Prospective manipulation of the gut microbiome with microbial ecosystem therapeutic 4 (MET4) in HPV-related locoregionally-advanced oropharyngeal cancer squamous cell carcinoma (LA-OPSCC) undergoing primary chemoradiation: ROMA2 study.

BACKGROUND: Gut microbiome modulation to boost antitumor immune responses is under investigation. METHODS: ROMA-2 evaluated the microbial ecosystem therapeutic (MET)-4 oral consortia, a mixture of cultured human stool-derived immune-responsiveness associated bacteria, given with chemoradiation (CRT) in HPV-related oropharyngeal cancer patients. Co-primary endpoints were safety and changes in stool cumulative MET-4 taxa relative abundance (RA) by 16SRNA sequencing. Stools and plasma were collected pre/post-MET-4 intervention for microbiome and metabolome analysis. RESULTS: Twenty-nine patients received ≥1 dose of MET-4 and were evaluable for safety: drug-related adverse events (AEs) occurred in 13/29 patients: all grade 1-2 except one grade 3 (diarrhea). MET-4 was discontinued early in 7/29 patients due to CRT-induced toxicity, and in 1/29 due to MET-4 AEs. Twenty patients were evaluable for ecological endpoints: there was no increase in stool MET-4 RA post-intervention but trended to increase in stage III patients (p = 0.06). MET-4 RA was higher in stage III vs I-II patients at week 4 (p = 0.03) and 2-month follow-up (p = 0.01), which correlated with changes in plasma and stool targeted metabolomics. CONCLUSIONS: ROMA-2 did not meet its primary ecologic endpoint, as no engraftment was observed in the overall cohort. Exploratory findings of engraftment in stage III patients warrants further investigation of microbiome interventions in this subgroup.

Transoral Robotic Surgery and Radiation Volume Deintensification in Unknown Primary Squamous Cell Carcinoma of the Neck: The Phase 2 FIND Nonrandomized Controlled Trial.

Importance: Patients with unknown primary squamous cell carcinoma (CUP) with cervical metastases typically receive comprehensive radiotherapy (RT) of the pharynx and bilateral neck. Typically, these patients receive comprehensive RT of the pharynx and bilateral neck that may produce treatment-related toxic effects. Objective: To determine whether localization of occult oropharyngeal cancers with transoral robotic surgery (TORS) combined with reduced pharyngeal and neck RT volumes provides acceptable disease control. Design, Setting, and Participants: This phase 2, single-group nonrandomized controlled trial at a single institution accrued 32 prospective participants with p16-positive CUP without a primary squamous cell carcinoma on examination and imaging from 2017 to 2019, and 24-month follow-up. The data analysis was conducted from January 2021 to June 2022. Intervention: Diagnostic- (n = 13) or therapeutic-intent (n = 9) TORS, with pharyngeal-sparing radiotherapy (PSRT) prescribed for negative margins or pT0, and unilateral neck RT (UNRT) prescribed for unilateral lymphadenopathy with lateralized primary tumor or pT0. Main Outcomes and Measures: Out-of-radiation treatment volume failure (<15% was hypothesized to be acceptable) and reports of local and regional recurrence, overall survival, toxic effects, swallowing outcomes (per the MD Anderson Dysphagia Inventory), and videofluoroscopic swallow (per Dynamic Imaging Grade of Swallowing Toxic Effects [DIGEST]) ratings. Results: The study sample comprised 22 patients (mean [SD] age, 59.1 [5.7] years; 3 [14%] females and 19 [86%] male) with CUP. Of these, 19 patients (86%) had tumor stage cN1; 2 (9%), cN2; and 1 (5%), cN3. Five patients (23%), 14 patients (64%), and 3 patients (13%) had 0, 1, or 2 primary tumors, respectively. Twenty patients received RT; of these, 9 patients (45%) underwent PSRT and 10 patients (50%), UNRT. In the diagnostic-intent group, 8 patients (62%) and 5 patients (38%) underwent RT and RT-concurrent chemotherapy, respectively. In the therapeutic-intent group, 6 patients (67%) and 1 patient (11%) received adjuvant RT-concurrent chemotherapy, respectively; 2 patients declined RT. Two-year out-of-radiation treatment volume failure, locoregional control, distant metastasis control, and overall survival were 0%, 100%, 95%, and 100%, respectively. Grade 3 or 4 surgical, acute, and late toxic effects occurred in 2 (9%), 5 (23%), and 1 (5%) patients, respectively. PSRT was associated with lower RT dose to superior constrictors (37 vs 53 Gy; mean difference, 16 Gy; 95% CI, 6.4, 24.9), smaller decline in swallowing scores during treatment (19.3 vs 39.7; mean difference, -20.4; 95% CI, -34.1 to -6.1), and fewer patients with worsening DIGEST grade on findings of videofluoroscopic swallow studies at 2 years (0% vs 60%; difference, 60%; 95% CI, 30% to 90%). Conclusions and Relevance: These findings indicate that TORS for p16-positive CUP allows RT volume deintensification with excellent outcomes and support future investigation in randomized clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT03281499.

