Eocene greenhouse climate revealed by coupled clumped isotope-Mg/Ca thermometry.

Past greenhouse periods with elevated atmospheric CO2 were characterized by globally warmer sea-surface temperatures (SST). However, the extent to which the high latitudes warmed to a greater degree than the tropics (polar amplification) remains poorly constrained, in particular because there are only a few temperature reconstructions from the tropics. Consequently, the relationship between increased CO2, the degree of tropical warming, and the resulting latitudinal SST gradient is not well known. Here, we present coupled clumped isotope (Δ47)-Mg/Ca measurements of foraminifera from a set of globally distributed sites in the tropics and midlatitudes. Δ47 is insensitive to seawater chemistry and therefore provides a robust constraint on tropical SST. Crucially, coupling these data with Mg/Ca measurements allows the precise reconstruction of Mg/Casw throughout the Eocene, enabling the reinterpretation of all planktonic foraminifera Mg/Ca data. The combined dataset constrains the range in Eocene tropical SST to 30-36 °C (from sites in all basins). We compare these accurate tropical SST to deep-ocean temperatures, serving as a minimum constraint on high-latitude SST. This results in a robust conservative reconstruction of the early Eocene latitudinal gradient, which was reduced by at least 32 ± 10% compared with present day, demonstrating greater polar amplification than captured by most climate models.

A high burden of asymptomatic genital tract infections undermines the syndromic management approach among adolescents and young adults in South Africa: implications for HIV prevention efforts.

BACKGROUND: Youth in southern Africa, particularly adolescent girls and young women, are a key population for HIV prevention interventions. Untreated genital tract infections (GTIs) increase both HIV transmission and acquisition risks. South African GTI treatment guidelines employ syndromic management, which relies on individuals to report GTI signs and symptoms. Syndromic management may, however, underestimate cases, particularly among youth. We compared genital tract infection (GTI) prevalence by symptom-based and laboratory assessment among sexually-experienced youth in South Africa, overall and stratified by sex. METHODS: Interviewer-administered surveys assessed socio-demographics, behaviors, and GTI symptoms among 352 youth (16-24 yrs., HIV-negative or unknown HIV status at enrollment) enrolled in community-based cohorts in Durban and Soweto (2014-2016). Laboratory tests assessed HIV, Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Trichomonas vaginalis (TV) infections and, among females, bacterial vaginosis (BV) and Candida species. Youth with genital ulcers were tested for HSV-2 and syphilis. We assessed sensitivity (and specificity) of symptom-based reporting in identifying laboratory-confirmed GTIs. RESULTS: At baseline, 16.2% of females (32/198) and < 1% (1/154) of males reported ≥1 GTI symptom. However, laboratory tests identified ≥1 GTI in 70.2% and 10.4%, respectively. Female CT prevalence was 18.2%, NG 7.1%, MG 9.6%, TV 8.1%, and 5.1% were newly diagnosed with HIV. BV prevalence was 53.0% and candidiasis 9.6%. One female case of herpes was identified (0 syphilis). Male CT prevalence was 7.8%, NG 1.3%, MG 3.3%, TV < 1%, and 2.0% were newly diagnosed with HIV. Overall, 77.8% of females and 100% of males with laboratory-diagnosed GTIs reported no symptoms or were asymptomatic. Sensitivity (and specificity) of symptom-based reporting was 14% (97%) among females and 0% (99%) among males. CONCLUSION: A high prevalence of asymptomatic GTIs and very poor sensitivity of symptom-based reporting undermines the applicability of syndromic GTI management, thus compromising GTI control and HIV prevention efforts among youth. Syndromic GTI management does not meet the sexual health needs of young people. Policy changes incorporating innovations in GTI diagnostic testing are needed to reduce GTIs and HIV-associated risks among youth.

Population-Level Immune-Mediated Adaptation in HIV-1 Polymerase during the North American Epidemic.

