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Controlled liquid exchange is a fundamental requirement for numerous biological and chemical experimental protocols. However, achieving constant-volume exchange often requires electronic syringe pumps that are cost-prohibitive and time-consuming to set up in a push-pull configuration. To address these limitations, we developed the Push2Pull syringe holder, a simple 3D-printed device that mechanically synchronizes two syringes to simultaneously add and extract equal fluid volumes. This device is compatible with standard disposable syringes from 1 to 60 mL in size and operates without electricity or additional hardware, making it ideal for both laboratory and field settings. Validation experiments demonstrate an exchange accuracy within ±2%v/v across the whole travel range, while fluid exchange efficiency was calculated for various use cases using CFD simulations. The Push2Pull syringe holder offers an accessible, open-source solution for precise fluid handling, for a material cost of less than $10.

Nerve agents such as VX, Tabun (GA), and Cyclosarin (GF) are lethal even in trace amounts. Their lethality in addition to being difficult to detect makes nerve agents a significant global security problem. To address this problem, surface-enhanced Raman spectroscopy (SERS) has in recent years shown to be a promising technique for rapid and non-destructive field detection of various harmful substances. However, the application of SERS to nerve agents has so far been limited by their inherently poor Raman scattering cross-section, with nerve agents such as GF being particularly difficult to detect. In this work, we present a novel custom-designed Raman probe molecule: 4-Thiophene Pyridine Amido Oxime (TPAO), which enables selective detection and discrimination of live nerve agents in liquid media at trace concentrations, even using portable Raman instruments with limited spectral resolution. We report a detection limit for GF downward 1 ppm, much lower than that found in previous studies. We further find that TPAO allows us to detect VX and GA at 1.1 and 2.1 ppm, respectively, in line with or slightly surpassing previous benchmarks. To demonstrate the specificity with which TPAO can be used to sense and discriminate between nerve agents, we use principal component analysis to quantify spectral differences between nerve agents and simulants. Lastly, we show the robustness of TPAO, demonstrating its ability to detect nerve agents in complex backgrounds and after rinsing procedures. Altogether, this work represents a significant step forward towards achieving rapid and selective field detection of live nerve agents using SERS.

According to glymphatic system theory, cerebrospinal fluid (CSF) perfuses the brain’s interstitial space to support waste clearance, but the magnitude of this flow and the outflow pathway of interstitial fluid (ISF) in humans remain uncertain. To achieve flow quantification, we applied a compartment-model approach applied in conjunction with serial quantitative MRI data acquired after intrathecal gadolinium administration. Using the method, we estimated CSF-to-ISF inflow to 45 ± 20 mL/h, in patients with suspected idiopathic normal pressure hydrocephalus. Tissue-specific contributions were 34 ± 14 mL/h in cortical gray matter, 11±6 mL/h in white matter, and 0.4 ± 0.3 mL/h in subcortical gray matter, suggesting that CSF perfusion occurs primarily in superficial regions near the subarachnoid space. A lack of correlation between inflow and total craniospinal system outflow (r = 0.03, P = 0.91) suggested that ISF recirculates back into CSF rather than exiting the craniospinal system via a separate route. Independent experiments in healthy older individuals using intravenous gadolinium administration supported ISF-to-CSF recirculation, where contrast material that presumably crossed the blood–brain barrier subsequently appeared in the subarachnoid space, allowing ISF-to-CSF flow quantification. These findings provide a quantitative framework for studying brain clearance in humans and support subarachnoid space recirculation as an important efflux route.

Growing biofilms of thermophilic (heat-loving) and psychrotrophic (cold-tolerant) bacteria pose several challenges due to specific environmental requirements. Thermophilic bacteria typically grow between 45 and 80 C, while psychrotrophic bacteria thrive between 0 and 15 C. Maintaining the precise temperature and fluid conditions required for biofilm growth can be technically challenging. To overcome these challenges, we designed the Bio-Rocker, a temperature- and shear stress-controlled rocker platform for biofilm incubation. The platform supports temperatures between − 9 and 99 C, while its digital controller can adjust the rocking speed from 1 to 99/s and set rocking angles up to ±19. This ability, together with data from analytical models and multi-physics simulations, provides control over the shear stress distribution at the growth surfaces, peaking at 2.4 N/m. Finally, we evaluated the system’s ability to grow bacteria at different temperatures, shear stress, and materials by looking at the coverage and thickness of the biofilm, as well as the total biomass. A step-by-step guide, 3D CAD files, and controller software is provided for easy replication of the Bio-Rocker, using mostly 3D-printed and off-the-shelf components. We conclude that the Bio-Rocker’s performance is comparable to high-end commercial systems like the Enviro-Genie (Scientific Industries) yet costs less than $350 dollars to produce.

