Torrey Pines Institute For Molecular Studies

Catalytic reactions involving multiple cycles are common in many key organic transformations. We report a detailed mechanistic analysis of one such network of "nested" cycles, the Ni-catalyzed cross-coupling of alkyl sulfonyl hydrazides with (hetero)aryl halides, employing a battery of techniques, including Temperature Scanning Reaction (TSR) calorimetry coupled with Reaction Progress Kinetic Analysis (RPKA), quantitative NMR time-course analysis, kinetic modeling, linear free energy analysis, and density functional theory (DFT) quantum chemical calculations. TSR/RPKA studies yield the simultaneous determination of the thermodynamic enthalpy of the reaction, the kinetic rate law, and the activation parameters (ΔH‡, ΔS‡, ΔG‡), for the rate-determining step. Expanding the analysis to the coupling of an array of electron-poor and electron-rich sulfonyl hydrazides (each readily accessible on the decagram scale) with a range of (hetero)aryl halide coupling partners introduces the concept of "kinetics matching," and demonstrates that optimal yields may be obtained when the reactivities of the separate cycles are tuned to work in unison. These requirements for reaction networks involving nested catalytic cycles are showcased by several real-world examples. Our work highlights the predictive power of this kinetics-focused mechanistic approach, which may be extended to other nested catalytic networks to select appropriate catalyst and substrate combinations for optimal outcomes.

CLOCK, BMAL1, and HIFs are basic helix-loop-helix and Per-Arnt-Sim domain (bHLH-PAS) proteins, which function as transcription factors. bHLH-PAS proteins are designated in two classes. Many class I proteins are regulated by environmental signals via their PAS domains, but such signals have not been identified for all. Class II (ARNTs and BMALs) are partners for Class I and can be regulated by synthetic PAS ligands. Previous studies suggested restricted dimerization for bHLH-PAS proteins. BMAL1 and BMAL2 were believed to dimerize only with CLOCK and NPAS2, while ARNT was thought to dimerize with most Class I proteins except for CLOCK and NPAS2. The logic underlying these assumptions was flawed, and evidence supports dimerization of both HIF1α and HIF2α with BMAL1.

The doctrine of eternal generation sometimes draws the objection that it sounds like it is covertly telling a story about how the Son came to be. This objection, called here the story objection, can be answered in several ways. Trinitarian theology will never be able to preempt the raising of the story objection, because Scripture itself reveals the second person as the Son, and the Nicene Creed rightly models the classical theological practice of unfolding that revealed term by the use of action verbs: if Son, then generated. Thomas Aquinas is a key example of a trinitarian theologian who accepts both the biblical name and its Nicene unfolding as action that does not reduce to story.

Neuroimmune signals can regulate neuronal function and affect behavior through mechanisms that are not yet fully understood. Here we investigated the action of interleukin 13 (IL-13), a cytokine that can be produced in the brain by both microglia and neurons. We show that dopamine-containing neurons in the ventral tegmental area (VTA) predominantly express the IL-13 receptor alpha 1 (IL-13Rα1) and exhibit presynaptic vesicular localization of neuronal IL-13. Exogenous application of IL-13, or its endogenous mobilization by optogenetics, reduced the activity of VTA dopaminergic neurons and opposed the stimulatory effects of nicotine on these neurons in rodents. These actions required IL-13Rα1, activation of the PI3K/AKT pathway, and functional hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Consistently, local infusion of IL-13 into the VTA markedly reduced nicotine self-administration in rodents. Collectively, these findings demonstrate that IL-13 acts in a neuromodulator-like fashion on mesolimbic dopamine neurons expressing IL-13Rα1. Our data also indicate that IL-13Rα1 signaling regulates the stimulatory actions of nicotine, suggesting a potential role for this neuronal immune signaling in reward processing and the addictive properties of nicotine.

