Short Incubation Times Research Articles

Protein Stacking on the APTES-Functionalized Pyrochlore Bi2Ru2O7 Clusters for Ultrasensitive and Selective Immunosensing.

With their unique physicochemical properties, such as metallic-like conductivity, favorable (electro)catalytic properties, electrochemical stability, and ease of functionalization, pyrochlores have found applications in various fields such as solid oxide fuel cells, batteries, thick film resistors, and temperature sensors; however, there are no reports on their application in electrochemical immunosensing. In this study, we exploited the (electro)catalytic nature and stability of the pyrochlore Bi2Ru2O7 clusters silanized with (3-aminopropyl)triethoxysilane (APTES) to demonstrate their potential for the effective stacking of functional proteins. Characterization of the clusters by XPS disclosed a dual environment of Bi, also indicating the presence of Bi2O3 alongside APTES-Bi2Ru2O7 clusters and, importantly, the predominant involvement of pyrochlore moieties in subsequent protein stacking. After stacking protein A and antibodies, the immunosensor revealed a nearly interference-free operation, high sensitivity, a detection limit of 118 fM SARS-CoV-2 spike protein, and operation in a wide examined concentration range of 10-5-10-1 μg mL-1 with an r2 of 0.98. In combination with a short incubation time of 30 min, the pyrochlore-based immunosensor provides a solid platform for future point-of-need applications.

Solid-State Precipitation of Silver Nanoparticles Nucleated during Al Anodizing: Mechanism and Antibacterial Properties.

This study presents an innovative approach to creating antibacterial aluminum surfaces by combining the antibacterial properties of silver nanoparticles (Ag NPs) with the nanoarchitecture of anodized aluminum oxide in one step. An Al-Ag alloy containing 10 wt % Ag was synthesized and anodized in 0.3 M oxalic acid. Ag NPs precipitated in the solid state during anodization, resulting in a porous nanocomposite structure. Comprehensive characterization using SEM, TEM, and EDS revealed a 43 μm thick oxide layer with uniformly distributed nanopores of approximately 100 nm in diameter. Ag NPs with diameters ranging from 2 to 14 nm precipitated dispersed on the surface, inside pores, and within the Al2O3 matrix. Antibacterial properties were evaluated against Escherichia coli. The anodized Al-Ag surface demonstrated robust antibacterial activity after short incubation times (up to 1 × 108 CFU/ml after 3 h). The enhanced antibacterial properties are attributed to the optimal size and distribution of Ag NPs and the potential physical bactericidal effect of the nanoporous structure. This strategy for the precipitation of Ag NPs in the solid state could be used to fabricate high-touch surfaces in hospitals.

Polystyrene nanoparticles induce DNA damage and apoptosis in HeLa cells.

Nanoplastics (NPs) are plastic particles, typically less than 100nm in size, that result from daily life products as well as the degradation of larger plastic debris. Due to their small size and chemical composition, they can interact with biological systems in ways that larger plastic particles cannot. Humans are continuously exposed to NPs and several studies showed the potentially toxic effects of these latter on health. Polystyrene nanoplastics (PS-NPs) are the prevalent form of nanoparticles found in the environment and their cellular uptake can cause cytotoxicity and structural alteration of biomolecules. Thus, there is an urgent need for evaluation of the genotoxic effects of PS-NPs on human cell models. Through different and complementary experimental approaches, we investigated the potential genotoxic and cytotoxic effects of PS-NPs exposure on HeLa cell lines. We highlighted the genotoxic effects of polystyrene nanoplastics by showing the formation of multinuclei and micronuclei in all the studied concentrations and time points, also at short incubation time (6h) and low concentration. At higher concentrations, we demonstrate the presence of apoptotic and necrotic cells outlining the acute cytotoxic effects of nanoplastics. The genotoxic potential is further highlighted by the presence of low molecular weight DNA fragments in PS-NPs treated cells, and by the relationship between polystyrene nanoplastics and γ-H2AX. Thus, our data provide important insights at a cellular level into the possible risks produced by these nanoparticles and recommend further deeper research studies to address the impacts of nanoplastics on human health.