UNLABELLED: Human leukocyte antigen (HLA) class I-associated polymorphisms in HIV-1 that persist upon transmission to HLA-mismatched hosts may spread in the population as the epidemic progresses. Transmission of HIV-1 sequences containing such adaptations may undermine cellular immune responses to the incoming virus in future hosts. Building upon previous work, we investigated the extent of HLA-associated polymorphism accumulation in HIV-1 polymerase (Pol) through comparative analysis of linked HIV-1/HLA class I genotypes sampled during historic (1979 to 1989; n = 338) and modern (2001 to 2011; n = 278) eras from across North America (Vancouver, BC, Canada; Boston, MA; New York, NY; and San Francisco, CA). Phylogenies inferred from historic and modern HIV-1 Pol sequences were star-like in shape, with an inferred most recent common ancestor (epidemic founder virus) sequence nearly identical to the modern North American subtype B consensus sequence. Nevertheless, modern HIV-1 Pol sequences exhibited roughly 2-fold-higher patristic (tip-to-tip) genetic distances than historic sequences, with HLA pressures likely driving ongoing diversification. Moreover, the frequencies of published HLA-associated polymorphisms in individuals lacking the selecting HLA class I allele was on average ∼2.5-fold higher in the modern than in the historic era, supporting their spread in circulation, though some remained stable in frequency during this time. Notably, polymorphisms restricted by protective HLA alleles appear to be spreading to a greater relative extent than others, though these increases are generally of modest absolute magnitude. However, despite evidence of polymorphism spread, North American hosts generally remain at relatively low risk of acquiring an HIV-1 polymerase sequence substantially preadapted to their HLA profiles, even in the present era. IMPORTANCE: HLA class I-restricted cytotoxic T-lymphocyte (CTL) escape mutations in HIV-1 that persist upon transmission may accumulate in circulation over time, potentially undermining host antiviral immunity to the transmitted viral strain. We studied >600 experimentally collected HIV-1 polymerase sequences linked to host HLA information dating back to 1979, along with phylogenetically reconstructed HIV-1 sequences dating back to the virus' introduction into North America. Overall, our results support the gradual spread of many-though not all-HIV-1 polymerase immune escape mutations in circulation over time. This is consistent with recent observations from other global regions, though the extent of polymorphism accumulation in North America appears to be lower than in populations with high seroprevalence, older epidemics, and/or limited HLA diversity. Importantly, the risk of acquiring an HIV-1 polymerase sequence at transmission that is substantially preadapted to one's HLA profile remains relatively low in North America, even in the present era.

Genotypic and functional impact of HIV-1 adaptation to its host population during the North American epidemic.

HLA-restricted immune escape mutations that persist following HIV transmission could gradually spread through the viral population, thereby compromising host antiviral immunity as the epidemic progresses. To assess the extent and phenotypic impact of this phenomenon in an immunogenetically diverse population, we genotypically and functionally compared linked HLA and HIV (Gag/Nef) sequences from 358 historic (1979-1989) and 382 modern (2000-2011) specimens from four key cities in the North American epidemic (New York, Boston, San Francisco, Vancouver). Inferred HIV phylogenies were star-like, with approximately two-fold greater mean pairwise distances in modern versus historic sequences. The reconstructed epidemic ancestral (founder) HIV sequence was essentially identical to the North American subtype B consensus. Consistent with gradual diversification of a "consensus-like" founder virus, the median "background" frequencies of individual HLA-associated polymorphisms in HIV (in individuals lacking the restricting HLA[s]) were ∼ 2-fold higher in modern versus historic HIV sequences, though these remained notably low overall (e.g. in Gag, medians were 3.7% in the 2000s versus 2.0% in the 1980s). HIV polymorphisms exhibiting the greatest relative spread were those restricted by protective HLAs. Despite these increases, when HIV sequences were analyzed as a whole, their total average burden of polymorphisms that were "pre-adapted" to the average host HLA profile was only ∼ 2% greater in modern versus historic eras. Furthermore, HLA-associated polymorphisms identified in historic HIV sequences were consistent with those detectable today, with none identified that could explain the few HIV codons where the inferred epidemic ancestor differed from the modern consensus. Results are therefore consistent with slow HIV adaptation to HLA, but at a rate unlikely to yield imminent negative implications for cellular immunity, at least in North America. Intriguingly, temporal changes in protein activity of patient-derived Nef (though not Gag) sequences were observed, suggesting functional implications of population-level HIV evolution on certain viral proteins.

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function.

UNLABELLED: HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24(Gag) (GQMVHQAIGag140-147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCE: Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.

Intersubtype differences in the effect of a rare p24 gag mutation on HIV-1 replicative fitness.

Certain immune-driven mutations in HIV-1, such as those arising in p24(Gag), decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24(Gag) M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24(Gag) codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.

Correlates of protective cellular immunity revealed by analysis of population-level immune escape pathways in HIV-1.

HLA class I-associated polymorphisms identified at the population level mark viral sites under immune pressure by individual HLA alleles. As such, analysis of their distribution, frequency, location, statistical strength, sequence conservation, and other properties offers a unique perspective from which to identify correlates of protective cellular immunity. We analyzed HLA-associated HIV-1 subtype B polymorphisms in 1,888 treatment-naïve, chronically infected individuals using phylogenetically informed methods and identified characteristics of HLA-associated immune pressures that differentiate protective and nonprotective alleles. Over 2,100 HLA-associated HIV-1 polymorphisms were identified, approximately one-third of which occurred inside or within 3 residues of an optimally defined cytotoxic T-lymphocyte (CTL) epitope. Differential CTL escape patterns between closely related HLA alleles were common and increased with greater evolutionary distance between allele group members. Among 9-mer epitopes, mutations at HLA-specific anchor residues represented the most frequently detected escape type: these occurred nearly 2-fold more frequently than expected by chance and were computationally predicted to reduce peptide-HLA binding nearly 10-fold on average. Characteristics associated with protective HLA alleles (defined using hazard ratios for progression to AIDS from natural history cohorts) included the potential to mount broad immune selection pressures across all HIV-1 proteins except Nef, the tendency to drive multisite and/or anchor residue escape mutations within known CTL epitopes, and the ability to strongly select mutations in conserved regions within HIV's structural and functional proteins. Thus, the factors defining protective cellular immune responses may be more complex than simply targeting conserved viral regions. The results provide new information to guide vaccine design and immunogenicity studies.

