Structural interplay between germline interactions and adaptive recognition determines the bandwidth of TCR-peptide-MHC cross-reactivity.

The T cell antigen receptor (TCR)-peptide-major histocompatibility complex (MHC) interface is composed of conserved and diverse regions, yet the relative contribution of each in shaping recognition by T cells remains unclear. Here we isolated cross-reactive peptides with limited homology, which allowed us to compare the structural properties of nine peptides for a single TCR-MHC pair. The TCR's cross-reactivity was rooted in highly similar recognition of an apical 'hot-spot' position in the peptide with tolerance of sequence variation at ancillary positions. Furthermore, we found a striking structural convergence onto a germline-mediated interaction between the TCR CDR1α region and the MHC α2 helix in twelve TCR-peptide-MHC complexes. Our studies suggest that TCR-MHC germline-mediated constraints, together with a focus on a small peptide hot spot, might place limits on peptide antigen cross-reactivity.

Exploiting spatiotemporal regulation of FZD5 during neural patterning for efficient ventral midbrain specification.

The Wnt/ß-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of ß-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/ß-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.

Human leukocyte antigen expression in paired primary lung tumors and brain metastases in non-small cell lung cancer.

Loss of human leukocyte antigen (HLA) class 1 expression is a mechanism of tumor immune escape and may contribute to resistance to immunotherapy. Patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors can have discordant responses between brain metastases and extracranial sites of disease. We sought to evaluate whether HLA class 1 expression was retained in metastatic NSCLC. Patients with paired primary NSCLC and brain metastases were identified from our institution's tissue registry. HLA class 1 cell membrane expression on tumor cells was determined by immunohistochemistry. Tumors with greater than the median of 10% HLA expression were considered positive. Agreement statistics (κ) were used to assess the congruence of HLA expression. 51 patients with paired primary NSCLC and brain lesions were identified. The median HLA class 1 expression was 20% in the primary tumors (IQR 0-65%) and 10% in the brain metastases (IQR 5-40%). 27 primary tumors and 24 brain metastases were positive for HLA expression. There was disagreement in HLA positivity between paired lesions in 11 patients (22%, 95% CI 12-35%) (κ = 0.57, 95% CI 0.35-0.79) (p = 0.0001). None of the patients received checkpoint inhibitors for treatment of these lesions. The results show that while there is moderate agreement in HLA class 1 expression between primary lung tumor and brain metastasis pairs, HLA expression is incongruent in nearly one quarter of patients. Loss of antigen presentation may represent one of the many potential mechanisms of discordant responses to checkpoint inhibitor therapy.

The molecular basis of TCR germline bias for MHC is surprisingly simple.

The elusive etiology of germline bias of the T cell receptor (TCR) for major histocompatibility complex (MHC) has been clarified by recent 'proof-of-concept' structural results demonstrating the conservation of specific TCR-MHC interfacial contacts in complexes bearing common variable segments and MHC allotypes. We suggest that each TCR variable-region gene product engages each type of MHC through a 'menu' of structurally coded recognition motifs that have arisen through coevolution. The requirement for MHC-restricted T cell recognition during thymic selection and peripheral surveillance has necessitated the existence of such a coded recognition system. Given these findings, a reconsideration of the TCR-peptide-MHC structural database shows that not only have the answers been there all along but also they were predictable by the first principles of physical chemistry.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.

T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex.

T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

Ensuring identifiability in hierarchical mixed effects Bayesian models.

Ecologists are increasingly familiar with Bayesian statistical modeling and its associated Markov chain Monte Carlo (MCMC) methodology to infer about or to discover interesting effects in data. The complexity of ecological data often suggests implementation of (statistical) models with a commensurately rich structure of effects, including crossed or nested (i.e., hierarchical or multi-level) structures of fixed and/or random effects. Yet, our experience suggests that most ecologists are not familiar with subtle but important problems that often arise with such models and with their implementation in popular software. Of foremost consideration for us is the notion of effect identifiability, which generally concerns how well data, models, or implementation approaches inform about, i.e., identify, quantities of interest. In this paper, we focus on implementation pitfalls that potentially misinform subsequent inference, despite otherwise informative data and models. We illustrate the aforementioned issues using random effects regressions on synthetic data. We show how to diagnose identifiability issues and how to remediate these issues with model reparameterization and computational and/or coding practices in popular software, with a focus on JAGS, OpenBUGS, and Stan. We also show how these solutions can be extended to more complex models involving multiple groups of nested, crossed, additive, or multiplicative effects, for models involving random and/or fixed effects. Finally, we provide example code (JAGS/OpenBUGS and Stan) that practitioners can modify and use for their own applications.

Factors contributing to student satisfaction with dental loupes and headlights.

