Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome.

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two sub-assemblies: Retriever composed of VPS35L, VPS26C, and VPS29; and the CCC complex which contains twelve subunits: COMMD1-COMMD10 and the coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy, and in silico predictions, we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilized by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a 16th subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

Discovery and mechanistic study of Imperatorin that inhibits HBsAg expression and cccDNA transcription.

Chronic hepatitis B virus (HBV) infection remains a significant global health challenge due to its link to severe conditions like HBV-related cirrhosis and hepatocellular carcinoma (HCC). Although current treatments effectively reduce viral levels, they have limited impact on certain HBV elements, namely hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA). This highlights the urgent need for innovative pharmaceutical and biological interventions that can disrupt HBsAg production originating from cccDNA. In this study, we identified a natural furanocoumarin compound, Imperatorin, which markedly inhibited the expression of HBsAg from cccDNA, by screening a library of natural compounds derived from Chinese herbal medicines using ELISA assay and qRT-PCR. The pharmacodynamics study of Imperatorin was explored on HBV infected HepG2-NTCP/PHHs and HBV-infected humanized mouse model. Proteome analysis was performed on HBV infected HepG2-NTCP cells following Imperatorin treatment. Molecular docking and bio-layer interferometry (BLI) were used for finding the target of Imperatorin. Our findings demonstrated Imperatorin remarkably reduced the level of HBsAg, HBV RNAs, HBV DNA and transcriptional activity of cccDNA both in vitro and in vivo. Additionally, Imperatorin effectively restrained the actions of HBV promoters responsible for cccDNA transcription. Mechanistic study revealed that Imperatorin directly binds to ERK and subsequently interfering with the activation of CAMP response element-binding protein (CREB), a crucial transcriptional factor for HBV and has been demonstrated to bind to the PreS2/S and X promoter regions of HBV. Importantly, the absence of ERK could nullify the antiviral impact triggered by Imperatorin. Collectively, the natural compound Imperatorin may be an effective candidate agent for inhibiting HBsAg production and cccDNA transcription by impeding the activities of HBV promoters through ERK-CREB axis.

Diversification of refugia types needed to secure the future of coral reefs subject to climate change.

Identifying locations of refugia from the thermal stresses of climate change for coral reefs and better managing them is one of the key recommendations for climate change adaptation. We review and summarize approximately 30 years of applied research focused on identifying climate refugia to prioritize the conservation actions for coral reefs under rapid climate change. We found that currently proposed climate refugia and the locations predicted to avoid future coral losses are highly reliant on  excess heat metrics, such as degree heating weeks. However, many existing alternative environmental, ecological, and life-history variables could be used to identify other types of refugia that lead to the desired diversified portfolio for coral reef conservation. To improve conservation priorities for coral reefs, there is a need to evaluate and validate the predictions of climate refugia with long-term field data on coral abundance, diversity, and functioning. There is also the need to identify and safeguard locations displaying resistance toprolonged exposure to heat waves and the ability to recover quickly after thermal exposure. We recommend using more metrics to identify a portfolio of potential refugia sites for coral reefs that can avoid, resist, and recover from exposure to high ocean temperatures and the consequences of climate change, thereby shifting past efforts focused on avoidance to a diversified risk-spreading portfolio that can be used to improve strategic coral reef conservation in a rapidly warming climate.

Diversificación de los tipos de refugio necesarios para asegurar el futuro de los arrecifes de coral sujetos al cambio climático Resumen Una de las principales recomendaciones para la adaptación al cambio climático es identificar los refugios de los arrecifes de coral frente al estrés térmico del cambio climático y mejorar su gestión. Revisamos y resumimos ∼30 años de investigación aplicada centrada en la identificación de refugios climáticos para priorizar las acciones de conservación de los arrecifes de coral bajo un rápido cambio climático. Descubrimos que los refugios climáticos propuestos actualmente y las ubicaciones que pueden evitarlos dependen en gran medida de métricas de exceso de calor, como las semanas de calentamiento en grados (SCG). Sin embargo, existen muchas variables alternativas de historia vital, ambientales y ecológicas que podrían utilizarse para identificar otros tipos de refugios que resulten en el acervo diversificado que se desea para la conservación de los arrecifes de coral. Para mejorar las prioridades de conservación de los arrecifes de coral, es necesario evaluar y validar las predicciones sobre refugios climáticos con datos de campo a largo plazo sobre abundancia, diversidad y funcionamiento de los corales. También es necesario identificar y salvaguardar lugares que muestren resistencia a la exposición climática prolongada a olas de calor y la capacidad de recuperarse rápidamente tras la exposición térmica. Recomendamos utilizar más métricas para identificar un acervo de posibles lugares de refugio para los arrecifes de coral que puedan evitar, resistir y recuperarse de la exposición a las altas temperaturas oceánicas y las consecuencias del cambio climático, para así desplazar los esfuerzos pasados centrados en la evitación hacia un acervo diversificado de riesgos que pueda utilizarse para mejorar la conservación estratégica de los arrecifes de coral en un clima que se calienta rápidamente.

