Joint analysis of land, carbon and nitrogen reveals diverging trends in the sustainability of organic crops in Spain.

The world's top ten Organic Farming (OF) countries by converted area include several Mediterranean countries, including Spain. Despite this, little is known about the consequences of OF on crop production and environmental sustainability in this country. In this article, we conduct an agronomic analysis of Spanish considerable conversion rate to OF, which tends to concentrate in certain provinces and crops. Indeed, in the case of various crops and in several provinces, the organic share of total agricultural land exceeds 20-30 %. This concentration makes it possible to compare information obtained from farmers through interviews and provincial statistical information. The study data consisted of information collected from interviews of a representative sample of organic farmers conducted in 2004 and 2020 as well as official statistical information. The results showed that no yield gap between OF and conventional farming was found for vegetables and fruit trees, while it showed an increasing trend in arable crops. Presumably, the reason is that fruit trees and vegetables generate and incorporate high levels of carbon (C) flows into the soil and have a low land cost per unit of incorporated nitrogen (N) (or can be paid for), allowing to meet crop needs and to increase soil organic matter (SOM). Conversely, in the case of rainfed arable crops, the soil C and N inputs are deficient due to the low crop residues and the high land cost of N. Consequently, SOM destruction and N deficit progressively broaden the yield gap, undermining the agroecosystem sustainability. To reverse the situation, among other measures, it is necessary to implement agricultural policies designed to make rotations with high legume ratios viable and to plant varieties presenting higher production of residues and roots, such as traditional varieties.

Systematic under-ascertainment of anaplastic large cell lymphoma cases in cancer registries: report from a comprehensive cancer center.

INTRODUCTION: Breast implant-associated anaplastic large cell lymphoma (ALCL) has been rapidly rising in the US and around the world, leading to a mandated "black-box" label on all silicone- and saline-filled implants by the Food and Drug Administration (FDA). Because regulatory decisions in the US and around the world have been influenced primarily by risk estimates derived from cancer registries, it is important to determine their validity in identifying cases of ALCL. METHOD: We reviewed all cases of ALCL submitted to the New York State Cancer Registry from a large comprehensive cancer center in New York City from 2007 to 2019. To determine the possibility of misdiagnosis or under-diagnosis of ALCL cases reported to cancer registries, we accessed the sensitivity and specificity of the ICD-O-3 codes 9714 (ALCL) and 9702 (Mature T-cell lymphoma, not otherwise specified [T-NOS]) to identify pathologically-proven ALCL. RESULTS: We reviewed 2286,164 pathology reports from 47,466 unique patients with primary cancers. Twenty-eight cases of histologically-proven ALCL were identified. The sensitivity and specificity of the ICD-O-3 code 9714 (ALCL) were 82% and 100%, respectively. The sensitivity of the combined codes 9714/9702 (ALCL/T-NOS) was 96% and the specificity was 44%. CONCLUSION: Previous epidemiological studies that influenced regulatory decisions by the FDA may have systematically underestimated the risk of ALCL by at least 20%. We encourage updated global risk estimates of breast ALCL using methods that ensure adequate case ascertainment.

Feeding Practices of Stable Term and Late Preterm Neonates Born at a Tertiary Hospital in the Philippines during the COVID-19 Pandemic.

