Structural basis for DNA proofreading.

DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved 'bolt-action' mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps.

Analysis and Visualization of Longitudinal Genomic and Clinical Data from the AACR Project GENIE Biopharma Collaborative in cBioPortal.

International cancer registries make real-world genomic and clinical data available, but their joint analysis remains a challenge. AACR Project GENIE, an international cancer registry collecting data from 19 cancer centers, makes data from >130,000 patients publicly available through the cBioPortal for Cancer Genomics (https://genie.cbioportal.org). For 25,000 patients, additional real-world longitudinal clinical data, including treatment and outcome data, are being collected by the AACR Project GENIE Biopharma Collaborative using the PRISSMM data curation model. Several thousand of these cases are now also available in cBioPortal. We have significantly enhanced the functionalities of cBioPortal to support the visualization and analysis of this rich clinico-genomic linked dataset, as well as datasets generated by other centers and consortia. Examples of these enhancements include (i) visualization of the longitudinal clinical and genomic data at the patient level, including timelines for diagnoses, treatments, and outcomes; (ii) the ability to select samples based on treatment status, facilitating a comparison of molecular and clinical attributes between samples before and after a specific treatment; and (iii) survival analysis estimates based on individual treatment regimens received. Together, these features provide cBioPortal users with a toolkit to interactively investigate complex clinico-genomic data to generate hypotheses and make discoveries about the impact of specific genomic variants on prognosis and therapeutic sensitivities in cancer. SIGNIFICANCE: Enhanced cBioPortal features allow clinicians and researchers to effectively investigate longitudinal clinico-genomic data from patients with cancer, which will improve exploration of data from the AACR Project GENIE Biopharma Collaborative and similar datasets.

Molecular basis for maternal inheritance of human mitochondrial DNA.

Uniparental inheritance of mitochondrial DNA (mtDNA) is an evolutionary trait found in nearly all eukaryotes. In many species, including humans, the sperm mitochondria are introduced to the oocyte during fertilization1,2. The mechanisms hypothesized to prevent paternal mtDNA transmission include ubiquitination of the sperm mitochondria and mitophagy3,4. However, the causative mechanisms of paternal mtDNA elimination have not been defined5,6. We found that mitochondria in human spermatozoa are devoid of intact mtDNA and lack mitochondrial transcription factor A (TFAM)-the major nucleoid protein required to protect, maintain and transcribe mtDNA. During spermatogenesis, sperm cells express an isoform of TFAM, which retains the mitochondrial presequence, ordinarily removed upon mitochondrial import. Phosphorylation of this presequence prevents mitochondrial import and directs TFAM to the spermatozoon nucleus. TFAM relocalization from the mitochondria of spermatogonia to the spermatozoa nucleus directly correlates with the elimination of mtDNA, thereby explaining maternal inheritance in this species.

Prospective study of factors associated with asthma attack recurrence (ATTACK) in children from three Ecuadorian cities during COVID-19: a study protocol.

INTRODUCTION: Asthma is a growing health problem in children in marginalised urban settings in low-income and middle-income countries. Asthma attacks are an important cause of emergency care attendance and long-term morbidity. We designed a prospective study, the Asthma Attacks study, to identify factors associated with recurrence of asthma attacks (or exacerbations) among children and adolescents attending emergency care in three Ecuadorian cities. METHODS AND ANALYSIS: Prospective cohort study designed to identify risk factors associated with recurrence of asthma attacks in 450 children and adolescents aged 5-17 years attending emergency care in public hospitals in three Ecuadorian cities (Quito, Cuenca and Portoviejo). The primary outcome will be rate of asthma attack recurrence during up to 12 months of follow-up. Data are being collected at baseline and during follow-up by questionnaire: sociodemographic data, asthma history and management (baseline only); recurrence of asthma symptoms and attacks (monthly); economic costs of asthma to family; Asthma Control Test; Pediatric Asthma Quality of life Questionnaire; and Newcastle Asthma Knowledge Questionnaire (baseline only). In addition, the following are being measured at baseline and during follow-up: lung function and reversibility by spirometry before and after salbutamol; fractional exhaled nitric oxide (FeNO); and presence of IgG antibodies to SARS-CoV-2 in blood. Recruitment started in 2019 but because of severe disruption to emergency services caused by the COVID-19 pandemic, eligibility criteria were modified to include asthmatic children with uncontrolled symptoms and registered with collaborating hospitals. Data will be analysed using logistic regression and survival analyses. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Hospital General Docente de Calderon (CEISH-HGDC 2019-001) and Ecuadorian Ministry of Public Health (MSP-CGDES-2021-0041-O N° 096-2021). The study results will be disseminated through presentations at conferences and to key stakeholder groups including policy-makers, postgraduate theses, peer-review publications and a study website. Participants gave informed consent to participate in the study before taking part.