Understanding variation in impacts from private protected areas across regions and protection mechanisms to inform organizational practices.

Private land protection is an important and growing tool to address biodiversity loss and climate change. Thus, better empirical evidence on the effectiveness of private land protection and organizational practices, such as targeting of lands for protection and choice of protection mechanism (i.e., fee simple land acquisition and conservation easements), is needed. We addressed this gap by estimating the impacts of The Nature Conservancy's (TNC) (a large nongovernmental organization with relatively decentralized management) conservation land acquisitions and easements from 1988 to 2016 in three regions of the United States (Mid-Atlantic, New England and New York, and California). We estimated impact in terms of avoided conversion by comparing natural land cover on 3179 protected parcels with matched unprotected parcels. Nineteen of 21 ecoregional plans used threats of agriculture and development to identify priorities for protection. When regions and protection mechanisms were pooled, on average there was no evidence of avoided conversion from 1988 to 2016. Accounting for mechanisms, TNC land acquisitions avoided conversion and easements did not. TNC's easements on parcels acquired by conservation partners did avoid conversion. Limitations of these results include focus on a single measure of impact, inability to capture future avoided conversion, and low land cover change accuracy in California. Our results suggest that private land protection managers who seek to avoid land conversion in the near to medium term should increase focus on areas with higher threats. Special attention should be paid to strengthening accountability and the role of partners, improving or clarifying how easements are used, and facilitating the flow of resources to work with the greatest potential impact.

Comprensión de la variación en el impacto de las áreas protegidas privadas sobre las regiones y los mecanismos de protección para guiar las prácticas organizativas Resumen La protección de terreno privado es una herramienta importante, aunque su naturaleza voluntaria puede sesgar la protección hacia parcelas menos amenazadas­lo que resulta en que la conversión no se evite o se evite muy poco. Además, muchos programas de protección privada tienen una supervisión limitada y pocos reportes de sus resultados. Por lo tanto, se necesitan mejores evidencias empíricas de la efectividad de la protección en suelo privado y las prácticas organizativas, como el enfoque en tierras para protección y la selección de los mecanismos de protección (adquisición de terrenos a título oneroso y servidumbres de conservación). Abordamos esta brecha con la estimación de impactos de las adquisiciones de suelo de The Nature Conservancy (TNC, una gran organización no gubernamental con un manejo relativamente descentralizado) y las servidumbres implementadas entre 1988 y 2016 en tres regiones de los Estados Unidos: Atlántico Medio, Nueva Inglaterra y Nueva York, y California. Estimamos el impacto en términos de la conversión que se evitó al comparar la cobertura de suelo en 3,179 parcelas protegidas con parcelas desprotegidas equivalentes. Diecinueve de los 21 planes eco­regionales usaron las amenazas a la agricultura y al desarrollo para identificar las prioridades de protección. Cuando agrupamos las regiones y los mecanismos de protección, en promedio no hubo impacto alguno. Si se consideran los mecanismos, las adquisiciones de suelo de la TNC tuvieron un impacto mientras que las servidumbres no. Las servidumbres de la TNC en las parcelas adquiridas por socios de conservación sí tuvieron un impacto, aunque esta manera de proteger sólo se presentó en las regiones del Atlántico Medio y de Nueva Inglaterra y Nueva York. Nuestros resultados sugieren que la protección privada, especialmente mediante servidumbres, puede estar sesgada hacia suelos no amenazados. Los gestores que buscan evitar la conversión del suelo a mediano o corto plazo deberían enfocarse más en las áreas con más amenazas. Se debería prestar especial atención al fortalecimiento del papel y las responsabilidades de los socios, a la mejora o aclaración de cómo se usan las servidumbres y la facilitación del flujo de recursos para trabajar con el mayor impacto potencial.

Chlorocholine chloride exposure induced spermatogenic dysfunction via iron overload caused by AhR/PERK axis-dependent ferritinophagy activation.

