Washing Steps Research Articles

Autohydrolysis of sugarcane bagasse with water reuse: Impacts on residues’ composition and enzymatic hydrolysis

This work presents a new sequential approach to the sugarcane bagasse autohydrolysis process in a way that the liquor from the previous reaction was reused in the next one, and the makeup water for the next batch was used to wash the solid fraction before being added to the liquor in the next batch. This approach was suggested first as a way of reducing the water usage in the process, and second as a way of concentrating the liquor in any interesting component, working with the possibility of losing efficiency towards the glucose production through a cellulose loss to the liquor or enzymatic efficiency loss from a higher inhibitor concentration. Two sets of five sequential batches were performed: one washing the solids with the makeup water prior to the enzymatic step (Set 2), and the other one without the washing step (Set 1), looking forward to the effects of the water reuse over the glucose production and the liquor composition. Autohydrolysis pretreatment removed most of the hemicellulose (∼94 %), with liquor recycling improving its removal until the third batch and stabilizing after that. Although the washing step showed little impact on the composition of the solids, it was determinant to the success of the enzymatic hydrolysis, since it was possible to maintain the cellulose to glucose yield around 53% throughout the batches. There was a 64 % reduction in the water used in the sequential reactions without interfering in the glucose production, which indicates that the proposed strategy could be successfully used to reduce costs and environmental impacts associated with the pretreatment of lignocellulosic biomass.

Boron nanoparticles (BNPs) produced by ns-laser ablation in water: synthesis and characterization

Boron nanoparticles (BNPs) are attractive nanomaterials for their employment in many applications, such as neutron detection, boron neutron capture therapy, proton boron capture therapy and nuclear fusion. Depending on the specific application, 10B or 11B isotopes can be used.Nevertheless, there are significant challenges in developing suitable BNPs using both conventional chemical synthesis routes and dry fabrication techniques. In this study we report BNPs directly synthetised in water by pulsed Laser Ablation in Liquid (PLAL). Nanoparticles of elemental boron have been generated by laser ablation of a sintered 10B target in MilliQ water by employing ns laser pulse. The ablation resulted in BNPs and boron target micro-fragments with hydrogen gas and boric acid as by-products. Simple washing steps were used to obtain clean BNPs in water. The BNPs showed a narrow size distribution between 3 and 4 nm and their stability in water was induced by a thin layer of boron oxide surrounds BNPs. The BNPs were fully characterized by the chemical and structural point of view employing several techniques. A discussion on boron chemical reactions during laser ablation in water and after the NPs were released in solution was done.

Multi enzyme production from mixed untreated agricultural residues applied in enzymatic saccharification of lignocellulosic biomass for biofuel

Lignocellulose recalcitrance essentially dictates high cost and low efficiency of its enzymatic saccharification. The complex lignocellulosic structure is a major obstacle to enzymatic saccharification and green ionic liquid pretreatment methods face difficulty of water washing prior enzymatic hydrolysis. Ionic liquid tolerant lignocellulolytic enzymes overcome the inhibition of enzymes during enzymatic saccharification and eliminated water washing step after pretreatment of lignocellulosic biomass. The cell wall composition cellulose, hemicellulose, and lignin, three main wall polymers of various agricultural residues and food waste lignocellulose substrates also affected the enzyme activities secreted by the B. subtilis strain. This study explores the potential of the halophilic, alkalophilic, and ionic liquid (IL)-tolerant strain Bacillus subtilis BC-001 for the simultaneous production of hydrolytic enzymes essential for lignocellulosic bio-refinery processes. BC-001 produced cellulase, amylase, xylanase, pectinase and protease are 70.41, 87.14, 65.50, 122.55 and 66.48 U/mL, respectively under optimized fermentation conditions. Cellulase produced by BC-001 retained more than 80% activity after 72 hours in 20% w/w of different ILs tested and enzymes retained more than 68% activities after 12 h in 50% w/w ILs. Employing such IL-stable cellulase, enzymatic saccharification is conducted without water washing on rice straw (RS) that has been pretreated with various ILs, including 1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium chloride, and choline chloride. This research is novel as the study marks the first instance of multi-hydrolytic enzyme production from a novel alkalophilic, halophilic, thermophilic, and IL-tolerant bacterial strain using a mixture of untreated agricultural residues.

