Solvent-mediated browning of proteins and amino acids.

Proteins or amino acids are subjected to the Maillard (browning) reaction in the presence of reducing sugars and catalyzed by high temperature. The reaction occurs in daily-life cooking and in the human body. Although the reaction is ubiquitous and has been known for over 100 years, it still intrigues researchers across disciplines. Here we report an unexpected finding where proteins and amino acids turn brown in a mixture of two common solvents: dimethyl sulfoxide and acetone. The browning reaction proceeds at room temperature, without the presence of any sugars. This novel browning reaction was confirmed by a series of investigation on a protein-based gel, 3 proteins and 20 amino acids. The browning is spontaneous, regardless of whether the protein or amino acid was dissolved or not. The kinetic study reveals a fast reaction with a formation half-life of about 4 h. Notably, the reactivity is bell-shaped, with the maximal catalytic effect occurring at an acetone-to-DMSO volume ratio of 0.1-0.3. Among the 20 amino acids tested, tryptophan, lysine, and proline are the most susceptible amino acids to the solvent-mediated browning reaction.

A nano-platform harnessing synergistic amino acid browning for biomedical applications.

Amino acids show promise as versatile biomolecules for creating a variety of functional biomaterials. Previously, we discovered a novel amino acid reaction, in which a single amino acid can form browning species in a simple solvent mixture comprising DMSO and acetone at room temperature. In the present study, we initially conducted a comprehensive analysis of 190 pairs of binary amino acids (i.e., all the possible pairwise combinations out of 20 amino acids) and identified several surprising combinations that exhibited synergistic browning effects. Particularly, cysteine-lysine and cysteine-arginine pairs exhibited pronounced browning in DMSO/acetone cosolvent solutions. We hypothesize that the coloured species result from the formation of extended, hydrophobic molecules with highly conjugated systems, arising from extensive condensation reactions between amino acids. Subsequently, we aimed at developing a nano-platform based on this newly discovered amino acid reaction. We demonstrate that through a nanoprecipitation process (solvent-shifting), spherical nanoparticles with sizes ranging from 100 to 200 nm can be produced, in the presence of ferric ions added to the water phase. Through systematic optimization and comprehensive characterization, the final product is a zwitterionic, charge-reversible nanoparticle featuring three functional groups on its surface: carboxylates, amines, and thiols. Furthermore, it possesses mild antioxidant activity, making it a new type of nano-antioxidant. Finally, we present preliminary results highlighting the potential of using this new nanomaterial as a delivery system for polynucleotides. In conclusion, the paper introduces a novel class of amino acid-derived nanoparticles with significant promise for future biomedical applications.

Microbiome and Otic Quinolone Levels Following Tympanoplasty Assessed by Gelatin Sponge Analysis.

OBJECTIVE: To determine if absorbable gelatin sponge (AGS) can be used to assess the posttympanoplasty microbiome and otic antibiotic exposure. STUDY DESIGN: Prospective. SETTING: Tertiary hospital. METHODS: Patients undergoing tympanoplasty were prospectively enrolled. Intraoperatively, AGS was applied to the medial ear canal/tympanic membrane (TM) for 1 minute after canal incision, then saved for analysis. Ear canals were packed with AGS at the end of surgery. Otic ofloxacin was administered until the first postoperative visit, when AGS was collected. Microbial presence was assessed by culture. Ofloxacin levels were assessed by liquid-chromatography mass-spectrometry. RESULTS: Fifty-three patients were included. AGS was collected in 92.9% of patients seen within 21 days compared to 70.8% of those seen at 22 to 35 days. At surgery, AGS yielded bacteria and fungi in 81% and 11%, respectively, including Staphylococcus species (55%) and Pseudomonas species (25%). Postoperatively, AGS yielded bacteria in 71% and fungi in 21% at the meatus, (staphylococci 57% and pseudomonas 25%). TM samples yielded bacteria in 69%, fungi in 6%, staphylococci in 53%, and pseudomonas in 19%. Ofloxacin concentration at the meatus was 248 µg/mL (95% confidence interval [CI]: 119-377) and at the TM was 126 µg/mL (95% CI: 58-194). Ofloxacin-resistant colonies were found in 75% of patients. CONCLUSION: Analysis of AGS is a viable technique for noninvasively studying healing metrics posttympanoplasty, including the microbiome and otic antibiotic exposure. Despite exposure to a high concentration of quinolones, the tympanoplasty wound is far from sterile, which may impact healing outcomes.

Preclinical pharmacokinetic studies of villocarine A, an active Uncaria alkaloid.

