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Chronic kidney disease (CKD) imposes a strong and independent risk for peripheral artery disease (PAD). While solutes retained in CKD patients (uremic solutes) inflict vascular damage, their role in PAD remains elusive. Here, we show that the dietary tryptophan-derived uremic solutes including indoxyl sulfate (IS) and kynurenine (Kyn) at concentrations corresponding to those in CKD patients suppress β-catenin in several cell types, including microvascular endothelial cells (ECs), inhibiting Wnt activity and proangiogenic Wnt targets in ECs. Mechanistic probing revealed that these uremic solutes downregulated β-catenin in a manner dependent on serine 33 in its degron motif and through the aryl hydrocarbon receptor (AHR). Hindlimb ischemia in adenine-induced CKD and IS solute-specific mouse models showed diminished β-catenin and VEGF-A in the capillaries and reduced capillary density, which correlated inversely with blood levels of IS and Kyn and AHR activity in ECs. An AHR inhibitor treatment normalized postischemic angiogenic response in CKD mice to a non-CKD level. In a prospective cohort of PAD patients, plasma levels of tryptophan metabolites and plasma's AHR-inducing activity in ECs significantly increased the risk of future adverse limb events. This work uncovers the tryptophan metabolite/AHR/β-catenin axis as a mediator of microvascular rarefaction in CKD patients and demonstrates its targetability for PAD in CKD models.

The authors show that NMDA receptor activation induces a rapid protein phosphatase 1–dependent dephosphorylation and downregulation of the neuron-specific potassium-chloride co-transporter KCC2. Deficits in KCC2 activity have been observed in disease states associated with pathophysiological glutamate release. Blocking dephosphorylation reduces the glutamate-induced degradation of KCC2 and maintains hyperpolarizing GABAergic inhibition. KCC2 is a neuron-specific K + -Cl − co-transporter that maintains a low intracellular Cl − concentration that is essential for hyperpolarizing inhibition mediated by GABA A receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release. However, the mechanisms by which elevated glutamate alters KCC2 function are unknown. The phosphorylation of KCC2 residue Ser940 is known to regulate its surface activity. We found that NMDA receptor activity and Ca 2+ influx caused the dephosphorylation of Ser940 in dissociated rat neurons, leading to a loss of KCC2 function that lasted longer than 20 min. Protein phosphatase 1 mediated the dephosphorylation events of Ser940 that coincided with a deficit in hyperpolarizing GABAergic inhibition resulting from the loss of KCC2 activity. Blocking dephosphorylation of Ser940 reduced the glutamate-induced downregulation of KCC2 and substantially improved the maintenance of hyperpolarizing GABAergic inhibition. Reducing the downregulation of KCC2 therefore has therapeutic potential in the treatment of neurological disorders.

We introduce machine learning (ML) to perform classification and quantitation of images of nuclei from human blood neutrophils. Here we assessed the use of convolutional neural networks (CNNs) using free, open source software to accurately quantitate neutrophil NETosis, a recently discovered process involved in multiple human diseases. CNNs achieved >94% in performance accuracy in differentiating NETotic from non-NETotic cells and vastly facilitated dose-response analysis and screening of the NETotic response in neutrophils from patients. Using only features learned from nuclear morphology, CNNs can distinguish between NETosis and necrosis and between distinct NETosis signaling pathways, making them a precise tool for NETosis detection. Furthermore, by using CNNs and tools to determine object dispersion, we uncovered differences in NETotic nuclei clustering between major NETosis pathways that is useful in understanding NETosis signaling events. Our study also shows that neutrophils from patients with sickle cell disease were unresponsive to one of two major NETosis pathways. Thus, we demonstrate the design, performance, and implementation of ML tools for rapid quantitative and qualitative cell analysis in basic science.

