Immunoglobulin constant regions provide stabilization to the paratope and enforce epitope specificity.

Constant domains in antibody molecules at the level of the Fab (CH1 and CL) have long been considered to be simple scaffolding elements that physically separate the paratope-defining variable (V) region from the effector function-mediating constant (C) regions. However, due to recent findings that C domains of different isotypes can modulate the fine specificity encoded in the V region, elucidating the role of C domains in shaping the paratope and influencing specificity is a critical area of interest. To dissect the relative contributions of each C domain to this phenomenon, we generated antibody fragments with different C regions omitted, using a set of antibodies targeting capsular polysaccharide from the fungal pathogen, Cryptococcus neoformans. Antigen specificity mapping and functional activity measurements revealed that V region-only antibody fragments exhibited poly-specificity to antigenic variants and extending to recognition of self-antigens, while measurable hydrolytic activity of the capsule was greatly attenuated. To better understand the mechanistic origins of the remarkable loss of specificity that accompanies the removal of C domains from identical paratopes, we performed molecular dynamics simulations which revealed increased paratope plasticity in the scFv relative to the corresponding Fab. Together, our results and provide insight into how the remarkable specificity of immunoglobulins is governed and maintained at the level of the Fab through the enforcement of structural restrictions on the paratope by CH1 domains.

Approaching the 1000 K energy barrier in high coordinate lanthanide single-ion magnets: Increasing Ueff in the [Dy(Tp2-py)F]+ moiety with tetrahydrofuran.

Using the existing architecture of [Dy(Tp2-py)F]+ that supports a terminal Dy-F bond, the synthesis and isolation of [Dy(Tp2-py)F(THF)2](PF6) and its diamagnetic yttrium analogue have been achieved. Alternating current magnetic susceptibility studies show an increased effective energy barrier of Ueff = 661(6) cm-1 (951(8) K), the highest yet reported for a high coordinate (n > 8) lanthanide single-ion magnet.

Methods of Cryptococcal Polysaccharide Analysis Using ELISA.

One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group-indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.

Insights on the G protein-coupled receptor helix 8 solution structure and orientation using a neurotensin receptor 1 peptide.

G-protein coupled receptors (GPCRs) are the largest class of membrane proteins encoded in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminal tail (Ctail). Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the Ctail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and Ctail (H8-Ctail) to investigate its structural stability, conformational dynamics, and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift-derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement NMR. Taken together, our studies reveal the H8-Ctail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.

Integrative multi-region molecular profiling of primary prostate cancer in men with synchronous lymph node metastasis.

Localized prostate cancer is frequently composed of multiple spatially distinct tumors with significant inter- and intra-tumoral molecular heterogeneity. This genomic diversity gives rise to many competing clones that may drive the biological trajectory of the disease. Previous large-scale sequencing efforts have focused on the evolutionary process in metastatic prostate cancer, revealing a potential clonal progression to castration resistance. However, the clonal origin of synchronous lymph node (LN) metastases in primary disease is still unknown. Here, we perform multi-region, targeted next generation sequencing and construct phylogenetic trees in men with prostate cancer with synchronous LN metastasis to better define the pathologic and molecular features of primary disease most likely to spread to the LNs. Collectively, we demonstrate that a combination of histopathologic and molecular factors, including tumor grade, presence of extra-prostatic extension, cellular morphology, and oncogenic genomic alterations are associated with synchronous LN metastasis.

Phospholipid isotope tracing suggests ß-catenin-driven suppression of phosphatidylcholine metabolism in hepatocellular carcinoma.