Chlorocholine chloride (CCC) is a plant growth regulator used worldwide that is detectable in cereals, fruits and animal products. The health effects of CCC exposure have raised public concern. Our previous research showed that CCC exposure decreased testosterone synthesis in pubertal rats. However, little is known about whether and how pubertal CCC exposure impacts spermatogenesis. In this study, we used BALB/c mice and spermatogonia-derived GC-1 cells to examine CCC-induced spermatogenic dysfunction. In vivo, pubertal CCC exposure led to decreased testicular weight, decreased testicular germ cells and poor sperm quality. This effect worsened after cessation of CCC exposure for the next 30 days. RNA-seq and western blot analysis revealed that CCC induced aryl hydrocarbon receptor (AhR) signaling, endoplasmic reticulum stress (ERS) and ferritinophagy. Increased iron content and lipid peroxidation levels were also observed in CCC-treated testes. In vitro, it was identified that iron overload mediated by enhanced ferritinophagy occurred in CCC-treated GC-1 cells, which might be attributed to the PERK pathway in ERS. Further, for the first time, our study elucidated the involvement of AhR in CCC-induced iron overload, which aggravated testicular oxidative damage via lipid peroxidation. Considering the adverse impact of CCC exposure on rodents, supportive evidence from GC-1 cells, and the critical importance of spermatogenesis on male development, the effects of CCC on the male reproduction warrant increased attention.

Elesclomol elevates cellular and mitochondrial iron levels by delivering copper to the iron import machinery.

Copper (Cu) and iron (Fe) are redox-active metals that serve as cofactors for many essential cellular enzymes. Disruption in the intracellular homeostasis of these metals results in debilitating and frequently fatal human disorders, such as Menkes disease and Friedreich's ataxia. Recently, we reported that an investigational anticancer drug, elesclomol (ES), can deliver Cu to critical mitochondrial cuproenzymes and has the potential to be repurposed for the treatment of Cu deficiency disorders. Here, we sought to determine the specificity of ES and the ES-Cu complex in delivering Cu to cuproenzymes in different intracellular compartments. Using a combination of yeast genetics, subcellular fractionation, and inductively coupled plasma-mass spectrometry-based metal measurements, we showed that ES and ES-Cu treatment results in an increase in cellular and mitochondrial Fe content, along with the expected increase in Cu. Using yeast mutants of Cu and Fe transporters, we demonstrate that ES-based elevation in cellular Fe levels is independent of the major cellular Cu importer but is dependent on the Fe importer Ftr1 and its partner Fet3, a multicopper oxidase. As Fet3 is metalated in the Golgi lumen, we sought to uncover the mechanism by which Fet3 receives Cu from ES. Using yeast knockouts of genes involved in Cu delivery to Fet3, we determined that ES can bypass Atx1, a metallochaperone involved in Cu delivery to the Golgi membrane Cu pump, Ccc2, but not Ccc2 itself. Taken together, our study provides a mechanism by which ES distributes Cu in cells and impacts cellular and mitochondrial Fe homeostasis.

Region-Specific Hepatitis B Virus Genome Exposure from Nucleocapsid Modulated by Capsid Linker Sequence and Inhibitor: Implications for Uncoating.

Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.

Regulation of Hepatitis B Virus Virion Release and Envelopment Timing by Nucleocapsid and Envelope Interactions.

Interactions between the N-terminal (assembly) domain (NTD), the linker region of the hepatitis B virus (HBV) capsid protein, and the large (L) envelope protein are required for virion formation, which occurs via budding of cytoplasmic mature nucleocapsids (NCs) containing the relaxed circular (RC) DNA genome into an intracellular membrane compartment containing viral envelope proteins. L-capsid interactions also negatively regulate covalently closed circular (CCC) DNA formation, which occurs after RC DNA release from mature NCs and nuclear import. We have now found that L could increase RC DNA in cytoplasmic mature NCs that are destabilized due to mutations in the NTD or the linker, even in those that apparently fail to support secretion of complete virions extracellularly. Other mutations in the capsid linker could block the effects of L on both cytoplasmic NC DNA and nuclear CCC DNA. Furthermore, the maturity of RC DNA in cytoplasmic NCs that was enhanced by L or found in secreted virions was modulated by the capsid linker sequence. The level and maturity of the cytoplasmic RC DNA were further influenced by the efficiency of extracellular virion secretion dependent on viral genotype-specific envelope proteins. These results suggest that interactions between the capsid and envelope proteins regulate one or more steps during virion secretion beyond initial capsid envelopment and highlight the critical role of the capsid linker in regulating capsid-envelope interaction, including the timing of envelopment during NC maturation. IMPORTANCE Hepatitis B virus (HBV) is a major human pathogen causing serious liver diseases, including cancer. Interactions between the HBV capsid and the large (L) envelope protein are required for formation of infectious viral particles and also negatively regulate formation of an HBV DNA episome in the host cell nucleus, which serves as the sole transcriptional template capable of supporting all viral gene expression to sustain HBV replication and, therefore, is the molecular basis of HBV persistence. Here, we report evidence indicating that L-capsid interactions modulate the timing of formation of infectious HBV particles during replication and facilitate extracellular release following their formation. Furthermore, a short linker sequence in the capsid protein plays a critical role in these processes as well as controls the amplification of the nuclear episome. These findings inform fundamental mechanisms of HBV replication as well as antiviral development targeting the HBV capsid and DNA episome.