Objective: We aimed to evaluate the impact of maternal COVID-19 infection and separation of the mother-baby dyad on feeding practices by determining modes of feeding upon discharge and following up at 2-3 days, 14 days, and 1 month post-discharge. Methods: This was a prospective observational cohort study conducted at the Philippine General Hospital, a tertiary government COVID-19 referral center in Manila. Mothers who delivered between the months of July and August 2021, and whose COVID-19 status was known were followed up on their baby's mode of feeding at 2-3 days, 14 days, and 1 month post-discharge via phone call. For babies of COVID-19 positive mothers, presence of any symptoms (up to the 14-day time point only) as well as adherence to infection prevention and control practices were also identified. Results: For all time points post-discharge, breastfeeding rates were higher in babies born to COVID-19-negative mothers and in those who were roomed in. However, the differences were not statistically significant. Of the 108 infants, 72.90% remained exclusively breastfed by 1 month of age, with 4.67% formula-fed and 22.43% on mixed feeding. Perception of insufficient milk supply was the most common reason for shifting to formula or mixed feeding. We found a significantly higher direct breastfeeding rate upon discharge in the roomed-in population compared to those admitted to the NICU. Risk factors affecting breastfeeding at 1 month of age was the presence of COVID-19 infection in the mother and mother-baby separation due to NICU admissionCOVID-19-positive mothers were 66.02% less likely (p=0.016, 95% CI 0.1411 to 0.8183) to still be breastfeeding at 1 month, and separation was not found to be a significant risk factor. Conclusions: Feeding practices can be affected not only by COVID-19 infection in the mother and its attendant difficulties such as prolonged hospital stay and physical and social isolation, but also by limitations in the hospital environment that can have an impact on breastfeeding education, support, and opportunities for mother-child bonding.

Zika emergence, persistence, and transmission rate in Colombia: a nationwide application of a space-time Markov switching model.

Zika, a viral disease transmitted to humans by Aedes mosquitoes, emerged in the Americas in 2015, causing large-scale epidemics. Colombia alone reported over 72,000 Zika cases between 2015 and 2016. Using national surveillance data from 1121 municipalities over 70 weeks, we identified sociodemographic and environmental factors associated with Zika's emergence, re-emergence, persistence, and transmission intensity in Colombia. We fitted a zero-state Markov-switching model under the Bayesian framework, assuming Zika switched between periods of presence and absence according to spatially and temporally varying probabilities of emergence/re-emergence (from absence to presence) and persistence (from presence to presence). These probabilities were assumed to follow a series of mixed multiple logistic regressions. When Zika was present, assuming that the cases follow a negative binomial distribution, we estimated the transmission intensity rate. Our results indicate that Zika emerged/re-emerged sooner and that transmission was intensified in municipalities that were more densely populated, at lower altitudes and/or with less vegetation cover. Warmer temperatures and less weekly-accumulated rain were also associated with Zika emergence. Zika cases persisted for longer in more densely populated areas with more cases reported in the previous week. Overall, population density, elevation, and temperature were identified as the main contributors to the first Zika epidemic in Colombia. We also estimated the probability of Zika presence by municipality and week, and the results suggest that the disease circulated undetected by the surveillance system on many occasions. Our results offer insights into priority areas for public health interventions against emerging and re-emerging Aedes-borne diseases.

AMPK regulates phagophore-to-autophagosome maturation.

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example, by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK-mediated inhibition of phagophore tethering to donor membrane. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

Connecting Clinical Capacity and Intervention Sustainability in Resource-Variable Pediatric Oncology Centers in Latin America.

Clinical capacity for sustainability, or the clinical resources needed to sustain an evidence-based practice, represent proximal determinants that contribute to intervention sustainment. We examine the relationship between clinical capacity for sustainability and sustainment of PEWS, an evidence-based intervention to improve outcomes for pediatric oncology patients in resource-variable hospitals. We conducted a cross-sectional survey among Latin American pediatric oncology centers participating in Proyecto Escala de Valoración de Alerta Temprana (EVAT), an improvement collaborative to implement Pediatric Early Warning Systems (PEWS). Hospitals were eligible if they had completed PEWS implementation. Clinicians were eligible to participate if they were involved in PEWS implementation or used PEWS in clinical work. The Spanish language survey consisted of 56 close and open-ended questions about the respondent, hospital, participants' assessment of clinical capacity to sustain PEWS using the clinical sustainability assessment tool (CSAT), and perceptions about PEWS and its use as an intervention. Results were analyzed using a multi-level modeling approach to examine the relationship between individual, hospital, intervention, and clinical capacity determinants to PEWS sustainment. A total of 797 responses from 37 centers in 13 countries were included in the analysis. Eighty-seven percent of participants reported PEWS sustainment. After controlling for individual, hospital, and intervention factors, clinical capacity was significantly associated with PEWS sustainment (OR 3.27, p < .01). Marginal effects from the final model indicate that an increasing capacity score has a positive influence (11% for every additional CSAT point) of predicting PEWS sustainment. PEWS is a sustainable intervention and clinical capacity to sustain PEWS contributes meaningfully to PEWS sustainment.

