A critical view on autoantibodies in lupus nephritis: Concrete knowledge based on evidence.

Deposition of autoantibodies in glomeruli is a key factor in the development of lupus nephritis (LN). For a long time, anti-dsDNA and anti-C1q antibodies were thought to be the main cause of the kidney damage. However, recent studies have shown that the list of autoantibidies that have renal tropism and deposit in the kidney in LN is increasing and the link between anti-dsDNA and renal pathology is weak due to potential confounders. Aspecific bindings of dsDNA with cationic antibodies and of anti-dsDNA with several renal antigens such as actinin, laminin, entactin, and annexinA2 raised doubts about the specific target of these antibodies in the kidney. Moreover, the isotype of anti-dsDNA in SLE and LN has never received adequate interest until the recent observation that IgG2 are preponderant over IgG1, IgG3 and IgG4. Based on the above background, recent studies investigated the involvement of anti-dsDNA IgG2 and of other antibodies in LN. It was concluded that circulating anti-dsDNA IgG2 levels do not distinguish between LN versus non-renal SLE, and, in patients with LN, their levels do not change over time. Circulating levels of other antibodies such as anti-ENO1 and anti-H2 IgG2 were, instead, higher in LN vs non-renal SLE at the time of diagnosis and decreased following therapies. Finally, new classes of renal antibodies that potentially modify the anti-inflammatory response in the kidney are emerging as new co-actors in the pathogenetic scenario. They have been defined as 'second wave antibodies' for the link with detoxifying mechanisms limiting the oxidative stress in glomeruli that are classically stimulated in a second phase of inflammation. These findings have important clinical implications that may modify the laboratory approach to LN. Serum levels of anti-ENO1 and anti-H2 IgG2 should be measured in the follow up of patients for designing the length of therapies and identify those patients who respond to treatments. Anti-SOD2 could help to monitor and potentiate the anti-inflammatory response in the kidney.

Genome-wide epitope mapping across multiple host species reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests.

Natural immunity to malaria preferentially targets the endothelial protein C receptor-binding regions of PfEMP1s.

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Control of maternal Zika virus infection during pregnancy is associated with lower antibody titers in a macaque model.

Introduction: Zika virus (ZIKV) infection during pregnancy results in a spectrum of birth defects and neurodevelopmental deficits in prenatally exposed infants, with no clear understanding of why some pregnancies are more severely affected. Differential control of maternal ZIKV infection may explain the spectrum of adverse outcomes. Methods: Here, we investigated whether the magnitude and breadth of the maternal ZIKV-specific antibody response is associated with better virologic control using a rhesus macaque model of prenatal ZIKV infection. We inoculated 18 dams with an Asian-lineage ZIKV isolate (PRVABC59) at 30-45 gestational days. Plasma vRNA and infectious virus kinetics were determined over the course of pregnancy, as well as vRNA burden in the maternal-fetal interface (MFI) at delivery. Binding and neutralizing antibody assays were performed to determine the magnitude of the ZIKV-specific IgM and IgG antibody responses throughout pregnancy, along with peptide microarray assays to define the breadth of linear ZIKV epitopes recognized. Results: Dams with better virologic control (n= 9) cleared detectable infectious virus and vRNA from the plasma by 7 days post-infection (DPI) and had a lower vRNA burden in the MFI at delivery. In comparison, dams with worse virologic control (n= 9) still cleared detectable infectious virus from the plasma by 7 DPI but had vRNA that persisted longer, and had higher vRNA burden in the MFI at delivery. The magnitudes of the ZIKV-specific antibody responses were significantly lower in the dams with better virologic control, suggesting that higher antibody titers are not associated with better control of ZIKV infection. Additionally, the breadth of the ZIKV linear epitopes recognized did not differ between the dams with better and worse control of ZIKV infection. Discussion: Thus, the magnitude and breadth of the maternal antibody responses do not seem to impact maternal virologic control. This may be because control of maternal infection is determined in the first 7 DPI, when detectable infectious virus is present and before robust antibody responses are generated. However, the presence of higher ZIKV-specific antibody titers in dams with worse virologic control suggests that these could be used as a biomarker of poor maternal control of infection and should be explored further.

The association between foot posture, single leg balance and running biomechanics of the foot.

