Real-Time Imaging of Calcium Dynamics in Human Sperm After Precise Single-Cell Stimulation.

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR and ddPCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10-5. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org ).

Prevalência de Influenza A, vírus sincicial respiratório eSARS-CoV-2 em pacientes com síndrome respiratóriaaguda grave em Passo Fundo, Rio Grande do Sul / Prevalence of Influenza A, respiratory syncytial virus andSARS-CoV-2 in patients with severe acute respiratorysyndrome in Passo Fundo, Rio Grande do Sul

A síndrome respiratória aguda grave (SRAG) é caracterizada por sintomas de febre alta, tosse e dispneia, e, na maioria dos casos, relacionada a uma quantidade reduzida de agentes infecciosos. O objetivo foi avaliar a prevalência dos vírus respiratórios Influenza A (FluA), vírus sincicial respiratório (RSV) e do novo coronavírus (SARS-CoV-2) em pacientes com internação hospitalar por SRAG. Estudo transversal, com pacientes em internação hospitalar com SRAG entre novembro de 2021 e maio de 2022. Dados sociodemográficos e clínicos e amostras da nasofaringe foram coletados/as, as quais foram submetidas à extração de RNA e testadas quanto à positividade para Influenza A, RSV e SARS-CoV-2 por meio da técnica de PCR em tempo real pelo método SYBR Green. Foram incluídos 42 pacientes, sendo 59,5% do sexo feminino, 57,1% idosos, 54,8% com ensino fundamental. A maior parte dos pacientes reportou hábito tabagista prévio ou atual (54,8%), não etilista (73,8%) e 83,3% deles apresentavam alguma comorbidade, sendo hipertensão arterial sistêmica e diabetes mellitus tipo 2 as mais prevalentes. Um total de 10,5% dos pacientes testou positivo para FluA, nenhuma amostra positiva para RSV e 76,3% positivos para SARS-CoV-2. Na população estudada, SRAG com agravo hospitalar foi observado em maior proporção, em mulheres, idosos e pessoas com comorbidades, embora sem significância estatística, sendo o novo coronavírus o agente etiológico mais relacionado, o que evidencia a patogenicidade desse agente e suas consequências ainda são evidentes após quase 2 anos de período pandêmico.

Severe acute respiratory syndrome (SARS) is characterized by symptoms of high fever, cough and dyspnea, and is in most cases related to a reduced amount of infectious agents. The objective was to assess the prevalence of respiratory viruses Influenza A (FluA), respiratory syncytial virus (RSV) and the new coronavirus (SARS-CoV-2) in patients hospitalized for SARS. Cross-sectional study, with patients hospitalized with SARS between November 2021 and May 2022. Sociodemographic and clinical data and nasopharyngeal samples were collected, which were subjected to RNA extraction and tested for positivity for Influenza A, RSV and SARS-CoV-2 using the real-time PCR technique using the SYBR Green method. 42 patients were included, 59.5% female, 57.1% elderly, 54.8% with primary education. Most patients reported previous or current smoking habits (54.8%), non-drinkers (73.8) and 83.3% of them had some comorbidity, with systemic arterial hypertension and type 2 diabetes mellitus being the most prevalent. A total of 10.5% of patients tested positive for FluA, no samples positive for RSV, and 76.3% positive for SARS-CoV-2. In the studied population, SARS with hospital injury was observed more frequently in women and the elderly, with associated comorbidities, with the new coronavirus being the most related etiological agent, which shows, although not statistically significant, that the pathogenicity of this agent and its consequences are still evident after almost 2 years of period pandemic.

Development and Implementation of a Pediatric Clinical Teaching Case Library Based on Massive Real-Time Data.