How Do Kidney Disease Clinicians View Kidney Supportive Care and Palliative Care? A Qualitative Study.

RATIONALE & OBJECTIVE: Kidney supportive care (KSC) is a developing area in medicine that integrates the expertise of kidney and palliative care practitioners to improve symptoms and quality of life for people with advanced kidney disease. The intersection of the practical aspects of KSC (including care activities and clinical referrals) with palliative and end-of-life care (EOLC) are largely unknown. The aim of this study was to explore kidney disease clinicians' experiences of KSC, palliative care, and EOLC. STUDY DESIGN: An exploratory qualitative study using semistructured focus groups. SETTING & PARTICIPANTS: Kidney disease clinicians (18 physicians, 3 trainees, and 33 kidney disease nurses) from 5 public hospitals were recruited across Victoria, Australia. ANALYTICAL APPROACH: Thematic analysis of focus group transcripts. RESULTS: The 2 overarching themes highlighted by clinicians were their perception that their health care systems insufficiently addressed the needs of people with advanced kidney disease, as well as their aspirations to develop KSC services to improve health care experiences. Three subthemes were identified related to limitations in health care systems: (1) variation in the clinical scope of KSC, (2) limited integration of palliative care, and (3) experiences of challenging and compromised provision of EOLC. The second theme described aspirations for future KSC services to be more inclusive, seamless, and collaborative across health care providers with capacity to respond to meet changing palliative care needs. LIMITATIONS: Findings may not be transferable to contexts outside of Victoria, Australia; data were collected in 2017-2018 and may not reflect current or future experiences. CONCLUSIONS: Kidney clinicians described systemic challenges and compromises in care experiences and the need for development of KSC services. They expressed that this development would require a consistent and systematic approach that integrates palliative care and embeds KSC as part of kidney health service delivery.

Circular cell culture for sustainable food production using recombinant lactate-assimilating cyanobacteria that supplies pyruvate and amino acids.

Recently, we reported a circular cell culture (CCC) system using microalgae and animal muscle cells for sustainable culture food production. However, lactate accumulation excreted by animal cells in the system characterized by medium reuse was a huge problem. To solve the problem, as an advanced CCC, we used a lactate-assimilating cyanobacterium Synechococcus sp. PCC 7002, using gene-recombination technology that synthesises pyruvate from lactate. We found that the cyanobacteria and animal cells mutually exchanged substances via their waste media: (i) cyanobacteria used lactate and ammonia excreted by animal muscle cells, and (ii) the animal cells used pyruvate and some amino acids excreted by the cyanobacteria. Because of this, animal muscle C2C12 cells were amplified efficiently without animal serum in cyanobacterial culture waste medium in two cycles (first cycle: 3.6-fold; second cycle: 3.9-fold/three days-cultivation) using the same reuse medium. We believe that this advanced CCC system will solve the problem of lactate accumulation in cell culture and lead to efficient cultured food production.

Conceptual frameworks and key questions for assessing the contribution of marine protected areas to shark and ray conservation.

Marine protected areas (MPAs) are key tools in addressing the global decline of sharks and rays, and marine parks and shark sanctuaries of various configurations have been established to conserve shark populations. However, assessments of their efficacy are compromised by inconsistent terminology, lack of standardized approaches to assess how MPAs contribute to shark and ray conservation, and ambiguity about how to integrate movement data in assessment processes. We devised a conceptual framework to standardize key terms (e.g., protection, contribution, potential impact, risk, threat) and used the concept of portfolio risk to identify key attributes of sharks and rays (assets), the threats they face (portfolio risk), and the specific role of MPAs in risk mitigation (insurance). Movement data can be integrated into the process by informing risk exposure and mitigation through MPAs. The framework is operationalized by posing 8 key questions that prompt practitioners to consider the assessment scope, MPA type and purpose, range of existing and potential threats, species biology and ecology, and management and operational contexts. Ultimately, MPA contributions to shark and ray conservation differ according to a complex set of human and natural factors and interactions that should be carefully considered in MPA design, implementation, and evaluation.