Telemedicine-enabled biofeedback electropalatography rehabilitation (TEBER): A pilot study for patients treated with surgery for oral cavity carcinoma.

BACKGROUND: To address the rehabilitative barriers to frequency and precision of care, we conducted a pilot study of a biofeedback electropalatography (EPG) device paired with telemedicine for patients who underwent primary surgery +/- adjuvant radiation for oral cavity carcinoma. We hypothesized that lingual optimization followed by telemedicine-enabled biofeedback electropalatography rehabilitation (TEBER) would further improve speech and swallowing outcomes after "standard-of-care" SOC rehabilitation. METHOD: Pilot prospective 8-week (TEBER) program following 8 weeks of (SOC) rehabilitation. RESULTS: Twenty-seven patients were included and 11 completed the protocol. When examining the benefit of TEBER independent of standard of care, "range-of-liquids" improved by +0.36 [95% CI, 0.02-0.70, p = 0.05] and "range-of-solids" improved by +0.73 [95% CI, 0.12-1.34, p = 0.03]. There was a positive trend toward better oral cavity obliteration; residual volume decreased by -1.2 [95% CI, -2.45 to 0.053, p = 0.06], and "nutritional-mode" increased by +0.55 [95% CI, -0.15 to 1.24, p = 0.08]. CONCLUSION: This pilot suggests that TEBER bolsters oral rehabilitation after 8 weeks of SOC lingual range of motion.

A phylogenetic method linking nucleotide substitution rates to rates of continuous trait evolution.

Genomes contain conserved non-coding sequences that perform important biological functions, such as gene regulation. We present a phylogenetic method, PhyloAcc-C, that associates nucleotide substitution rates with changes in a continuous trait of interest. The method takes as input a multiple sequence alignment of conserved elements, continuous trait data observed in extant species, and a background phylogeny and substitution process. Gibbs sampling is used to assign rate categories (background, conserved, accelerated) to lineages and explore whether the assigned rate categories are associated with increases or decreases in the rate of trait evolution. We test our method using simulations and then illustrate its application using mammalian body size and lifespan data previously analyzed with respect to protein coding genes. Like other studies, we find processes such as tumor suppression, telomere maintenance, and p53 regulation to be related to changes in longevity and body size. In addition, we also find that skeletal genes, and developmental processes, such as sprouting angiogenesis, are relevant.

Boom-bust population dynamics drive rapid genetic change.