Villocarine A is a bioactive indole alkaloid isolated from the Uncaria genus. It has demonstrated vasorelaxation activity and potential to protect the central nervous system. To identify the pharmacokinetic properties of villocarine A, a series of in vitro and in vivo studies have been performed. Villocarine A was found to be highly permeable (15.6 ± 1.6*10-6 cm/s) across human colorectal adenocarcinoma cell monolayer with high protein binding (>91%) in both rat and human plasma. Hepatic extraction ratio of villocarine A was 0.1 in pooled rat liver and 0.2 in human liver microsomes and was found stable in rat plasma at 37°C. Due to the high permeability and low rate of metabolism properties, villocarine A was initially considered suitable for preclinical development and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification (linearity: 1-150 ng/ml) in rat plasma was developed and validated for in vivo studies. Essential pharmacokinetic parameters included the volume of distribution and clearance of villocarine A, which were found to be 100.3 ± 15.6 L/kg and 8.2 ± 1.1 L/h/kg, respectively, after intravenous administration in rats. Following oral dosing, villocarine A exhibited rapid absorption as the maximum plasma concentration (53.2 ± 10.4 ng/ml) occurred at 0.3 ± 0.1 h, post-dose. The absolute oral bioavailability of villocarine A was 16.8 ± 0.1%. To our knowledge, this was the first pharmacokinetic study of villocarine A, which demonstrated the essential pharmacokinetic properties of villocarine A: large volume distribution, high clearance, and low oral bioavailability in rats.

Pharmacokinetic Interaction of Kratom and Cannabidiol in Male Rats.

Kratom and cannabidiol products are used to self-treat a variety of conditions, including anxiety and pain, and to elevate mood. Research into the individual pharmacokinetic properties of commercially available kratom and cannabidiol products has been performed, but there are no studies on coadministration of these products. Surveys of individuals with kratom use history indicate that cannabidiol use is one of the strongest predictors of both lifetime and past month kratom use. The purpose of this study was to determine if there are changes in pharmacokinetic properties when commercially available kratom and cannabidiol products are administered concomitantly. It was found that with concomitant administration of cannabidiol, there was a 2.8-fold increase in the exposure of the most abundant kratom alkaloid, mitragynine, and increases in the exposure of other minor alkaloids. The results of this work suggest that with cannabidiol coadministration, the effects of kratom may be both delayed and increased due to a delay in time to reach maximum plasma concentration and higher systemic exposure of the psychoactive alkaloids found in kratom.

Comparative Pharmacokinetics of Commercially Available Cannabidiol Isolate, Broad-Spectrum, and Full-Spectrum Products.

BACKGROUND AND OBJECTIVES: A wide variety of products containing cannabidiol (CBD) are available on the commercial market. One of the most common products, CBD oil, is administered to self-treat a variety of conditions. These oils are available as CBD isolate, broad-spectrum [all terpenes and minor cannabinoids except Δ-9-tetrahydrocannabinol (THC)], or full-spectrum (all terpenes and minor cannabinoids with THC < 0.3% dried weight) products. A systematic pharmacokinetic study was performed to determine whether there are differences in the pharmacokinetic parameters and systemic exposure of CBD after oral dosing as an isolate, broad-spectrum, or full-spectrum product. METHODS: Male and female Sprague Dawley rats were treated with a single, equivalent oral dose of CBD delivered as isolate, broad-spectrum, or full-spectrum product. An additional study using an in-house preparation of CBD isolate plus 0.2% THC was performed. A permeability assay was also conducted to investigate whether the presence of THC alters the intestinal permeability of CBD. RESULTS: There was an increase in the oral bioavailability of CBD (12% and 21% in male and female rats, respectively) when administered as a full-spectrum product compared with the isolate and broad-spectrum products. There was no difference in the bioavailability of CBD between the commercially available full-spectrum formulation (3.1% CBD; containing 0.2% THC plus terpenes and other minor cannabinoids) versus the in-house preparation of CBD full-spectrum (CBD isolate 3.2% plus 0.2% THC isolate). In vitro permeability assays demonstrated that the presence of THC increases permeability of CBD while also decreasing efflux through the gut wall. CONCLUSIONS: The presence of 0.2% THC increased the oral bioavailability of CBD in male and female rats, indicating that full-spectrum products may produce increased effectiveness of CBD due to a greater exposure available systemically.

Development of transgenic watermelon resistant to Cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct.