Background: With the development of information technology, establishing a clinical teaching case library based on vast real-time data resources has become a new educational approach. Nevertheless, a robust theoretical underpinning for harnessing real-time data to enhance clinical education remains elusive. The current body of research frequently falls short of a coherent theoretical structure, and has yet to delve deeply into the intrinsic worth and obstacles that real-time data presents in the educational sphere. Objective: To construct a real-time data resource set for pediatric clinical cases. Methods: This study was conducted within the framework of a university-level medical data center where advanced data de-identification protocols and encryption technologies were employed. The inclusion criteria for cases were determined based on their distinctive clinical characteristics and educational relevance aligning with established curriculum standards. These cases were then incorporated into the case library. To ensure ongoing enrichment and relevance of the pediatric clinical teaching case library, a two-phase evaluation system focused on aspects of storage-use and quality-availability was implemented. Results: This study successfully established a pediatric clinical teaching case library, supported by substantial real-time data. This database has been seamlessly incorporated into various facets of pediatric education, including classroom instruction in Pediatrics, serving as a resource for educational material and facilitating in practical teaching scenarios. Conclusion: This case library provides an authentic and dynamic data foundation for clinical teaching by leveraging a vast repository of real-time clinical data. It not only facilitates access to high-quality educational resources but also promotes the exploration and adoption of interdisciplinary teaching methodologies. Future research should clarify the theoretical foundation for the application of real-time data, fill existing theoretical gaps, and explore its applicability in various educational environments.

Quality assurance for online adaptive radiotherapy: a secondary dose verification model with geometry-encoded U-Net.

In online adaptive radiotherapy (ART), quick computation-based secondary dose verification is crucial for ensuring the quality of ART plans while the patient is positioned on the treatment couch. However, traditional dose verification algorithms are generally time-consuming, reducing the efficiency of ART workflow. This study aims to develop an ultra-fast deep-learning (DL) based secondary dose verification algorithm to accurately estimate dose distributions using computed tomography (CT) and fluence maps (FMs). We integrated FMs into the CT image domain by explicitly resolving the geometry of treatment delivery. For each gantry angle, an FM was constructed based on the optimized multi-leaf collimator apertures and corresponding monitoring units. To effectively encode treatment beam configuration, the constructed FMs were back-projected to 30 cm away from the isocenter with respect to the exact geometry of the treatment machines. Then, a 3D U-Net was utilized to take the integrated CT and FM volume as input to estimate dose. Training and validation were performed on 381 prostate cancer cases, with an additional 40 testing cases for independent evaluation of model performance. The proposed model can estimate dose in ∼ 15 ms for each patient. The average γ passing rate ( 3 % / 2 mm , 10 % threshold) for the estimated dose was 99.9% ± 0.15% on testing patients. The mean dose differences for the planning target volume and organs at risk were 0.07 % ± 0.34 % and 0.48 % ± 0.72 % , respectively. We have developed a geometry-resolved DL framework for accurate dose estimation and demonstrated its potential in real-time online ART doses verification.

Infectious Coryza in Pennsylvania.

Infectious coryza (IC) is a respiratory disease of chickens, including pullets, layers, and broilers, caused by the bacteria Avibacterium paragallinarum (AP), which was previously known as Hemophilus gallinarum. IC classically causes production decreases and mortality in chickens, frequently paired with swelling of the sinuses, mucoid nasal discharge, and respiratory rales. Although IC is considered an endemic disease of chickens in California, it has been unusual to rare in commercial chickens in Pennsylvania. The last reported IC case in Pennsylvania was in 2002, involving broiler breeders. However, between December 2018 and December 2019, 68 farms were affected by IC in Pennsylvania, involving approximately 14 million birds. Several farms had multiple flocks affected. Most affected farms housed layer chickens (37/68), but a smaller number of broiler farms, pullet farms, and layer breeder farms have been affected. Ages of affected birds and duration of disease were variable between flocks, as were the severity of clinical signs, pathologic lesions, and rates of mortality. PCR testing has greatly aided and sped diagnostic efforts in addition to traditional bacterial culture. In eight layer cases and five broiler cases, bacterial culture of the sinus or choanal cleft proved unrewarding, whereas culture of trachea, air sacs, lungs, heart, or liver were diagnostic. Although cases of IC in commercial Pennsylvania poultry continue, they have been greatly reduced because of implementation of a successful vaccination program. In this case series report we detail epidemiologic, clinical, and pathologic aspects of this outbreak and discuss vaccination as a control measure of IC in the state of Pennsylvania.

