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BACKGROUND: CRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection. METHODS: We developed SHERLOCK assays capable of detecting all Plasmodium species known to cause human malaria and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We further tested these assays using a diverse panel of clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. In addition, we developed a prototype SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine resistance. FINDINGS: The suite of Plasmodium assays achieved analytical sensitivities ranging from 2*5-18*8 parasites per reaction when tested against laboratory strain genomic DNA. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity during testing of 123 clinical samples. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities. INTERPRETATION: These novel SHERLOCK assays demonstrate the versatility of CRISPR-based diagnostics and their potential as a new generation of molecular tools for malaria diagnosis and surveillance. FUNDING: National Institutes of Health (T32GM007092, R21AI148579, K24AI134990, R01AI121558, UL1TR002489, P30CA016086).

Aiming at improving the transport of biotechnologically relevant carboxylic acids in engineered microbial cell factories, the focus of this work was to study plasma membrane transporters belonging to the Acetate Uptake Transporter (AceTr) family. Ato1 and SatP, members of this family from Saccharomyces cerevisiae and Escherichia coli, respectively, are the main acetate transporters in these species. The analysis of conserved amino acid residues within AceTr family members combined with the study of Ato1 3D model based on SatP, was the rationale for selection of site-directed mutagenesis targets. The library of Ato1-GFP mutant alleles was functionally analysed in the S. cerevisiae IMX1000 strain which shows residual growth in all carboxylic acids tested. A gain of function phenotype was found for mutations in the residues F98 and L219 located at the central constrictive site of the pore, enabling cells to grow on lactic and on succinic acid. This phenotype was associated with an increased transport activity for these substrates. A dominant negative acetic acid hypersensitivity was induced in S. cerevisiae cells expressing the E144A mutant, which was associated with an increased acetic acid uptake. By utilizing computer-assisted 3D-modelling tools we highlight structural features that explain the acquired traits found in the analysed Ato1 mutants. Additionally, we achieved the proper expression of the Escherichia coli SatP, a homologue of Ato1, in S. cerevisiae. To our knowledge, this constitutes the first report of a fully functional bacterial plasma membrane transporter protein in yeast cells. ; This work was supported by the strategic programme UID/BIA/04050/2019 funded by Portuguese funds through the FCT-IP, the project TransAcids (PTDC/BIAMIC/5184/2014) funded by FCT-IP and ERDF by COMPETE 2020-POCI and the project River2Ocean NORTE-01-0145-FEDER-000068, co-financed by the European Regional Development Fund (ERDF) , through Programa Opera-cional Regional do Norte (NORTE 2020) as well as the European Union's ...

International audience ; BACKGROUND:Recently, there has been a re-emergence of cutaneous leishmaniasis in endemic countries and an increase in imported cases in non-endemic countries by travelers, workers, expatriates, immigrants, and military force personnel. Old World cutaneous leishmaniasis is caused primarily by Leishmania major, L. tropica and L. aethiopica. Despite their low sensitivity, diagnosis traditionally includes microscopic and histopathological examinations, and in vitro cultivation. Several conventional PCR techniques have been developed for species identification, which are time-consuming and labour-intensive. Real-time PCR using SYBR green dye, although provides rapid detection, may generate false positive signals. Therefore, a rapid and easy method such as a FRET-based real-time PCR would improve not only the turn-around time of diagnosing Old World cutaneous Leishmania species but will also increase its specificity and sensitivity.METHODS:A FRET-based real-time PCR assay which amplifies the cathepsin L-like cysteine protease B gene encoding a major Leishmania antigen was developed to differentiate L. major, L. tropica, and L. aethiopica in one single step using one set of primers and probes. Assay performance was tested on cutaneous and visceral strains of Leishmania parasite cultures and isolates of other protozoan parasites as well as human biopsy specimen.RESULTS:The assay readily differentiates between the three Old World cutaneous leishmaniasis species based on their melting curve characteristics. A single Tm at 55.2 ± 0.5 °C for L. aethiopica strains was distinguished from a single Tm at 57.4 ± 0.2 °C for L. major strains. A double curve with melting peaks at 66.6 ± 0.1 °C and 48.1 ± 0.5 °C or 55.8 ± 0.6 °C was observed for all L. tropica strains. The assay was further tested on biopsy specimens, which showed 100% agreement with results obtained from isoenzyme electrophoresis and Sanger sequencing.CONCLUSION:Currently, there are no published data on real-time PCR using FRET technology ...