Analyzing microscopy images of large growing cell samples using traditional methods is a complex and time-consuming process. In this work, we have developed an attention-driven UNet-enhanced model using deep learning techniques to efficiently quantify the position, area, and circularity of bacterial spores and vegetative cells from images containing more than 10,000 bacterial cells. Our attention-driven UNet algorithm has an accuracy of 96%, precision of 82%, sensitivity of 81%, and specificity of 98%. Therefore, it can segment cells at a level comparable to manual annotation. We demonstrate the efficacy of this model by applying it to a live-dead decontamination assay. The model is provided in three formats: Python code, a Binder that operates within a web browser without needing installation, and a Flask Web application for local use.

Purpose: Hypochlorite-based formulations are widely used for surface disinfection. However, the efficacy of hypochlorite against spore-forming bacteria varies significantly in the literature. Although neutral or low pH hypochlorite solutions are effective sporicides due to the formation of hypochlorous acid (HOCl), their optimal conditions and the specific role of pH in disinfection remain unclear. These conditions also increase the solution’s corrosiveness and compromise its shelf life. Therefore, further research is needed to identify the pH conditions that balance solution stability and effective hypochlorite-based spore disinfection.

Results: This study investigates the impact of neutral to alkaline pH on the sporicidal efficiency of hypochlorite against a pathogenic Bacillus cereus strain. We apply a 5,000 ppm hypochlorite formulation for 10-min across a pH range of 7.0-12.0, simulating common surface decontamination practices. Our results demonstrate that hypochlorite is largely ineffective at pH levels above 11.0, showing less than 1-log reduction in spore viability. However, there is a significant increase in sporicidal efficiency between pH 11.0 and 9.5, with a 4-log reduction in viability. This pH level corresponds to 2 - 55 ppm of the HOCl ionic form of hypochlorite. Further reduction in pH slightly improves the disinfection efficacy. However, the shelf life of hypochlorite solution decreases exponentially below pH 8.5. To explore the pH-dependent efficacy of hypochlorite, Raman spectroscopy and fluorescence imaging were used to investigate the biochemical mechanisms of spore decontamination. Results showed that lower pH enhances spore permeability and promotes calcium dipicolinic acid (CaDPA) release from the core.

Conclusion: Our results highlight the complex relationship between pH, sporicidal efficacy of hypochlorite, and its shelf life. While lower pH enhances the sporicidal efficiency, it compromises the solution’s shelf life. A pH of 9.5 offers a balance, significantly improving shelf life compared to previously suggested pH ranges 7.0-8.0 while maintaining effective spore inactivation. Our findings challenge the common practice of diluting sodium hypochlorite with water to a 5,000 ppm solution, as this highly alkaline solution (pH of 11.9), is insufficient for eliminating B. cereus spores, even after a 10-min exposure. These findings are critical for improving disinfection practices, highlighting the importance of optimizing sodium hypochlorite effectiveness through pH adjustments before application.

Spores of species belonging to the Bacillus cereus sensu lato (s.l.) group are common contaminants in food processing environments due to their ability to adhere to surfaces and resist cleaning procedures. These spores are equipped with pilus-like endospore appendages (ENAs), which are believed to promote surface adhesion. We investigated the role of ENAs in spore adhesion to abiotic surfaces using a wild-type (WT) Bacillus paranthracis strain and isogenic mutants lacking ENAs or an intact exosporium. WT spores expressing both short and long ENAs (S+L+) adhered significantly more to stainless steel (SS) and polypropylene (PP) compared to bald spores (S-L-) and spores of an exosporium-deficient mutant (Delta exsY), whereas adhesion to polystyrene (PS) and glass was not significantly affected by the presence of ENAs. The Delta exsY mutant also showed the lowest adhesion across all tested surfaces, a pattern similarly observed for vegetative cells. The strongest adhesion to PP was observed when both fiber types were present. A clear trend also emerged: on PP, WT remained adhered for at least an hour, while bald spores tended to detach within that time. Under saline conditions and at different pH levels, bald spores adhered strongly to SS. However, in the presence of a non-ionic surfactant or a concentrated protein solution, WT spores adhered more. Our results highlight the crucial role of ENAs in B. cereus spp. spore adhesion to industrially relevant surfaces, providing mechanistic insight into spore persistence. These insights support the design of surface treatments to prevent contamination, spoilage, and foodborne illnesses.IMPORTANCEBacteria belonging to the Bacillus cereus sensu lato group represent a persistent challenge in food production due to their highly resilient endospores (spores), which withstand cleaning, disinfection, and food processing. Understanding spore adhesion is essential for designing effective surface treatments that reduce chemical use, enhance food safety and quality, and minimize environmental impact. This study underscores the important role of endospore appendages (ENAs) in spore adhesion to common materials in food processing and laboratory environments. Wild-type spores expressing both S-ENA and L-ENA adhered significantly more than mutants lacking ENAs or the exosporium, highlighting ENAs as potential targets for disrupting spore adhesion. Time-dependent adhesion assays on polypropylene revealed strong, sustained attachment by wild-type spores, contrasting with weaker, transient adhesion by ENA-depleted mutants. These findings offer valuable insights into B. paranthracis spore adhesion dynamics, guiding the development of tailored cleaning protocols to improve contamination control and sustainability.

