Bioanalysis Research Articles

Universal smartphone-assisted label-free CRISPR/Cas12a-DNAzyme chemiluminescence biosensing platform for on-site detection of nucleic acid and non-nucleic acid targets

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) system enables sensitive and specific detection of biomolecules, thanks to its programmability, high fidelity, and powerful signal amplification capabilities. Herein, a universal smartphone-assisted label-free G-quadruplex (G4) DNAzyme-based chemiluminescence CRISPR/Cas12a biosensing platform (G4CLCas) is firstly described that achieves on-site, ultrasensitive visual detection of nucleic acid and non-nucleic acid targets. The G4CLCas-based sensing platform relies on Cas12a trans-cleavage activation that triggers the cleavage of the G4 DNAzyme, resulting in chemiluminescence signals off/on compared to that of the control. Chemiluminescence signals are captured as images that are quantitatively analyzed and visualized using a smartphone-assisted imaging cartridge. Under optimal conditions, G4CLCas achieves a low limit of detection (LOD) of 8.6 aM (∼5.2 copies/μL) for monkeypox virus (MPXV) DNA within the linear concentration range of 10–300 aM and can accurately quantify viral DNA in spiked samples. G4CLCas can also detect non-nucleic acid targets, whereby it achieves a low LOD value of 84.3 nM for adenosine triphosphate (ATP) within the linear concentration range of 2–2000 μM. Here, a label-free, portable, on-site CRISPR/Cas12a chemiluminescence biosensing platform based on the G4 DNAzyme substrates is proposed with potential applications in clinical detection and bioanalytical chemistry research.

A pilot study on non-invasive in situ detection of phytochemicals and plant endogenous status using fiber optic infrared spectroscopy

Traditional methods for assessing plant health often lack the necessary attributes for continuous and non-destructive monitoring. In this pilot study, we present a novel technique utilizing a customized fiber optic probe based on attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) with a contact force control unit for non-invasive and continuous plant health monitoring. We also developed a normalized difference mid-infrared reflectance index through statistical analysis of spectral features, enabling differentiation of drought and age conditions in plants. Our research aims to characterize phytochemicals and plant endogenous status optically, addressing the need for improved analytical measurement methods for in situ plant health assessment. The probe configuration was optimized with a triple-loop tip and a 3 N contact force, allowing sensitive measurements while minimizing leaf damage. By combining polycrystalline and chalcogenide fiber probes, a comprehensive wavenumber range analysis (4000–900 cm−1) was achieved. Results revealed significant variations in phytochemical composition among plant species, for example, red spinach with the highest polyphenolic content and green kale with the highest lignin content. Petioles displayed higher lignin and cellulose absorbance values compared to veins. The technique effectively monitored drought stress on potted green bok choy plants in situ, facilitating the quantification of changes in water content, antioxidant activity, lignin, and cellulose levels. This research represents the first demonstration of the potential of fiber optic ATR-FTIR probes for non-invasive and rapid plant health measurements, providing insights into plant health and advancements in quantitative monitoring for indoor farming practices, bioanalytical chemistry, and environmental sciences.

Plasmon-induced immobilization of xanthene chemosensors toward repurposing as SERS nanotags

Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful tool in biophysics, bioanalytical chemistry, and biomedicine due to its unique ability to monitor extracellular chemical activity using fiber optics and multiplexed detection, which are essential for the study of complex biological systems. However, the exceptionally limited choice of SERS nanotags has hindered the practical application of SERS sensing in the fields. Enormous efforts and time are needed to develop new nanotag lines from ab initio for different targeted analytes. This study is inspired by the extensive range of fluorescent chemosensors already in use for various applications and takes a pioneering step toward repurposing them as the SERS nanotags. More specifically, this study explores the potential of plasmon-induced aromatic radical substitution reaction to immobilize xanthene-based chemosensors onto plasmonic nanostructures using a chemical linker without pre-modifying the chemical structures of the chemosensors or compromising the functionality of their binding groups towards specific analytes. Using this proposed method, we immobilize two primary xanthene-based chemosensors and demonstrate their functionality as SERS chemosensors.