Fabrication and Characterisation of Sustainable 3D-Printed Parts Using Post-Consumer PLA Plastic and Virgin PLA Blends

Sustainable manufacturing practices are becoming increasingly necessary due to the growing concerns regarding climate change and resource scarcity. Consequently, material recycling technologies have gradually become preferred over conventional processes. This study aimed to recycle waste polylactic acid (PLA) from household-disposed cups and lids to create 3D-printed parts and promote sustainable manufacturing practices. To achieve this, the current study utilised virgin and post-consumer PLA (PC-PLA) (sourced from household waste) blends. The PC-PLA wastes were shredded and sorted by size with the aid of a washing step, resulting in a filament with a 1.70 ± 0.5 mm diameter without fragmentation or dissolution. A 50:50 wt.% blend of virgin PLA (vPLA) and PC-PLA was selected as the standard recycling percentage based on previous research and resource conservation goals. The study investigated the impact of three 3D printing parameters (layer height (LH), infill density (I), and nozzle temperature (NT)) on the quality of 3D-printed parts using a three-level L9 Taguchi orthogonal array. The findings revealed that blending PC-PLA with vPLA led to significant improvements in tensile, flexural, and impact strengths by 18.40%, 8%, and 9.15%, respectively, compared to those of recycled PLA (rPLA). This conclusion was supported by the investigation of the fracture surface area, which revealed fractographic features associated with printing parameters, such as plastic deformation and interfilament debonding. An ANOVA analysis revealed a positive influence of a greater layer height and high nozzle temperature on mechanical properties. Subsequently, the optimal printing parameters (LH: 0.3 mm, I: 100%, and NT: 215 °C) were determined using the S/N ratio, and a confirmation test using the optimum printing parameters exhibited a strong correlation with the statistically predicted outcomes. Finally, the study used optimum printing parameters to fabricate 100% PC-PLA 3D-printed parts, demonstrating their potential for low-strength applications. The findings suggest that employing vPLA and PC-PLA blended filaments for fabricating 3D-printed components presents an effective means of promoting plastic recycling within a closed-loop recycling system and achieving a circular economy.

Connaissances et pratiques des étapes préalables à la stérilisation des dispositifs médicaux thermorésistants : étude au centre hospitalo-universitaire Sahloul, Sousse-Tunisie – 2022

The standard process of central sterilization is crucial for the optimal functioning of the operating room. The outcome of this process is closely linked to the steps preceding the steps prior to the sterilization step itself. These steps include pre-disinfection carried out in the operating rooms and other stages, namely washing, drying and packaging, which must be performed in the central sterilization unit. In this context, this study aimed to describe the knowledge of the staff in the operating rooms and the central sterilization unit at Sahloul University Hospital in Sousse (Tunisia) in 2022, regarding the steps prior to the sterilization of reusable thermoresistant medical devices and to describe their practices in terms of compliance with these steps. A descriptive study was conducted from January 2022to June 2022with the aforementioned staff, using a self-administered questionnaire to assess their knowledge of the pre-sterilization steps and a direct observation audit of their practices with regard to these steps. Both measurement instruments were pre-tested. Out of 102self-administered questionnaires (knowledge assessment) distributed to the staff concerned, only 80were returned and correctly filled out, giving a response rate of 78.4%. Participants' responses regarding the order of steps prior to the sterilization were incorrect in 64% of cases. With regard to the evaluation of professional practices, 224observations were made in the study area (practice audit). In 82% of these observations, the pre-disinfection step was confused with the washing step. The use of Betadine brushes and scrubbing pads for device washing was noted in 89.3%, along with the absence of swabbing of the canals and hollow parts in 9.4% of cases and the absence of drying of the canals with compressed air. Mastery of the steps prior to sterilisation of reusable thermoresistant medical devices was insufficient in our institution, suggesting the importance of reinforcing the implementation of the procedure through a continuous training program followed by action plans.

