Oxidative cyclization reagents reveal tryptophan cation-π interactions.

Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.

Correlation Between Skeletal Muscle Mass and Fat-Free Mass in Infants Born Very Preterm.

Assessing growth quality in preterm infants present challenges, particularly with the use of the standard fat-free mass measurement. We report here a moderate correlation between indirect skeletal muscle mass measurements using the D3-creatine dilution method and fat-free mass measured with air-displacement plethysmography. Skeletal muscle mass could serve as an indicator of growth quality.

A Pilot Graduate Student-Led Near-Peer Mentorship Program for Transfer Students Provides a Supportive Network at an R1 Institution.

The undergraduate transfer process has well-documented challenges, especially for those who identify with groups historically excluded from science, technology, engineering, and mathematics (STEM) programs. Because transfer students gain later access to university networking and research opportunities than first-time-in-college students, transfer students interested in pursuing postbaccalaureate degrees in chemistry have a significantly shortened timeline in which to conduct research, a crucial component in graduate school applications. Mentorship programs have previously been instituted as effective platforms for the transfer of community cultural wealth within large institutions. We report here the design, institution, and assessment of a near-peer mentorship program for transfer students, the Transfer Student Mentorship Program (TSMP). Founded in 2020 by graduate students, the TSMP pairs incoming undergraduate transfer students with current graduate students for personalized mentorship and conducts discussion-based seminars to foster peer relationships. The transfer student participants have access to a fast-tracked networking method during their first transfer semester that can serve as a route for acquiring undergraduate research positions. Program efficacy was assessed via surveys investigating the rates of research participation and sense of belonging of transfer students. We observed that respondents that participated in the program experienced an overall improvement in these measures compared to respondents who did not. Having been entirely designed, instituted, and led by graduate students, we anticipate that this program will be highly tractable to other universities looking for actionable methods to improve their students' persistence in pursuing STEM degrees.

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4.

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. By identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

A Chemiluminescent Probe for HNO Quantification and Real-Time Monitoring in Living Cells.

Azanone (HNO) is a reactive nitrogen species with pronounced biological activity and high therapeutic potential for cardiovascular dysfunction. A critical barrier to understanding the biology of HNO and furthering clinical development is the quantification and real-time monitoring of its delivery in living systems. Herein, we describe the design and synthesis of the first chemiluminescent probe for HNO, HNOCL-1, which can detect HNO generated from concentrations of Angeli's salt as low as 138 nm with high selectivity based on the reaction with a phosphine group to form a self-cleavable azaylide intermediate. We have capitalized on this high sensitivity to develop a generalizable kinetics-based approach, which provides real-time quantitative measurements of HNO concentration at the picomolar level. HNOCL-1 can monitor dynamics of HNO delivery in living cells and tissues, demonstrating the versatility of this method for tracking HNO in living systems.

Early and exclusive enteral nutrition in infants born very preterm.

OBJECTIVE: To characterise the effects of early and exclusive enteral nutrition with either maternal or donor milk in infants born very preterm (280/7-326/7 weeks of gestation). DESIGN: Parallel-group, unmasked randomised controlled trial. SETTING: Regional, tertiary neonatal intensive care unit. PARTICIPANTS: 102 infants born very preterm between 2021 and 2022 (51 in each group). INTERVENTION: Infants randomised to the intervention group received 60-80 mL/kg/day within the first 36 hours after birth. Infants randomised to the control group received 20-30 mL/kg/day (standard trophic feeding volumes). MAIN OUTCOME MEASURES: The primary outcome was the number of full enteral feeding days (>150 mL/kg/day) in the first 28 days after birth. Secondary outcomes included growth and body composition at the end of the first two postnatal weeks, and length of hospitalisation. RESULTS: The mean birth weight was 1477 g (SD: 334). Half of the infants were male, and 44% were black. Early and exclusive enteral nutrition increased the number of full enteral feeding days (+2; 0-2 days; p=0.004), the fat-free mass-for-age z-scores at postnatal day 14 (+0.5; 0.1-1.0; p=0.02) and the length-for-age z-scores at the time of hospital discharge (+0.6; 0.2-1.0; p=0.002). Hospitalisation costs differed between groups (mean difference favouring the intervention group: -$28 754; -$647 to -$56 861; p=0.04). CONCLUSIONS: In infants born very preterm, early and exclusive enteral nutrition increases the number of full enteral feeding days. This feeding practice may also improve fat-free mass accretion, increase length and reduce hospitalisation costs. TRIAL REGISTRATION NUMBER: NCT04337710.

Total Fluid Administration and Weight Loss during the First 2 Weeks in Infants Randomized to Early Enteral Feeding after Extremely Preterm Birth.

