Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase.

The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate-distinct from that between Mcm2/5 used for origin loading-is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication.

Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α-primase DNA polymerase activity in replication and show that Pol α-primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.

Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC.

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.

A neurotransmitter produced by gut bacteria modulates host sensory behaviour.

Animals coexist in commensal, pathogenic or mutualistic relationships with complex communities of diverse organisms, including microorganisms1. Some bacteria produce bioactive neurotransmitters that have previously been proposed to modulate nervous system activity and behaviours of their hosts2,3. However, the mechanistic basis of this microbiota-brain signalling and its physiological relevance are largely unknown. Here we show that in Caenorhabditis elegans, the neuromodulator tyramine produced by commensal Providencia bacteria, which colonize the gut, bypasses the requirement for host tyramine biosynthesis and manipulates a host sensory decision. Bacterially produced tyramine is probably converted to octopamine by the host tyramine ß-hydroxylase enzyme. Octopamine, in turn, targets the OCTR-1 octopamine receptor on ASH nociceptive neurons to modulate an aversive olfactory response. We identify the genes that are required for tyramine biosynthesis in Providencia, and show that these genes are necessary for the modulation of host behaviour. We further find that C. elegans colonized by Providencia preferentially select these bacteria in food choice assays, and that this selection bias requires bacterially produced tyramine and host octopamine signalling. Our results demonstrate that a neurotransmitter produced by gut bacteria mimics the functions of the cognate host molecule to override host control of a sensory decision, and thereby promotes fitness of both the host and the microorganism.

Mechanism of eukaryotic origin unwinding is a dual helicase DNA shearing process.

DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA polymerases. In bacteria, origin DNA is presumed to be melted by accessory proteins that allow loading of two ring-shaped replicative helicases around single-strand DNA (ssDNA) for bidirectional unwinding and DNA replication. In eukaryotes, by contrast, two replicative CMG (Cdc45-Mcm2-7-GINS) helicases are initially loaded head to head around origin double-strand DNA (dsDNA), and there does not appear to be a separate origin unwinding factor. This led us to investigate whether head-to-head CMGs use their adenosine triphosphate (ATP)-driven motors to initiate duplex DNA unwinding at the origin. Here, we show that CMG tracks on one strand of the duplex while surrounding it, and this feature allows two head-to-head CMGs to unwind dsDNA by using their respective motors to pull on opposite strands of the duplex. We further show that while CMG is capable of limited duplex unwinding on its own, the extent of unwinding is greatly and rapidly stimulated by addition of the multifunctional CMG-binding protein Mcm10 that is critical for productive initiation of DNA replication in vivo. On the basis of these findings, we propose that Mcm10 is a processivity or positioning factor that helps translate the work performed by the dual CMG motors at the origin into productive unwinding that facilitates bidirectional DNA replication.

Adaptive Capacity of a DNA Polymerase Clamp-loader ATPase Complex.

The ability of mutations to facilitate adaptation is central to evolution. To understand how mutations can lead to functional adaptation in a complex molecular machine, we created a defective version of the T4 clamp-loader complex, which is essential for DNA replication. This variant, which is ∼5,000-fold less active than the wild type, was made by replacing the catalytic domains with those from another phage. A directed-evolution experiment revealed that multiple substitutions to a single negatively charged residue in the chimeric clamp loader-Asp 86-restore fitness to within ∼20-fold of wild type. These mutations remove an adventitious electrostatic repulsive interaction between Asp 86 and the sliding clamp. Thus, the fitness decrease of the chimeric clamp loader is caused by a reduction in affinity between the clamp loader and the clamp. Deep mutagenesis shows that the reduced fitness of the chimeric clamp loader is also compensated for by lysine and arginine substitutions of several DNA-proximal residues in the clamp loader or the sliding clamp. Our results demonstrate that there is a latent capacity for increasing the affinity of the clamp loader for DNA and the sliding clamp, such that even single-point mutations can readily compensate for the loss of function due to suboptimal interactions elsewhere.

Context-dependent reversal of odorant preference is driven by inversion of the response in a single sensory neuron type.