Increasing environmental threats and more extreme environmental perturbations place species at risk of population declines, with associated loss of genetic diversity and evolutionary potential. While theory shows that rapid population declines can cause loss of genetic diversity, populations in some environments, like Australia's arid zone, are repeatedly subject to major population fluctuations yet persist and appear able to maintain genetic diversity. Here, we use repeated population sampling over 13 y and genotype-by-sequencing of 1903 individuals to investigate the genetic consequences of repeated population fluctuations in two small mammals in the Australian arid zone. The sandy inland mouse (Pseudomys hermannsburgensis) experiences marked boom-bust population dynamics in response to the highly variable desert environment. We show that heterozygosity levels declined, and population differentiation (FST) increased, during bust periods when populations became small and isolated, but that heterozygosity was rapidly restored during episodic population booms. In contrast, the lesser hairy-footed dunnart (Sminthopsis youngsoni), a desert marsupial that maintains relatively stable population sizes, showed no linear declines in heterozygosity. These results reveal two contrasting ways in which genetic diversity is maintained in highly variable environments. In one species, diversity is conserved through the maintenance of stable population sizes across time. In the other species, diversity is conserved through rapid genetic mixing during population booms that restores heterozygosity lost during population busts.

Properties of packings and dispersions of superellipse sector particles.

Superellipse sector particles (SeSPs) are segments of superelliptical curves that form a tunable set of hard-particle shapes for granular and colloidal systems. SeSPs allow for continuous parametrization of corner sharpness, aspect ratio, and particle curvature; rods, circles, rectangles, and staples are examples of shapes SeSPs can model. We compare three computational processes: pair-wise Monte Carlo simulations that explore particle-particle geometric constraints, Monte Carlo simulations that reveal how these geometric constraints play out over dispersions of many particles, and Molecular Dynamics simulations that form random loose and close packings. We investigate the dependence of critical random loose and close packing fractions on particle parameters, finding that both values increase with opening aperture and decrease with increasing corner sharpness. The identified packing fractions are compared with the mean-field prediction of the random contact model; we find deviations from the model's prediction due to correlations between particle orientations. The complex interaction of spatial proximity and orientational alignment is also explored with a generalized spatioorientational distribution area (SODA) plot, which shows how higher density packings are achieved through particles assuming a small number of preferred configurations that depend sensitively on particle shape and system preparation.

COVID-19 Severity and Mortality in Veterans with Chronic Lung Disease.

INTRODUCTION: Chronic lung disease (CLD) has been associated with risk for more severe manifestations and death with COVID-19. However, few studies have evaluated the risk overall and by type of CLD for severity of COVID-19 outcomes in a US national cohort. METHODS: Using data from the Veterans Health Administration, we determined the risk associated with CLDs including COPD (mild/severe), asthma (mild/active/severe), idiopathic pulmonary fibrosis (IPF), sarcoidosis and other interstitial lung diseases (ILDs) for outcomes among veterans with SARS-CoV-2 positive tests between 3/1/2020-4/30/2021. We used multinomial regression to estimate risk of four mutually exclusive COVID-19 outcomes within 30-days: outpatient management, hospitalization, hospitalization with indicators of critical illness, or death. We calculated the overall proportion with each outcome, the absolute risk difference and risk ratios for each outcome between those with and without CLD. We also describe clinical and laboratory abnormalities by CLD in those hospitalized. RESULTS: We included 208,283 veterans with COVID-19; 35,587 (17%) had CLD. Compared to no CLD, veterans with CLD were older and had more comorbidities. Hospitalized veterans with CLD were more likely to have low temperature, mean arterial pressure, oxygen saturation, leukopenia and thrombocytopenia, and more likely to receive oxygen, mechanical ventilation and vasopressors. Veterans with CLD were significantly less likely to have mild COVID-19 (-4.5%, adjusted risk ratio [aRR] 0.94, 95% confidence interval [CI] 0.94-0.95), and more likely to have a moderate (+2.5%, aRR 1.21, 95% CI 1.18-1.24), critical (+1.4%, aRR 1.38, 95% CI 1.32-1.45) or fatal (+0.7%, aRR 1.15, 95% CI 1.10-1.20) outcome. IPF was most strongly associated with COVID-19 severity, especially mortality (+3.2%, aRR 1.69, 95% CI 1.46-1.96), followed by other ILDs and COPD, whereas asthma was less likely to be associated with severity of COVID-19. In veterans under age 65, worse COVID-19 outcomes were generally more likely with IPF, sarcoidosis, and other ILDs. CONCLUSIONS: Veterans who had CLD, particularly IPF, other ILDs and COPD, had an increased probability of more severe 30-day outcomes with COVID-19. These results provide insight into the absolute and relative risk of different CLDs with severity of COVID-19 outcomes and can help inform considerations of healthcare utilization and prognosis.