Despite effective antiretroviral therapy, HIV co-morbidities remain where central nervous system (CNS) neurocognitive disorders and cardiovascular disease (CVD)-pathology that are linked with myeloid activation are most prevalent. Comorbidities such as neurocogntive dysfunction and cardiovascular disease (CVD) remain prevalent among people living with HIV. We sought to investigate if cardiac pathology (inflammation, fibrosis, cardiomyocyte damage) and CNS pathology (encephalitis) develop together during simian immunodeficiency virus (SIV) infection and if their co-development is linked with monocyte/macrophage activation. We used a cohort of SIV-infected rhesus macaques with rapid AIDS and demonstrated that SIV encephalitis (SIVE) and CVD pathology occur together more frequently than SIVE or CVD pathology alone. Their co-development correlated more strongly with activated myeloid cells, increased numbers of CD14+CD16+ monocytes, plasma CD163 and interleukin-18 (IL-18) than did SIVE or CVD pathology alone, or no pathology. Animals with both SIVE and CVD pathology had greater numbers of cardiac macrophages and increased collagen and monocyte/macrophage accumulation, which were better correlates of CVD-pathology than SIV-RNA. Animals with SIVE alone had higher levels of activated macrophage biomarkers and cardiac macrophage accumulation than SIVnoE animals. These observations were confirmed in HIV infected individuals with HIV encephalitis (HIVE) that had greater numbers of cardiac macrophages and fibrosis than HIV-infected controls without HIVE. These results underscore the notion that CNS and CVD pathologies frequently occur together in HIV and SIV infection, and demonstrate an unmet need for adjunctive therapies targeting macrophages.

The Escherichia coli (E. coli) ribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains in vitro but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high-resolution cryo-EM structures of the ribosome with each of these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A-site). The structures reveal how the aromatic ring of each monomer sterically blocks the positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the bound water network that is believed to facilitate formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a mechanistic rationale for differences in reactivity of aminobenzoic acid derivatives relative to l-α-amino acids and each other and identify stereochemical constraints on the size and geometry of non-monomers that can be accepted efficiently by wild-type ribosomes.

Background Kolb’s Cycle of Learning Theory acts as a foundational framework for the evolution of knowledge gained by learners throughout their education. Through Kolb’s cycle of experiential learning, one’s preferred way of learning could impact academic achievement in the pre-clinical years of medical education. Methods The medical student classes of 2020 and 2021 at a public university in the southeastern U.S. were invited to complete Kolb’s Learning Style Inventory (LSI). For those participants completing the LSI, examination results for their pre-clinical blocks were obtained and matched to the LSI results. Examination scores (locally-developed examinations and customized National Board of Medical Examiners (NBME) final examinations) were compared by LSI classification for each examination using Kruskal-Wallis Test. Results Out of 360 possible participants, 314 (87.2%) completed the Learning Style Inventory. Convergers and Assimilators made up 84.1% [Convergers ( n  = 177, 56.4%), Assimilators ( n  = 87, 27.7%)]. Accommodators ( n  = 25, 7.9%) and Divergers ( n  = 25, 7.9%) made up the remaining sample. Accomodators’ scores were significantly lower on locally-developed examinations in Principles of Medicine, Hematology, and Gastrointestinal System. The only NBME examination that demonstrated a significant difference across learning styles was from the Cardiovascular block. Conclusions Upon reviewing Kolb’s LSI, our study indicated that performance on the customized NBME examinations minimized the variance in performance compared to locally-developed examinations. The lack of variance across learning styles for all but one NBME final examination appears to provide a more equitable assessment strategy.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products with potent antibiotic, antiviral, and anticancer properties. RiPP enzymes known as cyclodehydratases and dehydrogenases work together to catalyze intramolecular, inter-residue condensation and dehydrogenation reactions that install oxazoline/oxazole and thiazoline/thiazole heterocycles within ribosomally produced polypeptide chains. Here, we show that the previously reported enzymes MicD-F and ArtGox accept backbone-modified monomersincluding aminobenzoic acid derivatives and beta-amino acidswithin leader-free polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The products are sequence-defined chemical polymers with multiple, diverse non-α-amino acid subunits. We show further that MicD-F and ArtGox can install heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bondboth features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