Genome Nexus: A Comprehensive Resource for the Annotation and Interpretation of Genomic Variants in Cancer.

PURPOSE: Interpretation of genomic variants in tumor samples still presents a challenge in research and the clinical setting. A major issue is that information for variant interpretation is fragmented across disparate databases, and aggregation of information from these requires building extensive infrastructure. To this end, we have developed Genome Nexus, a one-stop shop for variant annotation with a user-friendly interface for cancer researchers and clinicians. METHODS: Genome Nexus (1) aggregates variant information from sources that are relevant to cancer research and clinical applications, (2) allows high-performance programmatic access to the aggregated data via a unified application programming interface, (3) provides a reference page for individual cancer variants, (4) provides user-friendly tools for annotating variants in patients, and (5) is freely available under an open source license and can be installed in a private cloud or local environment and integrated with local institutional resources. RESULTS: Genome Nexus is available at https://www.genomenexus.org. It displays annotations from more than a dozen resources including those that provide variant effect information (variant effect predictor), protein sequence annotation (Uniprot, Pfam, and dbPTM), functional consequence prediction (Polyphen-2, Mutation Assessor, and SIFT), population prevalences (gnomAD, dbSNP, and ExAC), cancer population prevalences (Cancer hotspots and SignalDB), and clinical actionability (OncoKB, CIViC, and ClinVar). We describe several use cases that demonstrate the utility of Genome Nexus to clinicians, researchers, and bioinformaticians. We cover single-variant annotation, cohort analysis, and programmatic use of the application programming interface. Genome Nexus is unique in providing a user-friendly interface specific to cancer that allows high-performance annotation of any variant including unknown ones. CONCLUSION: Interpretation of cancer genomic variants is improved tremendously by having an integrated resource for annotations. Genome Nexus is freely available under an open source license.

Mechanisms of mitochondrial promoter recognition in humans and other mammalian species.

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for > 50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of the DNA in the initiation complex and permitted investigation of the sequence-specific protein-DNA interactions. We determined the molecular basis of promoter recognition by mtRNAP and TFB2M, which cooperatively recognize bases near TSS in a species-specific manner. Our findings reveal a role of mitochondrial transcription machinery in mitonuclear coevolution and speciation.

Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL.

The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.

OncoTree: A Cancer Classification System for Precision Oncology.

PURPOSE: Cancer classification is foundational for patient care and oncology research. Systems such as International Classification of Diseases for Oncology (ICD-O), Systematized Nomenclature of Medicine Clinical Terms (SNOMED-CT), and National Cancer Institute Thesaurus (NCIt) provide large sets of cancer classification terminologies but they lack a dynamic modernized cancer classification platform that addresses the fast-evolving needs in clinical reporting of genomic sequencing results and associated oncology research. METHODS: To meet these needs, we have developed OncoTree, an open-source cancer classification system. It is maintained by a cross-institutional committee of oncologists, pathologists, scientists, and engineers, accessible via an open-source Web user interface and an application programming interface. RESULTS: OncoTree currently includes 868 tumor types across 32 organ sites. OncoTree has been adopted as the tumor classification system for American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE), a large genomic and clinical data-sharing consortium, and for clinical molecular testing efforts at Memorial Sloan Kettering Cancer Center and Dana-Farber Cancer Institute. It is also used by precision oncology tools such as OncoKB and cBioPortal for Cancer Genomics. CONCLUSION: OncoTree is a dynamic and flexible community-driven cancer classification platform encompassing rare and common cancers that provides clinically relevant and appropriately granular cancer classification for clinical decision support systems and oncology research.