Optimizing the Composition of the Substrate Enhances the Performance of Peroxidase-like Nanozymes in Colorimetric Assays: A Case Study of Prussian Blue and 3,3'-Diaminobenzidine.

One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3'-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5-2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2.

Concentrated ionic liquids for proteomics: Caveat emptor!

The use of concentrated ionic liquids (ILs) in the bioanalytical chemistry of proteins is sparse; typically, dilute aqueous IL solutions are used. Concentrated ILs have unique properties that may allow researchers to dissolve previously insoluble protein analytes, to increase the depth and robustness of sample preparation and the analysis of proteins. Previous research using concentrated ILs for this purpose is sparse and there is a need to systematically investigate the structure-activity relationship between the IL structure and its capacity to solubilise proteins. Here, bovine serum albumin was dissolved in various ionic liquids and monitored over time by light microscopy and SDS-PAGE. While qualitative, these measures provide a good estimate of, respectively, the dissolving power of an IL towards the given protein and the retained integrity of the protein. Hydrophilic ILs show the best solubilisation capacity and higher temperatures (in a restricted sense) improve the solubility of the protein. Higher temperatures and longer reaction times reduce the molecular weight of the protein, which could inhibit their applicability in proteomics, unless the conditions are judiciously controlled. Researchers should exercise caution when using concentrated ILs for protein analysis until the full scope and limitations are known, an aspect we are presently investigating.

Metallomics as an Essential Analytical Tool for the Development of Potential Metallodrugs

Metallomics as an Essential Analytical Tool for the Development of Potential Metallodrugs

Lanthanide‐FRET Molecular Beacons for microRNA Biosensing, Logic Operations, and Physical Unclonable Functions

AbstractTime‐resolved or time‐gated (TG) biosensing and bioimaging with luminescent lanthanide probes and Förster resonance energy transfer (FRET) have significantly advanced bioanalytical chemistry. However, the development of lanthanide‐based molecular beacons (MBs) has been rather limited. Here, we designed DNA stem‐loop MB probes against two different microRNAs (miR‐21 and miR‐27b) using Tb and Eu FRET donors and quenching (BHQ2) and fluorescent (Cy3) FRET acceptors. Limits of detection down to 190 pM and duplexed miR‐21/miR‐27b quantification at low nanomolar concentrations with Tb‐BHQ2 and Eu‐BHQ2 TG‐FRET MBs demonstrated the versatility and high analytical performance of lanthanide‐based MBs. The particular donor‐acceptor distances in the Tb‐Cy3 MB resulted in inverted nucleic acid target concentration‐dependent TG PL intensities in short (e. g., 0 to 40 μs) and long (e. g., 0.1 to 2.1 ms) TG detection windows after pulsed excitation. We showed that this specific feature of our TG‐FRET MBs can be adapted to the design of molecular logic devices (NOR, OR, NAND, AND, XNOR, XOR, IMPLEMENT, and INHIBIT). Moreover, the almost unlimited choice of TG detection windows and the distinct spectral features of Tb and Cy3 over a broad visible spectral range could be exploited to devise biophotonic physical unclonable functions for highly secure authentication and identification. Our study manifests the versatility of lanthanides for advanced biophotonic applications.

Environment-sensitive indolenine-based hemisquaraine dyes: Synthesis, molecular structure, and spectral properties

Environment-sensitive fluorescent dyes are widely used in biomedical research, clinical diagnostics, molecular and cellular biology, analytical and bioanalytical chemistry. Many sensing dyes in the series of cyanines, styryls, BODIPY, squaraines, fluoresceins, and rhodamines among others have been developed and employed for these applications. Meanwhile, push-pull hemisquaraine dyes have been only moderately explored as the environment sensors. Herein, we developed a series of hemisquaraine dyes containing methylamino (MeH), dimethylamino (MeMe), phenylamino (PhH), phenylmethylamino (PhMe), and diphenylamino (PhPh) groups. These substituents were found to strongly affect sensitivity of the dyes towards H-bond formation, polarity, pH, viscosity, and the presence of bovine serum albumin (BSA), which aids the development of sensors for particular applications.