Obtaining reliable estimates of demographic parameters is critical to effective wildlife conservation and management. Densities of Mojave Desert Tortoises (Gopherus agassizii) were historically derived from capture–mark–recapture surveys on small, often strategically placed demography plots, or demographic study areas, that also provided information on demographic composition and vital rates. After protection was afforded to Desert Tortoises under the US Endangered Species Act in 1990, monitoring shifted mostly to line-distance sampling across broad areas for estimating densities of primarily adult tortoises to inform long-term population trends. However, that approach is incapable of providing data about other demographic characteristics important to population growth and viability. We surveyed a previously unsampled demography plot in the western Mojave Desert, California, USA, during 2022 and applied spatial capture–recapture (SCR) models to estimate spatially explicit Desert Tortoise density and sex-by-size class compositions. Directly accounting for spatiotemporally varying survey effort in SCR models via hazard-based adjustment reduced the estimated detection rate by 91% and increased the estimated density by 17%. Estimated spatial mean Desert Tortoise density across a 2.53-km2 area was 18.53 tortoises/km2 (95% confidence interval [CI] = 12.36–27.77). The SCR model–estimated size class ratio was skewed toward prereproductive tortoises (64% prereproductive; 36% adults), whereas the adult sex ratio was female biased (61% females; 39% males). Those ratios corresponded to densities of 11.86 prereproductive tortoises/km2 (95% CI = 7.91–17.77), 4.08 adult female tortoises/km2 (95% CI = 2.72–6.11), and 2.59 adult male tortoises/km2 (95% CI = 1.73–3.89). Estimated tortoise density and demographic composition collectively support a high potential for population growth. Our study provides an illustrative example of using SCR models to directly estimate spatially explicit local Desert Tortoise densities and demographic composition that can be used for long-term monitoring and comparisons with other demography plots to inform conservation.

Glycans are now recognized as essential biomolecules for life, and an increasing number of investigators and studies continue to reveal the myriad ways in which these post-translational modifications regulate important biological events. Chief among the ways in which glycans carry out their roles is by engaging in binding interactions with glycan-binding proteins (GBPs). Such interactions are important for proper physiology or in the activation of disease. Thus, achieving a precise molecular understanding of these interactions can pave new avenues for synthetic control and potentially therapeutic strategies to regulate disease. In this Account, we discuss our efforts toward revealing the collective interactions between glycans, protein glycoconjugates, and GBPs in cells, with an eye toward identifying the specific glycan-carrying proteins that interact with the GBPs in a functional manner.While the importance of studying glycan-GBP interactions has long been established, prior to our work, much of glycoscience had been occupied with the systematic assignment of the structural features of glycans required for recognition by GBPs in vitro, often using homogeneous glycan arrays. This important body of work enabled the preliminary identification of principal GBP-glycan binding preferences and allowed the rational design of functionalized glycan molecules to use as competitive inhibitors. Equipped with these two sets of important tools, expanding our understanding of glycan-GBP interactions from in vitro glycan binding preferences toward that of glycoprotein-GBP interactions in vivo has become timely and possible. We describe the conceptual motivation behind developing proximity tagging technologies to capture these glycoprotein-GBP interactions and how we used these interactome data sets to guide the discovery of functional interactors or create new hypotheses. Furthermore, we describe how proximity tagging can continuously be used toward application in our work, and the potential routes of investigation it may facilitate in the coming years.

DNA double strand breaks (DSBs) are the effective lesion of cancer radiotherapy and induce gene editing. 53BP1 accumulates at DSBs and is implicated in end joining (EJ) repair, but its influence on DSB repair is distinct from canonical non-homologous end joining (C-NHEJ). We sought to define the genetic interplay of 53BP1 with C-NHEJ, focusing on the DNA-PKcs kinase. We examined Cas9 DSBs, which largely generates blunt DSBs, since blunt DSB EJ is dependent on C-NHEJ. Loss of 53BP1 does not affect blunt DSB EJ, but causes a reduction in such repair in DNA-PKcs deficient cells. In contrast, disrupting 53BP1 and DNA-PKcs, both alone and together, has similar effects on the type of deletion mutation (increase in microhomology deletions). We found similar effects on EJ with RIF1 loss, which is a downstream effector of 53BP1. Thus, 53BP1/RIF1 appear to play a backup role for DNA-PKcs during blunt DSB EJ, but function in the same pathway to suppress microhomology deletions. In contrast, 53BP1 and DNA-PKcs function independently to suppress homology-directed repair. Finally, DNA-PKcs kinase inhibition causes marked radiosensitivity, which is not additive with loss of 53BP1 and RIF1. Altogether, 53BP1/RIF1 and DNA-PKcs show distinct genetic interactions with diverse DSB repair outcomes.