OBJECTIVES: A mal-aligned foot posture (high-arched and flat feet) and poor single leg balance ability have been separately associated with foot injuries during running. Therefore, clinicians assess these routinely. However, the extent to which foot posture and single-leg balance ability affect actual foot biomechanics during running is not known. This study aims to investigate the association of foot posture, single-leg balance ability, and foot biomechanics during running. METHOD: This is a cross sectional study of sixty-nine participants who had their foot postures and single-leg balance ability assessed. The Foot Posture Index and Balance Error Scoring System were used. Their foot kinetics was measured as they ran on an instrumented treadmill and foot kinematics was processed using a 3D motion capture system. Multiple-regression was used to analyse the variance of foot biomechanics explained by foot posture and single-leg balance ability. RESULTS: Foot posture and single-leg balance ability were found to account significantly for the variance in rearfoot eversion (24%) and forefoot dorsiflexion (7%). Two regression equations were derived, where rearfoot eversion and forefoot dorsiflexion during running may be predicted. CONCLUSION: Foot posture and single-leg balance ability can predict rearfoot eversion and forefoot dorsiflexion only during running. Based on the regression equations, individuals with the same foot posture but different single-leg balance ability may exhibit different foot kinematics. However, the angular differences are small. The equations may be useful for clinicians working in places where running gait analysis equipment are not readily accessible. Further studies with larger sample sizes are required to validate these equations. In addition, further studies are necessary to investigate the effect of these two variables under different running conditions e.g. with footwear and with orthoses.

Discovery of anti-Formin-like 1 protein (FMNL1) antibodies in membranous nephropathy and other glomerular diseases.

Evidence has shown that podocyte-directed autoantibodies can cause membranous nephropathy (MN). In the present work we investigated sera of MN patients using a high-density peptide array covering the whole coding sequences of the human genome encompassing 7,499,126 tiled peptides. A panel of 21 proteins reactive to MN sera were identified. We focused our attention on Formin-like 1 (FMNL1), a protein expressed by macrophages in MN patients tissues. High levels of anti-FMNL1 IgG4 were demonstrated in sera of MN patients with an orthogonal methodology (ELISA) contemporary demonstrating FMNL1 positive cells in kidney co-staining with CD68 in glomeruli. High levels of circulating anti-FMNL1 IgG4 were associated with lack of remission of proteinuria, potentially indicating that autoantibodies directed against cells other than podocytes, involved in tissue repair, might play a role in MN disease progression. High serum levels of anti-FMNL1 IgGs were also observed in other non-autoimmune glomerolonephrites, i.e. idiopathic and genetic FSGS, IgAGN. These findings are suggestive of a broader role of those autoantibodies in other glomerular disease conditions.

An Analysis of the Effects of Spaceflight and Vaccination on Antibody Repertoire Diversity.

Ab repertoire diversity plays a critical role in the host's ability to fight pathogens. CDR3 is partially responsible for Ab-Ag binding and is a significant source of diversity in the repertoire. CDR3 diversity is generated during VDJ rearrangement because of gene segment selection, gene segment trimming and splicing, and the addition of nucleotides. We analyzed the Ab repertoire diversity across multiple experiments examining the effects of spaceflight on the Ab repertoire after vaccination. Five datasets from four experiments were analyzed using rank-abundance curves and Shannon indices as measures of diversity. We discovered a trend toward lower diversity as a result of spaceflight but did not find the same decrease in our physiological model of microgravity in either the spleen or bone marrow. However, the bone marrow repertoire showed a reduction in diversity after vaccination. We also detected differences in Shannon indices between experiments and tissues. We did not detect a pattern of CDR3 usage across the experiments. Overall, we were able to find differences in the Ab repertoire diversity across experimental groups and tissues.

Epitope-Specific Antibody Responses to a Plasmodium falciparum Subunit Vaccine Target in a Malaria-Endemic Population.

Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.

Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination.

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.

Oral dextrose reduced procedural pain without altering cellular ATP metabolism in preterm neonates: a prospective randomized trial.

OBJECTIVE: To examine the effects of 30% oral dextrose on biochemical markers of pain, adenosine triphosphate (ATP) degradation, and oxidative stress in preterm neonates experiencing a clinically required heel lance. STUDY DESIGN: Utilizing a prospective study design, preterm neonates that met study criteria (n = 169) were randomized to receive either (1) 30% oral dextrose, (2) facilitated tucking, or (3) 30% oral dextrose and facilitated tucking 2 min before heel lance. Plasma markers of ATP degradation (hypoxanthine, uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the premature infant pain profile-revised (PIPP-R). RESULTS: Oral dextrose, administered alone or with facilitated tucking, did not alter plasma markers of ATP utilization and oxidative stress. CONCLUSION: A single dose of 30% oral dextrose, given before a clinically required heel lance, decreased signs of pain without increasing ATP utilization and oxidative stress in premature neonates.

Oxidative Stress Biomarker Decreased in Preterm Neonates Treated With Kangaroo Mother Care.