Gene variants for the WNT pathway are associated with severity in periodontal disease.

OBJECTIVE: Studies of Wnt variants-related to bone resorption in periodontitis are limited. The aim of this study was to establish the genotype and allele frequency of gene variants associated with the Wnt pathway in systemically healthy individuals with and without periodontitis (PD). MATERIALS AND METHODS: One hundred fifty-seven systemically healthy individuals were evaluated, 90 with PD and 67 without PD. Periodontal clinical indexes, serological and clinical indices of inflammation, and the following variants associated with the Wnt pathway: DKK, SOST, LRP5, and KREMEN were analyzed by high resolution melting and confirmed by Sanger sequencing. RESULTS: In the PD-free group, 67.2% of the individuals presented the variant for DKKrs1896367 (p = 0.008) and 82.6% had the variant for KREMEN rs132274 (p = 0.016). The heterozygous variant for the DKK rs1896367 polymorphism was associated with the absence of PD and lower severity OR: 0.33 (CI95% 0.15-0.70) and OR: 0.24 (CI95% 0.11-0.53), respectively. Similarly, KREMEN rs132274 was the homozygous variant associated with the absence of PD (OR: 0.33 (CI95% 0.13-0.88)). On the contrary, 85.6% of individuals with PD presented a variant for DKK rs1896368 (p = 0.042), all suffering severe forms of periodontitis. CONCLUSION: The presence of DKKrs1896367 and KREMENrs132274 variants in individuals without PD suggests that these single nucleotide polymorphisms could be protective factors for bone loss in PD. A very interesting finding is that the DKKrs1896368 variant was found in a high percentage of severe cases, suggesting that the presence of this variant may be related to the severe bone loss observed in PD.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center in 2023.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) is a Bioinformatics Resource Center funded by the National Institutes of Health with additional funding from the Wellcome Trust. VEuPathDB supports >600 organisms that comprise invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Since 2004, VEuPathDB has analyzed omics data from the public domain using contemporary bioinformatic workflows, including orthology predictions via OrthoMCL, and integrated the analysis results with analysis tools, visualizations, and advanced search capabilities. The unique data mining platform coupled with >3000 pre-analyzed data sets facilitates the exploration of pertinent omics data in support of hypothesis driven research. Comparisons are easily made across data sets, data types and organisms. A Galaxy workspace offers the opportunity for the analysis of private large-scale datasets and for porting to VEuPathDB for comparisons with integrated data. The MapVEu tool provides a platform for exploration of spatially resolved data such as vector surveillance and insecticide resistance monitoring. To address the growing body of omics data and advances in laboratory techniques, VEuPathDB has added several new data types, searches and features, improved the Galaxy workspace environment, redesigned the MapVEu interface and updated the infrastructure to accommodate these changes.

Development of an open access, virtual bioinformatics lab for students in medical laboratory sciences.

OBJECTIVES: Despite the growing importance of bioinformatics in molecular diagnostics, not all medical laboratory sciences (MLS) programs provide instruction in this field. We developed and assessed a virtual laboratory learning unit to introduce basic bioinformatics concepts and tools to MLS students. METHODS: The unit included a video tutorial, written instructions for the online laboratory activity, and a postactivity review video. The effectiveness of the instruction was evaluated using preassessment and postassessment questions, performance of the online tasks, and a survey assessing the students' attitudes toward the learning unit. RESULTS: A prototype of the module was tested with 32 graduate and undergraduate students. Modifications were made based on the pilot test results and student feedback, and the refined version was subsequently evaluated with a different group of 20 undergraduate students. The participants responded favorably to the learning unit and successfully achieved the learning objectives, gaining familiarity with fundamental bioinformatics concepts and terminology, effectively employing basic computational tools, and developing an appreciation for the field. CONCLUSIONS: Our learning unit is a promising tool for introducing MLS students to the field of bioinformatics. As an open educational resource, it has the potential to be integrated into molecular biology education for MLS programs anywhere.