Activating mutations in the CTNNB1 gene encoding ß-catenin are among the most frequently observed oncogenic alterations in hepatocellular carcinoma (HCC). Profound alterations in lipid metabolism, including increases in fatty acid oxidation and transformation of the phospholipidome, occur in HCC with CTNNB1 mutations, but it is unclear what mechanisms give rise to these changes. We employed untargeted lipidomics and targeted isotope tracing to measure phospholipid synthesis activity in an inducible human liver cell line expressing mutant ß-catenin, as well as in transgenic zebrafish with activated ß-catenin-driven HCC. In both models, activated ß-catenin expression was associated with large changes in the lipidome including conserved increases in acylcarnitines and ceramides and decreases in triglycerides. Lipid isotope tracing analysis in human cells revealed a reduction in phosphatidylcholine (PC) production rates as assayed by choline incorporation. We developed lipid isotope tracing analysis for zebrafish tumors and observed reductions in phosphatidylcholine synthesis by both the CDP-choline and PEMT pathways. The observed changes in the ß-catenin-driven HCC phospholipidome suggest that zebrafish can recapitulate conserved features of HCC lipid metabolism and may serve as a model for identifying future HCC-specific lipid metabolic targets.

AAV-mediated gene therapy for Sialidosis.

Sialidosis (Mucolipidosis I) is a glycoprotein storage disease, clinically characterized by a spectrum of systemic and neurological phenotypes. The primary cause of the disease is deficiency of the lysosomal sialidase NEU1, resulting in accumulation of sialylated glycoproteins/ oligosaccharides in tissues and body fluids. Neu1-/- mice recapitulate the severe, early-onset forms of the disease, affecting visceral organs, muscles, and the nervous system, with wide-spread lysosomal vacuolization evident in most cell types. Sialidosis is considered an orphan disorder with no therapy currently available. Here, we assessed the therapeutic potential of AAV-mediated gene therapy for the treatment of sialidosis. Neu1-/- mice were co-injected with two scAAV2/8 vectors, expressing human NEU1 and its chaperone PPCA. Treated mice were phenotypically indistinguishable from their WT controls. NEU1 activity was restored to different extent in most tissues, including the brain, heart and muscle, and visceral organs. This resulted in diminished/absent lysosomal vacuolization in multiple cell types and reversal of sialyl-oligosacchariduria. Lastly, normalization of lysosomal exocytosis in the CSF and serum of treated mice, coupled to diminished neuroinflammation, were measures of therapeutic efficacy. These findings point to AAV-mediated gene therapy as a suitable treatment for sialidosis and possibly other diseases, associated with low NEU1 expression.

Structural analysis of human IgE monoclonal antibody epitopes on dust mite allergen Der p 2.

BACKGROUND: Human IgE (hIgE) mAbs against major mite allergen Der p 2 developed using human hybridoma technology were used for IgE epitope mapping and analysis of epitopes associated with the hIgE repertoire. OBJECTIVE: We sought to elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2. METHODS: X-ray crystallography was used to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human high-affinity IgE receptor (FcεRIα)-transgenic mouse model of passive systemic anaphylaxis. RESULTS: The structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 Å. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by 3 overlapping hIgE mAbs (1B8, 5D10, and 2G1). Compared with wild-type Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human FcεRIα-transgenic mice sensitized by hIgE mAbs, which were susceptible to anaphylaxis when challenged with wild-type Der p 2, could no longer cross-link FcεRI to induce anaphylaxis when challenged with the epitope mutants. CONCLUSIONS: These data establish the structural basis of allergenicity of 2 hIgE mAb nonoverlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics.

Revealing rural motivations in pharmacy students pursuing a graduate certificate program.