Fast preparing bioelectrode with conductive bioink for nitrite detection in high sensitivity and stability

Electrochemically active biofilms (EABs) for nitrite detection have high specificity, rapid response, operational simplicity, and extended lifespan advantages. However, their scale production remains challenging due to time-consuming and uniform preparation. In this study, a novel approach was proposed to fast fabricate an EAB biosensor with a synthetic biofilm electrode for nitrite detection. The biofilm electrode was prepared by coating bioinks with varying conductive materials onto the surface of the graphite sheets, showing short incubation time and good reproducibility. Incorporating conductive materials into the bioinks remarkably enhanced the maximum voltage of the first cycle of bioelectrode incubation, with an increase of up to 633% for carbon nanofibers. The nitrite reduction current was amplified by a factor of 2.97, due to the enhancement of extracellular electron transfer (EET). The developed nitrite biosensor exhibited a detection range of 0.1–15 mg NO2--N L−1, with a high sensitivity of 610.8 μA mM−1 cm−2, and a stabilization operation time of at least 280 cycles. This study not only provided valuable insights into conductive materials for synthetic biofilms but also presented a practical approach for the rapid preparation, scale production, and optimization of highly sensitive and stable EAB sensors.

3-Benzylmenadiones and their Heteroaromatic Analogues Target the Apicoplast of Apicomplexa Parasites: Synthesis and Bioimaging Studies.

The apicoplast is an essential organelle for the viability of apicomplexan parasites Plasmodium falciparum or Toxoplasma gondii, which has been proposed as a suitable drug target for the development of new antiplasmodial drug-candidates. Plasmodione, an antimalarial redox-active lead drug is active at low nM concentrations on several blood stages of Plasmodiumsuch as early rings and gametocytes. Nevertheless, its precise biological targets remain unknown. Here, we described the synthesis and the evaluation of new heteroaromatic analogues of plasmodione, active on asexual blood P. falciparum stages and T. gondii tachyzoites. Using a bioimaging-based analysis, we followed the morphological alterations of T. gondii tachyzoites and revealed a specific loss of the apicoplast upon drug treatment. Lipidomic and fluxomic analyses determined that drug treatment severely impacts apicoplast-hosted FASII activity in T. gondii tachyzoites, further supporting that the apicoplast is a primary target of plasmodione analogues. To follow the drug localization, "clickable" analogues of plasmodione were designed as tools for fluorescence imaging through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Short-time incubation of two probes with P. falciparum trophozoites and T. gondii tachyzoites showed that the clicked products localize within, or in the vicinity of, the apicoplast of both Apicomplexa parasites. In P. falciparum, the fluorescence signal was also associated with the mitochondrion, suggesting that bioactivation and activity of plasmodione and related analogues are potentially associated with these two organelles in malaria parasites.

PSVIII-6 Effect of adding plant extracted saponin (PES) on accumulative gas production kinetics and methane emission of the prairie blend pelleted feed products in ruminant system

Abstract The objectives of this study were to study the effect of adding plant extracted saponin (PES) as phytochemical feed additive on in vitro accumulative gas production kinetics and methane (CH4) emission of blend pelleted feed products in ruminant system. Four levels of PES (0, 1, 2, 3%) were added to canola meal and pea mixtures with two different ratios (CMP1: 50:50; CMP2: 70:30) which were used to make Prairie blend pellet feed products. The accumulative gas production kinetics and CH4 were determined using in vitro systems. Ruminal fluid for in vitro studies was obtained from rumen fistulated lactation dairy cows. The experimental design was a randomized complete block design with the PES level as a fixed effect and in vitro run with fistulated animals as a random effect. The data were analyzed using Mixed Model Procedure of SAS. Polynomial contract was used to determine linear and quadratic relationship. The results showed that adding PES did not significantly affect the asymptotic production (a) with an average of 104.06 ± 12.38 (SD) mL/g DM, the gas production fractional rate (c) with an average of 1.00 ± 0.90 %/h, the average production at one-half of asymptotic production (AP) with an average 11.26 ± 0.87 mL and lag time with an average of 2.08 ± 0.81 h. As to CH4 emission at different incubation times, the result showed that at short incubation times (2 h, 4 h, 12 h), adding PES showed numerically reduced CH4 from 277 to 1.08 mL/g (at 2 h) and 4.39 to 1.59 mL/g (at 4 h). With increasing PES level, CH4 was significantly linearly reduced (P = 0.019) from 8.31 to 6.30 mL/g at 12h incubation time. At long incubation (24 h), adding PES did not have significant impact on CH4. In conclusion, adding PES at the studied levels to Prairie blend pellet feed products did not significantly affect accumulative gas production kinetics but it had a high potential to decrease CH4 emission at short incubation times.