The valence and salience of individual odorants are modulated by an animal's innate preferences, learned associations, and internal state, as well as by the context of odorant presentation. The mechanisms underlying context-dependent flexibility in odor valence are not fully understood. Here, we show that the behavioral response of Caenorhabditis elegans to bacterially produced medium-chain alcohols switches from attraction to avoidance when presented in the background of a subset of additional attractive chemicals. This context-dependent reversal of odorant preference is driven by cell-autonomous inversion of the response to these alcohols in the single AWC olfactory neuron pair. We find that while medium-chain alcohols inhibit the AWC olfactory neurons to drive attraction, these alcohols instead activate AWC to promote avoidance when presented in the background of a second AWC-sensed odorant. We show that these opposing responses are driven via engagement of distinct odorant-directed signal transduction pathways within AWC. Our results indicate that context-dependent recruitment of alternative intracellular signaling pathways within a single sensory neuron type conveys opposite hedonic valences, thereby providing a robust mechanism for odorant encoding and discrimination at the periphery.

CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism.

The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to "preload" CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATPγS and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATPγS compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATPγS for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding.

SV40 T-antigen uses a DNA shearing mechanism to initiate origin unwinding.

Duplication of DNA genomes requires unwinding of the double-strand (ds) DNA so that each single strand (ss) can be copied by a DNA polymerase. The genomes of eukaryotic cells are unwound by two ring-shaped hexameric helicases that initially encircle dsDNA but transition to ssDNA for function as replicative helicases. How the duplex is initially unwound, and the role of the two helicases in this process, is poorly understood. We recently described an initiation mechanism for eukaryotes in which the two helicases are directed inward toward one another and shear the duplex open by pulling on opposite strands of the duplex while encircling dsDNA [L. D. Langston, M. E. O'Donnell, eLife 8, e46515 (2019)]. Two head-to-head T-Antigen helicases are long known to be loaded at the SV40 origin. We show here that T-Antigen tracks head (N-tier) first on ssDNA, opposite the direction proposed for decades. We also find that SV40 T-Antigen tracks directionally while encircling dsDNA and mainly tracks on one strand of the duplex in the same orientation as during ssDNA translocation. Further, two inward directed T-Antigen helicases on dsDNA are able to melt a 150-bp duplex. These findings explain the "rabbit ear" DNA loops observed at the SV40 origin by electron microscopy and reconfigure how the DNA loops emerge from the double hexamer relative to earlier models. Thus, the mechanism of DNA shearing by two opposing helicases is conserved in a eukaryotic viral helicase and may be widely used to initiate origin unwinding of dsDNA genomes.

Origin recognition complex harbors an intrinsic nucleosome remodeling activity.

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)2 tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

Efficacy of Intravesical Nadofaragene Firadenovec for Patients With Bacillus Calmette-Guérin-Unresponsive Nonmuscle-Invasive Bladder Cancer: 5-Year Follow-Up From a Phase 3 Trial.

PURPOSE: Nadofaragene firadenovec-vncg is a nonreplicating adenoviral vector-based gene therapy for bacillus Calmette-Guérin (BCG)-unresponsive carcinoma in situ (CIS) with/without high-grade Ta/T1. We report outcomes following 5 years of planned follow-up. MATERIALS AND METHODS: This open-label phase 3 trial (NCT02773849) enrolled patients with BCG-unresponsive nonmuscle-invasive bladder cancer in 2 cohorts: CIS ± Ta/T1 (CIS; n = 107) and Ta/T1 without CIS (Ta/T1 cohort; n = 50). Patients received 75 mL (3 × 1011 vp/mL) nadofaragene firadenovec intravesically once every 3 months with cystoscopy and cytology assessments, with continued treatment offered to those remaining high grade recurrence-free (HGRF). RESULTS: One hundred fifty-seven patients were enrolled from 33 US sites (n = 151 included in efficacy analyses). Median follow-up was 50.8 months (interquartile range 39.1-60.0), with 27% receiving ≥ 5 instillations and 7.6% receiving treatment for ≥ 57 months. Of patients with CIS 5.8% (95% CI 2.2-12.2) were HGRF at month 57, and 15% (95% CI 6.1-27.8) of patients with high-grade Ta/T1 were HGRF at month 57. Kaplan-Meier-estimated HGRF survival at 57 months was 13% (95% CI 6.9-21.5) and 33% (95% CI 19.5-46.6) in the CIS and Ta/T1 cohorts, respectively. Cystectomy-free survival at month 60 was 49% (95% CI 40.0-57.1): 43% (95% CI 32.2-53.7) in the CIS cohort and 59% (95% CI 43.1-71.4) in the Ta/T1 cohort. Overall survival at 60 months was 80% (71.0, 86.0): 76% (64.6-84.5) and 86% (70.9-93.5) in the CIS and Ta/T1 cohorts, respectively. Only 5 patients (4 with CIS and 1 with Ta/T1) experienced clinical progression to muscle-invasive disease. CONCLUSIONS: At 60 months, nadofaragene firadenovec-vncg allowed bladder preservation in nearly half of the patients and proved to be a safe option for BCG-unresponsive nonmuscle-invasive bladder cancer.