PURPOSE: Surgical loupes and headlights are becoming standard equipment for dental professionals and students. Students often purchase these relatively expensive instruments with limited knowledge of which features will affect their satisfaction with use. The purpose of this study was to measure levels of satisfaction with loupes and headlights amongst dental and dental hygiene students after 1, 2 and 3 years of use, and use those findings to guide first-year students in making better purchasing decisions. METHODS: An electronic survey was sent to all second, third and fourth year dental students as well as second-year dental hygiene students at the University of Nebraska Medical Center (UNMC) in October 2018. The survey asked students to respond to multiple questions with a 5-level Likert scale measurement of satisfaction and provide open-ended responses to questions about their loupes and headlights. RESULTS: Ninety-three percent of eligible students responded. Overall, 68% of students were very satisfied with their loupes and 56% were very satisfied with their headlights. Sixty-seven percent of dental students chose 3.5× magnification or higher and 96% of dental hygiene students chose 2.5×. CONCLUSIONS: Of all characteristics evaluated for loupes, customer service and sales representatives received the lowest satisfaction ratings. For headlights, battery-related factors (battery life in hours, rate of recharging, size or weight and on/off controls) received the lowest satisfaction scores. Information collected and reported in this way can help future students make better decisions when selecting products, vendors and features that will best meet their needs.

Biomarkers of hyperprogression and pseudoprogression with immune checkpoint inhibitor therapy.

Hyperprogression and pseudoprogression are two atypical responses to immune checkpoint inhibitor therapy that affect therapeutic decisions and prognosis. Identification of predictive biomarkers for atypical responses either before or during treatment remains a huge unmet need in cancer immunotherapy. Many studies have looked at potential biomarkers, including clinical factors and laboratory findings (e.g., peripheral blood counts, circulating tumor DNA, cytokine levels). The results of these studies have been inconsistent, possibly due to small sample sizes, different tumor types and heterogeneity of the definition of these atypical responses.

Short communication: Effect of abomasal inorganic phosphorus infusion on phosphorus absorption in large intestine, milk production, and phosphorus excretion of dairy cattle.

The objective of the study was to evaluate the effect of inorganic phosphorus (Pi) infusion on P absorption in large intestine, milk production, and phosphorus excretion. Four ruminally and ileally cannulated crossbred cows were used in a 4 × 4 Latin square with 21-d periods. Cows were fed a total mixed ration containing 0.21% P, providing 50% of the cows' P requirement. Cobalt-EDTA was used as marker to measure large intestine digesta flow. On d 13 to 21 of each period, each cow was infused daily with 0, 20.1, 40.2, or 60.3 g of Pi into the abomasum and total collection was conducted on d 18 to 21. Ileal samples were collected every 9 h on d 18 to 21. Feed, digesta, and fecal samples were analyzed for total P and Pi using the molybdovanadate yellow method and blue method, respectively. All data were analyzed using PROC GLIMMIX in SAS 9.3 (SAS Institute Inc., Cary, NC) using contrasts to evaluate linear, quadratic, and cubic effects of Pi infusion dose. Dry matter intake, apparent dry matter digestibility, milk yield, and milk total P were unaffected by Pi infusion. Ileal flow and fecal excretion of total P and Pi increased linearly with increasing infused Pi. In the large intestine, net absorption of TP and Pi was increased linearly with increasing infused Pi. The magnitude of absorption from the large intestine was greater than reflected in current models, raising questions that could be evaluated with longer infusion periods or dietary alteration.

Discovery and SAR of a novel series of potent, CNS penetrant M4 PAMs based on a non-enolizable ketone core: Challenges in disposition.

This Letter describes the chemical optimization of a novel series of M4 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M4 PAM activity.

Short communication: Comparison of predicted dietary phosphorus balance using bioavailabilities from the NRC (2001) and Virginia Tech model.

The objective of the current work was to use digestion coefficients from the Feng et al. (2015) model (Virginia Tech model) to calculate P bioavailability of common feeds used in dairy production. Compared with the bioavailability calculated by the Virginia Tech model, using the NRC (2001) P absorption coefficient of 0.64 for forages would underestimate the bioavailabilities of alfalfa hay, alfalfa silage, corn silage, grass hay, and mixed mainly legume silage. For concentrates, using the NRC (2001) P absorption coefficient of 0.70 would overestimate the bioavailabilities of corn grain but underestimate bioavailability of high-moisture corn. Two dairy diets were formulated using nutrient values from the NRC (2001): a standard diet that includes minimal by-products, and a by-product diet that has reduced corn and soybean meal which was replaced with corn gluten feed, distillers grains, hominy feed, and wet brewers grains. For each diet, total bioavailable P was calculated using availability values from the NRC (2001) and the Virginia Tech model. Comparison of P balance (the difference between required and bioavailable P) for each diet was made using the 2 sets of bioavailabilities for a reference cow weighing 682 kg, producing 38.6 kg of milk/d (3.5% fat and 3.0% true protein, 100 d in milk), and consuming 23 kg of dry matter/d, yielding an absorbed P requirement of 59.4 g/d. The standard diet supplied 56.69 and 53.52 g of bioavailable P per day using bioavailabilities from the NRC (2001) and Virginia Tech models, respectively, resulting in a P balance of -2.71 and -5.88±0.26 g/d. The by-product diet provided 75.75 and 78.47 g/d of bioavailable P, yielding P balances of 16.35 and 19.07±0.37 g per day, respectively, using the 2 sets of bioavailabilities. Using P bioavailabilities for individual ingredients that were based on the Virginia Tech model resulted in different bioavailable P levels thus resulted in differing dietary P balances in a field setting.