Coriza infecciosa en Pensilvania. La coriza infecciosa (CI) es una enfermedad respiratoria de las gallinas, incluyendo pollas de reemplazo, gallinas de postura y pollo de engorde, causada por la bacteria Avibacterium paragallinarum (AP), que anteriormente era conocida como Hemophilus gallinarum. Clásicamente, la coriza infecciosa causa disminución en la producción y aumento de la mortalidad en los pollos, frecuentemente es acompañada de inflamación de los senos nasales, secreción nasal mucoide y estertores respiratorios. Aunque la coriza infecciosa se considera una enfermedad endémica en la avicultura en California, ha sido inusual o esporádica en las aves comerciales de Pensilvania. El último caso notificado de coriza infecciosa en Pensilvania ocurrió en el 2002 y afectó a reproductoras pesadas. Sin embargo, entre diciembre del 2018 y diciembre del 2019, 68 granjas se vieron afectadas por esta enfermedad en Pensilvania, lo que afectó a aproximadamente 14 millones de aves. Varias granjas tuvieron múltiples parvadas afectadas. La mayor'ia de las granjas afectadas albergaban gallinas de postura (37/68), pero un número menor de granjas de pollos de engorde, de pollas de reemplazo y de reproductoras de aves de postura se han visto afectadas. Las edades de las aves afectadas y la duración de la enfermedad variaron entre parvadas, al igual que la severidad de los signos cl'inicos, las lesiones patológicas y las tasas de mortalidad. Las pruebas de PCR han ayudado acelerado enormemente los esfuerzos de diagnóstico además del cultivo bacteriano tradicional. En ocho casos de ponedoras y cinco de pollos de engorde, el cultivo bacteriano de senos respiratorios o de la hendidura coanal resultó infructuoso, mientras que el cultivo de tráquea, alvéolos, pulmones, corazón o h'igado fueron de utilidad diagnóstica. Aunque la presentación de casos de coriza infecciosa en aves comerciales de Pensilvania continúa, se han reducido considerablemente gracias a la implementación de un programa de vacunación exitoso. En esta serie de reportes de casos, se detallan los aspectos epizootiológicos, cl'inicos y patológicos de este brote y se analiza la vacunación como medida de control contra la coriza infecciosa en el estado de Pensilvania.

An Automated Clubbed Fingers Detection System Based on YOLOv8 and U-Net: A Tool for Early Prediction of Lung and Cardiovascular Diseases.

Background/Objectives: Lung and cardiovascular diseases are leading causes of mortality worldwide, yet early detection remains challenging due to the subtle symptoms. Digital clubbing, characterized by the bulbous enlargement of the fingertips, serves as an early indicator of these diseases. This study aims to develop an automated system for detecting digital clubbing using deep-learning models for real-time monitoring and early intervention. Methods: The proposed system utilizes the YOLOv8 model for object detection and U-Net for image segmentation, integrated with the ESP32-CAM development board to capture and analyze finger images. The severity of digital clubbing is determined using a custom algorithm based on the Lovibond angle theory, categorizing the condition into normal, mild, moderate, and severe. The system was evaluated using 1768 images and achieved cloud-based and real-time processing capabilities. Results: The system demonstrated high accuracy (98.34%) in real-time detection with precision (98.22%), sensitivity (99.48%), and specificity (98.22%). Cloud-based processing achieved slightly lower but robust results, with an accuracy of 96.38%. The average processing time was 0.15 s per image, showcasing its real-time potential. Conclusions: This automated system provides a scalable and cost-effective solution for the early detection of digital clubbing, enabling timely intervention for lung and cardiovascular diseases. Its high accuracy and real-time capabilities make it suitable for both clinical and home-based health monitoring.

Wearable network for multilevel physical fatigue prediction in manufacturing workers.

Manufacturing workers face prolonged strenuous physical activities, impacting both financial aspects and their health due to work-related fatigue. Continuously monitoring physical fatigue and providing meaningful feedback is crucial to mitigating human and monetary losses in manufacturing workplaces. This study introduces a novel application of multimodal wearable sensors and machine learning techniques to quantify physical fatigue and tackle the challenges of real-time monitoring on the factory floor. Unlike past studies that view fatigue as a dichotomous variable, our central formulation revolves around the ability to predict multilevel fatigue, providing a more nuanced understanding of the subject's physical state. Our multimodal sensing framework is designed for continuous monitoring of vital signs, including heart rate, heart rate variability, skin temperature, and more, as well as locomotive signs by employing inertial motion units strategically placed at six locations on the upper body. This comprehensive sensor placement allows us to capture detailed data from both the torso and arms, surpassing the capabilities of single-point data collection methods. We developed an innovative asymmetric loss function for our machine learning model, which enhances prediction accuracy for numerical fatigue levels and supports real-time inference. We collected data on 43 subjects following an authentic manufacturing protocol and logged their self-reported fatigue. Based on the analysis, we provide insights into our multilevel fatigue monitoring system and discuss results from an in-the-wild evaluation of actual operators on the factory floor. This study demonstrates our system's practical applicability and contributes a valuable open-access database for future research.