A unique Malpighian tubule architecture in Tribolium castaneum informs the evolutionary origins of systemic osmoregulation in beetles.

Maintaining internal salt and water balance in response to fluctuating external conditions is essential for animal survival. This is particularly true for insects as their high surface-to-volume ratio makes them highly susceptible to osmotic stress. However, the cellular and hormonal mechanisms that mediate the systemic control of osmotic homeostasis in beetles (Coleoptera), the largest group of insects, remain largely unidentified. Here, we demonstrate that eight neurons in the brain of the red flour beetle Tribolium castaneum respond to internal changes in osmolality by releasing diuretic hormone (DH) 37 and DH47-homologs of vertebrate corticotropin-releasing factor (CRF) hormones-to control systemic water balance. Knockdown of the gene encoding the two hormones (Urinate, Urn8) reduces Malpighian tubule secretion and restricts organismal fluid loss, whereas injection of DH37 or DH47 reverses these phenotypes. We further identify a CRF-like receptor, Urinate receptor (Urn8R), which is exclusively expressed in a functionally unique secondary cell in the beetle tubules, as underlying this response. Activation of Urn8R increases K+ secretion, creating a lumen-positive transepithelial potential that drives fluid secretion. Together, these data show that beetle Malpighian tubules operate by a fundamentally different mechanism than those of other insects. Finally, we adopt a fluorescent labeling strategy to identify the evolutionary origin of this unusual tubule architecture, revealing that it evolved in the last common ancestor of the higher beetle families. Our work thus uncovers an important homeostatic program that is key to maintaining osmotic control in beetles, which evolved parallel to the radiation of the "advanced" beetle lineages.

Empirical audit and review and an assessment of evidentiary value in research on the psychological consequences of scarcity.

Empirical audit and review is an approach to assessing the evidentiary value of a research area. It involves identifying a topic and selecting a cross-section of studies for replication. We apply the method to research on the psychological consequences of scarcity. Starting with the papers citing a seminal publication in the field, we conducted replications of 20 studies that evaluate the role of scarcity priming in pain sensitivity, resource allocation, materialism, and many other domains. There was considerable variability in the replicability, with some strong successes and other undeniable failures. Empirical audit and review does not attempt to assign an overall replication rate for a heterogeneous field, but rather facilitates researchers seeking to incorporate strength of evidence as they refine theories and plan new investigations in the research area. This method allows for an integration of qualitative and quantitative approaches to review and enables the growth of a cumulative science.

Biocultural and social determinants of ill health and early mortality in a New Mexican paediatric autopsy sample.

RESULTS.: Hispanic children have higher odds of growth stunting than non-Hispanic White children. Native American children die younger and have higher odds of respiratory diseases and porous lesions than Hispanic and non-Hispanic Whites. Rural/urban location does not significantly impact age at death, but housing type does. Individuals who lived in trailers/mobile homes had earlier ages at death. When intersections between housing type and housing location are considered, children who were poor and from impoverished areas lived longer than those who were poor from relatively well-off areas. CONCLUSIONS.: Children's health is shaped by factors outside their control. The children included in this study embodied experiences of social and ELS and did not survive to adulthood. They provide the most sobering example of the harm that social factors (structural racism/discrimination, socioeconomic, and political structures) can inflict.

Inflammatory myofibroblastic tumor of the urinary bladder: A systematic review of the literature and report of a case.