Magnetic iron-based nanoparticles encapsulated in graphene/reduced graphene oxide: Synthesis, functionalization and cytotoxicity tests

Nanomaterials for suitable particle sizes, shapes, surface properties, biocompatibility, magnetic properties, and chemical stability are candidates for biomedical applications. Among these nanomaterials, iron-based ones are highly interested in their morphological and magnetic properties for potential utilizations in biomedicine. However, iron-based nanoparticles lose their chemical stability in body fluids because of their oxide formations and transformations. Their use in biomedical applications, especially in imaging, may be less effective if they are oxidized and have lower magnetization values. Thus, the idea of coating them with a protective layer has recently emerged to prevent magnetic nanoparticles from degrading in human fluids and losing their magnetic properties. However, the biological effects of these coated nanoparticles on human cells are poorly understood. In this paper, the synthesis of multilayer graphene (MLG) encapsulated iron-based nanoparticles was investigated by solvothermal and chemical vapor deposition (CVD) methods followed by purification. Subsequently, their surface modification was conducted with pyrene end-functional POEGMA obtained by atom transfer radical polymerization (ATRP). Cytotoxicities of synthesized nanoparticles were evaluated in MCF7 cell lines, which is a commonly used model for breast cancer research. We also compare the results with those obtained from bare iron oxide nanoparticles (IONPs) and iron oxides that were embedded in reduced graphene oxide (rGO) or partially coated with it. We aim to evaluate the safety and efficiency of these nanoparticles and increase their chemical stability as a multifunctional nano platform for cancer diagnosis and treatment. Characterization techniques such as XRD, XPS, SEM, TEM, DTA/TG, DLS, zeta potential, BET, NMR, FTIR, and VSM were performed on the nanoparticles. Cytotoxicity assessments on MCF-7 cell lines indicated the potential of these graphene-based magnetic nanoparticles for biomedical applications, particularly drug delivery, due to their small size, soft ferromagnetic properties, high chemical stability, and cytocompatibility at concentrations below 500 μg/mL over short incubation times.

499 Effect of adding plant extracted hydrolysable tanning and saponin combination to prairie blend pelleted feed products on accumulative gas production kinetics and methane emission in ruminant systems

Abstract The objectives of this study were to study the effect of adding plant extracted hydrolysable tanning (HT) and saponin (S) combination as a combined phytochemical feed additive (HTS) on in vitro accumulative gas production kinetics and methane (CH4) emission of blend pelleted feed products in ruminant systems. Four combination levels [0 (control), 1.5 (1% HT + 0.5% S), 3 (2% HT + 1%S), 4.5 (3% HT + 1.5% S)] were added to canola meal and pea mixtures with two different ratios (CMP1: 50:50; CMP2: 70:30) which were used to make Prairie blend pellet feed products. The accumulative gas production kinetics and CH4 were determined using in vitro systems. The ruminal fluid was obtained from rumen fistulated lactation dairy cows. The experimental design was a randomized complete block design with the HTS level as a fixed effect and in vitro run with fistulated animals as a random effect. The data were analyzed using SAS Mixed Procedure. Polynomial contract was used to determine linear and quadratic relationship. The results showed that adding HTS level linearly reduced asymptotic production a) from 110.5 to 82.7 mL/g DM (P = 0.043) and average production at one-half of asymptotic production (AP) from 11.9 to 9.5 mL (P = 0.069) without significantly affecting gas production fractional rate (c, %/h) with an average of 0.58 ± 0.49 %/h and lag time with an average of 1.95 ± 0.50 h. As to CH4 emission at different incubation times, the result showed that at short incubation times (2 h, 4 h, 12 h), adding HTS had numerically reduced methane emission (CH4) from 2.77 to 0.98 mL/g (at 2 h) and 4.39 to 1.38 mL/g (at 4 h). with increasing HTS level, CH4 was significantly quadratically reduced (P = 0.028) at 12 h incubation time from 8.31 to 5.76 mL/g. After long incubation (24 h), adding HTS did not have significant impact on CH4. In conclusion, adding HTS to prairie blend pellet feed products significantly affected gas production kinetics (the asymptotic production and average production at one-half of asymptotic production). Adding HTS had an increased potential to decrease CH4 emission at short incubation times.