INTRODUCTION: To assist recruitment and retention efforts and influence the need for an increased supply of future rural pharmacists, this study examines perceptions and key motivators of pharmacy students who chose to participate in the Rural Pharmacy Health Certificate (RPHC) program and pursue pathways to rural practice. METHODS: We interviewed six RPHC students prior to or shortly after beginning their first semester in the RPHC program. Interview questions assessed applicants' reasons for pursuing the RPHC, perceptions of living in and providing healthcare in rural and small communities, awareness of barriers and health disparities in rural areas, and qualities needed to be a successful rural pharmacist. We analyzed data with the Sort and Sift, Think and Shift method, a common approach to qualitative data analysis. RESULTS: Interest in pursuing rural pharmacy grew out of growing up in a rural area, as well as a desire to serve and help others. Students expected that completing the RPHC would strengthen their skillsets to provide the best care by addressing barriers such as difficulty accessing care and health literacy. Being a learner of one's community was the primary quality identified as necessary to be a successful rural pharmacist. CONCLUSION: This study identified primary motivators and perceptions that led students to pursue a rural health program at one US pharmacy school. The results can be used to identify and train good candidates for rural pharmacy practice, strengthening the rural pharmacy workforce to better meet communities' needs.

State rumination predicts inhibitory control failures and dysregulation of default, salience, and cognitive control networks in youth at risk of depressive relapse: Findings from the RuMeChange trial.

Background: Trait rumination is a habitual response to negative experiences that can emerge during adolescence, increasing risk of depression. Trait rumination is correlated with poor inhibitory control (IC) and altered default mode network (DMN) and cognitive control network (CCN) engagement. Provoking state rumination in high ruminating youth permits investigation of rumination and IC at the neural level, highlighting potential treatment targets. Methods: Fifty-three high-ruminating youth were cued with an unresolved goal that provoked state rumination, then completed a modified Sustained Attention to Response Task (SART) that measures IC (commissions on no-go trials) in a functional MRI study. Thought probes measured state rumination about that unresolved goal and task-focused thoughts during the SART. Results: Greater state rumination during the SART was correlated with more IC failures. CCN engagement increased during rumination (relative to task-focus), including left dorsolateral prefrontal cortex and dorsalmedial prefrontal cortex. Relative to successful response suppression, DMN engagement increased during IC failures amongst individuals with higher state and trait rumination. Exploratory analyzes suggested more bothersome unresolved goals predicted higher left DLPFC activation during rumination. Limitations: The correlational research design did not permit a direct contrast of causal accounts of the relationship between rumination and IC. Conclusions: State rumination was associated with impaired IC and disrupted modulation of DMN and CCN. Increased CCN engagement during rumination suggested effortful suppression of negative thoughts, and this was greater for more bothersome unresolved goals. Relative task disengagement was observed during rumination-related errors. DMN-CCN dysregulation in high-ruminating youth may be an important treatment target.

The Role of Metallurgical Features in the Microbially Influenced Corrosion of Carbon Steel: A Critical Review.

Microbially influenced corrosion (MIC) is a potentially critical degradation mechanism for a wide range of materials exposed to environments that contain relevant microorganisms. The likelihood and rate of MIC are affected by microbiological, chemical, and metallurgical factors; hence, the understanding of the mechanisms involved, verification of the presence of MIC, and the development of mitigation methods require a multidisciplinary approach. Much of the recent focus in MIC research has been on the microbiological and chemical aspects, with less attention given to metallurgical attributes. Here, we address this knowledge gap by providing a critical synthesis of the literature on the metallurgical aspects of MIC of carbon steel, a material frequently associated with MIC failures and widely used in construction and infrastructure globally. The article begins by introducing the process of MIC, then progresses to explore the complexities of various metallurgical factors relevant to MIC in carbon steel. These factors include chemical composition, grain size, grain boundaries, microstructural phases, inclusions, and welds, highlighting their potential influence on MIC processes. This review systematically presents key discoveries, trends, and the limitations of prior research, offering some novel insights into the impact of metallurgical factors on MIC, particularly for the benefit of those already familiar with other aspects of MIC. The article concludes with recommendations for documenting metallurgical data in MIC research. An appreciation of relevant metallurgical attributes is essential for a critical assessment of a material's vulnerability to MIC to advance research practices and to broaden the collective knowledge in this rapidly evolving area of study.

Immune checkpoint inhibitor associated epidermal necrosis, beyond SJS and TEN: a review of 98 cases.