Targeting Oral Squamous Cell Carcinoma with Combined Polo-Like-Kinase-1 Inhibitors and γ-Radiation Therapy.

Polo-like-kinase-1 (PLK-1) is a serine/threonine kinase that regulates the cell cycle and acts as an oncogene in multiple cancers, including oral squamous cell carcinoma (OSCC). The loss of PLK-1 can inhibit growth and induce apoptosis, making it an attractive therapeutic target in OSCC. We evaluated the efficacy of PLK-1 inhibitors as novel, targeted therapeutics in OSCC. PLK-1 inhibition using BI6727 (volasertib) was found to affect cell death at low nanomolar concentrations in most tested OSCC cell lines, but not in normal oral keratinocytes. In cell lines resistant to volasertib alone, pre-treatment with radiotherapy followed by volasertib reduced cell viability and induced apoptosis. The combinatorial efficacy of volasertib and radiotherapy was replicated in xenograft mouse models. These findings highlight the potential of adding PLK-1 inhibitors to adjuvant therapy regimens in OSCC.

Genetic Basis and Evolution of Structural Color Polymorphism in an Australian Songbird.

Island organisms often evolve phenotypes divergent from their mainland counterparts, providing a useful system for studying adaptation under differential selection. In the white-winged fairywren (Malurus leucopterus), subspecies on two islands have a black nuptial plumage whereas the subspecies on the Australian mainland has a blue nuptial plumage. The black subspecies have a feather nanostructure that could in principle produce a blue structural color, suggesting a blue ancestor. An earlier study proposed independent evolution of melanism on the islands based on the history of subspecies divergence. However, the genetic basis of melanism and the origin of color differentiation in this group are still unknown. Here, we used whole-genome resequencing to investigate the genetic basis of melanism by comparing the blue and black M. leucopterus subspecies to identify highly divergent genomic regions. We identified a well-known pigmentation gene ASIP and four candidate genes that may contribute to feather nanostructure development. Contrary to the prediction of convergent evolution of island melanism, we detected signatures of a selective sweep in genomic regions containing ASIP and SCUBE2 not in the black subspecies but in the blue subspecies, which possesses many derived SNPs in these regions, suggesting that the mainland subspecies has re-evolved a blue plumage from a black ancestor. This proposed re-evolution was likely driven by a preexisting female preference. Our findings provide new insight into the evolution of plumage coloration in island versus continental populations, and, importantly, we identify candidate genes that likely play roles in the development and evolution of feather structural coloration.

Gene flow and an anomaly zone complicate phylogenomic inference in a rapidly radiated avian family (Prunellidae).