Genetic hallmarks of recurrent/metastatic adenoid cystic carcinoma.

BACKGROUNDAdenoid cystic carcinoma (ACC) is a rare malignancy arising in salivary glands and other sites, characterized by high rates of relapse and distant spread. Recurrent/metastatic (R/M) ACCs are generally incurable, due to a lack of active systemic therapies. To improve outcomes, deeper understanding of genetic alterations and vulnerabilities in R/M tumors is needed.METHODSAn integrated genomic analysis of 1,045 ACCs (177 primary, 868 R/M) was performed to identify alterations associated with advanced and metastatic tumors. Intratumoral genetic heterogeneity, germline mutations, and therapeutic actionability were assessed.RESULTSCompared with primary tumors, R/M tumors were enriched for alterations in key Notch (NOTCH1, 26.3% vs. 8.5%; NOTCH2, 4.6% vs. 2.3%; NOTCH3, 5.7% vs. 2.3%; NOTCH4, 3.6% vs. 0.6%) and chromatin-remodeling (KDM6A, 15.2% vs. 3.4%; KMT2C/MLL3, 14.3% vs. 4.0%; ARID1B, 14.1% vs. 4.0%) genes. TERT promoter mutations (13.1% of R/M cases) were mutually exclusive with both NOTCH1 mutations (q = 3.3 × 10-4) and MYB/MYBL1 fusions (q = 5.6 × 10-3), suggesting discrete, alternative mechanisms of tumorigenesis. This network of alterations defined 4 distinct ACC subgroups: MYB+NOTCH1+, MYB+/other, MYBWTNOTCH1+, and MYBWTTERT+. Despite low mutational load, we identified numerous samples with marked intratumoral genetic heterogeneity, including branching evolution across multiregion sequencing.CONCLUSIONThese observations collectively redefine the molecular underpinnings of ACC progression and identify further targets for precision therapies.FUNDINGAdenoid Cystic Carcinoma Research Foundation, Pershing Square Sohn Cancer Research grant, the PaineWebber Chair, Stand Up 2 Cancer, NIH R01 CA205426, the STARR Cancer Consortium, NCI R35 CA232097, the Frederick Adler Chair, Cycle for Survival, the Jayme Flowers Fund, The Sebastian Nativo Fund, NIH K08 DE024774 and R01 DE027738, and MSKCC through NIH/NCI Cancer Center Support Grant (P30 CA008748).

Confluent paths: Research and community participation to protect the right to health among transgender women in Peru.

The recognition of transgender women (TGW) as the most vulnerable population to HIV/AIDS in Peru and their inclusion as a specific key affected population in health research was the outcome of an extended process that culminated when TGW community organisations succeeded in articulating themselves as a population separate from men who have sex with men (MSM) and, in alliance with some academic research groups, documented their HIV prevalence and vulnerability factors. Prior to that process, TGW remained subsumed under the epidemiological category of men who have sex with men (MSM), invisible in the context of public health policies. Based on a growing body of academic research evidence, coupled with the increasing number and capacities of TGW representatives in technical and policy-related gatherings, a consensus emerged for the establishment of TGW health statistics separate from MSM by 2010. During the past decade, social and health research has contributed conclusive evidence on the living conditions of TGW and the structural barriers they face, beyond the focus of HIV/AIDS research. Despite such progress, pervasive barriers in public policies continue to hinder the use of existing research evidence and community experience in the development of sensitive HIV prevention and care strategies as part of a comprehensive health model for TGW in Peru.

Oncogenic Signaling Pathways in The Cancer Genome Atlas.