Electro-responsive Molecularly Imprinted Polymers (E-MIPs) for Rapid Detection of Progesterone in Human Serum Samples.

Rapid and reagent-free detection of progesterone (P4) is crucial in point-of-care (POC) measurement due to its important role in the human endocrine and central nervous system. Currently available technologies for P4 detection are often not rapid or require reagents, limiting their use in POC settings. In this work, a self-signaling electrochemical sensing platform for rapid detection of P4 is developed by using electroactive molecularly imprinted polymers (E-MIPs). E-MIPs possess the ability to transduce the molecular recognition event into a measurable electrochemical signal. The E-MIPs are prepared by bulk copolymerization of acrylic acid (AA) and ferrocenylmethyl methacrylate (FcMA) in the presence of P4 followed by its removal from the polymer matrix. By incorporation of ferrocene moieties, the MIP gains intrinsic electroactivity, enabling label-free and direct electrochemical detection of P4 levels. Electrochemical techniques, including cyclic voltammetry and electrochemical impedance spectroscopy, were employed to elucidate the electron transport reaction responsible for the MIP's intrinsic electroactivity. Additionally, the predetermined complementary binding sites confined to the small working area of the screen-printed carbon electrode (SPCE) within the E-MIP matrix facilitate rapid P4 binding, achieving completion within 120 s. The developed E-MIP sensing platform was used for the rapid detection of P4 levels in human serum samples. The short incubation times reported for the E-MIP sensing platform would be beneficial in minimizing the nonspecific binding in the real-world samples. We believe that the proposed E-MIP sensing platform can be applied to clinical samples for detecting fluctuations in P4 levels.

Short-Term Exposure to Foodborne Xenoestrogens Affects Breast Cancer Cell Morphology and Motility Relevant for Metastatic Behavior In Vitro.

Breast cancer is highly susceptible to metastasis formation. During the time of disease progression, tumor pathophysiology can be impacted by endogenous factors, like hormonal status, as well as by environmental exposures, such as those related to diet and lifestyle. New lines of evidence point toward a potential role for foodborne endocrine disruptive chemicals in this respect; however, mechanistic understanding remains limited. At the molecular level, crucial steps toward metastasis formation include cell structural changes, alteration of adhesion, and reorganization of cytoskeletal proteins involved in motility. Hence, this study investigates the potential of dietary xenoestrogens to impact selected aspects of breast cancer cell mechanotransduction. Taking the onset of the metastatic cascade as a model, experiments focused on cell-matrix adhesion, single-cell migration, and adaptation of cell morphology. Dietary mycoestrogens alternariol (AOH, 1 μM) and α-zearalenol (α-ZEL, 10 nM), soy isoflavone genistein (GEN, 1 μM), and food packaging plasticizer bisphenol A (BPA, 10 nM) were applied as single compounds or in mixtures. Pursuing the hypothesis that endocrine active molecules could affect cell functions beyond the estrogen receptor-dependent cascade, experiments were performed comparing the MCF-7 cell line to the triple negative breast cancer cells MDA MB-231. Indeed, the four compounds functionally affected the motility and the adhesion of both cell types. These responses were coherent with rearrangements of the actin cytoskeleton and with the modulation of the expression of integrin β1 and cathepsin D. Mechanistically, molecular dynamics simulations confirmed a potential interaction with fragments of the α1 and β1 integrin subunits. In sum, dietary xenoestrogens proved effective in modifying the motility and adhesion of breast cancer cells, as predictive end points for metastatic behavior in vitro. These effects were measurable after short incubation times (1 or 8 h) and contribute to shed novel light on the activity of compounds with hormonal mimicry potential in breast cancer progression.