BACKGROUND: Resolving the phylogeny of rapidly radiating lineages presents a challenge when building the Tree of Life. An Old World avian family Prunellidae (Accentors) comprises twelve species that rapidly diversified at the Pliocene-Pleistocene boundary. RESULTS: Here we investigate the phylogenetic relationships of all species of Prunellidae using a chromosome-level de novo assembly of Prunella strophiata and 36 high-coverage resequenced genomes. We use homologous alignments of thousands of exonic and intronic loci to build the coalescent and concatenated phylogenies and recover four different species trees. Topology tests show a large degree of gene tree-species tree discordance but only 40-54% of intronic gene trees and 36-75% of exonic genic trees can be explained by incomplete lineage sorting and gene tree estimation errors. Estimated branch lengths for three successive internal branches in the inferred species trees suggest the existence of an empirical anomaly zone. The most common topology recovered for species in this anomaly zone was not similar to any coalescent or concatenated inference phylogenies, suggesting presence of anomalous gene trees. However, this interpretation is complicated by the presence of gene flow because extensive introgression was detected among these species. When exploring tree topology distributions, introgression, and regional variation in recombination rate, we find that many autosomal regions contain signatures of introgression and thus may mislead phylogenetic inference. Conversely, the phylogenetic signal is concentrated to regions with low-recombination rate, such as the Z chromosome, which are also more resistant to interspecific introgression. CONCLUSIONS: Collectively, our results suggest that phylogenomic inference should consider the underlying genomic architecture to maximize the consistency of phylogenomic signal.

Genomes, fossils, and the concurrent rise of modern birds and flowering plants in the Late Cretaceous.

The phylogeny and divergence timing of the Neoavian radiation remain controversial despite recent progress. We analyzed the genomes of 124 species across all Neoavian orders, using data from 25,460 loci spanning four DNA classes, including 5,756 coding sequences, 12,449 conserved nonexonic elements, 4,871 introns, and 2,384 intergenic segments. We conducted a comprehensive sensitivity analysis to account for the heterogeneity across different DNA classes, leading to an optimal tree of Neoaves with high resolution. This phylogeny features a novel Neoavian dichotomy comprising two monophyletic clades: a previously recognized Telluraves (land birds) and a newly circumscribed Aquaterraves (waterbirds and relatives). Molecular dating analyses with 20 fossil calibrations indicate that the diversification of modern birds began in the Late Cretaceous and underwent a constant and steady radiation across the KPg boundary, concurrent with the rise of angiosperms as well as other major Cenozoic animal groups including placental and multituberculate mammals. The KPg catastrophe had a limited impact on avian evolution compared to the Paleocene-Eocene Thermal Maximum, which triggered a rapid diversification of seabirds. Our findings suggest that the evolution of modern birds followed a slow process of gradualism rather than a rapid process of punctuated equilibrium, with limited interruption by the KPg catastrophe. This study places bird evolution into a new context within vertebrates, with ramifications for the evolution of the Earth's biota.

Multimodal detection of molecular residual disease in high-risk locally advanced squamous cell carcinoma of the head and neck.

Up to 30% of patients with locally advanced head and neck squamous cell carcinoma (LA-HNSCC) relapse. Molecular residual disease (MRD) detection using multiple assays after definitive therapy has not been reported. In this study, we included patients with LA-HNSCC (stage III Human Papilloma virus (HPV)-positive, III-IVB HPV-negative) treated with curative intent. Plasma was collected pre-treatment, at 4-6 weeks (FU1) and 8-12 weeks (FU2) post-treatment. Circulating tumor DNA (ctDNA) was analyzed using a tumor-informed (RaDaR®) and a tumor-naïve (CAPP-seq) assay. HPV DNA was measured using HPV-sequencing (HPV-seq) and digital PCR (dPCR). A total of 86 plasma samples from 32 patients were analyzed; all patients with at least 1 follow-up sample. Most patients were stage III HPV-positive (50%) and received chemoradiation (78%). No patients had radiological residual disease at FU2. With a median follow-up of 25 months, there were 7 clinical relapses. ctDNA at baseline was detected in 15/17 (88%) by RaDaR and was not associated with recurrence free survival (RFS). Two patients relapsed within a year after definitive therapy and showed MRD at FU2 using RaDaR; detection of ctDNA during follow-up was associated with shorter RFS (p < 0.001). ctDNA detection by CAPP-seq pre-treatment and during follow-up was not associated with RFS (p = 0.09). HPV DNA using HPV-seq or dPCR during follow-up was associated with shorter RFS (p < 0.001). Sensitivity and specificity for MRD at FU2 using RaDaR was 40% and 100% versus 20 and 90.5% using CAPP-seq. Sensitivity and specificity for MRD during follow-up using HPV-seq was 100% and 91.7% versus 50% and 100% using dPCR. In conclusion, HPV DNA and ctDNA can be detected in LA-HNSCC before definitive therapy. The RaDaR assay but not CAPP-seq may detect MRD in patients who relapse within 1 year. HPV-seq may be more sensitive than dPCR for MRD detection.