International audience ; BACKGROUND:Recently, there has been a re-emergence of cutaneous leishmaniasis in endemic countries and an increase in imported cases in non-endemic countries by travelers, workers, expatriates, immigrants, and military force personnel. Old World cutaneous leishmaniasis is caused primarily by Leishmania major, L. tropica and L. aethiopica. Despite their low sensitivity, diagnosis traditionally includes microscopic and histopathological examinations, and in vitro cultivation. Several conventional PCR techniques have been developed for species identification, which are time-consuming and labour-intensive. Real-time PCR using SYBR green dye, although provides rapid detection, may generate false positive signals. Therefore, a rapid and easy method such as a FRET-based real-time PCR would improve not only the turn-around time of diagnosing Old World cutaneous Leishmania species but will also increase its specificity and sensitivity.METHODS:A FRET-based real-time PCR assay which amplifies the cathepsin L-like cysteine protease B gene encoding a major Leishmania antigen was developed to differentiate L. major, L. tropica, and L. aethiopica in one single step using one set of primers and probes. Assay performance was tested on cutaneous and visceral strains of Leishmania parasite cultures and isolates of other protozoan parasites as well as human biopsy specimen.RESULTS:The assay readily differentiates between the three Old World cutaneous leishmaniasis species based on their melting curve characteristics. A single Tm at 55.2 ± 0.5 °C for L. aethiopica strains was distinguished from a single Tm at 57.4 ± 0.2 °C for L. major strains. A double curve with melting peaks at 66.6 ± 0.1 °C and 48.1 ± 0.5 °C or 55.8 ± 0.6 °C was observed for all L. tropica strains. The assay was further tested on biopsy specimens, which showed 100% agreement with results obtained from isoenzyme electrophoresis and Sanger sequencing.CONCLUSION:Currently, there are no published data on real-time PCR using FRET technology to differentiate between Old World cutaneous Leishmania species. In summary, our assay based on specific hybridization addresses the limitations of previous PCR technology and provides a single step, reliable method of species identification and rapid diagnostic applications.

BackgroundPesticide-environment interactions are bidirectional. The environment alters pesticides by metabolism and photodegradation, and pesticides in turn change the environment through nontarget or secondary effects.ObjectivesApproximately 900 currently used commercial pesticides of widely diverse structures act by nearly a hundred mechanisms to control insects, weeds, and fungi, usually with minimal disruption of nature's equilibrium. Here I consider some aspects of the discovery, development, and use of ecofriendly or green pesticides (i.e., pesticides that are safe, effective, and biodegradable with minimal adverse secondary effects on the environment). Emphasis is given to research in my laboratory.DiscussionThe need for understanding and improving pesticide-environment interactions began with production of the first major insecticide approximately 150 years ago: The arsenical poison Paris Green was green in color but definitely not ecofriendly. Development and use of other pesticides has led to a variety of problems. Topics considered here include the need for high purity [e.g., hexachlorocyclohexane and polychloroborane isomers and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)], environmental degradation and the bioactivity of resulting photoproducts and metabolites, pesticide photochemistry (including the use of structural optimization, photostabilizers, and photosensitizers to achieve suitable persistence), the presence of multiple active ingredients in botanical insecticides, the need to consider compounds with common mechanisms of action, issues related to primary and secondary targets, and chemically induced or genetically modified changes in plant biochemistry. Many insecticides are bird, fish, and honeybee toxicants, whereas herbicides and fungicides pose fewer environmental problems.ConclusionSix factors have contributed to the greening of pesticide-environment interactions: advances in pesticide chemistry and toxicology, banning of many chlorinated hydrocarbons, the development of new biochemical targets, increased reliance on genetically modified crops that reduce the amount and variety of pesticides applied, emphasis on biodegradability and environmental protection, and integrated pest- and pesticide-management systems.