Genetic alterations in signaling pathways that control cell-cycle progression, apoptosis, and cell growth are common hallmarks of cancer, but the extent, mechanisms, and co-occurrence of alterations in these pathways differ between individual tumors and tumor types. Using mutations, copy-number changes, mRNA expression, gene fusions and DNA methylation in 9,125 tumors profiled by The Cancer Genome Atlas (TCGA), we analyzed the mechanisms and patterns of somatic alterations in ten canonical pathways: cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGFß signaling, p53 and ß-catenin/Wnt. We charted the detailed landscape of pathway alterations in 33 cancer types, stratified into 64 subtypes, and identified patterns of co-occurrence and mutual exclusivity. Eighty-nine percent of tumors had at least one driver alteration in these pathways, and 57% percent of tumors had at least one alteration potentially targetable by currently available drugs. Thirty percent of tumors had multiple targetable alterations, indicating opportunities for combination therapy.

Unifying cancer and normal RNA sequencing data from different sources.

Driven by the recent advances of next generation sequencing (NGS) technologies and an urgent need to decode complex human diseases, a multitude of large-scale studies were conducted recently that have resulted in an unprecedented volume of whole transcriptome sequencing (RNA-seq) data, such as the Genotype Tissue Expression project (GTEx) and The Cancer Genome Atlas (TCGA). While these data offer new opportunities to identify the mechanisms underlying disease, the comparison of data from different sources remains challenging, due to differences in sample and data processing. Here, we developed a pipeline that processes and unifies RNA-seq data from different studies, which includes uniform realignment, gene expression quantification, and batch effect removal. We find that uniform alignment and quantification is not sufficient when combining RNA-seq data from different sources and that the removal of other batch effects is essential to facilitate data comparison. We have processed data from GTEx and TCGA and successfully corrected for study-specific biases, enabling comparative analysis between TCGA and GTEx. The normalized datasets are available for download on figshare.

Cbp3 and Cbp6 are dispensable for synthesis regulation of cytochrome b in yeast mitochondria.

Cytochrome b (Cytb) is the only mitochondrial encoded subunit from the bc1 complex. Cbp3 and Cbp6 are chaperones necessary for translation of the COB mRNA and Cytb hemylation. Here we demonstrate that their role in translation is dispensable in some laboratory strains, whereas their role in Cytb hemylation seems to be universally conserved. BY4742 yeast requires Cbp3 and Cbp6 for efficient COB mRNA translation, whereas the D273-10b strain synthesizes Cytb at wildtype levels in the absence of Cbp3 and Cbp6. Steady-state levels of Cytb are close to wildtype in mutant D273-10b cells, and Cytb forms non-functional, supercomplex-like species with cytochrome c oxidase, in which at least core 1, cytochrome c1, and Rieske iron-sulfur subunits are present. We demonstrated that Cbp3 interacts with the mitochondrial ribosome and with the COB mRNA in both BY4742 and D273-10b strains. The polymorphism(s) causing the differential function of Cbp3, Cbp6, and the assembly feedback regulation of Cytb synthesis is of nuclear origin rather than mitochondrial, and Smt1, a COB mRNA-binding protein, does not seem to be involved in the observed differential phenotype. Our results indicate that the essential role of Cbp3 and Cbp6 is to assist Cytb hemylation and demonstrate that in the absence of heme b, Cytb can form non-functional supercomplexes with cytochrome c oxidase. Our observations support that an additional protein or proteins are involved in Cytb synthesis in some yeast strains.

The DEAD-box helicase Mss116 plays distinct roles in mitochondrial ribogenesis and mRNA-specific translation.

Members of the DEAD-box family are often multifunctional proteins involved in several RNA transactions. Among them, yeast Saccharomyces cerevisiae Mss116 participates in mitochondrial intron splicing and, under cold stress, also in mitochondrial transcription elongation. Here, we show that Mss116 interacts with the mitoribosome assembly factor Mrh4, is required for efficient mitoribosome biogenesis, and consequently, maintenance of the overall mitochondrial protein synthesis rate. Additionally, Mss116 is required for efficient COX1 mRNA translation initiation and elongation. Mss116 interacts with a COX1 mRNA-specific translational activator, the pentatricopeptide repeat protein Pet309. In the absence of Mss116, Pet309 is virtually absent, and although mitoribosome loading onto COX1 mRNA can occur, activation of COX1 mRNA translation is impaired. Mutations abolishing the helicase activity of Mss116 do not prevent the interaction of Mss116 with Pet309 but also do not allow COX1 mRNA translation. We propose that Pet309 acts as an adaptor protein for Mss116 action on the COX1 mRNA 5΄-UTR to promote efficient Cox1 synthesis. Overall, we conclude that the different functions of Mss116 in the biogenesis and functioning of the mitochondrial translation machinery depend on Mss116 interplay with its protein cofactors.