Passive Solid Chemosensor as Saliva Point of Need Analysis for Ammonium Determination by Using a Smartphone

Point-of-need analysis is of great interest nowadays. It refers to the timely analysis or detection of a specific parameter or substance at the location or moment it is needed, often with the aim of providing rapid and on-site results for informed decision-making or immediate interventions. This approach has gained interest in various fields but has not been extensively explored in bioanalytical chemistry. In order to contribute in this way, the analysis of ammonium in saliva as a biological fluid is proposed here. For that purpose, a passive solid sensor of 1,2-naphthoquinone-4-sulfonic acid sodium salt (NQS) embedded in polydimethylsiloxane (PDMS) doped with silica nanoparticles and an ionic liquid was proposed. The assay was developed by delivering ammonia from saliva in a confined atmosphere containing the sensor for 20 to 45 min. Measurements were carried out by absorbance from a benchtop diffuse reflectance spectrophotometer and a fiber optic miniaturized portable spectrometer coupled to a smartphone for point-of-need analysis. Another option for this kind of analysis was the use of the color intensity from digitalized images obtained by a smartphone by isolating the intensity in the color planes R (red), G (green), and B (blue). Good figures of merit were obtained for all three types of instruments, bearing in mind the ammonium content in saliva. Results for 30 samples of male and female volunteers (n = 30) demonstrated the usefulness of the assay, values of mg NH4+/mL saliva between 0.02 and 0.27 were found, and no matrix effect was present. Recoveries for spiked samples were around 100% for all methodologies. Selectivity was demonstrated from spectra obtained from benchtop instruments and the fiber optic mini spectrometer. Two applications were applied for directly determining the ammonium concentration in saliva.

Gels in Microscale Electrophoresis.

Gel matrices are fundamental to electrophoresis analyses of biopolymers in microscale channels. Both capillary gel and microchannel gel electrophoresis systems have produced fundamental advances in the scientific community. These analytical techniques remain as foundational tools in bioanalytical chemistry and are indispensable in the field of biotherapeutics. This review summarizes the current state of gels in microscale channels and provides a brief description of electrophoretic transport in gels. In addition to the discussion of traditional polymers, several nontraditional gels are introduced. Advances in gel matrices highlighted include selective polymers modified to contain added functionality as well as thermally responsive gels formed through self-assembly. This review discusses cutting-edge applications to challenging areas of discovery in DNA, RNA, protein, and glycan analyses. Finally, emerging techniques that result in multifunctional assays for real-time biochemical processing in capillary and three-dimensional channels are identified.

THU-199 - The prevelance of hepatitis C virus infection in high-risk patients with high normal alanine aminotransferase (ALT) in Israel: a bio bank analysis

THU-199 - The prevelance of hepatitis C virus infection in high-risk patients with high normal alanine aminotransferase (ALT) in Israel: a bio bank analysis

Bioorthogonal Metabolic Labeling Experiments to Introduce Undergraduate Students to Interdisciplinarity at the Interface of Chemistry and Biology