Evaluation of Screening Results for Vancomycin-Resistant Enterococci: Three-Year Surveillance.

Background: Enterococci are facultative anaerobic, binary, or chained Gram-positive cocci. The gastrointestinal colonization of hospitalized patients is the most important reservoir of vancomycin-resistant enterococci. We aimed to evaluate retrospectively the screening results of vancomycin-resistant enterococci, studied by the simultaneous (real-time) polymerase chain reaction method on rectal swabs of adult and pediatric patients hospitalized in our hospital in 2019-2021. Methods: Adult and pediatric patients were included in our study between Jan 2019 and Dec 2021. The results of vancomycin-resistant enterococci, studied with the real-time polymerase chain reaction method from rectal swabs sent from intensive care units and services, were analyzed retrospectively. Isolation of the samples was performed using the Fluorion VRE QLP 1.0 real-time polymerase chain reaction kit (Iontek, Turkey), and detection was performed with the Fluorion Detection System (Iontek, Turkey) real-time polymerase chain reaction device. Results: Overall, 31,725 patients were included in our study. When evaluated in order of years, in 2019, 379 (7%) of 5,389 adults, 322 (7.4%) of 4,003 children, 234 (5.5%) of 4,185 adults in 2020, 157 (2.4%) of 6,499 children, and in 2021, vancomycin-resistant enterococci were detected in 469 (7.5%) of 6,232 adults and 224 (4.1%) of 5,417 children. Conclusion: The prevalence of vancomycin-resistant enterococci is greater in adults, particularly in intensive care units, compared to children. Infection control precautions and training be augmented in high-risk clinics, while the unnecessary utilization of glycopeptides should be limited.

Investigation of Leptospirosis Agents in Cattle and Humans.

Background: Leptospirosis, a global zoonotic disease, causes serious morbidity and mortality generally in low-income societies. This study aimed to investigate the prevalence of Leptospira serovars in cattle and high-risk human populations. Methods: This study investigated the presence of pathogenic Leptospira serogroups in the blood and kidney samples of cattle arriving at the Erzurum Meat and Milk Institution for slaughter between April and July, and between September and December 2022, and in the serum samples of humans. Kidney and serum samples from 289 cattle and serum samples from 100 individuals from at-risk occupational groups (58 farmers, 25 veterinarians and 17 butchers) were collected. As a control, 100 human blood samples were collected from civil servants who had no contact with animals. Microagglutination testing was used to investigate Leptospira serogroups in bovine sera, real-time polymerase chain reaction (PCR) for Leptospira DNA in kidney samples, and microagglutination testing and enzyme-linked immunosorbent assays for Leptospira antibodies in human blood serum samples. Results: Microagglutination test in 4.5% of cattle; Leptospira DNA was positive in 0.7%. Six strains of Leptospira interrogans, two of Bratislava, one of Pomana and one of Icterohaemorrhagiae were found in cattle, as well as one strain of Leptospira kirschneri Dadas. In humans, two Icterohaemorrhagiae, one Hebdomadis and one Dadas serovar were detected in both the risk group and the control group. Using ELISA, antibody positivity was found to be 14.0% in the risk group and 11.0% in the control group. Conclusion: The seroprevalence of Leptospira spp. in cattle in Erzurum, Türkiye, is relatively high. In this region, the risk of encountering Leptospira in the normal population is as high as in the risk group.

Oral Bacteria Counter Using Dielectrophoretic Impedance Measurement: Usefulness and Usage Considerations.