Next Generation Chemiluminescent Probes for Antimalarial Drug Discovery.

Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [3H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark β-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (β-D-galactoside) by a transgenic β-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.

Base de données Enthalpies : évidence et théorie appliquée à la pratique en stérilisation

In order to share their knowledge, sterilization unit managers publish the results of their work in journals or at scientific conferences. The aim of Enthalpies is to list all such work in a single database. The work presented describes the construction of this database and the assessment of its feasibility for the washing step. The first step was to carry out a literature review. Only articles published over 10years (2013-2023) in connection with the ten-year washing stage were included. Then, each publication was categorized, coded, graded and summarized by a pharmacist-internal pair. All this data was collated in an Excel® spreadsheet, and from the data categorization a tree structure was created, enabling the interface between the user and the platform hosting Enthalpies to be understood. Eighty-one publications were identified. These were categorized into 6 themes and 27 sub-themes. A reading grid was drawn up for each publication. Genially®, an online platform, was chosen to host our database. Enthalpies was not designed to issue recommendations for best practice. However, by collating published scientific data, this tool can be used to assist decision-making. It represents an innovative solution for providing a literature review in the field of hospital sterilization.

DETERMINING GOOD DNA QUALITY OF FFPE CRC TISSUES VIA A MACRODISSECTION-DNA EXTRACTION PROTOCOL

A good DNA quality and quantity from a formalin-fixed paraffin-embedded (FFPE) tissue sample is crucial for a better downstream genomic analysis. Previous research has focused on comparing FFPE DNA extraction kits, but the improvement in kit outcome has yet to be studied. This study aimed to make some modifications to a selected Qiagen DNA extraction manual protocol using a manual macrodissected FFPE colorectal cancer (CRC) tissue sample. A variety of DNA extraction protocol-related variables, such as washing steps, tissue sections, and FFPE tissue age have been investigated to determine the DNA quality and quantity of the FFPE tissues. The prechilled absolute ethanol washing step showed the highest DNA concentration with good purity ratios. One, two, or four tissue sections using the washing step were sufficient to obtain adequate DNA concentrations with acceptable DNA purity ratios. 1% or 2% agarose gel electrophoresis with the pre-casting method allowed better visualisation of FFPE DNA. Based on the ideal quality and quantity, we chose a manual macrodissected DNA extraction protocol employing 4 x 10 µm FFPE tissue sections generated in recent years with an optimised prechilled absolute ethanol washing step.

Abstract 7023: Application of exclusive liquid repellency for low input CUT&Tag histone analyses in prostate cancer spheroids

Abstract Introduction/Background: Epigenetic alterations, including histone modifications and DNA methylation, drive treatment resistance and lethal prostate cancer. However, epigenetic assays typically require thousands of cells and are difficult to scale for patient tumor biopsies. While identifying these alterations is possible in cell lines, these analytes are more difficult to follow in clinical settings. Patient-derived organoids have emerged as viable intermediaries to investigate the changing chromatin landscape during the progression toward treatment-emergent prostate cancer. This report seeks to adapt the Cleavage Under Targets and Tagmentation (CUT&Tag) assay for use on a microfluidic technology to eliminate cell loss associated with complex, multi-step epigenetic assays to facilitate analysis of low input prostate cancer spheroids. Methods: The Oil Immersed Lossless Total Analysis System (OIL-TAS) is an exclusive liquid repellency (ELR) platform that passes magnetic bead-bound analytes through immiscible phases to separate and extract analytes of interest while eliminating traditional centrifugation and wash steps that lead to sample loss. This platform enables adaptation of complex molecular assays such as CUT&Tag for lossless sample manipulation. We focus on the analysis of histone modifications in rare cells utilizing antibodies for H3K27 acetylation and methylation. Comparison of LNCaP prostate cancer cell lines harvested from either 2D cell culture or 3D spheroids was performed to evaluate the sensitivity and specificity of this integrated assay. Results: The macroscale CUT&Tag assay has been performed with inputs of 25,000 nuclei from LNCaP spheroids with greater yields than similar inputs from 2D LNCaP cell culture. Yield increases have also been seen when performing the assay with inputs <10,000 nuclei in microscale on the OIL-TAS relative to the macroscale. Utilizing the OIL-TAS platform with spheroid inputs will allow for more sensitive investigation of histone modifications. Conclusions: Integration of 3D, humanized models of tumor growth into the CUT&Tag assay will enable discovery of differential epigenetic regulation within patient-derived organoids or complex co-culture studies. Establishing baseline epigenetic landscapes among these intermediary models will enable a new platform for drug testing. Utilization of the OIL-TAS platform will allow for miniaturization of the assay down to 1,000 cell inputs and enable multiparametric RNA-seq, EM-seq, and CUT&Tag-seq of the same patient samples for longitudinal study. Citation Format: Zachary J. Kauffman, Kevin Koesser, Kyle T. Helzer, Jamie M. Sperger, Marina N. Sharifi, Chao Li, Erika Heninger, Xavier Hazelberg, Cole Gilsdorf, Shuang G. Zhao, Duane S. Juang, David J. Beebe, Joshua M. Lang. Application of exclusive liquid repellency for low input CUT&Tag histone analyses in prostate cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7023.