Following a bait-preference pilot study on captive feral pigs, a series of field studies assessed the attractiveness and target-specificity of a prototype manufactured feral pig bait (PIGOUT®). Two promising test baits and fresh meat reference baits were biomarked with iophenoxic acid and aerially distributed in 100-km2 blocks of land infested with feral pigs in western Queensland to assess field uptake and target-specificity without prefeeding. Uptake was assessed by measuring blood iodine levels in aerially shot feral pigs. In all, 80% of feral pigs sampled in a non-toxic PIGOUT®-baited area had significantly elevated blood iodine, compared with 52% of sampled feral pigs in a meat-baited area (although slightly different baiting strategies were employed). No age or sex bias was evident in PIGOUT®-consuming feral pigs. No monitored manufactured baits were consumed by non-target species in 500 bait-nights. Attractiveness and target-specificity trials of ground-laid, unfenced PIGOUT® baits compared with reference baits were subsequently undertaken in several regions of eastern Australia. Results showed that PIGOUT® was consumed readily by feral pigs at all sites, and that it offered significant improvement in target specificity when compared with unfenced wheat or meat baits. However, the baits were consumed by small numbers of macropods, birds and possums. Available evidence indicates that the target-specificity of PIGOUT® bait is highest in the rangelands, reducing slightly in temperate areas and subalpine forests, where abundance of small animals is higher.

Abstract We directly compared chimpanzee infants and human infants for categorical representations of three global‐like categories (mammals, furniture and vehicles), using the familiarization–novelty preference technique. Neither species received any training during the experiments. We used the time that participants spent looking at the stimulus object while touching it as a measure. During the familiarization phase, participants were presented with four familiarization objects from one of three categories (e.g. mammals). Then, they were tested with a pair of novel objects, one was a familiar‐category object and another was a novel‐category object (e.g. vehicle) in the test phase. The chimpanzee infants did not show significant habituation, whereas human infants did. However, most important, both species showed significant novelty‐preference in the test phase. This indicates that not only human infants, but also chimpanzee infants formed categorical representations of a global‐like level. Implications for the shared origins and species‐specificity of categorization abilities, and the cognitive operations underlying categorization, are discussed.