BackgroundClaudin (CLDN) 18.2 is a transmembrane tight junction protein that is expressed in stomach epithelia. CLDN18.2 expression is significantly elevated in gastric and pancreatic adenocarcinomas. Loss of cell polarity in tumors results in CLDN18.2 localization to surfaces that are more readily accessible to biologics and effector cells. This expression pattern makes it an excellent target for immune stimulating antibody conjugate (ISACs), which combine the specificity of a tumor-targeting antibody with potent immune stimulation. The delivery of ISACs to the tumor microenvironment triggers the innate and adaptive immune system to attack CLDN18.2-expressing tumors. T cell priming following phagocytosis of CLDN18.2-expressing tumor cells in the context of immune stimulation results in epitope spreading and the targeting of CLDN18.2-negative tumors cells with durable immunologic memory. These mechanisms differ from other cytotoxic payloads, which rely on the induction of apoptosis or cell death to kill tumor cells. Herein, we describe the development of a Claudin 18.2 ISAC with a TLR7/8 linker-payload.MethodsFor human in vitro assessment of ISACs, PBMCs or myeloid APCs were isolated from human healthy donor blood and activation was measured by flow cytometry, cytokine-bead array, and other ELISA-based methods. In vivo assessment of antitumor activity was performed using the PATU-8889s (endogenously expressing CLDN18.2) and MC38-muCLDN18.2 (engineered to express mouse CLDN18.2). Tolerability of a mouse CLDN18.2 binding ISAC was performed in healthy C57BL/6 mice.ResultsCLDN18.2 ISACs elicit robust tumor antigen-dependent activation of the immune system as measured by the secretion of proinflammatory cytokines TNFa and IL-12p70. Furthermore, a CLDN18.2 ISAC significantly inhibited tumor growth in a syngeneic model with CLDN18.2 expression levels consistent with those measured in the clinical setting. Interestingly, tumor regression was also observed in a model where only approximately 15% of tumor cells expressed CLDN18.2. Furthermore, the CLDN18.2 ISAC elicited T cell-dependent immunological memory with epitope spreading, as evidenced by a lack of tumor growth upon rechallenge with the original tumor cell line lacking CLDN18.2 expression. An exploratory mouse toxicity study revealed that the CLDN18.2 ISAC was well tolerated following two doses at 60 mg/kg, and the MTD was not reached in this study.ConclusionsWe believe that this is the first reported CLDN18.2 ISAC that demonstrates potent anti-tumor activity, induction of immunologic memory with epitope spreading, and an acceptable safety profile in preclinical studies. A CLDN18.2 ISAC may offer benefits beyond other ADCs in development.

KCC2 is a neuron-specific [K.sup.+]-[Cl.sup.-] co-transporter that maintains a low intracellular [Cl.sup.-] concentration that is essential for hyperpolarizing inhibition mediated by GABAA receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release. However, the mechanisms by which elevated glutamate alters KCC2 function are unknown. The phosphorylation of KCC2 residue Ser940 is known to regulate its surface activity. We found that NMDA receptor activity and [Ca.sup.2+] influx caused the dephosphorylation of Ser940 in dissociated rat neurons, leading to a loss of KCC2 function that lasted longer than 20 min. Protein phosphatase 1 mediated the dephosphorylation events of Ser940 that coincided with a deficit in hyperpolarizing GABAergic inhibition resulting from the loss of KCC2 activity. Blocking dephosphorylation of Ser940 reduced the glutamate-induced downregulation of KCC2 and substantially improved the maintenance of hyperpolarizing GABAergic inhibition. Reducing the downregulation of KCC2 therefore has therapeutic potential in the treatment of neurological disorders.

Chronic kidney disease (CKD) imposes a strong and independent risk for peripheral artery disease (PAD). While solutes retained in CKD patients (uremic solutes) inflict vascular damage, their role in PAD remains elusive. Here, we show that the dietary tryptophan-derived uremic solutes including indoxyl sulfate (IS) and kynurenine (Kyn) at concentrations corresponding to those in CKD patients suppress ß-catenin in several cell types, including microvascular endothelial cells (ECs), inhibiting Wnt activity and proangiogenic Wnt targets in ECs. Mechanistic probing revealed that these uremic solutes downregulated ß-catenin in a manner dependent on serine 33 in its degron motif and through the aryl hydrocarbon receptor (AHR). Hindlimb ischemia in adenine-induced CKD and IS solute-specific mouse models showed diminished ß-catenin and VEGF-A in the capillaries and reduced capillary density, which correlated inversely with blood levels of IS and Kyn and AHR activity in ECs. An AHR inhibitor treatment normalized postischemic angiogenic response in CKD mice to a non-CKD level. In a prospective cohort of PAD patients, plasma levels of tryptophan metabolites and plasma's AHR-inducing activity in ECs significantly increased the risk of future adverse limb events. This work uncovers the tryptophan metabolite/AHR/ß-catenin axis as a mediator of microvascular rarefaction in CKD patients and demonstrates its targetability for PAD in CKD models.