BACKGROUND: The oral cavity hosts numerous bacteria that are associated with various systemic diseases. The Oral Bacteria Counter (PHC Corporation, Tokyo, Japan), a microorganism quantitative analyzer that utilizes dielectrophoretic impedance measurements, enables rapid bacterial counting and is widely used in dental practice in Japan. However, it may also detect nonviable bacteria. This study aimed to assess the impact of disinfectants, electrolytes, and viscosity on the accuracy of the Oral Bacteria Counter and to determine whether it measures non-viable bacteria similarly to viable bacteria. METHODS: To evaluate the effect of the disinfectants, samples of 7% povidone-iodine (PV-I), 0.2% benzethonium chloride, 5% chlorhexidine (CHX), 0.2% CHX, 0.05% CHX, sterile water, and saline were measured using the Oral Bacteria Counter. The effect of viscosity was assessed by mixing sterile water with glycerol in various ratios and measuring the dielectrophoretic impedance of the bacterial counts at different viscosities. For the electrolyte effects, samples of Staphylococcus aureus diluted in sterile water or saline were measured using the Oral Bacteria Counter. Additionally, samples of 7% PV-I or 5% CHX diluted in sterile water or saline were measured. Bacterial counts were then measured and compared using the Oral Bacteria Counter, our developed delayed real-time polymerase chain reaction (DR-PCR) method (which quantifies only viable bacteria), and culture methods. RESULTS: Disinfectants such as 5% CHX and 7% PV-I produced high readings on the Oral Bacteria Counter, even when no viable bacteria were present. Higher glycerol concentrations, which increased the viscosity, resulted in lower bacterial counts. The presence of electrolytes, particularly saline, led to higher readings on the Oral Bacteria Counter, which detected both viable and non-viable bacteria, whereas DR-PCR and culture methods did not detect non-viable bacteria. CONCLUSION: The Oral Bacteria Counter may be influenced by disinfectants, viscosity, and electrolytes, leading to potential inaccuracies in bacterial quantification. For accurate bacterial measurements, it is essential to consider these factors and ideally combine the results from the Oral Bacteria Counter with methods such as DR-PCR for more reliable assessment.

Deep learning-based surgical step recognition for laparoscopic right-sided colectomy.

PURPOSE: Understanding the complex anatomy and surgical steps involved in laparoscopic right-sided colectomy (LAP-RC) is essential for standardizing the surgical procedure. Deep-learning (DL)-based computer vision can achieve this. This study aimed to develop a step recognition model for LAP-RC using a dataset of surgical videos with annotated step information and evaluate its recognition performance. METHODS: This single-center retrospective study utilized a video dataset of laparoscopic ileocecal resection (LAP-ICR) and laparoscopic right-sided hemicolectomy (LAP-RHC) for right-sided colon cancer performed between January 2018 and March 2022. The videos were split into still images, which were divided into training, validation, and test sets using 66%, 17%, and 17% of the data, respectively. Videos were manually classified into eight main steps: 1) medial mobilization, 2) central vascular ligation, 3) dissection of the superior mesenteric vein, 4) retroperitoneal mobilization, 5) lateral mobilization, 6) cranial mobilization, 7) mesocolon resection, and 8) intracorporeal anastomosis. In a simpler version, consecutive surgical steps were combined, resulting in five steps. Precision, recall, F1 scores, and overall accuracy were assessed to evaluate the model's performance in the surgical step classification task. RESULTS: Seventy-eight patients were included; LAP-ICR and LAP-RHC were performed in 35 (44%) and 44 (56%) patients, respectively. The overall accuracy was 72.1% and 82.9% for the eight-step and combined five-step classification tasks, respectively. CONCLUSIONS: The automatic surgical step-recognition model for LAP-RCs, developed using a DL algorithm, exhibited a fairly high classification performance. A model that understands the complex steps of LAP-RC will aid the standardization of the surgical procedure.

Relationships between fecal indicator abundance in water and sand and the presence of pathogenic genes in sand of recreational beaches.

For decades, the risk of exposure to infectious diseases in recreational beaches has been evaluated through the quantification of fecal indicator bacteria in water samples using culture methods. The analyses of sand samples have recently been developed as a complement to the monitoring of recreational waters in beach quality assessments. The growing use of molecular techniques for environmental monitoring allows for the rapid detection of pathogenic genes, thus providing more accurate information regarding the health risk of exposure to contaminated sand. The aim of this work was to determine the relationship between the fecal indicators abundance in water and sand and the presence of Shiga toxin-producer Escherichia coli (STEC) in sand by analyzing samples from touristic beaches using culture-dependent (fecal coliforms assay) and culture-independent (real-time PCR of stx1, stx2, and eae genes) techniques. We found a high concentration of coliform bacteria in water and sand in several beaches in eastern Uruguay, with different levels of sanitation networks and levels of urbanization. The presence of STEC virulence genes (mainly stx1) was confirmed in 8 out of 20 sand samples. The recreational use of sandy beaches may imply a risk to the health of its users, especially near streams and creek outflows, thus highlighting the need of monitoring sand bacteriological quality and pathogens using molecular tools.