Chemists have always designed and deployed cross-disciplinary approaches that interface chemistry with other disciplines to proffer solutions to many human problems. We designed a laboratory experiment that implements chemistry concepts in a biological model to prime undergraduate students to appreciate interdisciplinarity as an approach to solving complex problems in contemporary society. The experiments can also be implemented with graduate students transitioning to chemical biology research. The experiment introduces students to classical and emerging chemistry and molecular biology techniques─mammalian cell culture, bioorthogonal metabolic labeling, azide–alkyne cycloaddition reaction (“click” chemistry), microplate photometry, gel electrophoresis, and far-western blot analysis─that are routinely used in research at the interface of chemistry and biology. The students cultured a Chinese hamster ovary cell line (CHO-K1) and metabolically labeled the cellular sialoglycans with azide groups. Next, they used a click chemistry reaction to bioconjugate a fluorogenic molecule to the labeled sialoglycans, enabling the detection of the glycans using microplate photometry or far-western blot analysis. The experiment created a context for a first-hand demonstration of bioorthogonal chemical reaction, a concept novel to most undergraduates with traditional chemistry (organic, inorganic, physical chemistry, and analytical) background. The experience allows the students to apply and connect organic chemistry, bioanalytical chemistry, and molecular biology concepts while learning techniques such as microplate reading, SDS PAGE, and western blot imaging that are indispensable to the growing fields of drug discovery, chemical toxicology, chemical biology, and molecular biology. This experiment is ideal for preparing and motivating students to pursue future research opportunities at the interface of chemistry and biology.

Peptide-derived coordination frameworks for biomimetic and selective separation.

Peptide-derived metal-organic frameworks (PMOFs) have emerged as a class of biomimetic materials with attractive performances in analytical and bioanalytical chemistry. The incorporation of biomolecule peptides gives the frameworks conformational flexibility, guest adaptability, built-in chirality, and molecular recognition ability, which greatly accelerate the applications of PMOFs in enantiomeric separation, affinity separation, and the enrichment of bioactive species from complicated samples. This review focuses on the recent advances in the engineering and applications of PMOFs in selective separation. The unique biomimetic size-, enantio-, and affinity-selective performances for separation are discussed along with the chemical structures and functions of MOFs and peptides. Updates of the applications of PMOFs in adaptive separation of small molecules, chiral separation of drug molecules, and affinity isolation of bioactive species are summarized. Finally, the promising future and remaining challenges of PMOFs for selective separation of complex biosamples are discussed.

Coacervate-Based Plexcitonic Assembly toward Peroxidase-like Activity and Ultrasensitive Glucose Sensing

Inbuilt catalytic centers anchored inside the confined architecture of artificial nanoreactors have gained tremendous attention owing to their vast applicability in various catalytic transformations. However, designing homogeneously distributed catalytic units with exposed surfaces in confined environment is a challenging task. Here, we have utilized quantum dot (QD)-embedded coacervate droplets (QD-Ds) as a confined compartment for the in situ synthesis of gold nanoparticles (Au NPs) without any additional reducing agent. High-resolution transmission electron microscopy images reveal homogeneous distribution of 5.6 ± 0.2 nm-sized Au NPs inside the QD-Ds (Au@QD-Ds). The in situ synthesized Au NPs are found to be stable over a period of 28 days without any agglomeration. Control experiments reveal that the free surface carboxylic acid groups of embedded QDs simultaneously act as reducing and stabilizing agents for Au NPs. Notably, these Au@QD-Ds exhibit superior peroxidase-like activity compared to bulk aqueous Au NPs and Au@QDs under similar experimental conditions. The observed peroxidase-like activity follows the classical Michaelis–Menten model inside the Au@QD-Ds via the fast electron-transfer pathway. The enhanced peroxidase-like activity has been explained by considering confinement, mass action, and the ligand-free surface of embedded Au NPs. The present plexcitonic nanocomposites exhibit excellent recyclability over several consecutive cycles without any compromise in their catalytic activity. Finally, a cascade reaction with glucose oxidase (GOx)-loaded Au@QD-Ds have been utilized for colorimetric detection of glucose with a limit of detection of 272 nM in solution as well as on filter paper. The present work highlights a facile and robust methodology for the fabrication of optically active functional hybrid plexcitonic assemblies and may find importance in various fields including bioanalytical chemistry and optoelectronics.