In addition to the large quantities of solid waste rich in protein, most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic hydrogen sulphide. The current worldwide legislation on environmental requires tanneries to reduce pollution and to replace conventional processes by greener technologies. Enzymes are a technological tool of interest for industry because are able to achieve a high reaction rate under soft pH, temperature, and pressure conditions, besides a high specificity of reaction, biodegradability, non-toxic nature and non-polluting effluent generation. In leather industry enzymes are principally used in pre-tanning operations (soaking, dehairing, bating, and degreasing) and waste treatment. Particularly, proteases have been chosen as a promising eco-friendly alternative to lime and sodium sulphide dehairing. Extracts rich in cysteine proteases with high proteolytic activity (CU) have been obtained from fruits of Bromeliaceae species: Bromelia balansae (Bb), B. hieronymi (Bh), and Pseudananas macrodontes (Pm). In this work, Bb, Bh, and Pm have been studied for application in leather industry compared with commercial enzyme, focusing in their dehairing properties. Enzymatic activities against representative substrates of skin proteins were spectrophotometrically measured at 25, 35, and 55ºC (Tris-HCl, 0.1 M, pH 8, Cys 20 mM). Keratin Azure (KA), Elastin-Congo Red (E), epidermis substrate (EP), and Hide Powder Azure (HPA) were used as representative substrates of keratin, elastin, epidermis, and collagen, respectively. Ability to dehairing was evaluated by incubating soaked cow skins with different concentrations of extracts at 25ºC and pH 8 during 24 h. Grain surface and cross section of skins were observed by scanning electron microscopy. Extracts were able to degrade representative substrates of skin proteins and when compared to the same CU showed similar activity on collagen and epidermis; however, Bh and Pm were the most actives against keratin, while Bh was the only active against elastin. Extracts showing different proteolytic activity (Bb required 1 CU/ml, Bh 1.5 CU/ml, and Pm 0.5 CU/ml) were able to depilate cow skin after a gentle scraping. Although depilated skins with Bb, Bh, and Pm showed different surface aspects, desirable characteristics of dehairing were observed for all extracts since hair pores did not show residual hair, grain surface were clean and intact, and collagen fiber bundles of dermis were not damaged. In conclusion, results here presented show that proteolytic extracts of Bromeliaceae species are promising eco-compatible tools for leather industry, principally in treatment of their waste and dehairing process. ; Centro de Investigación de Proteínas Vegetales ; Comisión de Investigaciones Científicas de la provincia de Buenos Aires

Background Monogenea van Beneden, 1858 is a group of parasitic flatworms, commonly found infecting bony fish. Several genera, such as Cichlidogyrus Paperna, 1960, are reported to include potential pathogenic species that can negatively impact aquaculture fish stocks. They can switch from introduced to native fish and vice versa. In Africa (and all over the world), fish species belonging to Cichlidae are often kept in aquaculture and represent a major source of food. Thus, research on the biodiversity and occurrence of monogenean species on these fish is of importance for aquaculture and conservation. The present study is a survey of the diversity of species of Cichlidogyrus in the south of the Democratic Republic of the Congo (DRC) on three cichlid species: Orthochromis sp. 'Lomami', Serranochromis cf. macrocephalus, and Tilapia sparrmanii Smith, 1840. Methods Specimens of Cichlidogyrus were isolated from the gills and mounted on glass slides with Hoyer's medium. The genital and haptoral hard parts were measured and drawn using interference contrast. Results In total, six species of Cichlidogyrus were found, all new to science: C. bulbophallus n. sp. and C. pseudozambezensis n. sp. on S. cf. macrocephalus, C. flagellum n. sp. and C. lobus n. sp. on T. sparrmanii, C. ranula n. sp. on S. cf. macrocephalus and Orthochromis sp. 'Lomami', and C. maeander n. sp. found on Orthochromis sp. 'Lomami' and T. sparrmanii. The first four species are considered to be strict specialists, C. ranula n. sp. an intermediate generalist and C. maeander n. sp. a generalist. These parasite species show morphological similarities to species found in the Lower Guinea and Zambezi ichthyofaunal provinces, which might be explained by past river capture events between river systems of the Congo Province and both these regions. Conclusions Serranochromis cf. macrocephalus and Orthochromis sp. 'Lomami' can harbour respectively three and two species of Cichlidogyrus, all described in this study. Tilapia sparrmanii can harbour seven species, of which three are described in the present study. These results highlight the species diversity of this parasite genus in the Congo Basin. ; Peer reviewed