Early changes in tumor-naive cell-free methylomes and fragmentomes predict outcomes in pembrolizumab-treated solid tumors.

Early kinetics of circulating tumor DNA (ctDNA) in plasma predict response to pembrolizumab, but typically requires sequencing of matched tumor tissue or fixed gene panels. We analyzed genome-wide methylation and fragment length profiles using cell-free methylated DNA immunoprecipitation and sequencing (cfMeDIP-seq) in 204 plasma samples from 87 patients before and during treatment with pembrolizumab from a pan-cancer phase II investigator-initiated trial (INSPIRE). We trained a pan-cancer methylation signature using independent methylation array data from The Cancer Genome Atlas to quantify a cancer-specific methylation (CSM) and fragment length score (FLS) for each sample. CSM and FLS are strongly correlated with tumor-informed ctDNA levels. Early kinetics of CSM predict overall survival and progression-free survival, independently of tumor type, PD-L1, and tumor mutation burden. Early kinetics of FLS are associated with overall survival independently of CSM. Our tumor-naïve mutation-agnostic ctDNA approach integrating methylomics and fragmentomics could predict outcomes in patients treated with pembrolizumab.

The ATCC genome portal: 3,938 authenticated microbial reference genomes.

The ATCC Genome Portal (AGP, https://genomes.atcc.org/) is a database of authenticated genomes for bacteria, fungi, protists, and viruses held in ATCC's biorepository. It now includes 3,938 assemblies (253% increase) produced under ISO 9000 by ATCC. Here, we present new features and content added to the AGP for the research community.

Clinical Validation of Human Papilloma Virus Circulating Tumor DNA for Early Detection of Residual Disease After Chemoradiation in Cervical Cancer.

PURPOSE: Most cervical cancers are caused by human papilloma virus (HPV), and HPV circulating tumor DNA (ctDNA) may identify patients at highest risk of relapse. Our pilot study using digital polymerase chain reaction (dPCR) showed that detectable HPV ctDNA at the end of chemoradiation (CRT) is associated with inferior progression-free survival (PFS) and that a next-generation sequencing approach (HPV-seq) may outperform dPCR. We aimed to prospectively validate HPV ctDNA as a tool for early detection of residual disease. METHODS: This prospective, multicenter validation study accrued patients with stage IB-IVA cervical cancer treated with CRT between 2017 and 2022. Participants underwent phlebotomy at baseline, end of CRT, 4-6 weeks post-CRT, and 3 months post-CRT for HPV ctDNA levels. Plasma HPV genotype-specific DNA levels were quantified using both dPCR and HPV-seq. The primary end point was 2-year PFS. RESULTS: With a median follow-up of 2.2 (range, 0.5-5.5) years, there were 24 PFS events among the 70 patients with HPV+ cervical cancer. Patients with detectable HPV ctDNA on dPCR at the end of CRT, 4-6 weeks post-CRT, and 3 months post-CRT had significantly worse 2-year PFS compared with those with undetectable HPV ctDNA (77% v 51%, P = .03; 82% v 15%, P < .001; and 82% v 24%, P < .001, respectively); the median lead time to recurrence was 5.9 months. HPV-seq showed similar results as dPCR. On multivariable analyses, detectable HPV ctDNA on dPCR and HPV-seq remained independently associated with inferior PFS. CONCLUSION: Persistent HPV ctDNA after CRT is independently associated with inferior PFS. HPV ctDNA testing can identify, as early as at the end of CRT, patients at high risk of recurrence for future treatment intensification trials.

slimr: An R package for tailor-made integrations of data in population genomic simulations over space and time.