Abstract 3254: Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines

Abstract mRNA-based therapeutics have shown clinical efficacy, evidenced by the success of the mRNA vaccines for SARS-CoV-2. Personalized mRNA vaccines containing patient-derived tumor mutations are now entering the clinic and have shown promise. Delivery of mRNA-encoded therapeutic proteins (e.g., antibodies) is also under development. mRNA drug substance is typically generated by in vitro transcription (IVT). One byproduct and contaminant of IVT is the formation of double-stranded RNA (dsRNA). dsRNA is highly immunogenic, can induce inflammation and cell death, and inhibit protein translation, including the protein encoded by the therapeutic mRNA. As such, dsRNA must be measured for all IVT products to ensure safety and efficacy. However, existing dot bot and ELISA technology to detect dsRNA is neither sensitive nor quantitative. Herein, we describe the development of novel assays for sensitive and specific quantitation of dsRNA in IVT products and following encapsulation of IVT products in lipid nanoparticles (LNPs). First, NanoBiT® split luciferase technology was applied to detect dsRNA in IVT products. dsRNA binding domains were genetically attached to either SmBiT or LgBiT. In the presence of dsRNA, the SmBiT and LgBiT constructs bind via the dsRNA binding domains, resulting in dimerization and the complementation of functional NanoBiT® luciferase. NanoBiT® then generates light which is proportional to the amount of dsRNA in the sample. The assay detects dsRNA greater than or equal to 30 base pairs and is independent of the size and sequence of the dsRNA contaminant. The assay does not cross-react with single-stranded RNA or DNA and is highly sensitive, with a limit of detection less than 100 pg/ml. As a complementary tool to the biochemical assay, we developed a cell-based bioassay to detect dsRNA encapsulated in LNPs. dsRNA/LNPs are taken up by endocytosis where Toll-like receptor 3 (TLR3) detects dsRNA. Activation of TLR3 initiates a signaling pathway that is detected by a TLR3 pathway-specific promoter driving luciferase expression. This pathway likely represents the most physiologically-relevant readout of dsRNA bioactivity. Both assays are developed in “add-mix-read” format with no wash steps or media transfer, and exhibit robust, sensitive, and repeatable performance for specific detection of dsRNA in mixed solutions. Citation Format: Rich Moravec, Jun Wang, Jim Hartnett, Mei Cong, Jamison Grailer. Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3254.