Immune checkpoint inhibitor (ICI) therapies carry the risk of major immune-related adverse events (irAEs). Among the most severe irAEs is epidermal necrosis that may clinically mimic Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN). The aim of this study was to provide a summary of the clinical and histological features of ICI-associated epidermal necrosis, with a special focus on factors associated with fatal outcomes in cases of extensive disease. A total of 98 cases, 2 new cases and 96 reported on PubMed and in the literature, of ICI-associated epidermal necrosis were assessed. Development of epidermal necrosis occurred between 1 day and 3 years after starting ICI therapy, with an average onset of 13.8 weeks for patients with limited (< 30% BSA) and 11.3 weeks for those with extensive (≥ 30% BSA) involvement, and a median onset of 5.8 weeks and 4 weeks respectively. A preceding rash was seen in 52 cases and was more common in extensive cases. Mucosal involvement was only reported in 65% of extensive cases but was significantly associated with fatal reactions. Co-administration of cytotoxic chemotherapy was associated with more extensive disease. Recovery was observed in 96% and 65% of those with limited and extensive involvement respectively and no specific therapy was associated with improved survival. Young age was significantly associated with poor outcomes in extensive disease, the average age of surviving patients was 64.5 years old versus 55.1 years old for deceased patients, p < 0.01. Both superficial perivascular and interface/lichenoid inflammatory infiltrates were commonly seen. These findings suggest that ICI-associated epidermal necrosis should be considered a distinct clinical entity from drug-induced SJS/TEN.

Sleep interventions for adults admitted to psychiatric inpatient settings: A systematic scoping review.

Sleep disturbances are common, affecting over half of adults with a mental disorder. For those admitted to a psychiatric ward, difficulties with sleep, particularly insomnia, are compounded by factors relating to the inpatient setting. We conducted a scoping review of sleep intervention studies involving adults admitted to psychiatric settings. We categorised the different types of sleep interventions and identified the effects on sleep and other mental and physical health outcomes. Instruments used to measure sleep were also examined. The search strategy yielded 4780 studies, of which 28 met the inclusion criteria. There was evidence of more non-pharmacological than pharmacological interventions having been tested in inpatient settings. Results indicated that non-pharmacological interventions based on cognitive behaviour therapy for insomnia improve sleep and may improve mental and physical health. Several distinct sleep measures were used in the studies. Gaps in the literature were identified, highlighting the importance of research into a wider range of sleep interventions tested against robust controls, using validated measures of sleep with evaluation of additional mental and physical health outcomes among a large sample size of adults in the psychiatric inpatient settings.

Radiation-associated Vestibular Schwannomas: Case Series and Literature Review.

INTRODUCTION: Radiation treatment, particularly at a young age, creates theoretical risk for long term adverse radiation effects, including the development of malignancy. The literature is sparse on radiation-induced vestibular schwannomas (VS). METHODS: A retrospective review was performed for cases of suspected radiation-induced VS at two high volume centers. Only cases where radiation included coverage of the posterior fossa were included with those diagnosed within 3 years of radiation treatment being excluded. Patient and tumor characteristics were collected. A systematic literature review was also performed for any previously published series on radiation-induced VS. RESULTS: Eight cases of radiation-induced VS were identified with a median follow-up 125 months (range 7-131). Median age at incident radiation was 15 years (range 2-46). Median age at VS diagnosis was 57-years (range 26-83) with median interval from radiation to diagnosis of 51-years (range 15-66). Median tumor size was 6 mm (range 3-21). Two patients underwent surgical resection. Lesions were described as soft and highly vascular, with medium to high adherence to the facial nerve. Five articles with a total of 52 patients were identified, median age at VS diagnosis was 42-years (range 23-73) with a median interval from radiation to diagnosis of 19 years (range 15-23). CONCLUSIONS: The development of VS following radiation exposure appears rare and our understanding of the condition remains incomplete. Further studies are required to determine the best management of these patients and determine whether there is a causative relationship between radiation exposure and the development of VS.