Marcos conceptuales y preguntas clave para evaluar la contribución de las áreas marinas protegidas a la conservación de tiburones y rayas Resumen Las áreas marinas protegidas (AMP) son herramientas importantes para manejar la declinación mundial de tiburones y rayas, por lo que se han establecido parques marinos y santuarios de diversas configuraciones para conservar las poblaciones de tiburones. Sin embargo, el análisis de su eficiencia está compuesto por una terminología inconstante, la falta de estrategias estandarizadas para evaluar cómo las AMP contribuyen a la conservación de tiburones y rayas, y una ambigüedad sobre cómo integrar la información sobre movimientos en los procesos de evaluación. Diseñamos un marco conceptual para estandarizar los términos más importantes (p. ej.: protección, contribución, impacto potencial, amenaza, riesgo) y usamos el concepto de riesgo de portafolio para identificar los atributos clave de los tiburones y las rayas (activos), las amenazas que enfrentan (riesgo de portafolio) y el papel específico que juegan las AMP en la mitigación del riesgo (seguro). La información sobre los movimientos puede integrarse al proceso al guiar la exposición y mitigación del riesgo con las AMP. El marco conceptual es operado con el planteamiento de ocho preguntas clave que invitan a los practicantes a considerar el enfoque de la evaluación, el tipo de AMP y su propósito, gama de amenazas existentes y potenciales, la biología y ecología de las especies, y los contextos operativos y de manejo. Finalmente, las contribuciones que tienen las AMP a la conservación de los tiburones y las rayas difieren de acuerdo con un conjunto complejo de factores naturales y humanos e interacciones que deberían considerarse cuidadosamente en el diseño, implementación y evaluación de la AMP.

Application of decision tools to ethical analysis in biodiversity conservation.

Achieving ethically responsible decisions is crucial for the success of biodiversity conservation projects. We adapted the ethical matrix, decision tree, and Bateson's cube to assist in the ethical analysis of complex conservation scenarios by structuring these tools so that they can implement the different value dimensions (environmental, social, and animal welfare) involved in conservation ethics. We then applied them to a case study relative to the decision-making process regarding whether or not to continue collecting biomaterial on the oldest of the two remaining northern white rhinoceroses (Ceratotherium simum cottoni), a functionally extinct subspecies of the white rhinoceros. We used the ethical matrix to gather ethical pros and cons and as a starting point for a participatory approach to ethical decision-making. We used decision trees to compare the different options at stake on the basis of a set of ethical desiderata. We used Bateson's cube to establish a threshold of ethical acceptability and model the results of a simple survey. The application of these tools proved to be pivotal in structuring the decision-making process and in helping reach a shared, reasoned, and transparent decision on the best option from an ethical point of view among those available.

Que se logren decisiones éticamente responsables es crucial para el éxito de los proyectos de conservación de la biodiversidad. Adaptamos la matriz ética, el árbol de decisión y el cubo de Bateson para apoyar con el análisis ético de escenarios de conservación compleja mediante la estructuración de estas herramientas de tal manera que puedan ejecutar las diferentes dimensiones de valor (ambiental, social y bienestar animal) involucradas en la ética de la conservación. Después aplicamos las herramientas a un estudio de caso relacionado con el proceso de toma de decisiones respecto a si se debe seguir o no recolectando material biológico del rinoceronte blanco del norte (Ceratotherium simum cottoni) más viejo (una subespecie funcionalmente extinta) de los dos que existen. Usamos la matriz ética como un punto de partida para una estrategia participativa para la toma ética de decisiones y para recopilar los pros y contras éticos. Usamos el árbol de decisión para comparar las diferentes opciones en juego con base en un conjunto de deseos éticos. Usamos el cubo de Bateson para establecer un umbral de aceptación ética y modelar los resultados de una encuesta simple. La aplicación de estas herramientas demostró ser central en la estructuración del proceso de toma de decisiones y en el apoyo para lograr una decisión compartida, razonada y transparente sobre la mejor opción a partir de un punto de vista ético entre aquellos disponibles.

Contributions of the IUCN Red List of Ecosystems to risk-based design and management of protected and conserved areas in Africa.

Protected and conserved areas (PCAs) are key ecosystem management tools for conserving biodiversity and sustaining ecosystem services and social cobenefits. As countries adopt a 30% target for protection of land and sea under the Global Biodiversity Framework of the United Nations Convention on Biological Diversity, a critical question emerging is, which 30%? A risk-based answer to this question is that the 30% that returns the greatest reductions in risks of species extinction and ecosystem collapse should be protected. The International Union for Conservation of Nature (IUCN) Red List protocols provide practical methods for assessing these risks. All species, including humans, depend on the integrity of ecosystems for their well-being and survival. Africa is strategically important for ecosystem management due to convergence of high ecosystem diversity, intense pressures, and high levels of human dependency on nature. We reviewed the outcomes (e.g., applications of ecosystem red-list assessments to protected-area design, conservation planning, and management) of a symposium at the inaugural African Protected Areas Congress convened to discuss roles of the IUCN Red List of Ecosystems in the design and management of PCAs. Recent progress was made in ecosystem assessment, with 920 ecosystem types assessed against the IUCN Red List criteria across 21 countries. Although these ecosystems spanned a diversity of environments across the continent, the greatest thematic gaps were for freshwater, marine, and subterranean realms, and large geographic gaps existed in North Africa and parts of West and East Africa. Assessment projects were implemented by a diverse community of government agencies, nongovernmental organizations, and researchers. The assessments have influenced policy and management by informing extensions to and management of formal protected area networks supporting decision-making for sustainable development, and informing ecosystem conservation and threat abatement within boundaries of PCAs and in surrounding landscapes and seascapes. We recommend further integration of risk assessments in environmental policy and enhanced investment in ecosystem red-list assessment to fill current gaps.