Intestinal epithelial cells (IECs) uniquely express two IRE1 paralogues, IRE1α and IRE1β, whose roles in intestinal physiology are incompletely understood. We examined the individual and cooperative functions of IRE1α and IRE1β in IECs using mice using intestine-specific deletion of Ire1α or germline Ire1β deletion, and subsequently with double deleted Ire1α, Ire1β mice. At baseline, intestine-specific Ire1α deleted mice and mice with germline Ire1β deletion exhibited no morphologic changes in small intestine or colon, but double deleted Ire1α−/−Ire1β−/− mice developed progressive intestinal and colonic injury and tumorigenesis. In contrast to single-deleted IECs, RNA-Seq from Ire1α−/−Ire1β−/− IECs revealed decreased expression of defense-associated mRNAs, together with increased expression of inflammatory and pathogenic mRNAs. Utilizing orthogonal models of intestinal tumorigenesis, reflecting either inflammatory-mutagenic injury (AOM-DSS) or spontaneous polyposis (APCmin), we observed that loss of either intestinal epithelial Ire1α or of Ire1β alone produced a growth advantage, increasing tumor burden. IRE1α mediated splicing of Xbp1 mRNA was maintained following Ire1β deletion but not in double deleted Ire1α−/−Ire1β−/− mice. Increased expression of either Ire1α or Ire1β mRNA was associated with improved survival in patients with colorectal cancer. Taken together our findings suggest IRE1 paralogues utilize essential but distinct mechanisms to safeguard intestinal homeostasis and suppress tumorigenesis.

Activity-based protein profiling (ABPP) of stereoisomerically defined sets of electrophilic compounds ('stereoprobes') offers a versatile way to discover covalent ligands for proteins in native biological systems. Here we report the synthesis and chemical proteomic characterization of stereoprobes bearing a P(V)-oxathiaphospholane (OTP) reactive group. ABPP experiments identified numerous proteins in human cancer cells that showed stereoselective reactivity with OTP stereoprobes, and we confirmed several of these liganding events with recombinant proteins. OTP stereoprobes engaging the poorly characterized transmembrane protein TLCD1 impaired the incorporation of monounsaturated fatty acids into phosphatidylethanolamine lipids in cells, a lipidomic phenotype that mirrored genetic disruption of this protein. Using AlphaFold2, we found that TLCD1 structurally resembles the ceramide synthase and fatty acid elongase families of coenzyme A-dependent lipid processing enzymes. This structural similarity included conservation of catalytic histidine residues, the mutation of which blocked the OTP stereoprobe reactivity and lipid remodeling activity of recombinant TLCD1. Taken together, these data indicate that TLCD1 acts as a lipid acyltransferase in cells, and that OTP stereoprobes function as inhibitors of this enzymatic activity. Our findings thus illuminate how the chemical proteomic analysis of electrophilic compounds can facilitate the functional annotation and chemical inhibition of a key lipid metabolic enzyme in human cells.

Abstract The Nachwuchs Reaktionstechnik (NaWuReT) is an organization of early‐career chemical engineers in the DECHEMA/VDI subject division Reaction Engineering. In the spring of 2024, the annual colloquium was held as a series of online lectures featuring five distinguished late‐career academics and experts from industry. The speakers shared insights into how they transformed their efforts and ideas into successful careers in academia and industry. Additionally, they provided valuable recommendations for early‐career chemical engineers, offering a critical resource for professional development in the field.