Software for realistically simulating complex population genomic processes is revolutionizing our understanding of evolutionary processes, and providing novel opportunities for integrating empirical data with simulations. However, the integration between standalone simulation software and R is currently not well developed. Here, we present slimr, an R package designed to create a seamless link between standalone software SLiM >3.0, one of the most powerful population genomic simulation frameworks, and the R development environment, with its powerful data manipulation and analysis tools. We show how slimr facilitates smooth integration between genetic data, ecological data and simulation in a single environment. The package enables pipelines that begin with data reading, cleaning and manipulation, proceed to constructing empirically based parameters and initial conditions for simulations, then to running numerical simulations and finally to retrieving simulation results in a format suitable for comparisons with empirical data - aided by advanced analysis and visualization tools provided by R. We demonstrate the use of slimr with an example from our own work on the landscape population genomics of desert mammals, highlighting the advantage of having a single integrated tool for both data analysis and simulation. slimr makes the powerful simulation ability of SLiM directly accessible to R users, allowing integrated simulation projects that incorporate empirical data without the need to switch between software environments. This should provide more opportunities for evolutionary biologists and ecologists to use realistic simulations to better understand the interplay between ecological and evolutionary processes.

A miniaturized piezoelectric Mössbauer spectrometer with feedback control.

A Mössbauer spectrometer was built and evaluated using an amplified piezoelectric actuator under feedback control for the Doppler velocity drive. The actuator was driven with a quadratic displacement waveform, giving a linear velocity profile. The optimization of the piezoelectric Doppler drive under feedback control was performed with measurements from a laser Doppler vibrometer. 57Fe Mössbauer spectra of α-iron in transmission geometry show minimal peak distortions. The performance of this piezoelectric Doppler drive makes Mössbauer spectrometry possible in applications requiring small size, mass, and low cost.

Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Ancient duplication, coevolution, and selection at the MHC class IIA and IIB genes of birds.

Introduction: The Major Histocompatibility Complex (MHC) of vertebrates is a dynamically evolving multigene family primarily responsible for recognizing non-self peptide antigens and triggering a pathogen-specific adaptive immune response. In birds, the MHC was previously thought to evolve via concerted evolution with high degree of gene homogenization and the rapid loss of orthology. However, the discovery of two ancient avian MHC-IIB gene lineages (DAB1 and DAB2) originating before the radiation of extant birds indicated that despite the action of concerted evolution, orthology may be detectable for long evolutionary periods. Methods: Here, we take advantage of rapidly accumulating digital genomic resources to search for the signal of an ancient duplication at the avian MHC-IIA genes, as well as to compare phylogenetic distribution and selection between MHC-IIA and IIB gene lineages. Results: The analysis of MHC sequences from over 230 species representing ca. 70 bird families revealed the presence of two ancient MHC-IIA gene lineages (DAA1 and DAA2) and showed that their phylogenetic distribution matches exactly the distribution of DAB1 and DAB2 lineages, suggesting tight coevolution. The early post-duplication divergence of DAA1 and DAA2 was driven by positive selection fixing radical amino acid differences within the membrane-proximal domain and, most probably, being functionally related to the interactions between α2 and ß2 chains of the MHC-II heterodimer. We detected no evidence for an overall (gene-wide) relaxation or intensification of selection at either DAA1/DAB1 or DAA2/DAB2, but codon-specific differences in selection signature were found at the peptide-binding sites between the two gene lineages, perhaps implying specialization to different pathogen regimes. Discussion: Our results suggest that specific pairing of MHC-II α and ß chains may have an adaptive significance, a conclusion that advances knowledge on the macroevolution of the avian MHC-II and opens exciting novel directions for future research.