Assessing the energy trap of industrial agriculture in North America and Europe: 82 balances from 1830 to 2012.

Early energy analyses of agriculture revealed that behind higher labor and land productivity of industrial farming, there was a decrease in energy returns on energy (EROI) invested, in comparison to more traditional organic agricultural systems. Studies on recent trends show that efficiency gains in production and use of inputs have again somewhat improved energy returns. However, most of these agricultural energy studies have focused only on external inputs at the crop level, concealing the important role of internal biomass flows that livestock and forestry recirculate within agroecosystems. Here, we synthesize the results of 82 farm systems in North America and Europe from 1830 to 2012 that for the first time show the changing energy profiles of agroecosystems, including livestock and forestry, with a multi-EROI approach that accounts for the energy returns on external inputs, on internal biomass reuses, and on all inputs invested. With this historical circular bioeconomic approach, we found a general trend towards much lower external returns, little or no increases in internal returns, and almost no improvement in total returns. This "energy trap" was driven by shifts towards a growing dependence of crop production on fossil-fueled external inputs, much more intensive livestock production based on feed grains, less forestry, and a structural disintegration of agroecosystem components by increasingly linear industrial farm managements. We conclude that overcoming the energy trap requires nature-based solutions to reduce current dependence on fossil-fueled external industrial inputs and increase the circularity and complexity of agroecosystems to provide healthier diets with less animal products. Supplementary Information: The online version contains supplementary material available at 10.1007/s13593-023-00925-5.

AMPK Regulates Phagophore-to-Autophagosome Maturation.

Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK mediated inhibition of phagophores tethering to donor membranes. Our results clarify AMPK's regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.

miRNAs signature as potential biomarkers for cervical precancerous lesions in human papillomavirus positive women.

Biomarkers to identify women at risk of cervical cancer among those with high-risk HPV infection (hrHPV+) are needed. Deregulated expression of microRNAs (miRNAs) contributes to hrHPV-induced cervical carcinogenesis. We aimed at identifying miRNAs with the capacity to distinguish high (CIN2+) and low (≤ CIN1) grade cervical lesions. We sequenced miRNA libraries from Formalin-Fixed Paraffin-Embedded (FFPE) tissues from women with CIN2+ (n = 10) and age-matched women with ≤ CIN1 (n = 10), randomly and retrospectively selected from a trial that followed women for 24 months after a hrHPV+ test at the screening visit. Five miRNAs differentially expressed were validated by RT-qPCR in an independent set of FFPE tissues with a reviewed diagnosis of CIN2+ (n = 105) and ≤ CIN1 (n = 105). The Ingenuity Pathway Analysis (IPA) was conducted to identify mRNAs inversely correlated with the top 25 differentially expressed miRNAs. Inverse correlations with 401 unique mRNA targets were identified for fourteen of the top 25 differentially expressed miRNAs. Eleven of these miRNAs targeted 26 proteins of pathways deregulated by HPV E6 and E7 oncoproteins and two of them, miR-143-5p and miR-29a-3p, predicted CIN2+ and CIN3+ in the independent validation by RT-qPCR of FFPE tissues from hrHPV-positive women.

Evaluation of the Effect of Binary Fly Ash-Lime Mixture on the Bearing Capacity of Natural Soils: A Comparison with Two Conventional Stabilizers Lime and Portland Cement.