Microbial transglutaminase in cashew‐based vegan cheese: an innovative approach in achieving ideal texture and meltability

SummaryThe aim of this study was to investigate the effects of microbial transglutaminase (MTGase) on the textural properties and meltability of vegan cheese formulated using cashew paste, tapioca starch, coconut oil, and pea protein. The parameters were evaluated at various MTGase concentrations (0.1, 0.25, 0.5, 0.75, and 1%) and different incubation times (30, 60, and 90 min). A MTGase concentration of 0.25% resulted in higher meltability Index of 3.02 ± 0.14 and acceptable hardness of 3479.44 ± 14.49 g at 30 min of incubation time. However, prolonged incubation times were found to adversely affect the texture and meltability of the cheese. A similar adverse effect on texture was observed at an MTGase concentration of 0.1% at 60 and 90 min of incubation. The results revealed that lower MTGase concentrations with longer incubation times produced similar effects as higher MTGase concentrations with shorter incubation times. However, at a lower enzyme concentration of 0.1%, meltability was significantly higher (3.24 ± 0.33, P < 0.05) at 60 min of incubation compared to 90 min. The findings suggest that vegan cheese with improved texture, meltability, and sensory properties can be developed by combining cashew, tapioca starch, coconut oil, and pea protein in a ratio of 50:25:20:10, with 1% each of carrageenan and agar, and 60% water, and incubating the mixture with 0.25% MTGase for 30 min at 40 °C.

Quantifying non-transferrin-bound iron (NTBI) in human plasma: incorporating BODIPY-pyridylhydrazone (BODIPY-PH) within a thin green film linked to a portable fluorescence-based device.

Free iron in human serum or non-transferrin-bound iron (NTBI) can generate free radicals and lead to oxidative damage. Moreover, it is highly toxic to various tissues and a vital biomarker related to the iron-loading status of thalassemia and Alzheimer's patients. In NTBI in healthy individuals, NTBI levels are typically less than 1µM; current NTBI analysis usually requires advanced instrumentation and many-step sample pretreatment. To address this issue, we employed our invented BODIPY derivative, BODIPY-PH, as a fluorescence probe and trapped it onto the microcentrifuge tube lid using tapioca starch. The fluorescence intensity of BODIPY-PH increased with increasing NTBI concentration (turn-on). The developed portable reaction chamber facilitates rapid analysis (∼5min) using small sample volumes (10 μL sample in a total volume of 600 μL). Under optimum conditions, using the sample-developed portable fluorescence device and fluorescence spectrometer, we achieved impressive limits of detection (LOD) of 0.003 and 0.0015μM, respectively. Furthermore, the developed sensors show relatively high selectivity toward Fe3+ over other metal ions and biomolecules (i.e., Fe2+, Cr3+, Cu2+, and glucose). The sensor performance in serum samples of thalassemia patients exhibited no significant difference compared to the labeled value (obtained from standard methods). Overall, the developed fluorescence sensor is suitable for determining NTBI and offers high sensitivity, high selectivity, and a short incubation time (5min). Moreover, the method requires a limited number of reagents, is simple to use, and uses low-cost equipment to determine NTBI in human serum samples.

Kinetics of Riboflavin Production by Hyphopichia wangnamkhiaoensis under Varying Nutritional Conditions.

Riboflavin, an essential vitamin for humans, is extensively used in various industries, with its global demand being met through fermentative processes. Hyphopichia wangnamkhiaoensis is a novel dimorphic yeast species capable of producing riboflavin. However, the nutritional factors affecting riboflavin production in this yeast species remain unknown. Therefore, we conducted a kinetic study on the effects of various nutritional factors-carbon and energy sources, nitrogen sources, vitamins, and amino acids-on batch riboflavin production by H. wangnamkhiaoensis. Batch experiments were performed in a bubble column bioreactor to evaluate cell growth, substrate consumption, and riboflavin production. The highest riboflavin production was obtained when the yeast growth medium was supplemented with glucose, ammonium sulfate, biotin, and glycine. Using these chemical components, along with the mineral salts from Castañeda-Agullo's culture medium, we formulated a novel, low-cost, and effective culture medium (the RGE medium) for riboflavin production by H. wangnamkhiaoensis. This medium resulted in the highest levels of riboflavin production and volumetric productivity, reaching 16.68 mg/L and 0.713 mg/L·h, respectively, within 21 h of incubation. These findings suggest that H. wangnamkhiaoensis, with its shorter incubation time, could improve the efficiency and cost-effectiveness of industrial riboflavin production, paving the way for more sustainable production methods.

Improvement of bioanalytical parameters through automation: suitability of a hand-like robotic system.