A Novel Function of Pet54 in Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria.

Cytochrome c oxidase assembly requires the synthesis of the mitochondria-encoded core subunits, Cox1, Cox2, and Cox3. In yeast, Pet54 protein is required to activate translation of the COX3 mRNA and to process the aI5ß intron on the COX1 transcript. Here we report a third, novel function of Pet54 on Cox1 synthesis. We observed that Pet54 is necessary to achieve an efficient Cox1 synthesis. Translation of the COX1 mRNA is coupled to the assembly of cytochrome c oxidase by a mechanism that involves Mss51. This protein activates translation of the COX1 mRNA by acting on the COX1 5'-UTR, and, in addition, it interacts with the newly synthesized Cox1 protein in high molecular weight complexes that include the factors Coa3 and Cox14. Deletion of Pet54 decreased Cox1 synthesis, and, in contrast to what is commonly observed for other assembly mutants, double deletion of cox14 or coa3 did not recover Cox1 synthesis. Our results show that Pet54 is a positive regulator of Cox1 synthesis that renders Mss51 competent as a translational activator of the COX1 mRNA and that this role is independent of the assembly feedback regulatory loop of Cox1 synthesis. Pet54 may play a role in Mss51 hemylation/conformational change necessary for translational activity. Moreover, Pet54 physically interacts with the COX1 mRNA, and this binding was independent of the presence of Mss51.

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

The Pet309 pentatricopeptide repeat motifs mediate efficient binding to the mitochondrial COX1 transcript in yeast.

Mitochondrial synthesis of Cox1, the largest subunit of the cytochrome c oxidase complex, is controlled by Mss51 and Pet309, two mRNA-specific translational activators that act via the COX1 mRNA 5'-UTR through an unknown mechanism. Pet309 belongs to the pentatricopeptide repeat (PPR) protein family, which is involved in RNA metabolism in mitochondria and chloroplasts, and its sequence predicts at least 12 PPR motifs in the central portion of the protein. Deletion of these motifs selectively disrupted translation but not accumulation of the COX1 mRNA. We used RNA coimmunoprecipitation assays to show that Pet309 interacts with the COX1 mRNA in vivo and that this association is present before processing of the COX1 mRNA from the ATP8/6 polycistronic mRNA. This association was not affected by deletion of 8 of the PPR motifs but was undetectable after deletion of the entire 12-PPR region. However, interaction of the Pet309 protein lacking 12 PPR motifs with the COX1 mRNA was detected after overexpression of the mutated form of the protein, suggesting that deletion of this region decreased the binding affinity for the COX1 mRNA without abolishing it entirely. Moreover, binding of Pet309 to the COX1 mRNA was affected by deletion of Mss51. This work demonstrates an in vivo physical interaction between a yeast mitochondrial translational activator and its target mRNA and shows the cooperativity of the PPR domains of Pet309 in interaction with the COX1 mRNA.

Bidirectional crosstalk between periventricular endothelial cells and neural progenitor cells promotes the formation of a neurovascular unit.