Contribuciones de la Lista Roja de Ecosistemas de la UICN al diseño y manejo basados en riesgos de las áreas conservadas y protegidas en África Resumen Las áreas protegidas y conservadas (APC) son herramientas clave del manejo de ecosistemas para conservar la biodiversidad y mantener los servicios ambientales y los cobeneficios sociales. Conforme los países adoptan un objetivo de 30% para la protección del suelo y el mar bajo el Marco Mundial de Biodiversidad de la Convención sobre la Diversidad Biológica de las Naciones Unidas, surge una pregunta crítica: ¿cuál 30%? Una respuesta basada en riesgos a esta pregunta es que se debe proteger el 30% que rinda la mayor reducción del riesgo de extinción de especies y del colapso del ecosistema. Los protocolos de la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza (UICN) proporcionan métodos prácticos para evaluar estos riesgos. Todas las especies, incluidos los humanos, dependen de la integridad de los ecosistemas para su bienestar y supervivencia. África tiene una importancia estratégica para el manejo de ecosistemas debido a la convergencia de una gran diversidad de ecosistemas, presiones intensas y un nivel elevado de dependencia del humano hacia la naturaleza. Revisamos los resultados (p. ej.: aplicaciones de las valoraciones de las listas rojas de ecosistemas al diseño de áreas protegidas, planeación de la conservación y manejo) de un simposio en el primer Congreso de Áreas Protegidas Africanas convocado para discutir el papel de la Lista Roja de Ecosistemas de la UICN en el diseño y manejo de las APC. Existen avances recientes en la evaluación de los ecosistemas, con 920 tipos de ecosistemas evaluados bajo los criterios de la Lista Roja de la UICN en 21 países. Mientras estos ecosistemas comprenden una diversidad de ambientes en todo el continente, los principales vacíos temáticos los encontramos para los dominios subterráneos, de agua dulce y marina, además de que existe un gran vacío geográfico en el norte de África y en partes del este y oeste africano. Los proyectos de evaluación fueron implementados por una comunidad diversa de agencias gubernamentales, organizaciones no gubernamentales e investigadores. La influencia de las evaluaciones sobre las políticas y el manejo se da con la información que proveen a las extensiones y el manejo de las redes de áreas protegidas formales, el apoyo para la toma de decisiones de desarrollo sustentable y la guía para la conservación de ecosistemas y el abatimiento de amenazas dentro de los límites de las APC y en los paisajes terrestres y marinos adyacentes. Recomendamos una mayor integración de las evaluaciones de riesgo dentro de las políticas ambientales y más inversión para las evaluaciones de lista roja de los ecosistemas cubrir los vacíos existentes.

Comparison of four commercial dose calculation algorithms in different evaluation tests.

BACKGROUND: Accurate and fast dose calculation is crucial in modern radiation therapy. Four dose calculation algorithms (AAA, AXB, CCC, and MC) are available in Varian Eclipse and RaySearch Laboratories RayStation Treatment Planning Systems (TPSs). OBJECTIVES: This study aims to evaluate and compare dosimetric accuracy of the four dose calculation algorithms applying to homogeneous and heterogeneous media, VMAT plans (based on AAPM TG-119 test cases), and the surface and buildup regions. METHODS: The four algorithms are assessed in homogeneous (IAEA-TECDOCE 1540) and heterogeneous (IAEA-TECDOC 1583) media. Dosimetric evaluation accuracy for VMAT plans is then analyzed, along with the evaluation of the accuracy of algorithms applying to the surface and buildup regions. RESULTS: Tests conducted in homogeneous media revealed that all algorithms exhibit dose deviations within 5% for various conditions, with pass rates exceeding 95% based on recommended tolerances. Additionally, the tests conducted in heterogeneous media demonstrate high pass rates for all algorithms, with a 100% pass rate observed for 6 MV and mostly 100% pass rate for 15 MV, except for CCC, which achieves a pass rate of 94%. The results of gamma index pass rate (GIPR) for dose calculation algorithms in IMRT fields show that GIPR (3% /3 mm) for all four algorithms in all evaluated tests based on TG119, are greater than 97%. The results of the algorithm testing for the accuracy of superficial dose reveal variations in dose differences, ranging from -11.9% to 7.03% for 15 MV and -9.5% to 3.3% for 6 MV, respectively. It is noteworthy that the AXB and MC algorithms demonstrate relatively lower discrepancies compared to the other algorithms. CONCLUSIONS: This study shows that generally, two dose calculation algorithms (AXB and MC) that calculate dose in medium have better accuracy than other two dose calculation algorithms (CCC and AAA) that calculate dose to water.