Quantum Mechanical Assessment of Nitrosamine Potency.

Nitrosamines are in the cohort of concern (CoC) as determined by regulatory guidance. CoC compounds are considered highly potent carcinogens that need to be limited below the threshold of toxicological concern, 1.5 µg/day. Nitrosamines like NDMA and NDEA require strict control, while novel nitrosamine drug substance-related impurities (NDSRIs) may or may not be characterized as potent carcinogens. A risk assessment based on the structural features of NDSRIs is important in order to predict potency because they lack substance-specific carcinogenicity. Herein, we present a quantum mechanical (QM)-based analysis on structurally diverse sets of nitrosamines to better understand how structure influences the reactivity that could result in carcinogenicity. We describe the potency trend through activation energies corresponding to α-hydroxylation, aldehyde formation, diazonium intermediate formation, reaction with DNA base, and hydrolysis reactions, and other probable metabolic pathways associated with the carcinogenicity of nitrosamines. We evaluated activation energies for selected cases such as N-nitroso pyrrolidines, N-nitroso piperidines, N-nitroso piperazines, N-nitroso morpholines, N-nitroso thiomorpholine, N-methyl nitroso aromatic, fluorine-substituted nitrosamines, and substituted aliphatic nitrosamines. We compare these results to the recent framework of the carcinogenic potency characterization approach (CPCA) proposed by health authorities which is meant to give guidance on acceptable intakes (AI) for NDSRIs lacking substance-specific carcinogenicity data. We show examples where QM modeling and CPCA are aligned and examples where CPCA both underestimates and overestimates the AI. In cases where CPCA predicts high potency for NDSRIs, QM modeling can help better estimate an AI. Our results suggest that a combined mechanistic understanding of α-hydroxylation, aldehyde formation, hydrolysis, and reaction with DNA bases could help identify the structural features that underpin the potency of nitrosamines. We anticipate this work will be a valuable addition to the CPCA and provide a more analytical way to estimate AI for novel NDSRIs.

Inferring Stochastic Rates from Heterogeneous Snapshots of Particle Positions.

Many imaging techniques for biological systems-like fixation of cells coupled with fluorescence microscopy-provide sharp spatial resolution in reporting locations of individuals at a single moment in time but also destroy the dynamics they intend to capture. These snapshot observations contain no information about individual trajectories, but still encode information about movement and demographic dynamics, especially when combined with a well-motivated biophysical model. The relationship between spatially evolving populations and single-moment representations of their collective locations is well-established with partial differential equations (PDEs) and their inverse problems. However, experimental data is commonly a set of locations whose number is insufficient to approximate a continuous-in-space PDE solution. Here, motivated by popular subcellular imaging data of gene expression, we embrace the stochastic nature of the data and investigate the mathematical foundations of parametrically inferring demographic rates from snapshots of particles undergoing birth, diffusion, and death in a nuclear or cellular domain. Toward inference, we rigorously derive a connection between individual particle paths and their presentation as a Poisson spatial process. Using this framework, we investigate the properties of the resulting inverse problem and study factors that affect quality of inference. One pervasive feature of this experimental regime is the presence of cell-to-cell heterogeneity. Rather than being a hindrance, we show that cell-to-cell geometric heterogeneity can increase the quality of inference on dynamics for certain parameter regimes. Altogether, the results serve as a basis for more detailed investigations of subcellular spatial patterns of RNA molecules and other stochastically evolving populations that can only be observed for single instants in their time evolution.

Improving Appropriate Use of Peripherally Inserted Central Catheters Through a Statewide Collaborative Hospital Initiative: A Cost-Effectiveness Analysis.