A new stem group echinoid from the Triassic of China leads to a revised macroevolutionary history of echinoids during the end-Permian mass extinction.

The Permian-Triassic bottleneck has long been thought to have drastically altered the course of echinoid evolution, with the extinction of the entire echinoid stem group having taken place during the end-Permian mass extinction. The Early Triassic fossil record of echinoids is, however, sparse, and new fossils are paving the way for a revised interpretation of the evolutionary history of echinoids during the Permian-Triassic crisis and Early Mesozoic. A new species of echinoid, Yunnanechinus luopingensis n. sp. recovered from the Middle Triassic (Anisian) Luoping Biota fossil Lagerstätte of South China, displays morphologies that are not characteristic of the echinoid crown group. We have used phylogenetic analyses to further demonstrate that Yunnanechinus is not a member of the echinoid crown group. Thus a clade of stem group echinoids survived into the Middle Triassic, enduring the global crisis that characterized the end-Permian and Early Triassic. Therefore, stem group echinoids did not go extinct during the Palaeozoic, as previously thought, and appear to have coexisted with the echinoid crown group for at least 23 million years. Stem group echinoids thus exhibited the Lazarus effect during the latest Permian and Early Triassic, while crown group echinoids did not.

Three dimensional reconstructions of Nummulites tests reveal complex chamber shapes.

Larger benthic foraminifera (LBF) are important and prolific carbonate producers both in modern and ancient shallow tropical seas. During the Paleogene the genus Nummulites was particularly abundant with a global distribution, leading it to be frequently used in biostratigraphy. However, their evolution is poorly understood as classification is Europe-centered and mostly based on external characters and equatorial thin sections. New occurrences from regions outside the northern Tethys which poorly fit in thus reference frame, show that a more rigid framework for the classification of Nummulites is needed. Here we apply micro computed-tomographical scanning, a tool that recently has become available, to visualise 3D chamber shape of Nummulites djokdjokartae and compare these to traditional morphometrical characters. We find that despite the regular shape in equatorial and axial thin section the irregular 3D chamber shape is not predicted by these sections. We argue that 3D reconstructions of Nummulites tests will be a great aid in improving our understanding of lineages within the genus Nummulites, and to elucidate its evolutionary and biogeographical history.

Teen dating abuse: recognition and interventions.

Teen dating abuse, also known as teen dating violence, is a significant public health issue. Adolescents with a history of dating abuse may struggle academically and experience increased risk for serious injury or even death. They may engage in risky sexual behavior, substance abuse, and unhealthy dieting and exhibit suicidal behaviors. School nurses may be the first adults that teens confide in when experiencing dating abuse and may lack the knowledge and skills to intervene with teens involved in unhealthy dating relationships. Beginning in 2008, Dell Children s Medical Center in Austin, Texas, partnered with SafePlace (a local nonprofit that serves survivors of sexual and domestic violence) to address dating abuse. This collaboration is part of Start Strong Austin, one of 11 communities nationwide participating in the Start Strong: Building Healthy Teen Relationships Initiative funded by the Robert Wood Johnson Foundation. The Start Strong model employs innovative strategies in education, community engagement, policy change, and social marketing to prevent dating abuse before it starts.

HLA class I sequence-based typing using DNA recovered from frozen plasma.

We describe a rapid, reliable and cost-effective method for intermediate-to-high-resolution sequence-based HLA class I typing using frozen plasma as a source of genomic DNA. The plasma samples investigated had a median age of 8.5 years. Total nucleic acids were isolated from matched frozen PBMC (~2.5 million) and plasma (500 µl) samples from a panel of 25 individuals using commercial silica-based kits. Extractions yielded median [IQR] nucleic acid concentrations of 85.7 [47.0-130.0]ng/µl and 2.2 [1.7-2.6]ng/µl from PBMC and plasma, respectively. Following extraction, ~1000 base pair regions spanning exons 2 and 3 of HLA-A, -B and -C were amplified independently via nested PCR using universal, locus-specific primers and sequenced directly. Chromatogram analysis was performed using commercial DNA sequence analysis software and allele interpretation was performed using a free web-based tool. HLA-A, -B and -C amplification rates were 100% and chromatograms were of uniformly high quality with clearly distinguishable mixed bases regardless of DNA source. Concordance between PBMC and plasma-derived HLA types was 100% at the allele and protein levels. At the nucleotide level, a single partially discordant base (resulting from a failure to call both peaks in a mixed base) was observed out of >46,975 bases sequenced (>99.9% concordance). This protocol has previously been used to perform HLA class I typing from a variety of genomic DNA sources including PBMC, whole blood, granulocyte pellets and serum, from specimens up to 30 years old. This method provides comparable specificity to conventional sequence-based approaches and could be applied in situations where cell samples are unavailable or DNA quantities are limiting.