Watermelon, an important fruit crop worldwide, is prone to attack by several viruses that often results in destructive yield loss. To develop a transgenic watermelon resistant to multiple virus infection, a single chimeric transgene comprising a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein (CP) gene sequences of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) was constructed and transformed into watermelon (cv. Feeling) via Agrobacterium-mediated transformation. Single or multiple transgene copies randomly inserted into various locations in the genome were confirmed by Southern blot analysis. Transgenic watermelon R(0) plants were individually challenged with CMV, CGMMV or WMV, or with a mixture of these three viruses for resistance evaluation. Two lines were identified to exhibit resistance to CMV, CGMMV, WMV individually, and a mixed inoculation of the three viruses. The R(1) progeny of the two resistant R(0) lines showed resistance to CMV and WMV, but not to CGMMV. Low level accumulation of transgene transcripts in resistant plants and small interfering (si) RNAs specific to CMV and WMV were readily detected in the resistant R(1) plants by northern blot analysis, indicating that the resistance was established via RNA-mediated post-transcriptional gene silencing (PTGS). Loss of the CGMMV CP-transgene fragment in R1 progeny might be the reason for the failure to resistant CGMMV infection, as shown by the absence of a hybridization signal and no detectable siRNA specific to CGMMV in Southern and northern blot analyses. In summary, this study demonstrated that fusion of different viral CP gene fragments in transgenic watermelon contributed to multiple virus resistance via PTGS. The construct and resistant watermelon lines developed in this study could be used in a watermelon breeding program for resistance to multiple viruses.

Using Spheroids as Building Blocks Towards 3D Bioprinting of Tumor Microenvironment.

Cancer still ranks as a leading cause of mortality worldwide. Although considerable efforts have been dedicated to anticancer therapeutics, progress is still slow, partially due to the absence of robust prediction models. Multicellular tumor spheroids, as a major three-dimensional (3D) culture model exhibiting features of avascular tumors, gained great popularity in pathophysiological studies and high throughput drug screening. However, limited control over cellular and structural organization is still the key challenge in achieving in vivo like tissue microenvironment. 3D bioprinting has made great strides toward tissue/organ mimicry, due to its outstanding spatial control through combining both cells and materials, scalability, and reproducibility. Prospectively, harnessing the power from both 3D bioprinting and multicellular spheroids would likely generate more faithful tumor models and advance our understanding on the mechanism of tumor progression. In this review, the emerging concept on using spheroids as a building block in 3D bioprinting for tumor modeling is illustrated. We begin by describing the context of the tumor microenvironment, followed by an introduction of various methodologies for tumor spheroid formation, with their specific merits and drawbacks. Thereafter, we present an overview of existing 3D printed tumor models using spheroids as a focus. We provide a compilation of the contemporary literature sources and summarize the overall advancements in technology and possibilities of using spheroids as building blocks in 3D printed tissue modeling, with a particular emphasis on tumor models. Future outlooks about the wonderous advancements of integrated 3D spheroidal printing conclude this review.

Versatile composite hydrogels for drug delivery and beyond.

Hydrogels have extended applications across multiple fields. A novel hydrogel material is often evaluated for its properties and applications in either a wet or dry state, but not both. In this study, we investigated a protein-based, composite hydrogel system in both its wet and dry states. Bovine serum albumin (BSA) was used as the hydrogel base. With the assistance of organosilanes, BSA solutions became hydrogels under facile reaction conditions. In the first part, the wet gel was prepared in situ in a syringe; upon injecting through a needle, the gel retained its structure. The use of the nascent gel system as an injectable drug-delivery vehicle is of particular interest. We therefore developed a microplate platform that allows a "one-plate" study-i.e. gel preparation, payload loading and release-all being performed in a single plate. This one-plate method further enables a systematic study of various controlling parameters for drug release. For example, we can tune the release rate by simply adjusting the phosphate content in the hydrogel formulation. Besides, for low-releasing compounds, the release profile was also tunable while using the one-plate method. In the second part, we further demonstrate the versatility of our composite hydrogels. By simply varying the feed ratio of two organosilanes, (3-mercaptopropyl)methyldimethoxysilane and (3-mercaptopropyl)trimethoxysilane, and phosphate concentrations, dry gels exhibiting various absorption capacities towards water, organic solvents, and oil can be prepared. Further characterizations using SEM and 29Si NMR spectroscopy revealed porous structures and hybrid siloxane bridges within the composite material.

Caspase dependent apoptosis is required for anterior regeneration in Aeolosoma viride and its related gene expressions are regulated by the Wnt signaling pathway.

Although apoptosis has been widely observed during the regenerative process, the mechanisms by which it is regulated and its roles in regeneration remained unclear. In this study, we introduced Aeolosoma viride, a fresh water annelid with an extraordinary regenerative ability as our model organism to study the functions and regulations of apoptotic caspases. Here we showed that major events of apoptosis were detected near the wounded area and showed spatial correlation with the expression patterns of caspase gene namely Avi-caspase X and two apoptosis regulators namely Avi-Bax and Avi-Bcl-xL. Next, we investigated how Avi-caspase X gene expression and apoptosis influence regeneration following head amputation. RNA interference of Avi-caspase X reduced the amounts of apoptotic cells, as well as the percentage of successful regeneration, suggesting a critical role for apoptosis in anterior regeneration of A. viride. In addition, we also discovered that the expression of apoptotic caspases was regulated by the canonical Wnt signaling pathway. Together, our study showed that caspase dependent apoptosis was critical to the anterior regeneration of A. viride, and could be regulated by the canonical Wnt signaling pathway.