BACKGROUND: Randomized trials have not reported the effects of the early progression of feeding volumes on fluid balance and neurodevelopment among infants born extremely preterm (≤28 weeks). METHOD: Fluid, electrolyte, and neurodevelopment data of 60 extremely preterm infants randomly assigned to receive either 1 (early feeding group) or 4 days (late feeding group) of trophic feeding volumes at 20-24 mL/kg/day were analyzed. RESULTS: Infants randomized to the early feeding group received less parenteral fluids, generated lower urine volumes, and had less excessive weight loss during the first 14 days after birth. The 7-point difference in cognitive scores and the 0.5 difference in weight-for-age z-scores favoring the early feeding group did not reach statistical significance. CONCLUSIONS: In extremely preterm infants, early enteral feeding is associated with less total fluid administration and with less excessive weight loss during the first 2 weeks after birth. These short-term effects could have long-lasting benefits.

Early Human Milk Fortification in Infants Born Extremely Preterm: A Randomized Trial.

OBJECTIVES: Enteral nutrition with unfortified human milk during the first 2 postnatal weeks often leads to cumulative protein and energy deficits among preterm infants. Fortified human milk administered soon after birth could increase fat-free mass (FFM) and improve growth in these infants. METHODS: This was a masked, randomized trial. Starting on feeding day 2, extremely preterm infants 28 weeks or younger fed maternal or donor milk were randomized to receive either a diet fortified with a human-based product (intervention group) or a standard, unfortified diet (control group). This practice continued until the feeding day when a standard bovine-based fortifier was ordered. Caregivers were masked. The primary outcome was FFM-for-age z score at 36 weeks of postmenstrual age (PMA). RESULTS: A total of 150 infants were randomized between 2020 and 2022. The mean birth weight was 795±250 g, and the median gestational age was 26 weeks. Eleven infants died during the observation period. The primary outcome was assessed in 105 infants (70%). FFM-for-age z scores did not differ between groups. Length gain velocities from birth to 36 weeks PMA were higher in the intervention group. Declines in head circumference-for-age z score from birth to 36 weeks' PMA were less pronounced in the intervention group. CONCLUSIONS: In infants born extremely preterm, human milk diets fortified soon after birth do not increase FFM accretion at 36 weeks' PMA, but they may increase length gain velocity and reduce declines in head circumference-for-age z scores from birth to 36 weeks' PMA.

Activity-based sensing fluorescent probes for iron in biological systems.

Iron is an essential nutrient for life, and its capacity to cycle between different oxidation states is required for processes spanning oxygen transport and respiration to nucleotide synthesis and epigenetic regulation. However, this same redox ability also makes iron, if not regulated properly, a potentially dangerous toxin that can trigger oxidative stress and damage. New methods that enable monitoring of iron in living biological systems, particularly in labile Fe2+ forms, can help identify its contributions to physiology, aging, and disease. In this review, we summarize recent developments in activity-based sensing (ABS) probes for fluorescence Fe2+ detection.

A chemiluminescent probe for cellular peroxynitrite using a self-immolative oxidative decarbonylation reaction.

Peroxynitrite (ONOO-) is a highly reactive oxygen species which has been recognized as an endogenous mediator of physiological activities like the immune response as well as a damaging agent of oxidative stress under pathological conditions. While its biological importance is becoming clearer, many of the details of its production and mechanism of action remain elusive due to the lack of available selective and sensitive detection methods. Herein, we report the development, characterization, and biological applications of a reaction-based chemiluminescent probe for ONOO- detection, termed as PNCL. PNCL reacts with ONOO-via an isatin moiety through an oxidative decarbonylation reaction to initiate light emission that can be observed instantly with high selectivity against other reactive sulphur, oxygen, and nitrogen species. Detailed studies were performed to study the reaction between isatin and ONOO-, which confirm selectivity for ONOO- over NO2˙. PNCL has been applied for ONOO- detection in aqueous solution and live cells. Moreover, PNCL can be employed to detect cellular ONOO- generated in macrophages stimulated to mount an immune response with lipopolysaccharide (LPS). The sensitivity granted by chemiluminescent detection together with the specificity of the oxidative decarbonylation reaction provides a useful tool to explore ONOO- chemistry and biology.

Sensitivity of salivary hydrogen sulfide to psychological stress and its association with exhaled nitric oxide and affect.

BACKGROUND: Hydrogen sulfide (H2S) is the third gasotransmitter recently discovered after nitric oxide (NO) and carbon monoxide. Both NO and H2S are involved in multiple physiological functions. Whereas NO has been shown to vary with psychological stress, the influence of stress on H2S and the relationship between H2S and NO are unknown. We therefore examined levels of salivary H2S and NO in response to a stressful final academic exam period. METHODS: Measurements of stress, negative affect, and fraction of exhaled NO (FENO), were obtained from students (N=16) and saliva was collected at three time points: low-stress period in the semester, early exam period, and late exam period. Saliva was immediately analyzed for H2S with the fluorescent probe Sulfidefluor-4. RESULTS: H2S increased significantly during the early exam period and FENO decreased gradually towards the late exam period. H2S, FENO, negative affect, and stress ratings were positively associated with each other: as stress level and negative affect increased, values of H2S increased; in addition, as FENO levels decreased, H2S also decreased. Asthma status did not modify these associations. CONCLUSION: Sustained academic stress increases H2S and these changes are correlated with NO and the experience of stress and negative affect. These findings motivate research with larger samples to further explore the interaction and function of H2S and FENO during psychological stress.