Quantifying ecological memory in plant and ecosystem processes.

The role of time in ecology has a long history of investigation, but ecologists have largely restricted their attention to the influence of concurrent abiotic conditions on rates and magnitudes of important ecological processes. Recently, however, ecologists have improved their understanding of ecological processes by explicitly considering the effects of antecedent conditions. To broadly help in studying the role of time, we evaluate the length, temporal pattern, and strength of memory with respect to the influence of antecedent conditions on current ecological dynamics. We developed the stochastic antecedent modelling (SAM) framework as a flexible analytic approach for evaluating exogenous and endogenous process components of memory in a system of interest. We designed SAM to be useful in revealing novel insights promoting further study, illustrated in four examples with different degrees of complexity and varying time scales: stomatal conductance, soil respiration, ecosystem productivity, and tree growth. Models with antecedent effects explained an additional 18-28% of response variation compared to models without antecedent effects. Moreover, SAM also enabled identification of potential mechanisms that underlie components of memory, thus revealing temporal properties that are not apparent from traditional treatments of ecological time-series data and facilitating new hypothesis generation and additional research.

The effects of forage particle length and exogenous phytase inclusion on phosphorus digestion and absorption in lactating cows.

Accurate estimates of phosphorus (P) availability from feed are needed to allow P requirements to be met with reduced P intake, thus reducing P excretion by livestock. Exogenous phytase supplementation in poultry and swine diets improves bioavailability of P, and limited research suggests that this strategy may have some application in dairy cattle rations. The effects of exogenous phytase and forage particle length on site and extent of P digestion were evaluated with 5 ruminally and ileally cannulated lactating cows (188 ± 35 d in milk). Cows were assigned in a 2 × 2 factorial arrangement of treatments in 2 incomplete Latin squares with four 21-d periods. Diets contained P slightly in excess of National Research Council requirements with all P from feed sources. During the last 4d of each period, total mixed ration, refusals, omasal, ileal, and fecal samples were collected and analyzed for total P, inorganic P (Pi), and phytate (Pp). Total P intake was not influenced by dietary treatments but Pp intake decreased and Pi intake increased with supplemental phytase, suggesting rapid action of the enzyme in the total mixed ration after mixing. Omasal flow of Pi decreased with phytase supplementation, but we observed no effect of diet in ileal flow or small intestinal digestibility of any P fraction. Fecal excretion of total P was slightly higher and Pp excretion was lower for cows receiving diets supplemented with phytase. Milk yield and composition were unaffected by diets. When phytase was added to the mixed ration, dietary Pp was rapidly degraded before intake and total-tract Pp digestion was increased. The lack of effect of phytase supplementation on dietary P utilization was probably because these late-lactation cows had a low P requirement and were fed P-adequate diets.

Rifabutin-Induced Thrombocytopenia in a Patient With Uncontrolled HIV: A Case Report.

Uncontrolled HIV is associated with a wide range of hematologic abnormalities through direct suppressive effects, opportunistic infections, tumor marrow infiltration, or antiretroviral, antimicrobial, or antitumor therapy. We present a patient with a history of uncontrolled HIV presenting with acute severe thrombocytopenia shortly after starting treatment for disseminated Mycobacterium avium complex (MAC). While the thrombocytopenia was resistant to transfusion and intravenous immunoglobulin (IVIG), it mildly improved with dexamethasone after holding home medications. Etiologies for this patient's thrombocytopenia include uncontrolled HIV infection and medication-induced, likely secondary to rifabutin. We propose a possible combined effect of both factors. Clinicians should be aware of the increased risk of severe, acute medication-induced thrombocytopenia in patients with uncontrolled HIV, given their baseline susceptibility to hematologic abnormalities.

A Rare Case of Pseudo-Mirizzi Syndrome Presenting With Acute-on-Chronic Cholecystitis and Hepatic Abscesses.