Inhibitory activities of Ranunculus japonicus Thunb. ethanol extract against hepatitis B virus.

The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract. More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that R. japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that R. japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation.

Antiviral activities of Polygonum perfoliatum L. extract and related phenolic acid constituents against hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is an important public health problem. Polygonum perfoliatum L. is a traditional medicinal herb and has been reported to have pharmacological activities such as anti-inflammatory, antibacterial, and antiviral. In this study, the antiviral activities and mechanisms of Polygonum perfoliatum L. extract against HBV and the effective components were investigated. The results showed that the total extract of Polygonum perfoliatum L. reduced the levels of HBV e antigen (HBeAg) secretion and the viral covalently closed circular DNA (CCC DNA) formation, but had little or no negative effects on viral capsid assembly and pregenomic RNA packaging. Further fractionation showed that the water extract (WE) fraction exerted comparable anti-HBV activities with the total extract, especially in inhibiting the CCC DNA formation and HBeAg production, indicating that the effective antiviral components are mainly distributed in this fraction. Further study showed that the phenolic acids constituents, protocatechuic acid, and gallic acid, but not ethyl caffeate, which is reported enriched in the WE fraction, showed strong anti-HBV activities in inhibiting viral core DNA synthesis, CCC DNA formation, and HBeAg production. These results suggested that the Polygonum perfoliatum L. total extract and the related phenolic acids like protocatechuic acid and gallic acid could inhibit HBV replication and also indicated the potential utility of Polygonum perfoliatum L. and related constituents as sources of novel antivirals against HBV.

A circular cell culture system using microalgae and mammalian myoblasts for the production of sustainable cultured meat.

For sustainable production of cultured meat, we propose a novel circular cell culture (CCC) system in which microalgae are used as nutrient supply for the mammalian cell culture and as a waste-medium recycler. Chlorococcum littorale, RL34 hepatocytes, and C2C12 myoblasts were used as cell sources for microalgae, growth factor-producing cells, and muscle cells, respectively. In the first cycle, C2C12 cells were amplified 4.0-fold after 48 h of culture in an RL34 cell-conditioned medium. In the second cycle, C2C12 cells were cultured in the C. littorale culture waste medium to which the C. littorale-derived nutrients were added. The proliferation rates of C. littorale and C2C12 and the nutrient extraction efficiency from C. littorale were the same in the first and second cycles. Therefore, this CCC system, which works without additional grain-derived nutrients and animal sera, will help drastically reduce environmental load, resource/energy consumption, and costs in future cultured meat production.

A mixed methodology for evaluating use of evidence in conservation planning.

Conservation practitioners widely recognize the importance of making decisions based on the best available evidence. However, the effectiveness of evidence use in conservation planning is rarely assessed, which limits opportunities to improve evidence-based practice. We devised a mixed methodology for empirically evaluating use of evidence that applies social science tools to systematically appraise what kinds of evidence are used in conservation planning, to what effect, and under what limitations. We applied our approach in a case study of the Nature Conservancy of Canada (NCC), a leading land conservation organization. We conducted qualitative and quantitative analyses of 65 NCC planning documents (n = 13 in-depth) to identify patterns in evidence use, and surveyed 35 conservation planners to examine experiences of and barriers to using evidence. Although claims in plans contained a wide range of evidence types, 26% of claims were not referenced or associated with an identifiable source. Lack of evidence use was particularly apparent in claims associated with direct threats, particularly those identified as low (71% coded as insufficient or lacking evidence) or medium (45%) threats. Survey participants described relying heavily on practitioner experience and highlighted capacity limitations and disciplinary gaps in expertise among planning teams as barriers to using evidence effectively. We found that although time-intensive, this approach yielded actionable recommendations for improving evidence use in NCC conservation plans. Similar mixed-method assessments may streamline the process by including interviews and refining the document analysis frames to target issues or sections of concern. We suggest our method provides an accessible and robust point of departure for conservation practitioners to evaluate whether the use of conservation planning reflects in-house standards and more broadly recognized best practices.