OBJECTIVE: Due to physiological and metabolic immaturity, prematurely born infants are at increased risk because of maternal separation in many neonatal intensive care units (NICUs). The stress induced from maternal-infant separation can lead to well-documented short-term physiologic instability and potentially lifelong neurological, sociological, or psychological sequelae. Based on previous studies of kangaroo mother care (KMC) that demonstrated improvement in physiologic parameters, we examined the impact of KMC on physiologic measures of stress (abdominal temperature, heart rate, oxygen saturation, perfusion index, near-infrared spectrometry), oxidative stress, and energy utilization/conservation in preterm infants. METHODS: In this randomized, stratified study of premature neonates, we compared the effects on urinary concentrations of biomarkers of energy utilization and oxidative stress of 1 hr of KMC versus incubator care on Day 3 of life in intervention-group babies (n = 26) and control-group babies (n = 25), respectively. On Day 4, both groups received 1 hr of KMC. Urinary samples were collected 3 hr before and 3 hr after intervention/incubator care on both days. Energy utilization was assessed by measures of adenosine triphosphate (ATP) degradation (i.e., hypoxanthine, xanthine, and uric acid). Oxidative stress was assessed using urinary allantoin. Mixed-models analysis was used to assess differences in purine/allantoin. RESULTS: Mean allantoin levels over Days 3 and 4 were significantly lower in the KMC group than in the control group (p = .026). CONCLUSIONS: Results provide preliminary evidence that KMC reduces neonatal oxidative stress processes and that urinary allantoin could serve as an effective noninvasive marker for future studies.

Comprehensive Profiling of the Rheumatoid Arthritis Antibody Repertoire.

OBJECTIVE: Autoantibodies against citrullinated proteins are found in 64-89% of rheumatoid arthritis (RA) patients, with 88-99% specificity. This study was undertaken to create an unbiased, comprehensive profile of serum antibodies against the human proteome, including the citrullinome and the homocitrullinome, in RA patients, using a high-density peptide array. METHODS: Our high-density peptide array, consisting of >4.6 million peptides, contained the entire annotated human proteome. The 20,246 proteins were represented as overlapping 16-mer peptides. In addition to native peptides, citrullinated and homocitrullinated peptides were included, as substitutions for arginine and lysine, and provided a comprehensive screen against all possible epitopes. Twenty-six serum samples (from 8 controls and 18 RA patients) were profiled on the high-density peptide array. Using RA-specific epitopes, we constructed an 8-epitope diagnostic biomarker on a Gyrolab xPlore instrument with a cohort of 92 serum samples (from 29 controls and 63 RA patients). The diagnostic biomarker was further validated with an independent cohort of 181 serum samples (from 54 controls and 127 RA patients). RESULTS: In the initial cohort the diagnostic performance of the 8-epitope biomarker yielded 96.6% specificity and 92.1% sensitivity. The overall diagnostic performance in the validation cohort was 94.4% specificity and 85% sensitivity. In both cohorts, the performance of the 8-epitope diagnostic biomarker compared favorably against the Abnova cyclic citrullinated peptide 2 (CCP2) assay. Using data from the peptide array, we identified novel RA-specific epitopes and formed the basis of a new RA diagnostic assay. CONCLUSION: Comprehensive antibody profiling using a high-density peptide array not only identified novel RA-specific epitopes but also allowed us to construct a novel diagnostic biomarker that is as specific as and more sensitive than the Abnova CCP2 assay.

Antibodies to Peptides in Semiconserved Domains of RIFINs and STEVORs Correlate with Malaria Exposure.

The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria.IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.

Discovery and Structural Optimization of Acridones as Broad-Spectrum Antimalarials.

Malaria remains one of the deadliest diseases in the world today. Novel chemoprophylactic and chemotherapeutic antimalarials are needed to support the renewed eradication agenda. We have discovered a novel antimalarial acridone chemotype with dual-stage activity against both liver-stage and blood-stage malaria. Several lead compounds generated from structural optimization of a large library of novel acridones exhibit efficacy in the following systems: (1) picomolar inhibition of in vitro Plasmodium falciparum blood-stage growth against multidrug-resistant parasites; (2) curative efficacy after oral administration in an erythrocytic Plasmodium yoelii murine malaria model; (3) prevention of in vitro Plasmodium berghei sporozoite-induced development in human hepatocytes; and (4) protection of in vivo P. berghei sporozoite-induced infection in mice. This study offers the first account of liver-stage antimalarial activity in an acridone chemotype. Details of the design, chemistry, structure-activity relationships, safety, metabolic/pharmacokinetic studies, and mechanistic investigation are presented herein.

Contribution of non-HLA incompatibility between donor and recipient to kidney allograft survival: genome-wide analysis in a prospective cohort.