Commercial automation systems for small- and medium-sized laboratories, including research environments, are often complex to use. For liquid handling systems (LHS), development is required not only for the robot's movements but also for adapting the bioanalytical method to the automated system. This study investigates whether a more human-like automation strategy-using a robotic system (RS)-is more suitable for research laboratories than a professional automation approach utilizing a commercial automated LHS. We conducted a series of measurements for protein determination using a Bradford assay manually, with a fully automated LHS, and with our human-like RS. Although the hand-like RS approach requires more than twice the time of the LHS, it achieved the best standard deviation in this setup (RS = 0.5, manual = 0.71, LHS = 0.86). Due to the low limit of detection (LOD) and limit of quantification (LOQ), most protein samples could be quantified with the RS (samples below LOQ = 9.7%, LOD = 0.23; LOQ = 0.25) compared to manual (samples below LOQ = 28.8%, LOD = 0.24; LOQ = 0.26) and the LHS (samples below LOQ = 36.1%, LOD = 0.27; LOQ = 0.31). In another time-dependent enzymatic assay test, the RS achieved results comparable to the manual method and the LHS, although the required time could be a constraint for short incubation times. Our results demonstrate that a more hand-like automation system closely models the manual process, leading easier to accurate bioanalytical results. We conclude that such a system could be more suitable for typical research environments than a complex LHS.

Strukturaufklärung einer metagenomischen Urethanase und Verbesserung des Hydrolyseprofils durch Protein Engineering

AbstractWährend Kunststoffe wie Polyethylenterephthalat (PET) bereits effizient durch die Aktivität von Hydrolasen abgebaut werden können, sind andere synthetische Polymere wie Polyurethane (PUs) und Polyamide (PAs) weitgehend resistent gegenüber einem biologischen Abbau. In dieser Studie lösten wir die erste Kristallstruktur der metagenomischen Urethanase UMG‐SP‐1, identifizierten hochflexible Loopregionen, die Reste des aktiven Zentrums enthalten, und untersuchten insgesamt 20 potenzielle Hotspots mittels Sättigungsmutagenese. Die durch Protein Engineering erzeugten Einzelmutanten wiesen eine fast 3‐ bzw. 8‐fach verbesserte Aktivität gegenüber hochstabilen N‐Arylurethan‐ und Amidbindungen auf. Darüber hinaus konnte die Freisetzung der entsprechenden Monomere aus einem thermoplastischen Polyester‐PU und einem PA (Nylon 6) durch die Aktivität einer einzigen, metagenomischen Urethanase nach kurzer Inkubationszeit nachgewiesen werden. Dadurch konnte das Hydrolyseprofil von UMG‐SP‐1 über die bekannten niedermolekularen Carbamate hinaus erweitert werden und erschließt so neue Möglichkeiten für den enzymatischen Abbau und das Recycling von Kunststoffen und Plastikabfällen. Damit unterstützt diese Studie Bemühungen um die Verbesserung einer Kreislaufwirtschaft für synthetische Polymere.

Structural Elucidation of a Metagenomic Urethanase and Its Engineering Towards Enhanced Hydrolysis Profiles.

While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG-SP-1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site-saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3- and 8-fold improved activity against highly stable N-aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester-PU and a PA (nylon 6) by the activity of a single, metagenome-derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG-SP-1 beyond the reported low-molecular weight carbamates. Together, these findings promise advanced strategies for the bio-based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers.

The effects of queen mandibular pheromone on Nosema (Vairimorpha) ceranae infections in caged honey bees

Honey bees utilize queen mandibular pheromone (QMP) for maintaining social hierarchy and colony development. In controlled cage studies, synthetic QMP is often introduced to mimic natural conditions. However, questions have arisen about the effects of QMP on nosema disease studies. This short report identifies significant early-stage suppression effects of QMP on Nosema (Vairimorpha) ceranae infections. QMP was found to significantly lower infection rates below the reported infectious dose for 50 % infectivity (ID50) and to slow disease development in a dose-independent manner. These effects diminished at doses exceeding ID100. We recommend that studies investigating treatment effects using caged bees avoid QMP to ensure unambiguous results. Additionally, employing multiple infectious doses with shorter incubation times would be useful for evaluating other treatments that may have subtle effects. Furthermore, our findings support previous field studies suggesting that queen replacement reduces nosema disease at levels similar to treatment with fumagillin.