RESUMEN: Los practicantes de la conservación reconocen ampliamente la importancia de tomar decisiones con base en la mejor evidencia disponible. Sin embargo, pocas veces se evalúa la efectividad del uso de evidencias en la planeación de la conservación, lo que limita las oportunidades para mejorar la práctica basada en evidencias. Diseñamos una metodología mixta para evaluar empíricamente el uso de evidencias que aplica herramientas de las ciencias sociales para estimar cuáles son los tipos de evidencia que se usan en la planeación de la conservación, con cuál efecto y con cuáles limitaciones. Aplicamos nuestra estrategia a un estudio de caso de la Nature Conservancy of Canada (NCC), una organización puntera en la conservación del suelo. Realizamos el análisis cualitativo y cuantitativo de 65 documentos de planeación de NCC (n = 13 a fondo) para identificar patrones en el uso de evidencias y encuestamos a 35 planeadores de la conservación para examinar las barreras y las experiencias del uso de evidencias. Aunque las afirmaciones en los planes contenían una amplia gama de tipos de evidencia, el 26% de éstas no estuvo referenciado o asociado con una fuente identificable. La falta del uso de evidencias fue particularmente evidente en las afirmaciones asociadas con amenazas directas, particularmente aquellas identificadas como amenaza menor (71% codificado como insuficiente o falto de evidencias) o media (45%). Los participantes de la encuesta describieron una fuerte dependencia de la experiencia de los practicantes y resaltaron las limitaciones de capacidades y brechas disciplinarias en la experiencia entre los equipos de planeación como barreras para el uso efectivo de las evidencias. Descubrimos que, aunque lleva tiempo, esta estrategia produjo recomendaciones viables para mejorar el uso de evidencias en los planes de conservación de la NCC. Las evaluaciones similares de métodos mixtos pueden simplificar el proceso al incluir entrevistas y refinar los marcos de análisis documental para enfocarse en temas o secciones de interés. Sugerimos que nuestro método proporciona un punto de partida accesible y sólido para que los practicantes de la conservación evalúen si el uso de la planeación de la conservación refleja los estándares internos y las mejores prácticas reconocidas más ampliamente. Una Metodología Mixta para la Evaluación del Uso de Evidencias en la Planeación de la Conservación.

Integrated Differential Phase Contrast (IDPC)-STEM Utilizing a Multi-Sector Detector for Imaging Thick Samples.

The integrated differential phase contrast (IDPC) method is useful for generating the potential map of a thin sample. We evaluate theoretically the potential of IDPC imaging for thick samples by varying the focus at different sample thicknesses. Our calculations show that high defocus values result in enhanced anisotropy of the contrast transfer function (CTF) and uninterpretable images, if a quadrant detector is applied. We further show that applying a multi-sector detector can result in an almost isotropic CTF. By sector number-dependent calculations for both Cc/C3-corrected and C3-corrected scanning transmission electron microscopy (STEM), we show that the increase of detector sectors not only removes the anisotropy of the CTF, but also improves image contrast and resolution. For a proof-of-principle IDPC-STEM (uncorrected) experiment, we realize the functionality of a 12-sector detector from a physical quadrant detector and demonstrate the improvement in contrast and resolution on the example of InGaN/GaN quantum well structure.

Adaptation of Saccharomyces Species to High-Iron Conditions.

Iron is an indispensable element that participates as an essential cofactor in multiple biological processes. However, when present in excess, iron can engage in redox reactions that generate reactive oxygen species that damage cells at multiple levels. In this report, we characterized the response of budding yeast species from the Saccharomyces genus to elevated environmental iron concentrations. We have observed that S. cerevisiae strains are more resistant to high-iron concentrations than Saccharomyces non-cerevisiae species. Liquid growth assays showed that species evolutionarily closer to S. cerevisiae, such as S. paradoxus, S. jurei, S. mikatae, and S. arboricola, were more resistant to high-iron levels than the more distant species S. eubayanus and S. uvarum. Remarkably, S. kudriavzevii strains were especially iron sensitive. Growth assays in solid media suggested that S. cerevisiae and S. paradoxus were more resistant to the oxidative stress caused by elevated iron concentrations. When comparing iron accumulation and sensitivity, different patterns were observed. As previously described for S. cerevisiae, S. uvarum and particular strains of S. kudriavzevii and S. paradoxus became more sensitive to iron while accumulating more intracellular iron levels. However, no remarkable changes in intracellular iron accumulation were observed for the remainder of species. These results indicate that different mechanisms of response to elevated iron concentrations exist in the different species of the genus Saccharomyces.