Unveiling the impact of antibiotic stress on biofilm formation and expression of toxin-antitoxin system genes in Clostridium difficile clinical isolates.

OBJECTIVES: The study investigates how antibiotics affect biofilm formation and toxin gene expression in Clostridium difficile, which is essential for its survival and persistence. METHODS: The study confirmed 25 strains of C. difficile and assessed biofilm formation. The MIC of metronidazole and vancomycin was determined through agar dilution, and the impact of sub-MIC levels on biofilm formation and eradication was investigated. Additionally, Real-time PCR was used to analyze the expression levels of target genes related to antibiotic treatment. RESULTS: We found that certain genes, such as the ImmA/IrrE system, were associated with increased biofilm formation in isolates. Sub-MIC antibiotic levels influenced gene expression related to biofilm activities, particularly emphasizing the importance of toxin-antitoxin systems. The results suggest that antibiotics at sub-MIC levels may play a signaling role in promoting biofilm formation and gene expression in C. difficile. CONCLUSION: Our study suggests that toxin and antitoxin genes may impact C. difficile biofilm formation, while antibiotics could signal biofilm strengthening and gene expression increase.

A novel simple fluorometric protease assay for monitoring hydrolysis of proteins in real time.

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.

Initial feasibility evaluation of the RISES system: An innovative and activity-based closed-loop framework for spinal cord injury rehabilitation and recovery.

Background: Electrical stimulation of the spinal cord may improve rewiring of the affected pathways. Immediate modulation of stimulation parameters, and its effects of it on kinematics and electromyographic variables is unclear. Methods: This study piloted the safety and feasibility of the Reynolds Innovative Spinal Electrical Stimulation (RISES) technology with a focus on its novel closed-loop setting. This personalized, task-specific non-invasive stimulation system enables real-time stimulation parameter modulation and supports multi-data acquisition and storage. Four SCI participants underwent a clinical trial coupled with activity-based training. Primary safety outcome measures included adverse events (AEs) and skin integrity; secondary measures were vital signs, pain, and fatigue assessed at the pre, mid, and post-stimulation sessions. The trial included open-loop and closed-loop blocks of transcutaneous spinal cord stimulation (tSCS). Results: Results showed no serious adverse events, with skin integrity unaffected. Vital signs and pain showed no significant differences across session timepoints. Fatigue levels differed significantly with post-session > mid-session > pre-session. Comparisons between open-loop and closed-loop blocks showed no significant differences in setup time, vital signs, pain, or fatigue. Average stimulation duration per task was significantly longer for open-loop (467.6 sec) than Closed-loop (410.8 sec). Conclusions: RISES, demonstrated safety and feasibility. Further work will focus on clinical efficacy.

Fine-mapping of a QTL and identification of candidate genes associated with the lateral branch angle of peanuts (Arachis hypogaea L.) on chromosome B05.

Peanuts play a crucial role as an oil crop, serving not only as a primary source of edible oil but also offering ample protein and vitamins for human consumption. The lateral branch angle of peanuts is the angle between the main stem and the first pair of lateral branches, which is an important agronomic trait of peanuts, significantly impacts the peg penetration into the soil, plant growth, and pod yield. It is closely intertwined with planting density, cultivation techniques, and mechanized harvesting methods. Therefore, the lateral branch angle holds substantial importance in enhancing peanut yield and facilitating mechanization. In order to conduct in-depth research on the lateral branch angle of peanuts, this research is grounded in the QTL mapping findings, specifically focusing on the QTL qGH associated with the lateral branch angle of peanuts located on chromosome B05 (142610834-146688220). By using Jihua 5 and PZ42 for backcrossing, a BC1F2 population comprising 8000 individual plants was established. Molecular markers were then developed to screen the offspring plants, recombine individual plants, conduct fine mapping. he results showed that using the phenotype and genotype of 464 recombinant individual plants selected from 8000 offspring, narrow down the localization interval to 48kb, and designate it as qLBA. The gene Arahy.C4FM6Y, responsible for the F-Box protein, was identified within qLBA through screening. Real-time quantitative detection of Arahy.C4FM6Y was carried out using M130 and Jihua 5, revealing that the expression level of Arahy.C4FM6Y at the junction of the main stem and the first lateral branch of peanuts was lower in M130 compared to Jihua 5 during the growth period of the first lateral branch from 1 to 10 centimeters. Consequently, Arahy.C4FM6Y emerges as a gene that restrains the increase in the angle of the first lateral branch in peanuts. This investigation offers novel genetic reservoirs for peanut plant type breeding and furnishes a theoretical foundation for molecular marker-assisted peanut breeding.

