Leonard T. Rael

Sweet proteins trigger sweet taste perception through interactions with the human T1R2/R3 sweet taste receptor. To date, relatively few proteins have been identified as causing sweet taste perception, and the four most studied proteins: monellin, brazzein, thaumatin, and honey truffle active component (HT-AC), have minimal sequence homology or structural similarities aside from positively charged surface sites. Sweet taste perception has also been found to be readily perturbed by minor changes in the protein structure, such as natural isoforms inherent to heterologous expression of the protein, and synthetic amino acid substitutions. This study uses ab initio rigid-body docking to predict the interactions of known sweet proteins and variants with a recently resolved cryo-EM structure of the T1R2/R3 sweet taste receptor, incorporating comparative analyses between apo-, holo-, and a potentially transient conformation of the receptor. HT-AC mediated activation of the sweet taste receptor is confirmed by in vitro cell-based assays, and results from in silico docking of various sweet proteins are used to derive additional insights regarding sweet taste perception. Perturbations of HT-AC due to naturally occurring post-translational modifications and synthetic modifications are evaluated using in vitro and in silico methods to determine robustness of the interaction between T1R2/R3 and sweet proteins with primary focuses on HT-AC.

Honey truffle sweetener (HTS), a 121 amino acid protein is identified as a high-intensity sweetener found naturally occurring in the Hungarian Sweet Truffle Mattirolomyces terfezioides, an edible mushroom used in regional diets. The protein is intensely sweet, but the truffle is difficult to cultivate; therefore, the protein was systematically characterized, and the gene coding for the protein was expressed in a commonly used host yeast Komagataella phaffii. The heterologously expressed protein maintained the structural characteristics and sweet taste of the truffle. Preliminary safety evaluations for use as a food ingredient were performed on the protein including digestibility and in silico approaches for predicting the allergenicity and toxicity of the protein. HTS is predicted to be nonallergenic, nontoxic, and readily digestible. This protein is readily produced by precision fermentation of the host yeast, making it a potential replacement for both added sugars and small molecule high-intensity sweeteners in food.

There are few reliable markers for assessing traumatic brain injury (TBI). Elevated levels of oxidative stress have been observed in TBI patients. We hypothesized that oxidation‐reduction potential (ORP) could be a potent biomarker in TBI. Two types of ORP were measured in patient plasma samples: the static state of oxidative stress (sORP) and capacity for induced oxidative stress (icORP). Differences in ORP values as a function of time after injury, severity, and hospital discharge were compared using ANOVAs with significance at p ≤ 0.05. Logit regression analyses were used to predict acute outcome comparing ORP, Injury Severity Score (ISS), Abbreviated Injury Scale (AIS), and Glasgow Coma Scale (GCS). Antioxidant capacity (icORP) on day 4 was prognostic for acute outcomes (p < 0.05). An odds ratio of 4.08 was associated with poor acute outcome when icORP > 7.25 μC. IcORP was a better predictor than ISS, AIS, or GCS scores. sORP increased in those with the highest ISS values (p < 0.05). Based on these findings ORP is useful biomarker for severity and acute outcome in TBI patients. Changes in ORP values on day 4 after injury were the most prognostic, suggesting that patients’ response to brain injury over time is a factor that determines outcome.

The barriers to using plasma redox potential as a measure of oxidative stress in patients are the use of specialized equipment, necessity of sterilization between tests, and inconsistent electrode surfaces and cell geometry. These problems are resolved using disposable test strips with built in electrochemical cells. Measuring the electrode potential at a small current yields a stable value that is ∼50 mV higher for isolated traumatic brain injury (ITBI) patients than for healthy individuals. Even with natural variations within blood chemistry, this simple method may be clinically useful for assessing the degree of oxidative stress and injury severity in ITBI patients.