Microscopy examination has been the pillar of malaria diagnosis, being the recommended procedure when its quality can be maintained. However, the need for trained personnel and adequate equipment limits its availability and accessibility in malaria-endemic areas. Rapid, accurate, accessible diagnostic tools are increasingly required, as malaria control programs extend parasite-based diagnosis and the prevalence decreases. This paper presents an image processing and analysis methodology using supervised classification to assess the presence of malaria parasites and determine the species and life cycle stage in Giemsa-stained thin blood smears. The main differentiation factor is the usage of microscopic images exclusively acquired with low cost and accessible tools such as smartphones, a dataset of 566 images manually annotated by an experienced parasilogist being used. Eight different species-stage combinations were considered in this work, with an automatic detection performance ranging from 73.9% to 96.2% in terms of sensitivity and from 92.6% to 99.3% in terms of specificity. These promising results attest to the potential of using this approach as a valid alternative to conventional microscopy examination, with comparable detection performances and acceptable computational times. ; Financial support from North Portugal Regional Operational Programme (NORTE2020), Portugal 2020 and the European Regional Development Fund (ERDF) from the European Union through the project Deus ex Machina: Symbiotic Technology for Societal Efficiency Gains', NORTE-01-0145-FEDER-000026. ; N/A

13 páginas, 4 figuras, 4 tablas ; Despite representing only a small fraction of the ocean's dissolved organic matter pool, dissolved free amino acids (DFAA) have high turnover rates and are major nitrogen and carbon sources for bacterioplankton. Both phytoplankton and bacterioplankton assimilate and release DFAA, but their consumption and production are difficult to quantify in nature due to their short residence times (min) as dissolved monomers. We segregated DFAA production by phytoplankton and bacterial consumption by measuring individual DFAA concentrations in four axenic phytoplankton cultures during the exponential growth phase, and also after 4 d incubations in the presence of a natural bacterioplankton community. The amounts and composition of the DFAA pool varied widely among phytoplankton species. The proportion of dissolved organic carbon attributed to DFAA varied among cultures. The picoeukaryotic prasinophyte, Micromonas pusilla, released higher amounts of DFAA than the other species tested (diatoms and dinoflagellate), especially alanine, which has been reported as the dominant individual DFAA in some oligotrophic environments. Community structure of heterotrophic prokaryotes responded to differences in the quality of organic matter released among microalgal species, with Roseobacter-related bacteria responding strongly to exudate composition. Our results demonstrate the specificity of DFAA extracellular release among several algal species and their preferential uptake by members of bacterial communities ; This work was supported by projects STORM (CTM2009-09352/MAR) and ICARO (PIE 200830I120) and DOREMI (CTM2012-34294) funded by Spanish Ministerio de Economia y Competitividad. H.S. benefited from fellowships from the Spanish 'Ministerio de Educación y Ciencia' (JCI-2008-2727) and Brazilian 'Ciências sem Fronteiras' Program from CAPES (BJT 013/2012); C.R.-C. was funded by a I3P-CSIC predoctoral fellowship within the project MODIVUS, CTM2005-04795/MAR; J.P. was funded by the Crafoord Foundation and the Swedish governmental strong research program Ecochange; G.T.T. was partially supported by a sabbatical fellowship from the Spanish 'Ministerio de Educación y Ciencia' ; Peer reviewed

Human adenoviruses (AdV) have been implicated in a wide variety of diseases and are ubiquitous in populations worldwide. These agents are of concern particularly in immunocompromised patients, children, and military recruits, resulting in severe disease or death. Clinical diagnosis of AdV is usually achieved through routine viral cell culture, which can take weeks for results. Immunofluorescence and enzyme-linked immunosorbent assay-based techniques are more timely but lack sensitivity. The ability to distinguish between the six different AdV species (A to F) is diagnostically relevant, as infections with specific AdV species are often associated with unique clinical outcomes and epidemiological features. Therefore, we developed a multiplex PCR-enzyme hybridization assay, the Adenoplex, using primers to the fiber gene that can simultaneously detect all six AdV species A through F in a single test. The limit of detection (LOD) based on the viral 50% tissue culture infective dose/ml for AdV A, B, C, D, E, and F was 10−2, 10−1, 10−1, 10−2, 10−1, and 10−2, respectively. Similarly, the LOD for the six DNA controls ranged from 102 to 103 copies/ml. Twelve common respiratory pathogens were tested with the Adenoplex, and no cross-reactivity was observed. We also validated our assay using clinical specimens spiked with different concentrations of AdV strains of each species type and tested by multiplex PCR and culture. The results demonstrated an overall sensitivity and specificity of Adenoplex of 100%. This assay can be completed in as few as 5 h and provides a rapid, specific, and sensitive method to detect and subtype AdV species A through F.

