profiled, which laid a foundation on subsequently unveiling its role, while many challenging questions remain and require the assistance of advanced glycoproteomics. Study design, size, duration The original research and reviews in the PubMed and Web of Science databases were searched using terms regarding glycoproteome and male reproduction until September 2023. Participants/materials, setting, methods PubMed and Web of Science were used to search articles and reviews with the following main keywords pertaining to the glycoproteomics: “glycosylation”, “glycan”, “glycoprotein”, “glycome”, “glycoproteome”, “glycomics” and “glycoproteomics”, combined with “semen”, “spermatozoa”, “seminal plasma”, “epididymis”, “fertilization” and other key terms related to male reproduction. The search period included all publications until now (September 2023). The language was limited to English, and the species in most studies was limited to humans. Main results and the role of chance The precise site-specific glycoproteome of fertile human ejaculate spermatozoa and seminal plasma was generally established. Both are highly abundant in glycans and glycoproteins and share heavy fucosylation as the most significant feature. Their functions are mainly involved in immunomodulation, capacitation, and sperm-oocyte binding. Spatiotemporal regulation was found in glycosylation during male reproduction. Intracellular glycan synthesis occurs in testicular sperm, and a variety of glycan-modifying enzymes and extracellular vesicles are responsible for sperm maturation. Alterations in the semen glycoproteome were observed in male reproduction disorders and are associated with oligoasthenozoospermia, decreased sperm function, male accessory gland disease and immune dysregulation. Impaired glycosylation may be the result of genetic, endocrine, and oxidative stress and infection. Some idiopathic male infertility may be attributed to glycosylation abnormalities. Hence, glycoproteins have the potential to be biomarkers in male reproductive diseases and assisted reproductive technology. We point out that the mapping of the normal semen N-glycoproteome was the first step and there are many challenges such as the identification of the O-linked glycoproteome. Advanced glycoproteomic techniques developed recently have the potential to address them and promote the glycoproteome research in male reproduction. Limitations, reasons for caution There are no more than 20 articles regarding the glycomics or glycoproteomics of the semen and the samples processing is not ideal. Wider implications of the findings Our work hope to enlighten the biologists to depict the overall landscape of the male reproductive glycoproteome including spermatogenesis and maturation. Moreover, we provide adequate information for reproductive clinicians and technicians for the clinical translation of glyco-related biomarkers and therapeutic targets. Trial registration number not applicable

Longitudinal changes in cardiovascular disease-related proteins in welders.

Occupational exposure to welding fumes is linked to a higher risk of cardiovascular disease; however, the threshold exposure level is unknown. Here, we aimed to identify changes in proteins associated with cardiovascular disease in relation to exposure to welding fumes. Data were obtained from two timepoints six years apart for 338 non-smoking men (171 welders, 167 controls); of these, 174 (78 welders, 96 controls) had measurements available at both timepoints. Exposure was measured as personal respirable dust (adjusted for personal protective equipment), welding years, and cumulative exposure. Proximity extension assays were used to measure a panel of 92 proteins involved in cardiovascular processes in serum samples. Linear mixed models were used for longitudinal analysis. The biological functions and diseases related to the identified proteins were explored using the Ingenuity Pathway Analysis software. At both timepoints, the median respirable dust exposure was 0.7mg/m3for the welders. Seven proteins were differentially abundant between the welders and controls and increased incrementally with respirable dust: FGF23, CEACAM8, CD40L, PGF, CXCL1, CD84, and HO1. CD84 was significant after adjusting for multiple comparisons. These proteins have been linked to disorders of blood pressure, damage related to clogged blood vessels, and chronic inflammatory disorders. Exposure to mild steel welding fumes below current occupational exposure limits for respirable particles and welding fumes in Europe and the US (1-5mg/m3) was associated with changes in the abundance of proteins related to cardiovascular disease. Further research should evaluate the utility of these proteins as prospective biomarkers of occupational cardiovascular disease.

Engineering of a DNA/γPNA Hybrid Nanoreporter for ctDNA Mutation Detection via γPNA Urinalysis.