Mirizzi syndrome (MS) is an uncommon cause of gallstone disease caused by calculous cholecystitis resulting in extrinsic obstruction of the common bile duct, causing concurrent obstructive jaundice. An acalculous variant of MS, at times referred to as pseudo-MS, occurs even more rarely. We present the case of a patient who was found to have pseudo-MS complicated by several hepatic microabscesses. The patient was managed with an endoscopic retrograde cholangiopancreatography (ERCP) with sphincterotomy and eventual cholecystectomy, with histopathology of the gallbladder confirming chronic cholecystitis. To our knowledge, the case presented here is the first in literature that identified pseudo-MS in a patient with pathology-confirmed chronic cholecystitis, and the first to be associated with hepatic abscesses; which usually occur with calculous rather than acalculous biliary disease.

Distinct functional classes of CA1 hippocampal interneurons are modulated by cerebellar stimulation in a coordinated manner.

There is mounting evidence that the cerebellum impacts hippocampal functioning, but the impact of the cerebellum on hippocampal interneurons remains obscure. Using miniscopes in freely behaving animals, we find optogenetic stimulation of Purkinje cells alters the calcium activity of a large percentage of CA1 interneurons. This includes both increases and decreases in activity. Remarkably, this bidirectional impact occurs in a coordinated fashion, in line with interneurons' functional properties. Specifically, CA1 interneurons activated by cerebellar stimulation are commonly locomotion-active, while those inhibited by cerebellar stimulation are commonly rest-active interneurons. We additionally find that subsets of CA1 interneurons show altered activity during object investigations, suggesting a role in the processing of objects in space. Importantly, these neurons also show coordinated modulation by cerebellar stimulation: CA1 interneurons that are activated by cerebellar stimulation are more likely to be activated, rather than inhibited, during object investigations, while interneurons that show decreased activity during cerebellar stimulation show the opposite profile. Therefore, CA1 interneurons play a role in object processing and in cerebellar impacts on the hippocampus, providing insight into previously noted altered CA1 processing of objects in space with cerebellar stimulation. We examined two different stimulation locations (IV/V Vermis; Simplex) and two different stimulation approaches (7Hz or a single 1s light pulse) - in all cases, the cerebellum induces similar coordinated CA1 interneuron changes congruent with an explorative state. Overall, our data show that the cerebellum impacts CA1 interneurons in a bidirectional and coordinated fashion, positioning them to play an important role in cerebello-hippocampal communication. Significance Statement: Acute manipulation of the cerebellum can affect the activity of cells in CA1, and perturbing normal cerebellar functioning can affect hippocampal-dependent spatial processing, including the processing of objects in space. Despite the importance of interneurons on the local hippocampal circuit, it was unknown how cerebellar activation impacts CA1 inhibitory neurons. We find that stimulating the cerebellum robustly affects multiple populations of CA1 interneurons in a bidirectional, coordinated manner, according to their functional profiles during behavior, including locomotion and object investigations. Our work also provides support for a role of CA1 interneurons in spatial processing of objects, with populations of interneurons showing altered activity during object investigations.

Outcomes of a Near-Peer Intern Orientation Boot Camp.

BACKGROUND: Interns experience challenges in their transition from medical school to residency. Orientation is traditionally delivered by faculty and administrators and often does not address practical skills needed by interns during the transition. OBJECTIVES: The objective is to address traditional orientation gaps and improve incoming interns' transition experience.  Methods: We identified opportunities with our intern orientation using a quality improvement methodology. Plan Do Study Act (PDSA) cycle 1 consisted of a pilot boot camp. PDSA cycle 2 was conducted over two weeks, June 9-23, 2021, at the Detroit Medical Center, Detroit, MI. Participation was voluntary. Residents were assigned incoming interns on a 1:1 basis. Five virtual sessions were conducted addressing: daily workflow, documentation, presentation skills, and utilization of the Electronic Health Record (EHR). All participants received pre- and post-program surveys.  Results: Twenty-two rising second- and third-year residents (26%) and 22 incoming interns (58%) participated. There was a significant improvement in the understanding of daily workflow (mean improvement 0.957, p=0.003), and most tasks associated with EHR including comfort with the sign-out process (mean improvement 1.21; p=0.002), accessing specific team lists (mean improvement 1.75, p=0.001), writing orders (mean improvement 1.41; p=0.002), composing documentation (mean improvement 1.23; p=0.001). Writing notes improved significantly (mean improved by 0.52; p=0.04). Nearly all (93.2%) stated the program achieved its overall goals and believed (92.9%) the program should be continued for incoming intern classes. CONCLUSION: A targeted orientation bootcamp led by near-peers positively impacted the intern experience improving understanding of day-to-day responsibilities and comfort utilizing the electronic health record.