BACKGROUND: The introduction of HLA matching of donors and recipients was a breakthrough in kidney transplantation. However, half of all transplanted kidneys still fail within 15 years after transplantation. Epidemiological data suggest a fundamental role of non-HLA alloimmunity. METHODS: We genotyped 477 pairs of deceased donors and first kidney transplant recipients with stable graft function at three months that were transplanted between Dec 1, 2005, and April 30, 2015. Genome-wide genetic mismatches in non-synonymous single nucleotide polymorphisms (nsSNPs) were calculated to identify incompatibilities in transmembrane and secreted proteins. We estimated the association between nsSNP mismatch and graft loss in a Cox proportional hazard model, adjusting for HLA mismatch and clinical covariates. Customised peptide arrays were generated to screen for antibodies against genotype-derived mismatched epitopes in 25 patients with biopsy-confirmed chronic antibody-mediated rejection. FINDINGS: 59 268 nsSNPs affecting a transmembrane or secreted protein were analysed. The median number of nsSNP mismatches in immune-accessible transmembrane and secreted proteins between donors and recipients was 1892 (IQR 1850-1936). The degree of nsSNP mismatch was independently associated with graft loss in a multivariable model adjusted for HLA eplet mismatch (HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR). Each increase by a unit of one IQR had an HR of 1·68 (95% CI 1·17-2·41, p=0·005). 5-year death censored graft survival was 98% in the quartile with the lowest mismatch, 91% in the second quartile, 89% in the third quartile, and 82% in the highest quartile (p=0·003, log-rank test). Customised peptide arrays verified a donor-specific alloimmune response to genetically predicted mismatched epitopes. INTERPRETATION: Genetic mismatch of non-HLA haplotypes coding for transmembrane or secreted proteins is associated with an increased risk of functional graft loss independently of HLA incompatibility. As in HLA alloimmunity, donor-specific alloantibodies can be identified against genotype derived non-HLA epitopes. FUNDING: Austrian Science Fund, WWTF (Vienna Science and Technology Fund), and Ministry of Health of the Czech Republic.

Antibody responses to Zika virus proteins in pregnant and non-pregnant macaques.

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens.

Systems genetic analysis of inversion polymorphisms in the malaria mosquito Anopheles gambiae.

Inversion polymorphisms in the African malaria vector Anopheles gambiae segregate along climatic gradients of aridity. Despite indirect evidence of their adaptive significance, little is known of the phenotypic targets of selection or the underlying genetic mechanisms. Here we adopt a systems genetics approach to explore the interaction of two inversions on opposite arms of chromosome 2 with gender, climatic conditions, and one another. We measure organismal traits and transcriptional profiles in 8-d-old adults of both sexes and four alternative homokaryotypic classes reared under two alternative climatic regimes. We show that karyotype strongly influences both organismal traits and transcriptional profiles but that the strength and direction of the effects depend upon complex interactions with gender and environmental conditions and between inversions on independent arms. Our data support the suppressed recombination model for the role of inversions in local adaptation, and-supported by transcriptional and physiological measurements following perturbation with the drug rapamycin-suggest that one mechanism underlying their adaptive role may be the maintenance of energy homeostasis.

A Plasmodium yoelii HECT-like E3 ubiquitin ligase regulates parasite growth and virulence.

Infection of mice with strains of Plasmodium yoelii parasites can result in different pathology, but molecular mechanisms to explain this variation are unclear. Here we show that a P. yoelii gene encoding a HECT-like E3 ubiquitin ligase (Pyheul) influences parasitemia and host mortality. We genetically cross two lethal parasites with distinct disease phenotypes, and identify 43 genetically diverse progeny by typing with microsatellites and 9230 single-nucleotide polymorphisms. A genome-wide quantitative trait loci scan links parasite growth and host mortality to two major loci on chromosomes 1 and 7 with LOD (logarithm of the odds) scores = 6.1 and 8.1, respectively. Allelic exchange of partial sequences of Pyheul in the chromosome 7 locus and modification of the gene expression alter parasite growth and host mortality. This study identifies a gene that may have a function in parasite growth, virulence, and host-parasite interaction, and therefore could be a target for drug or vaccine development.Many strains of Plasmodium differ in virulence, but factors that control these distinctions are not known. Here the authors comparatively map virulence loci using the offspring from a P. yoelii YM and N67 genetic cross, and identify a putative HECT E3 ubiquitin ligase that may explain the variance.

Targeted LncRNA Sequencing with the SeqCap RNA Enrichment System.

Sequencing-based whole-transcriptome analysis (i.e., RNA-Seq) can be a powerful tool when used to measure gene expression, detect novel transcripts, characterize transcript isoforms, and identify sequence polymorphisms. However, this method can be inefficient when the goal is to study only one component of the transcriptome, such as long noncoding RNAs (lncRNAs), which constitute only a small fraction of transcripts in a total RNA sample. Here, we describe a target enrichment method where a total RNA sample is converted to a sequencing-ready cDNA library and hybridized to a complex pool of lncRNA-specific biotinylated long oligonucleotide capture probes prior to sequencing. The resulting sequence data are highly enriched for the targets of interest, dramatically increasing the efficiency of next-generation sequencing approaches for the analysis of lncRNAs.

Population Structure Shapes Copy Number Variation in Malaria Parasites.

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.