Ways of Long-Term Survival of Hydrocarbon-Oxidizing Bacteria in a New Biocomposite Material-Silanol-Humate Gel.

Immobilized bacterial cells are presently widely used in the development of bacterial preparations for the bioremediation of contaminated environmental objects. Oil hydrocarbons are among the most abundant pollutants. We have previously described a new biocomposite material containing hydrocarbon-oxidizing bacteria (HOB) embedded in silanol-humate gels (SHG) based on humates and aminopropyltriethoxysilane (APTES); high viable cell titer was maintained in this material for at least 12 months. The goal of the work was to describe the ways of long-term HOB survival in SHG and the relevant morphotypes using the techniques of microbiology, instrumental analytical chemistry and biochemistry, and electron microscopy. Bacteria surviving in SHG were characterized by: (1) capacity for rapid reactivation (growth and hydrocarbon oxidation) in fresh medium; (2) ability to synthesize surface-active compounds, which was not observed in the cultures stored without SHG); (3) elevated stress resistance (ability to grow at high Cu2+ and NaCl concentrations); (4) physiological heterogeneity of the populations, which contained the stationary hypometabolic cells, cystlike anabiotic dormant forms (DF), and ultrasmall cells; (5) occurrence of piles in many cells, which were probably used to exchange genetic material; (6) modification of the phase variants spectrum in the population growing after long-term storage in SHG; and (7) oxidation of ethanol and acetate by HOB populations stored in SHG. The combination of the physiological and cytomorphological properties of the cells surviving in SHG for long periods may indicate a new type of long-term bacterial survival, i.e., in a hypometabolic state.

Mapping flow-focusing microfluidic droplet formation to determine high-throughput droplet generation configurations

Droplet microfluidics enables the generation of monodisperse droplets of desired size using immiscible multiphase flows. These droplets serve as individual reactors in bio (chemical) analysis. In addition to monodispersity, high throughput is also necessary for many applications, especially in synthesizing nano- and microscale materials. Therefore, high-frequency droplet formation in the stable regimes (dripping and squeezing) is of great importance. In this work, the flow-focusing geometry is numerically investigated to determine the geometrical dimensions and flow rate ratio by which high-frequency, high mass flow rate, and monodispersed droplet formation are achievable. 3D numerical simulation has been performed with the commercial software ANSYS Fluent using the VOF method. The lowest and highest error values were 1.5% and 7% in the validation results, respectively, confirming the solver's reliability. The numerical model determined droplets' diameter and mass flow rate, and their generation frequency for a simple geometry that had been experimentally studied. The geometry, then, was changed both in dimension and junction, seeking the best performing configuration. Our results demonstrated that the highest mass flow rate and generation frequency are obtained with the flow rate ratio of 5 between the continuous and dispersed phases. Furthermore, by introducing a modified geometry using a rib, it was revealed that reducing the channel width in the simple geometry leads to much more improvement in the generation frequency and droplet diameter than using the modified geometry. Subsequently, the aspect ratio study showed that the aspect ratio 1 creates the highest production frequency in the simple geometry. Finally, taking into account all the previous findings, a relation is introduced which determines the required channel dimensions when a specific droplet diameter is of interest.

Mitochondria and cytochrome components released into the plasma of severe COVID-19 and ICU acute respiratory distress syndrome patients