Introduction: Inflammatory myofibroblastic tumors (IMTs) are intermediate-grade lesions that frequently recur and rarely metastasize. There are currently no guidelines on the management of bladder IMTs. This systematic review aims to describe the clinical presentation and compare the management options for bladder IMTs. Methods: A PubMed/Medline search was conducted, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, using the following Mesh terms: ("inflammatory myofibroblastic") AND ("tumor") OR ("tumor") AND ("bladder") AND ("case report"). A total of 75 case reports were included in the analysis. Results: The mean age of the patients was 36 years. 65% of the cases initially presented with hematuria. 68% of the tumors stained positive for anaplastic lymphoma kinase, and 20% invaded the muscularis. Patients underwent either transurethral resection of the bladder tumor (TURBT) only (34%), TURBT followed by complementary partial cystectomy (16%), or TURBT followed by radical cystectomy (4%). 36% and 9% of the cases underwent partial and radical cystectomy after the initial diagnosis, respectively. Cystectomies were performed using an open (74%), laparoscopic (14%), robotic-assisted (10%), or unknown (2%) approach. At a mean follow-up of 14 months, the recurrence and metastasis rates were about 9% and 4%, respectively. In addition, we present the case of a 49-year-old woman with a bladder IMT who underwent TURBT followed by laparoscopic partial cystectomy. The patient remains tumor free postoperatively (follow-up period of 12 months). Conclusion: A complete surgical excision of the bladder IMT is crucial for the optimal management of these cases. Proper differentiation of this tumor from sarcoma or leiomyosarcoma leads to the best outcomes.

Modulation of sensory perception by hydrogen peroxide enables Caenorhabditis elegans to find a niche that provides both food and protection from hydrogen peroxide.

Hydrogen peroxide (H2O2) is the most common chemical threat that organisms face. Here, we show that H2O2 alters the bacterial food preference of Caenorhabditis elegans, enabling the nematodes to find a safe environment with food. H2O2 induces the nematodes to leave food patches of laboratory and microbiome bacteria when those bacterial communities have insufficient H2O2-degrading capacity. The nematode's behavior is directed by H2O2-sensing neurons that promote escape from H2O2 and by bacteria-sensing neurons that promote attraction to bacteria. However, the input for H2O2-sensing neurons is removed by bacterial H2O2-degrading enzymes and the bacteria-sensing neurons' perception of bacteria is prevented by H2O2. The resulting cross-attenuation provides a general mechanism that ensures the nematode's behavior is faithful to the lethal threat of hydrogen peroxide, increasing the nematode's chances of finding a niche that provides both food and protection from hydrogen peroxide.

Efficacy and Safety of Mitomycin Gel (UGN-101) as an Adjuvant Therapy After Complete Endoscopic Management of Upper Tract Urothelial Carcinoma.

PURPOSE: We describe a novel application of the reverse thermal polymer gel of mitomycin C (UGN-101) as adjuvant therapy after complete endoscopic ablation of upper tract urothelial carcinoma. MATERIALS AND METHODS: We retrospectively reviewed patients treated with UGN-101 from 15 high-volume centers. Adjuvant therapy was defined as treatment administered following visually complete endoscopic ablation. Response at primary endoscopic evaluation was defined as no visual tumor or negative biopsy. Ipsilateral disease-free and progression-free survival were estimated by the Kaplan-Meier method. Ureteral stenosis and other adverse events were abstracted from the medical records. Ureteral stenosis was defined as a condition requiring ureteral stent or nephrostomy, or that would typically warrant stent or nephrostomy. RESULTS: Adjuvant UGN-101 after complete endoscopic ablation was used in 52 of 115 (45%) renal units in the oncologic analysis. At first endoscopic evaluation, 36/52 (69%) were without visible disease. At 6.8 months' median follow-up, the ipsilateral disease-free rate was 63%. Recurrence after adjuvant UGN-101 therapy was more likely in multifocal tumors compared to unifocal (HR 3.3, 95% CI 1.07-9.91). Compared with UGN-101 treatment for chemoablation of measurable disease, there were significantly fewer disease detections with adjuvant therapy (P < .001). Ureteral stenosis after UGN-101 was diagnosed in 10 patients (19%) undergoing adjuvant therapy compared to 17 (29%) undergoing chemoablative therapy (P = .28). CONCLUSIONS: In patients being considered for UGN-101, maximal endoscopic ablation prior to UGN-101 treatment may result in fewer patients with disease at first endoscopy and possibly fewer adverse events than primary chemoablative therapy. Longer follow-up is needed to determine if UGN-101 after complete endoscopic ablation will lead to durable disease-free interval.

Early and recent exposure to adversity, TLR-4 stimulated inflammation, and diurnal cortisol in women with interstitial cystitis/bladder pain syndrome: A MAPP research network study.