Selective monitoring of superior and inferior vena cava drainage flows in bicaval cannulation: Potential clinical benefits of proximal junction placement on the CPB side.

Background: In cardiac surgery, precision and efficiency are crucial, especially in managing venous drainage flows during complex procedures. This practical technique evaluates the benefits of placing the proximal junction on the cardiopulmonary bypass (CPB) side rather than on the surgical table in bicaval cannulation. Bicaval cannulation, involving both the superior vena cava (SVC) and inferior vena cava (IVC), ensures efficient venous return.Practical technique: Placing the junction on the CPB side allows for accurate, real-time control of venous drainage, allowing for swift responses to fluctuations and maintaining stable hemodynamics.Perspectives: This approach could minimize the risk of congestion in the liver, splanchnic system, and cerebral venous return, thereby preventing related complications. Another important potential benefit of this approach could be the ability to closely monitor and manage metabolic demands and oxygen consumption for SVC and IVC. Additionally, EmTec flow technology can be integrated for more precise management.

Intensive, Real-Time Data Collection of Psychological and Physiological Stress During a 96-Hour Field Training Exercise at a Senior Military College: Feasibility and Acceptability Cohort Study.

BACKGROUND: Poor physical fitness, stress, and fatigue are factors impacting military readiness, national security, and economic burden for the United States Department of Defense. Improved accuracy of wearable biosensors and remote field biologic sample collection strategies could make critical contributions to understanding how physical readiness and occupational stressors result in on-the-job and environment-related injury, sleep impairments, diagnosis of mental health disorders, and reductions in performance in war-fighters. OBJECTIVE: This study aimed to evaluate the feasibility and acceptability of intensive biomarker and biometric data collection to understand physiological and psychological stress in Army Reserved Officer Training Corps cadets before, during, and after a 96-hour field training exercise (FTX). METHODS: A prospective pilot study evaluated the feasibility and acceptability of multimodal field data collection using passive drool saliva sampling, sweat sensors, accelerometry, actigraphy, and photoplethysmography. In addition, physical fitness (Army Combat Fitness Test), self-reported injury, and psychological resilience (Brief Resilience Scale) were measured. RESULTS: A total of 22 cadets were included. Two were lost to follow-up due to injury during FTX, for a retention rate of 91%. Assessments of performance and psychological resilience were completed for all remaining participants, resulting in 100% testing adherence. All participants provided saliva samples before the FTX, with 98% adherence at the second time point and 91% at the third. For sweat, data collection was not possible. Average daily wear time for photoplethysmography devices was good to excellent, meeting a 70% threshold with data collected for ≥80% of person-days at all time points. Of the participants who completed the FTX and 12 completed a post-FTX acceptability survey for a response rate of 60%. Overall, participant acceptance was high (≥80%) for all metrics and devices. CONCLUSIONS: This study demonstrates that wearable biosensors and remote field biologic sample collection strategies during a military FTX have the potential to be used in higher stakes tactical environments in the future for some, but not all, of the strategies. Overall, real-time biometric and biomarker sampling is feasible and acceptable during field-based training and provides insights and strategies for future interventions on military cadet and active-duty readiness, environmental stress, and recovery.

Rapid detection of drugs in blood using "molecular hook" surface-enhanced Raman spectroscopy and artificial intelligence technology for clinical applications.

Accurate detection of cardiovascular drugs in blood is complicated by interference from serum biomolecules. This study introduces a novel surface-enhanced Raman spectroscopy (SERS) platform incorporating "molecular hooks" to capture small drug molecules while excluding larger biomolecules selectively. The self-assembled nanoparticles with the A13 molecule enhance Raman signals by creating dense electromagnetic "hotspot" regions, achieving detection limits of 10 pg/mL for dobutamine hydrochloride and 10 ng/mL for milrinone-substantially below therapeutic thresholds. Artificial intelligence (AI) integration enables automated spectral analysis, allowing rapid and precise drug detection in clinical blood samples. This approach offers a transformative solution for real-time diagnostics, significantly advancing personalized treatment strategies in clinical settings.