Abstract 724: MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling

Abstract The development of novel antibody drugs is a lengthy journey. Many factors can lead to failures during clinical development. According to reports, off-target binding of antibodies could also be a significant cause. It has been stated that about 25% of preclinical candidates might have off-target issues. With the emergence of new modalities like CD3 bispecific antibodies, ADCs, and CAR-T therapies, early-stage off-target screening of antibodies has become even more crucial. Although techniques such as immunohistochemistry, ELISA, and flow cytometry (FACS) have been used for off-target testing of antibodies, these methods still lack sensitivity and specificity, and their throughput is limited. To address the need for antibody target deconvolution and receptor identification, we developed a novel membrane protein screening array-AB5000 (MPSA-AB5000). MPSA-AB5000 is a high-throughput cell-based platform for identifying the targets of antibodies and other ligands that bind to membrane proteins. MPSA-AB5000 is an excellent solution for antibody specificity screening, which enables early off-target screening and can de-risk the antibody drug development program. The MPSA-AB5000 makes use of the full coverage of unique human membrane proteins, including receptors, transporters, enzymes, miscellaneous, and others. Each membrane protein is expressed in live cells to ensure native conformation. In addition, the same membrane protein clones with a C-terminal epitope tag were constructed, allowing confirmation of protein expression and cellular location. To provide the highest level of sensitivity, MPSA-AB5000 uses luciferase reporter cell line detection. The wash-free strategy reduces false negatives, and signal amplification makes remarkable discrimination. Compared to similar technologies, we possess significant advantages in the following aspects: a. High sensitivity and sigh throughput: our technology platform offers not only high sensitivity, capable of detecting subtle interactions but also supports high-throughput screening, allowing for the evaluation of a large number of samples in a short timeframe. b. Avoidance of false negative signals: by eliminating washing steps, our method reduces the risk of false negative signals, ensuring reliable detection of non-specific binding. c. Comprehensive validation approaches: We employ various validation methods such as FACS and ELISA to eliminate false positive signals and enhance the accuracy and credibility of our results. All the advantages ensure that MPSA-AB5000 is more rapid, simple, and highly sensitive than traditional assays. Citation Format: dan Li, Junyi Xiong, Jinying Ning, Feng Hao. MPSA-AB5000: A high-throughput membrane proteins screening array for therapeutic biologics specificity profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 724.

Ultra-stable and highly-bright CsPbBr3 perovskite/silica nanocomposites for miRNA detection based on digital single-nanoparticle counting

Single-nanoparticle counting (SNPC) based on fluorescent tag (FT) stands out for its capacity to achieve amplification-free and sensitive detection of biomarkers. The stability and luminescence of FT are important to the sensitivity and reliability of SPNC. In this work, we developed novel perovskite/silica nanocomposites by in-situ nanoconfined growth of CsPbBr3 nanocrystals inside mesoporous structure of silica nanoparticles. PbBr(OH) was formed in an alkaline-assisted reaction triggered by water on the surface of CsPbBr3 nanocrystals. The as-obtained nanocomposites, featuring dual protection from silica matrix and PbBr(OH), exhibited high absolute photoluminescence quantum yield (PLQY) of 86.5% and demonstrated outstanding PL stability confronting with water, heat, ultrasound and UV-irradiation, which is desired by SNPC-based biosensor. Thereafter, these nanocomposites were used to construct an operationally friendly SNPC assay for the amplification-free quantification of cancer-associated miRNA. Quantitative detection of miRNA could be accomplished by directly counting the number of nanocomposites using a flow cytometer in this assay. This strategy did not ask for multiple washing steps and demonstrated specific and sensitive detection of miRNA 21, which exhibited a dynamic range of 1–1000 pM and limit of detection of 79 amol. The employment of highly stable perovskite/silica nanocomposites improved the test reliability and stability of SNPC, revealing the vast potential of perovskites in biosensing.

A GMR enzymatic assay for quantifying nuclease and peptidase activity.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Investigating processing practices and microbiological quality of minimally processed vegetables in Brazil.