Performance of Flow Cytometry-Based Rapid Assay in Detection of Carbapenemase-Producing Enterobacterales.

Carbapenemase-producing Enterobacterales are increasingly being recognized in nosocomial infections. The performance of a flow cytometry-based rapid assay for their detection and differentiation was evaluated. This is a disruptive phenotypic technology, phenotypic and growth-independent, that searches for the lesions produced by drugs acting on cells after a short incubation time. Overall, 180 Gram-negative bacteria were studied, and results were compared with those obtained molecularly by PCR and phenotypically by 'KPC, MBL and OXA-48 Confirm Kit'. This phenotypic method was used as reference for comparison purposes. Susceptibility to carbapenems (imipenem, meropenem, and ertapenem) was determined by standard broth microdilution. Overall, 112 isolates (62.2%) were carbapenemase producers, 41 KPCs, 36 MβLs, and 31 OXA-48, and 4 strains were KPC + MβL co-producers. Sixty-eight isolates were carbapenemase-negative. The percentage of agreement, sensitivity, and specificity were calculated according to ISO 20776-2:2021. The FASTinov assay showed 97.7% agreement with the reference method for carbapenemase detection. Discrepant flow cytometry results were obtained in four isolates compared with both reference and PCR results. The sensitivity and specificity of this new technology were 95.3% and 98.5%, respectively, for KPCs, 97.6% and 99.3% for MβLs, and 96.9% and 98% for OXA-48 detection. In conclusion, we describe a rapid flow cytometry assay with high accuracy for carbapenemase detection and the differentiation of various carbapenemases, which should impact clinical microbiology laboratories and patient management.

Facile synthesis of intra-nanogap enhanced Raman tags with different shapes.

Hot spot engineering in plasmonic nanostructures plays a significant role in surface enhanced Raman scattering for bioanalysis and cell imaging. However, creating stable, reproducible, and strong SERS signals remains challenging due to the potential interference from surrounding chemicals and locating SERS-active analytes into hot-spot regions. Herein, we developed a straightforward approach to synthesize intra-gap nanoparticles encapsulating 4-nitrobenzenethiol (4-NBT) as a reporter molecule within these gaps to avoid outside interference. We made three kinds of intra-gap nanoparticles using nanorods, bipyramids, and nanospheres as cores, in which the nanorod based intra-gap nanoparticles exhibit the highest SERS activity. The advantage of our method is the ease of preparation of high-yield and stable intra-gap nanoparticles characterized by a short incubation time (10 mins) with 4-NBT and quick synthesis without requiring an additional step to centrifuge for the purification of core nanoparticles. The intense localized field in the synthesized hot spots of these plasmonic gap nanostructures holds great promise as a SERS substrate for a broad range of quantitative optical applications.

Copolymer-Coated Gold Nanoparticles: Enhanced Stability and Customizable Functionalization for Biological Assays.

Gold nanoparticles (AuNPs) play a vital role in biotechnology, medicine, and diagnostics due to their unique optical properties. Their conjugation with antibodies, antigens, proteins, or nucleic acids enables precise targeting and enhances biosensing capabilities. Functionalized AuNPs, however, may experience reduced stability, leading to aggregation or loss of functionality, especially in complex biological environments. Additionally, they can show non-specific binding to unintended targets, impairing assay specificity. Within this work, citrate-stabilized and silica-coated AuNPs (GNPs and SiGNPs, respectively) have been coated using N,N-dimethylacrylamide-based copolymers to increase their stability and enable their functionalization with biomolecules. AuNP stability after modification has been assessed by a combination of techniques including spectrophotometric characterization, nanoparticle tracking analysis, transmission electron microscopy and functional microarray tests. Two different copolymers were identified to provide a stable coating of AuNPs while enabling further modification through click chemistry reactions, due to the presence of azide groups in the polymers. Following this experimental design, AuNPs decorated with ssDNA and streptavidin were synthesized and successfully used in a biological assay. In conclusion, a functionalization scheme for AuNPs has been developed that offers ease of modification, often requiring single steps and short incubation time. The obtained functionalized AuNPs offer considerable flexibility, as the functionalization protocol can be personalized to match requirements of multiple assays.