BACKGROUND: Quality improvement (QI) programs require significant financial investment. The authors evaluated the cost-effectiveness of a physician-led, performance-incentivized, QI intervention that increased appropriate peripherally inserted central catheter (PICC) use. METHODS: The authors used an economic evaluation from a health care sector perspective. Implementation costs included incentive payments to hospitals and costs for data abstractors and the coordinating center. Effectiveness was calculated from propensity score-matched observations across two time periods for complications (venous thromboembolism [VTE], central line-associated bloodstream infection [CLABSI], and catheter occlusion): preintervention period (January 2015 through December 2016) and intervention period (January 2017 through December 2021). Cost-effectiveness was presented as the cost-offset per averted complication, reflecting the health care costs avoided due to having lower complication rates. RESULTS: Across 35 hospitals, this study sampled 17,418 PICCs placed preintervention and 26,004 placed during the intervention period. PICC complications decreased significantly following the intervention. CLABSIs decreased from 2.1% to 1.5%, VTEs from 3.2% to 2.3%, and catheter occlusions from 10.8% to 7.0% (all p < 0.01). Estimated number of complications prevented included 871 CLABSIs, 2,535 VTEs, and 8,743 catheter occlusions. Project implementation costs were $31.8 million, and the cost-offset related to avoided complications was $64.4 million. Each participating hospital averaged $932,073 in cost-offset over seven years, and the average cost-offset per complication averted was $2,614 (95% CI [confidence interval] $2,314-$3,003). CONCLUSION: A large-scale, multihospital QI initiative to improve appropriate PICC use yielded substantial return on investment from cost-offset of prevented complications.

Delineating memory reactivation in sleep with verbal and non-verbal retrieval cues.

Sleep supports memory consolidation via the reactivation of newly formed memory traces. One way to investigate memory reactivation in sleep is by exposing the sleeping brain to auditory retrieval cues; a paradigm known as targeted memory reactivation. To what extent the acoustic properties of memory cues influence the effectiveness of targeted memory reactivation, however, has received limited attention. We addressed this question by exploring how verbal and non-verbal memory cues affect oscillatory activity linked to memory reactivation in sleep. Fifty-one healthy male adults learned to associate visual stimuli with spoken words (verbal cues) and environmental sounds (non-verbal cues). Subsets of the verbal and non-verbal memory cues were then replayed during sleep. The voice of the verbal cues was either matched or mismatched to learning. Memory cues (relative to unheard control cues) prompted an increase in theta/alpha and spindle power, which have been heavily implicated in sleep-associated memory processing. Moreover, verbal memory cues were associated with a stronger increase in spindle power than non-verbal memory cues. There were no significant differences between the matched and mismatched verbal cues. Our findings suggest that verbal memory cues may be most effective for triggering memory reactivation in sleep, as indicated by an amplified spindle response.

Will a changing climate affect hatching success in cavity-nesting birds: A case study with Eastern Bluebirds (Sialia sialis)?

A significant body of evidence indicates that climate change is influencing many aspects of avian ecology. Yet, how climate change is affecting, and is expected to influence some aspects of the breeding ecology of cavity-nesting birds remains uncertain. To explore the potential linkage between timing of first clutch, and the influence of ambient temperature on hatching success, we used Eastern Bluebird (Sialia sialis) nest records over a nine-year period from Alabama, USA. We investigated changes to annual clutch initiation dates, as well as variability in hatching success associated with ambient air temperatures during the incubation period. Using a simple linear model, we observed earlier annual egg laying dates over the nine years of this study with a difference of 24 days between earliest egg-laying date of the season. Daily temperature minima increased 2 °C across the nine-year time frame of this study. These data also indicate that Eastern Bluebird hatching success was the highest when mean ambient air temperature during incubation was between 19 °C and 24 °C (78%, as opposed to 69% and 68% above and below this temperature range, respectively). Our findings of increasing maxima, earlier maxima each year, and the lower minima of temperatures within our study area could expand the breadth of temperatures experienced by nesting Eastern Bluebirds possibly exposing them to temperatures outside of what promotes nesting success. These findings with a cavity-nesting bird highlight an optimal range of ambient temperatures associated with highest hatching success, conditions likely to be affected by climate change.