Minimally processed vegetables (MPVs) are marketed as convenient and healthy choices for consumers. However, the absence of post-commercialization treatments raises concerns about their microbiological safety. This study investigated the processing practices of 28 Brazilian MPV plants and compared the microbiological quality of these products with fresh counterparts in the city of Sao Paulo, Brazil. Through cluster analysis, the processing plants were categorized into two groups: group 1 (nineteen plants) primarily uses chemical substances in the washing step, while group 2 (nine plants) avoids chemical use but employs similar rinsing practices. Microbiological analysis of 100 samples (49 unprocessed and 51 MPVs) revealed no significant differences in microbial group counts (Enterobacteriaceae, coliforms, and E. coli) between the in natura (unprocessed) and MPV products. However, the prevalence of E. coli was higher in natura vegetables than in MPVs. The results indicated the presence of Salmonella DNA (from either dead or live cells or residual DNA) in 4 samples (3 in natura and 1 MPV) using conventional PCR, suggesting the presence of the pathogen in these samples. Listeria monocytogenes was absent, but Listeria innocua was found in two unprocessed products. The study suggests that certain MPVs have microbial loads similar to unprocessed vegetables, potentially serving as carriers for pathogen transmission. These findings emphasize the importance of understanding practices in Brazilian MPV processing plants, informing the implementation of control measures to improve MPV safety and shelf-life, thusensuring microbiological safety.

Low-temperature and low-concentration sodium hydroxide pretreatment for enhanced enzyme hydrolysis rate from Quercus variabilis Blume

A surface response design was employed to develop a sodium hydroxide (NaOH) pretreatment method for Quercus variabilis Blume using low NaOH concentration at low temperature. Nevertheless, the persistent issues associated with alkaline pretreatment of lignocellulose, namely high-water consumption and wastewater generation, remain prevalent in this pretreatment process. To address these challenges, this study aimed to conduct enzymatic hydrolysis of NaOH-treated Q. variabilis Blume without the intermediary washing steps. The results revealed that, following pretreatment and solid-liquid separation, NaOH-treated Q. variabilis Blume could be directly subjected to cellulase-mediated hydrolysis with pH adjustment, eliminating the need for washing steps. The maximum enzymatic hydrolysis efficiency reached 95.9% under specific conditions (1.2% NaOH, 8.9 °C, 32.1 h). This approach offers a promising avenue to enhance the enzyme hydrolysis rate of NaOH-treated lignocellulose. Notably, the low-temperature and low-concentration NaOH treatment effectively removed a substantial portion of lignin and hemicelluloses, resulting in a higher crystallinity index of the cellulose-rich residue compared to substrates treated solely with steam explosion. The integration of direct pretreatment and alkaline treatment emerges as an environmentally friendly and economically viable method for producing glucose and high-purity lignin. The obtained lignin can be further transformed into high-value products within the biorefinery framework.

Sporopollenin Capsules as Biomimetic Templates for the Synthesis of Hydroxyapatite and β-TCP.

Pollen grains, with their resilient sporopollenin exine and defined morphologies, have been explored as bio-templates for the synthesis of calcium phosphate minerals, particularly hydroxyapatite (HAp) and β-tricalcium phosphate (TCP). Various pollen morphologies from different plant species (black alder, dandelion, lamb's quarters, ragweed, and stargazer lily) were evaluated. Pollen grains underwent acid washing to remove allergenic material and facilitate subsequent calcification. Ragweed and lamb's quarter pollen grains were chosen as templates for calcium phosphate salts deposition due to their distinct morphologies. The calcification process yielded well-defined spherical hollow particles. The washing step, intended to reduce the protein content, did not significantly affect the final product; thus, justifying the removal of this low-yield step from the synthesis process. Characterisation techniques, including X-ray diffraction, scanning electron microscopy, Fourier-transform infrared spectroscopy, and thermal gravimetric analysis, confirmed the successful calcification of pollen-derived materials, revealing that calcified grains were principally composed of calcium deficient HAp. After calcination, biphasic calcium phosphate composed of HAp and TPC was obtained. This study demonstrated the feasibility of using pollen grains as green and sustainable bio-templates for synthesizing biomaterials with controlled morphology, showcasing their potential in biomedical applications such as drug delivery and bone regeneration.

Heat 'n Beat: A Universal High-Throughput End-to-End Proteomics Sample Processing Platform in under an Hour.