IntroductionProteomic analysis of human plasma by LC–ESI–MS/MS has discovered a limited number of new cellular protein biomarkers that may be confirmed by independent biochemical methods. Analysis of COVID-19 plasma has indicated the re-purposing of known biomarkers that might be used as prognostic markers of COVID-19 infection. However, multiple molecular approaches have previously indicated that the SARS-COV2 infection cycle is linked to the biology of mitochondria and that the response to infections may involve the action of heme containing oxidative enzymes.MethodsHuman plasma from COVID-19 and ICU-ARDS was analyzed by classical analytical biochemistry techniques and classical frequency-based statistical approaches to look for prognostic markers of severe COVID-19 lung damage. Plasma proteins from COVID-19 and ICU-ARDS were identified and enumerated versus the controls of normal human plasma (NHP) by LC–ESI–MS/MS. The observation frequency of proteins detected in COVID-19 and ICU-ARDS patients were compared to normal human plasma, alongside random and noise MS/MS spectra controls, using the Chi Square (χ2) distribution.ResultsPCR showed the presence of MT-ND1 DNA in the plasma of COVID-19, ICU-ARDS, as well as normal human plasma. Mitochondrial proteins such as MRPL, L2HGDH, ATP, CYB, CYTB, CYP, NDUF and others, were increased in COVID-19 and ICU-ARDS plasma. The apparent activity of the cytochrome components were tested alongside NHP by dot blotting on PVDF against a purified cytochrome c standard preparation for H2O2 dependent reaction with luminol as measured by enhanced chemiluminescence (ECL) that showed increased activity in COVID-19 and ICU-ARDS patients.DiscussionThe results from PCR, LC–ESI–MS/MS of tryptic peptides, and cytochrome ECL assays confirmed that mitochondrial components were present in the plasma, in agreement with the established central role of the mitochondria in SARS-COV-2 biology. The cytochrome activity assay showed that there was the equivalent of at least nanogram amounts of cytochrome(s) in the plasma sample that should be clearly detectable by LC–ESI–MS/MS. The release of the luminol oxidase activity from cells into plasma forms the basis of a simple and rapid test for the severity of cell damage and lung injury in COVID-19 infection and ICU-ARDS.Graphical

Abstract 2114: Salivary glands shedding RNA into saliva: Challenges and opportunities

Abstract Liquid biopsies hold great promises as non-invasive, reproducible, cost-effective and accurate cancer screening techniques. Saliva is attractive due its simple, virtually risk-free collection. The oral cavity and its surroundings may shed contents to saliva and it is essential to choose specific alterations that can indicate cells of origin. Common confounding contaminants may include bacteria and food debris. This study aims to test different saliva collection and processing protocols to build a path for saliva-based RNA cancer markers, by improving RNA specificity for tissue of origin (here salivary gland) and by increasing the quality of saliva extracted RNA. Saliva collection from 3 healthy subjects was performed with 5 different methodologies: gargle, non-stimulation, stimulation with gum, tabasco or ascorbic acid. These samples were protected with sterile 0.9% Sodium Chloride solution, QIAzol (Qiagen), RNAprotect Cell Reagent (Qiagen) or OrageneRNA (DNA Genotek), or without a protection step. RNA was then extracted with 4 different kits mirVana (ThermoFisher), QIAzol (Qiagen), RNEasy (Qiagen) and TRIzol Reagent (ThermoFisher). Total RNA was converted to cDNA using qScript cDNA (QuantaBio). RNA quality was evaluated on Agilent’s 2100 Bio analyzer. RNA quantity was evaluated with RiboGreen Reagent (Invitrogen) on SpectraMax M2 (Molecular Devices). RNA specificity for salivary gland origin was assessed by qRT-PCR for 2 genes HTN3 and CA6, which are specifically highly expressed in salivary glands, normalized by GAPDH. Quantity assessment showed an increased amount of RNA collected from unstimulated samples when comparing collection methods. For gargle, unstimulated and tabasco groups had higher RNA yields with the pellet fraction, mirVana extraction protocol and Qiazol protection. For all collection methods, we observed the best quality with the whole fraction, RNEasy extraction reagent, except for gargle. RNA protect showed the highest RNA quality, except for tabasco stimulated samples. There was no variation for gene stability considering HTN3 (mean 30.97/SD 1.37) and CA6 (mean 34.97/SD 0.54) CTs. HTN3 amplified earlier and more consistently than CA6 gene. Analyzing by collection method, the whole fraction was the most specific by gargle and unstimulated samples. mirVana was the best performing extraction method shared between all collection protocols, expect for ascorbic acid saliva stimulation. And Qiazol was the best protection methodology. We obtained the best RNA quality from whole saliva fraction using the RNA protect method and RNEasy extraction protocol. Our findings may build the foundation for desperately needed molecular markers for deadly diseases related to the salivary glands such as salivary gland tumors, aiming for early detection and patient surveillance. Our results also support further validation of strategies to assess saliva as a promising liquid biopsy fluid option. Citation Format: Laura Palmieri, Dieila Giomo De Lima, Adrian D. Schubert, Yasmin Ghantous, Fernando T. Zamuner, Piotr T. Wysocki, Mariana Brait. Salivary glands shedding RNA into saliva: Challenges and opportunities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2114.