For population studies, the determination of age status is more important than the determination of calendar age. The research conducted in the biotopes of Precarpathian region indicates that according to the age structure the populations of Polygonatum multiflorum (L.) All. belong to the normal type. Self-maintenance of coenopopulations occurs in a combined way as a result of generative and vegetative reproduction, the intensity of which depends on local edaphic and coenotic conditions. The spectra of their age states and reproductive potential are determined by a set of biotope conditions and have species specificity. The nature of the spectrum of age conditions and the value of the recovery index are the most informative indicative markers. Left-sided spectra in combination with high recovery indices indicate the presence of favorable conditions and a high reproduction potential of cenopopulations. The biotopes of felling areas are the most favorable for the realization of the reproduction potential of most cenopopulations of the studied species; the least favorable are the mainline biotopes.

The conservation of tropical forests is recognized as one of the most important challenges for forestry, ecology and politics. Besides strict protection, the sustainable management of natural forests should be enhanced as a key part of the foundation for the maintenance of tropical rain forest ecosystems. Due to methodological reasons it has been complicated to attain reliable growth data to plan sustainable felling cycles and rotation periods. Tree ring analyses enable the estimation of growth rates over the entire life span of trees and their age as well as giving hints from forest dynamics in previous centuries. For tree ring analysis, stem disk samples were taken from three important commercial tree species (Cariniana micrantha, Caryocar villosum and Manilkara huberi) in the upland (terra firme) forests of the Precious Woods Amazon logging company near Itacoatiara, Brazil. Based on radiocarbon estimates of individual growth zones, the annual nature of tree rings was proven for the three species. Tree rings were measured and the results used together with height estimates to model diameter, height and volume growth. The age of the eldest tree, a C. micrantha, was 585 yrs with 165 cm in diameter. The species' diameter increments range from 0.20±0.12 cm yr-1 to 0.29±0.08 cm yr-1. At first sight, this is considerably lower than increments reported from other Amazonian or African timber species. Considering the respective wood density there is no significant difference in growth performance of dominant timber species across continents. The interpretation of lifetime tree ring curves indicate differences in shadow tolerance among species, the persistence of individuals in the understory for up to 150 years and natural stand dynamics without major disturbances. Management criteria should be adapted for the measured growth rates as they differed considerably from the Brazilian standards fixed by laws (felling cycle of 25–35 years and a common minimum logging diameter of 50 cm). Felling cycles should be increased to 32–51 years and minimum logging diameters to 63–123 cm depending on the species. © 2019 Worbes, Schöngart. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Little is known about the distribution of organisms in transitional areas, overall because of the lack of understanding regarding each species' specificity to the diversity of microhabitats found in these areas and effective ways to sample organisms in these regions. Therefore, in this study we aimed to inventory the bee species in a transitional Cerrado-Amazon area in the state of Pará, Brazil, evaluating the effects of three different sampling methods. We used six fragments, sampled in two different seasons with three sampling methods (arboreal pitfalls with urine, scent traps, and pan traps). The specimens were identified and we used accumulation curves to measure the effectiveness of each method. In total, we sampled 68 bee species, where 53 were exclusively from one single sampling method. The pitfall traps were those with reached the highest species richness among all three sampling methods analyzed, followed by the scent traps and pan traps, respectively. Despite the efficiency and generality of the bee groups we found, the pitfall method does not cover specific bee groups as the Euglossini bees, almost exclusively attracted to scent traps. Therefore, we suggest that studies using pitfalls with urine should be performed in other biomes to evaluate the efficiency of this method in future bee surveys.