Proteomic analysis by mass spectrometry of small (≤2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a nearly universal, rapid, and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centered on a one-tube homogenization and digestion method called Heat 'n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided into separate challenges. The initial sample washing and final peptide cleanup steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin embedded (FFPE). Third, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenization treatment, and reduction, alkylation, and proteolysis were optimized into a single step. The refinements produced near doubled peptide yield when compared to our earlier method ABLE delivered a consistently high digestion efficiency of 85-90%, reported by ProteinPilot, and required only 38 min for core processing in a single tube, with the total processing time being 53-63 min. The robustness of HnB was demonstrated on six organ types, a cell line, and a cancer biopsy. Its suitability for high-throughput applications was demonstrated on a set of 1171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 h, producing highly consistent peptide yield and quality for over 3513 MS runs.

Use of the dummy approach for the synthesis of ion imprinted polymers with Ni(II) or Zn(II) as template ion for the solid-phase extraction of Cu(II).

There is a strong interest in monitoring copper in environmental waters, but its direct analysis suffers from strong matrix interferences. This is why, a sample pretreatment based on solid-phase extraction (SPE) is often used but conventional sorbents usually lack specificity. It is overcome with ion-imprinted polymers (IIPs). This work evaluates for the first time the use of the dummy approach for the synthesis of Cu(II)-targeting IIPs. Two analog ions Ni(II) and Zn(II) were tested as templates and the resulting IIPs were packed in SPE cartridges. The SPE procedure was designed by optimizing a washing step following the sample percolation, to eliminate the interfering ions retained on the IIP by non-specific interactions. To optimize the washing step, solutions at different pH or containing tris(hydroxymethyl)aminomethane as a complexing agent at different concentrations were tested and combined. Zn-IIP appeared more promising than Ni-IIP, showing excellent specificity and a high selectivity. Its retention capacity was determined to be 100µg/g, and different isotherm models were evaluated to fit with the adsorption data. Finally, applications to mineral and sea waters were successfully completed and led to high and repeatable extraction recoveries for Cu(II) (88±1% and 83±3%, respectively).

Influence of Organic Matter from Native Fish on the Antimicrobial Efficacy of Sodium Hypochlorite (NaClO) in Reducing Salmonella spp. Population

Salmonella spp. is not part of the fish microbiota, being introduced through contaminated water or improper handling. In slaughterhouses, five parts per million (ppm) of free chlorine is recommended in the washing step to eliminate this pathogen. However, Salmonella spp. is still detected in fish processing plants due to persistent contamination as a result of chlorine–organic matter interactions that reduce chlorine effectiveness. Therefore, this study aimed to test whether organic matter contained in culture media and fish washes interferes with the efficacy of chlorine at 2 and 5 ppm, and what is the time of action required to inactivate ATCC and wild strains of S. typhimurium. For this, the elimination or survival of these strains was investigated when exposed to chlorinated solution for different durations (0, 5, 30, 60, 90 and 120 min) in culture medium, 0.85% saline solution and fish wash (in natura and sterilized). The results showed that the use of 5 ppm of free chlorine is efficient, even when the chlorine remains in contact with the organic matter (washed from the fish), as it reduced the bacterial population by ~5 log log10 CFU/mL of Salmonella spp., showing that this reduction was due to interactions, as long as the chlorine action time was 30 min.

Photocatalytic Aptamer Chemiluminescent System for a Homogeneous, Reliable, Label-Free, Generic Assay of Small Molecules.

Chemiluminescence is a powerful analytical technique with many advantages, while aptamers are well-known as good molecular recognition units. However, many aptamer-based chemiluminescence assays employed interface sensing, which often needed several immobilization, separation, and washing steps. To minimize the risks of contamination and false-positive, we for the first time proposed a photocatalytic aptamer chemiluminescent system for a homogeneous, label-free, generic assay of small molecules. After binding to a DNA aptamer, thioflavin T has a unique photocatalytic oxidase activity to activate the system's luminol chemiluminescence. Then, the specific binding between the aptamer and target molecules will compete with the above process. Therefore, we can realize the efficient assay of different analytes including estradiol and adenosine. Such a homogeneous chemiluminescent system allowed a direct assay of small molecules with limits of detection in a nM level. Several control tests were carried out to avoid possible false-positive results, which were originated from the interactions between analytes and sensing interfaces previously. This homogeneous chemiluminescent system provides a useful strategy to reliably assay various analytes in the pharmacy or biology field.