This study evaluates a binary mixture of fly ash and lime as a stabilizer for natural soils. A comparative analysis was performed on the effect on the bearing capacity of silty, sandy and clayey soils after the addition of lime and ordinary Portland cement as conventional stabilizers, and a non-conventional product of a binary mixture of fly ash and Ca(OH)2 called FLM. Laboratory tests were carried out to evaluate the effect of additions on the bearing capacity of stabilized soils by unconfined compressive strength (UCS). In addition, a mineralogical analysis to validate the presence of cementitious phases due to chemical reactions with FLM was performed. The highest UCS values were found in the soils that required the highest water demand for compaction. Thus, the silty soil added with FLM reached 10 MPa after 28 days of curing, which was in agreement with the analysis of the FLM pastes, where soil moistures higher than 20% showed the best mechanical characteristics. Furthermore, a 120 m long track was built with stabilized soil to evaluate its structural behavior for 10 months. An increase of 200% in the resilient modulus of the FLM-stabilized soils was identified, and a decrease of up to 50% in the roughness index of the FLM, lime (L) and Ordinary Portland Cement (OPC)-stabilized soils compared to the soil without addition, resulting in more functional surfaces.

TCAB1 prevents nucleolar accumulation of the telomerase RNA to facilitate telomerase assembly.

Localization of a variety of RNAs to non-membrane-bound cellular compartments such as nucleoli and Cajal bodies is critical for their stability and function. The molecular mechanisms that underly the recruitment and exclusion of RNAs from these phase-separated organelles is incompletely understood. Telomerase is a ribonucleoprotein composed of the reverse transcriptase protein telomerase reverse transcriptase (TERT), the telomerase RNA (TR), and several auxiliary proteins, including TCAB1. Here we show that in the absence of TCAB1, a large fraction of TR is tightly bound to the nucleolus, while TERT is largely excluded from the nucleolus, reducing telomerase assembly. This suggests that nuclear compartmentalization by the non-membrane-bound nucleolus counteracts telomerase assembly, and TCAB1 is required to retain TR in the nucleoplasm. Our work provides insight into the mechanism and functional consequences of RNA recruitment to organelles formed by phase separation and demonstrates that TCAB1 plays an important role in telomerase assembly.

Bioactive Compounds and Sensory Analysis of Freeze-Dried Prickly Pear Fruits from an Inter-Andean Valley in Peru.

Prickly pear fruits are seasonal and have shades ranging from pale green to deep purple. Their pigments are associated with bioactive compounds, being sensitive to thermal transformation processes for their conservation. The objective of this research was to evaluate the bioactive compounds and the sensory analysis of freeze-dried prickly pear fruits from an inter-Andean valley in Peru. The prickly pear fruits of the morada, anaranjada, and blanca ecotypes came from an inter-Andean valley in Peru at 2972 m altitude. The sliced fruits were freeze-dried at -40 °C and 0.25 mTorr, and the total polyphenol content (TPC), vitamin C, and antioxidant activity (AA) were determined, as well as the color L* a* b*, color index (CI*), FTIR spectra, and mineral content. In the same way, sensory analysis of preferences with nine scales was applied. It was observed that in the freeze-dried fruits, TPC, AA, and vitamin C increased significantly (p-value < 0.05), and their corresponding functional groups increased in intensity in their corresponding FTIR spectra; furthermore, trace elements such as Cu, Fe, Se, Zn, Si, and Mn were identified. On the other hand, freeze-drying provided deeper colors to the fruits, which most panelists said they "very much liked" during the sensory analysis, although the texture was not very well accepted, with most panelists reporting being "indifferent" towards it. The freeze-drying technique allows the bioactive and sensory attributes of prickly pear fruits from inter-Andean valleys to be preserved, making it a potential fruit for export and conservation due to its seasonality.

International health initiative: Development and pilot testing of a molecular diagnostics training program in Romania.

OBJECTIVES: The implementation of nucleic acid testing in laboratory medicine has revolutionized clinical diagnosis. Unfortunately, incorporation of these technologies in less developed countries remains a challenge. Despite Romania's recent economic growth, the country is in dire need of medical and laboratory staff trained in modern technologies. The aim of the study was to develop a curriculum that could easily be delivered to laboratory professionals in Romania and to pilot test the effectiveness of the training in increasing their understanding of molecular tests. METHODS: The program was developed in accordance with the US Centers for Disease Control and Prevention's (CDC's) quality training standards. It was offered to 50 laboratory professionals and consisted of online, asynchronous lectures and optional synchronous review sessions. Training effectiveness was evaluated using CDC guidelines based on pre- and postassessment questions answered anonymously. RESULTS: Forty-two people participated in the program, and 32 (81%) completed the training successfully. Based on 16 participants' self-assessment, the course was successful in improving learners' overall knowledge of molecular diagnostics-specifically, their understanding of molecular techniques and how to interpret results. Those participants were highly satisfied with the overall training. CONCLUSIONS: The piloted platform presented here is promising and can be a foundation for future larger-scale studies in countries with developing health systems.