Detection of circulating tumor DNA (ctDNA) mutations, which are molecular biomarkers present in bodily fluids of cancer patients, can be applied for tumor diagnosis and prognosis monitoring. However, current profiling of ctDNA mutations relies primarily on polymerase chain reaction (PCR) and DNA sequencing and these techniques require preanalytical processing of blood samples, which are time-consuming, expensive, and tedious procedures that increase the risk of sample contamination. To overcome these limitations, here the engineering of a DNA/γPNA (gamma peptide nucleic acid)hybrid nanoreporter is disclosed for ctDNA biosensing via in situ profiling and recording of tumor-specific DNA mutations. The low tolerance of γPNA to single mismatch in base pairing with DNA allows highly selective recognition and recording of ctDNA mutations in peripheral blood. Owing to their remarkable biostability, the detached γPNA strands triggered by mutant ctDNA will be enriched in kidneys and cleared into urine for urinalysis. It is demonstrated that the nanoreporter has high specificity for ctDNA mutation in peripheral blood, and urinalysis of cleared γPNA can provide valuable information for tumor progression and prognosis evaluation. This work demonstrates the potential of the nanoreporter for urinary monitoring of tumor and patient prognosis through in situ biosensing of ctDNA mutations.

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Benchmarking and Automating the Biotinylation Proteomics Workflow.

Protein biotinylation has been widely used in biotechnology with various labeling and enrichment strategies. However, different enrichment strategies have not been systematically evaluated due to the lack of a benchmarking model for fair comparison. Most biotinylation proteomics workflows suffer from lengthy experimental steps, non-specific bindings, limited throughput, and experimental variability. To address these challenges, we designed a two-proteome model, where biotinylated yeast proteins were spiked in unlabeled human proteins, allowing us to distinguish true enrichment from non-specific bindings. Using this benchmarking model, we compared common biotinylation proteomics methods and provided practical selection guidelines. We significantly optimized and shortened sample preparation from 3 days to 9 hours, enabling fully-automated 96-well plate sample processing. Next, we applied this optimized and automated workflow for proximity labeling to investigate the intricate interplay between mitochondria and lysosomes in living cells under both healthy state and mitochondrial damage. Our results suggested a time-dependent proteome remodeling and dynamic translocation within mitochondria and between mitochondria and lysosomes upon mitochondrial damage. This newly established benchmarking model and the fully-automated 9-hour workflow can be readily applied to the broad fields of protein biotinylation to study protein interaction and organelle dynamics.

Advantages of stereolithographic 3D printing in the fabrication of the Affiblot device for dot-blot assays

In stereolithographic (SLA) 3D printing, objects are constructed by exposing layers of photocurable resin to UV light. It is a highly user-friendly fabrication method that opens a possibility for technology sharing through CAD file online libraries. Here, we present a prototyping procedure of a microfluidics-enhanced dot-blot device (Affiblot) designed for simple and inexpensive screening of affinity molecule characteristics (antibodies, oligonucleotides, cell receptors, etc.). The incorporation of microfluidic features makes sample processing user-friendly, less time-consuming, and less laborious, all performed completely on-device, distinguishing it from other dot-blot devices. Initially, the Affiblot device was fabricated using CNC machining, which required significant investment in manual post-processing and resulted in low reproducibility. Utilization of SLA 3D printing reduced the amount of manual post-processing, which significantly streamlined the prototyping process. Moreover, it enabled the fabrication of previously impossible features, including internal fluidic channels. While 3D printing of sub-millimeter microchannels usually requires custom-built printers, we were able to fabricate microfluidic features on a readily available commercial printer. Open microchannels in the size range 200–300 μm could be fabricated with reliable repeatability and sealed with a replaceable foil. Economic aspects of device fabrication are also discussed.Graphical abstract

Highly Sensitive Spatial Glycomics at Near-Cellular Resolution by On-Slide Derivatization and Mass Spectrometry Imaging.

Glycans on proteins and lipids play important roles in maturation and cellular interactions, contributing to a variety of biological processes. Aberrant glycosylation has been associated with various human diseases including cancer; however, elucidating the distribution and heterogeneity of glycans in complex tissue samples remains a major challenge. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is routinely used to analyze the spatial distribution of a variety of molecules including N-glycans directly from tissue surfaces. Sialic acids are nine carbon acidic sugars that often exist as the terminal sugars of glycans and are inherently difficult to analyze using MALDI-MSI due to their instability prone to in- and postsource decay. Here, we report on a rapid and robust method for stabilizing sialic acid on N-glycans in FFPE tissue sections. The established method derivatizes and identifies the spatial distribution of α2,3- and α2,6-linked sialic acids through complete methylamidation using methylamine and PyAOP ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate). Our in situ approach increases the glycans detected and enhances the coverage of sialylated species. Using this streamlined, sensitive, and robust workflow, we rapidly characterize and spatially localize N-glycans in human tumor tissue sections. Additionally, we demonstrate this method's applicability in imaging mammalian cell suspensions directly on slides, achieving cellular resolution with minimal sample processing and cell numbers. This workflow reveals the cellular locations of distinct N-glycan species, shedding light on the biological and clinical significance of these biomolecules in human diseases.