Both early (ELA) and recent life adversity (RLA) have been linked with chronic pain conditions and persistent alterations of neuroendocrine and inflammatory responses. Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic urologic disorder characterized by bladder and/or pelvic pain, and excessive urinary frequency and/or urgency. IC/BPS has been associated with high levels of ELA as well as a distinct inflammatory signature. However, associations between ELA and RLA with inflammatory mechanisms in IC/BPS that might underlie the link between adversity and symptoms have not been examined. Here we investigated ELA and RLA in women with IC/BPS as potential risk factors for inflammatory processes and hypothalamic-pituitaryadrenal (HPA) abnormalities using data from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network. Women with IC/BPS and healthy controls (n = 154 and 32, respectively) completed surveys, collected salivary cortisol at awakening and bedtime for 3 days, and gave a blood sample which was analyzed for 7 LPS-stimulated cytokines and chemokines (IL-6, TNFα, IL-1ß, MIP1α, MCP1, IL-8, and IL-10). Two cytokine/chemokine composites were identified using principal components analysis. Patients with greater exposure to RLA or cumulative ELA and RLA of at least moderate severity showed elevated levels of a composite of all cytokines, adjusting for age, body mass index, and study site. Furthermore, there was a trending relationship between ELA and the pro-inflammatory composite score. Nocturnal cortisol and cortisol slope were not associated with ELA, RLA, or inflammation. The present findings support the importance of adverse events in IC/BPS via a biological mechanism and suggest that ELA and RLA should be assessed as risk factors for inflammation as part of a clinical workup for IC/BPS.

A genetic warning system for a hierarchically structured wildlife monitoring framework.

Genetic variation is a well-known indicator of population fitness yet is not typically included in monitoring programs for sensitive species. Additionally, most programs monitor populations at one scale, which can lead to potential mismatches with ecological processes critical to species' conservation. Recently developed methods generating hierarchically nested population units (i.e., clusters of varying scales) for greater sage-grouse (Centrocercus urophasianus) have identified population trend declines across spatiotemporal scales to help managers target areas for conservation. The same clusters used as a proxy for spatial scale can alert managers to local units (i.e., neighborhood-scale) with low genetic diversity, further facilitating identification of management targets. We developed a genetic warning system utilizing previously developed hierarchical population units to identify management-relevant areas with low genetic diversity within the greater sage-grouse range. Within this warning system we characterized conservation concern thresholds based on values of genetic diversity and developed a statistical model for microsatellite data to robustly estimate these values for hierarchically nested populations. We found that 41 of 224 neighborhood-scale clusters had low genetic diversity, 23 of which were coupled with documented local population trend decline. We also found evidence of cross-scale low genetic diversity in the small and isolated Washington population, unlikely to be reversed through typical local management actions alone. The combination of low genetic diversity and a declining population suggests relatively high conservation concern. Our findings could further facilitate conservation action prioritization in combination with population trend assessments and (or) local information, and act as a base-line of genetic diversity for future comparison. Importantly, the approach we used is broadly applicable across taxa.

Skeletal manifestations of disease experience: Length of illness and porous cranial lesion formation in a contemporary juvenile mortality sample.

OBJECTIVES: Porous lesions of the orbit (cribra orbitalia [CO]) and cranial vault (porotic hyperostosis [PH]) are used as skeletal indicators of childhood stress. Because they are understudied in contemporary populations, their relationship to disease experience is poorly understood. This paper examines the relationship between length of childhood illness and CO/PH formation in a clinically documented sample. "Turning points," which identify the window for lesion formation for CO/PH, are defined, implications for hidden heterogeneity in frailty are considered. METHODS: Data are from 333 (199 males; 134 females) pediatric postmortem computed tomography scans. Individuals died in New Mexico (2011-2019) and are 0.5 to 15.99 years (mean = 7.1). Length of illness was estimated using information from autopsy and field reports. Logistic regression was used to estimate predicted probabilities, odds ratios, and the temporal window for lesion formation. RESULTS: Illness, single bouts, or cumulative episodes lasting over 1 month is associated with higher odds of CO; individuals who were never sick have lower odds of having PH. This relationship was consistent for fatal and incidental illnesses that did not cause death. The developmental window for CO formation appears to close at 8 years. CONCLUSIONS: Those ill for over 1 month are more likely to have CO/PH than those with acute illnesses. Some individuals lived sufficiently long to form CO/PH but died of illness. Others with lesions died of circumstances unrelated to disease. This indicates hidden variation in robusticity even among ill individuals with CO/PH, which is vital in interpreting lesion frequencies in the archeological record.