Raman spectroscopy is a highly informative technique that is routinely used to analyze the chemical bonds in molecules, such as peptides. However, while Raman spectra of aqueous single amino acids are well-characterized, proteins and peptides are usually found only in solutions in the presence of ions for stability or further analysis. The impact of ions on the Raman spectra of amino acids and subsequently on peptides and proteins has not been systematically studied. In this work, the impact of different metal ions on the Raman spectra of four significant amino acids (glutamate, tyrosine, cysteine, and serine) and N-methylacetamide (a model of a peptide bond) in aqueous solution was studied using density functional theory simulations and Raman spectroscopy experimental validation. The research focused on analyzing band shifts and variations in peak intensity when different cations commonly used in experiments (Li+, Na+, K+, Ag+, Au+, Cu+, and Cu2+) were present. Experimental evidence on Raman spectra variation of Glutamate in the presence of different concentrations of Cu2+is also provided. Our results show that the ions stay close to the oxygen atoms in the amino acids tested. With regard to Raman spectra, we show Cu2+promotes Raman resonance. We have characterized and tabulated the shifts in the characteristic Raman peaks of the amino acids in response to metal ions with the aim of providing a reference for interpreting Raman spectra of proteins and other biological materials.

Bacterial spores are highly resilient and capable of surviving extreme conditions, making them a persistent threat in contexts such as disease transmission, food safety, and bioterrorism. Their ability to withstand conventional sterilization methods necessitates rapid and accurate detection techniques to effectively mitigate the risks they present. In this study, we introduce a surface-enhanced Raman spectroscopy (SERS) approach for detecting Bacillus thuringiensis spores by targeting calcium dipicolinate acid (CaDPA), a biomarker uniquely associated with bacterial spores. Our method uses probe sonication to disrupt spores, releasing their CaDPA, which is then detected by SERS on drop-dried supernatant mixed with gold nanorods. This simple approach enables the selective detection of CaDPA, distinguishing it from other spore components and background noise. We demonstrate detection of biogenic CaDPA from concentrations as low as 10³ spores/mL, with sensitivity reaching beyond CaDPA levels of a single spore. Finally, we show the method’s robustness by detecting CaDPA from a realistic sample of fresh milk mixed with spores. These findings highlight the potential of SERS as a sensitive and specific technique for bacterial spore detection, with implications for fields requiring rapid and reliable spore identification.

Many strains among spore-forming bacteria species are associated with food spoilage, foodborne disease, and hospital-acquired infections. Understanding the impact of environmental conditions and decontamination techniques on the metabolic activity, viability, and biomarkers of these spores is crucial for combatting them. To distinguish and track spores and to understand metabolic mechanisms, spores must be labeled. Staining or genetic modification are current methods for this, however, these methods can be time-consuming, and affect the viability and function of spore samples. In this work, we investigate the use of heavy water for permanent isotope labeling of spores and Raman spectroscopy for tracking sporulation/germination mechanisms. We also discuss the potential of this method in observing decontamination. We find that steady-state deuterium levels in the spore are achieved after only ∼48 h of incubation with 30% D2O-infused broth and sporulation, generating Raman peaks at cell silent region of 2200 and 2300 cm−1. These deuterium levels then decrease rapidly upon spore germination in non-deuterated media. We further find that unlike live spores, spores inactivated using various methods do not lose these Raman peaks upon incubation in growth media, suggesting these peaks may be used to indicate the viability of a spore sample. We further observe several Raman peaks exclusive to deuterated DPA, a spore-specific chemical biomarker, at e.g. 988 and 2300 cm−1, which can be used to track underlying changes in spores involving DPA. In conclusion, permanent spore labeling using deuterium offers a robust and non-invasive way of labeling bacterial spores for marking, viability determination, and characterising spore activity.