Post-secretory synthesis of a natural analog of iron-gall ink in the black nectar of Melianthus spp.

The black nectar produced by Melianthus flowers is thought to serve as a visual attractant to bird pollinators, but the chemical identity and synthesis of the black pigment are unknown. A combination of analytical biochemistry, transcriptomics, proteomics, and enzyme assays was used to identify the pigment that gives Melianthus nectar its black color and how it is synthesized. Visual modeling of pollinators was also used to infer a potential function of the black coloration. High concentrations of ellagic acid and iron give the nectar its dark black color, which can be recapitulated through synthetic solutions containing only ellagic acid and iron(III). The nectar also contains a peroxidase that oxidizes gallic acid to form ellagic acid. In vitro reactions containing the nectar peroxidase, gallic acid, hydrogen peroxide, and iron(III) fully recreate the black color of the nectar. Visual modeling indicates that the black color is highly conspicuous to avian pollinators within the context of the flower. Melianthus nectar contains a natural analog of iron-gall ink, which humans have used since at least medieval times. This pigment is derived from an ellagic acid-Fe complex synthesized in the nectar and is likely involved in the attraction of passerine pollinators endemic to southern Africa.

Polyaromatic hydrocarbons (PAHs) in the water environment: A review on toxicity, microbial biodegradation, systematic biological advancements, and environmental fate

Polycyclic aromatic hydrocarbons (PAHs) are considered a major class of organic contaminants or pollutants, which are poisonous, mutagenic, genotoxic, and/or carcinogenic. Due to their ubiquitous occurrence and recalcitrance, PAHs-related pollution possesses significant public health and environmental concerns. Increasing the understanding of PAHs' negative impacts on ecosystems and human health has encouraged more researchers to focus on eliminating these pollutants from the environment. Nutrients available in the aqueous phase, the amount and type of microbes in the culture, and the PAHs' nature and molecular characteristics are the common factors influencing the microbial breakdown of PAHs. In recent decades, microbial community analyses, biochemical pathways, enzyme systems, gene organization, and genetic regulation related to PAH degradation have been intensively researched. Although xenobiotic-degrading microbes have a lot of potential for restoring the damaged ecosystems in a cost-effective and efficient manner, their role and strength to eliminate the refractory PAH compounds using innovative technologies are still to be explored. Recent analytical biochemistry and genetically engineered technologies have aided in improving the effectiveness of PAHs' breakdown by microorganisms, creating and developing advanced bioremediation techniques. Optimizing the key characteristics like the adsorption, bioavailability, and mass transfer of PAH boosts the microorganisms' bioremediation performance, especially in the natural aquatic water bodies. This review's primary goal is to provide an understanding of recent information about how PAHs are degraded and/or transformed in the aquatic environment by halophilic archaea, bacteria, algae, and fungi. Furthermore, the removal mechanisms of PAH in the marine/aquatic environment are discussed in terms of the recent systemic advancements in microbial degradation methodologies. The review outputs would assist in facilitating the development of new insights into PAH bioremediation.