In Vivo Analysis of Heterogeneous Extracellular Vesicles Using a Red-Shifted Bioluminescence Resonance Energy Transfer Reporter Protein.

Current methods for characterizing the biodistribution of extracellular vesicles (EVs) are not sensitive enough to track EVs in vivo, despite significant advances over the past decade. Commonly used lipophilic fluorescent dyes are convenient, but lack specificity and yield inaccurate spatiotemporal images in the long-term tracking of EVs. In contrast, protein-based fluorescent or bioluminescent EV reporters have more accurately revealed their distribution in cells and mouse models. Here, we describe a red-shifted bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL, to analyze the trafficking of small EVs (<200 nm; sEVs) and medium/large EVs (>200 nm; m/lEVs) in mice. Its advantages are that (i) background signals in bioluminescence imaging (BLI) are negligible and (ii) the photons PalmReNL emits have spectral wavelengths longer than 600 nm and can more efficiently penetrate tissues than reporters emitting shorter wavelength light.

Magnetic Biochar Obtained by Chemical Coprecipitation and Pyrolysis of Corn Cob Residues: Characterization and Methylene Blue Adsorption.

Biochar is a carbonaceous and porous material with limited adsorption capacity, which increases by modifying its surface. Many of the biochars modified with magnetic nanoparticles reported previously were obtained in two steps: first, the biomass was pyrolyzed, and then the modification was performed. In this research, a biochar with Fe3O4 particles was obtained during the pyrolysis process. Corn cob residues were used to obtain the biochar (i.e., BCM) and the magnetic one (i.e., BCMFe). The BCMFe biochar was synthesized by a chemical coprecipitation technique prior to the pyrolysis process. The biochars obtained were characterized to determine their physicochemical, surface, and structural properties. The characterization revealed a porous surface with a 1013.52 m2/g area for BCM and 903.67 m2/g for BCMFe. The pores were uniformly distributed, as observed in SEM images. BCMFe showed Fe3O4 particles on the surface with a spherical shape and a uniform distribution. According to FTIR analysis, the functional groups formed on the surface were aliphatic and carbonyl functional groups. Ash content in the biochar was 4.0% in BCM and 8.0% in BCMFe; the difference corresponded to the presence of inorganic elements. The TGA showed that BCM lost 93.8 wt% while BCMFe was more thermally stable due to the inorganic species on the biochar surface, with a weight loss of 78.6%. Both biochars were tested as adsorbent materials for methylene blue. BCM and BCMFe obtained a maximum adsorption capacity (qm) of 23.17 mg/g and 39.66 mg/g, respectively. The obtained biochars are promising materials for the efficient removal of organic pollutants.

Quantitative analysis of autophagy reveals the role of ATG9 and ATG2 in autophagosome formation.

Autophagy is a catabolic pathway required for the recycling of cytoplasmic materials. To define the mechanisms underlying autophagy it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of cell lines expressing HaloTagged autophagy factors from their endogenous loci, we analyzed the abundance, single-molecule dynamics, and autophagosome association kinetics of autophagy proteins involved in autophagosome biogenesis. We demonstrate that autophagosome formation is inefficient and ATG2-mediated tethering to donor membranes is a key commitment step in autophagosome formation. Furthermore, our observations support the model that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles, and that the ULK1 complex and PI3-kinase form a positive feedback loop required for autophagosome formation. Finally, we demonstrate that the duration of autophagosome biogenesis is ∼110 s. In total, our work provides quantitative insight into autophagosome biogenesis and establishes an experimental framework to analyze autophagy in human cells.

Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.