Interactions between neural progenitor cells (NPC) and endothelial cells (EC) from adult vascular beds have been well explored previously. However, the factors and signaling mechanisms that regulate neurogenesis and angiogenesis are most prevalent during embryonic development. This study aimed to determine whether embryonic brain endothelial cells from the periventricular region (PVEC) present an advantage over adult brain EC in supporting NPC growth and differentiation. PVEC were isolated from E15 mouse brains, processed, and sorted with immunomagnetic beads using antibodies against CD31/PECAM. On immunofluorescence (IF) staining, nearly all cells were positive for EC markers CD31 and CD144/VE-Cadherin. In proliferation studies, NPC proliferation was highest in transwell co-culture with PVEC, approximately 2.3 fold increase compared to baseline versus 1.4 fold increase when co-cultured with adult brain endothelial cells (ABEC). These results correlated with the PVEC mediated delay in NPC differentiation, evidenced by high expression of progenitor marker Nestin evaluated by IF staining. Upon further characterization of PVEC in an angiogenesis assay measuring cord length, PVEC exhibited a high capacity to form cords in basal conditions compared to ABEC. This was enhanced in the presence of NPC, with both cell types displaying a preferential structural alignment resembling neurovascular networks. PVEC also expressed high Vegfa levels at baseline in comparison to NPC and ABEC. Vegfa levels increased when co-cultured with NPC. We demonstrate that PVEC and NPC co-cultures act synergistically to promote the formation of a neurovascular unit through dynamic and reciprocal communication. Our results suggest that PVEC/NPC could provide promising neuro-regenerative therapies for patients suffering brain injuries.

Phosphatidylcholine enrichment with medium chain fatty acids by immobilized phospholipase A(1) -catalyzed acidolysis.

Phospholipids are a biologically and industrially important class of compounds whose physical properties can be improved for diverse applications by substitution of medium-chain fatty acids for their native fatty acid chains. In this study, phosphatidylcholine (PC) was enriched with medium-chain fatty acids (MCFAs) by acidolysis with phospholipase A(1) (PLA(1) ) immobilized on Duolite A568. Response surface methodology was employed to evaluate the effects of the molar ratio of substrates (PC to free MCFAs), enzyme loading, and reaction temperature on the incorporation of free MCFAs into PC and on PC recovery. Enzyme loading and molar ratio of substrates contributed positively, but temperature negatively, to the incorporation of free MCFAs into PC. Increases in enzyme loading and the molar ratio of PC to free MCFAs led to increased incorporation of the latter into the former, but increased temperature had the opposite effect. By contrast, an increase in enzyme loading led to decreased PC recovery. Increased temperature had also a negative effect on PC recovery. Optimal conditions for maximum incorporation and PC recovery were molar ratio of PC to free MCFAs of 1:16, enzyme loading of 16%, and 50°C. Under these conditions, the incorporation of free MCFAs was 41% and the PC recovery was 53%.

Sexualidade e gênero na Amazônia urbana do Peru / Sexuality and gender in urban Peru's Amazon

O tema da sexualidade na região amazônica do Peru tem sido objeto de diversas elaborações discursivas desde os tempos coloniais, destacando-se certas ideias, como intensidade e desordem. Tais concepções sedimentaram-se em representações de ampla difusão e permanência no país, sendo a charapa ardiente, representação hipertextualizada da mulher amazônica, a mais paradigmática. A existência desses discursos e a escassez de literatura acadêmica sobre o tema da sexualidade na construção de si, entre mulheres da Amazônia urbana do Peru. Para tal foi efetuada uma revisão de fontes secundárias, dirigida a rastrear a origem desta representação e sua recriação, na história do país. A seguir, a partir de informações obtidas em entrevistas em profundidade com mulheres da região investigada, foram exploradas suas opinões acerca desta representação e a maneira como lidam com ela, em circurstâncias concretas da vida cotidiana. Os relatos evidenciaram tanto processos de negação como de reprodução e resignificação, em que as mulheres se encontram. Por outro lado, foram apreendidas as trajetórias afetivo-sexuais das informantes, por intermédio de entrevistas em profundidade , a partir de indagações sobre diversos temas, como iniciação sexual, infidelidade feminina, valoração da atividade sexual e trocas econômico-sexuais, entre outros. Foram identificados eixos estruturantes da vida sexual destas mulheres. Destaca-se um discurso relacional, que enaltece a reciprocidade como marco da vida sexual e, em segundo plano, comparece também uma retórica fisicalista, que evidenciou um importante papel da sexualidade como recurso feminino, no plano econômico, em estreita articulação com dimensões afetivas e considerações familiares. Trata-se de trocas econômico-sexuais que integram a dinâmica cotidiana de reciprocidade nos vínculos afetivos-sexuais.