The ability to rapidly and accurately evaluate bioactive compounds immobilized on porous particles is crucial in the discovery of drugs, diagnostic reagents, ligands, and catalysts. Existing options for solid phase screening of bioactive compounds, while highly effective and well established, can be cost-prohibitive for proof-of-concept and early stage work, limiting its applicability and flexibility in new research areas. Here, we present a low-cost microfluidics-based platform enabling automated screening of small porous beads from solid-phase peptide libraries with high sensitivity and specificity, to identify leads with high binding affinity for a biological target. The integration of unbiased computer assisted image processing and analysis tools, provided the platform with the flexibility of sorting through beads with distinct fluorescence patterns. The customized design of the microfluidic device helped with handling beads with different diameters (~100–300 µm). As a microfluidic device, this portable novel platform can be integrated with a variety of analytical instruments to perform screening. In this study, the system utilizes fluorescence microscopy and unsupervised image analysis, and can operate at a sorting speed of up to 125 beads/hr (~3.5 times faster than a trained operator) providing >90% yield and >90% bead sorting accuracy. Notably, the device has proven successful in screening a model solid-phase peptide library by showing the ability to select beads carrying peptides binding a target protein (human IgG). The introduction of solid-phase combinatorial libraries of synthetic compounds has resulted in an exponential growth of bioactive compounds. Peptides and peptide mimetics are among the most utilized class of molecules, owing to their innate affinity for biological targets10 and the rich variety of strategies and amino acid building blocks available for peptide synthesis14. Bioinformatic tools have also emerged that guide the design of peptide libraries in terms of peptide length, structure, and amino acid composition, based on molecular-level information on the biomolecular target and putative binding sites on its surface16. Together with library design, the technology for library screening is crucial to identify peptide sequences with high binding affinity. With the growth of basic research utilizing protein-binding ligands and bioactive compounds, a number of high-throughput systems for the screening of combinatorial libraries have been developed and made commercially available. This instrumentation, however, is often cost-prohibitive to most academic groups17, and alternative cost-effective solutions are highly sought after.The ability to rapidly identify small synthetic molecules capable of binding biological targets ((i) incubated with a labeled target, often in presence of other impurities, (ii) sorted using a detector that recognizes beads that have captured the labeled target, and finally (iii) analyzed to identify the peptide sequence they carry20. Commercial beads feature a polydispersed distribution of sub-millimeter diameters, and capture an amount of labeled target that likely depends not only on the binding affinity of the peptide they carry, but also on their particle diameter (~100 µm-300 µm) and pore size distribution. This inherent variability makes library screening and selection of candidate beads extremely labor intensive and reliant on the operator’s ability and subjective visual inspection. To streamline solid-phase screening and ensure rigorous peptide selection, fluorescence-activated cell sorting (FACS) has been previously used for screening peptide libraries21. However, when using large beads as solid substrates (~100–300 µm), sorting using FACS is not feasible. Instruments that address the size incompatibility issue from FACS screening, such as the Union Biometrica COPAS Flow Pilot system for library screening, have been made commercially available22. Hintersteiner et al. also developed a confocal nanoscanning and bead picking (CONA) platform for high-throughput screening of one-bead one compound libraries25. Their cost, accessibility, flexibility, and manufacturability, however, renders them prohibitive for academic research groups. Hintersteiner and coworkers cleverly sought to address this need with low-cost instrumentation for rapid picking from a monolayer of beads based on imaging by wide-field fluorescence microscopy17; this technology, however, does not allow for automated sorting and requires operator-directed selection of beads.Solid-phase peptide libraries comprise a large number (from thousands to millions) of small porous beads, wherein every bead carries multiple copies of a unique peptide sequence. Throughout the screening process, beads are 29. We therefore sought to develop a low-cost, accessible, and portable platform for inexpensive, automated sorting of library beads featuring equipment that is either present in academic laboratories or can be inexpensively fabricated, and yet that provides rigorous selection of peptide ligands with high affinity and selectivity for biological targets  acquisition of fluorescent bead images, followed by image-processing based analysis and sorting (to either the selection receptacle or to waste). Notably, the quantitative image analysis algorithm takes into account both intensity and radial distribution of fluorescence, which combined improves the sensitivity and specificity of the platform in selecting high-affinity peptides by reducing the risk of false positives and negatives. To ensure identification of peptides with high binding selectivity, the algorithm enables orthogonal dual-color sorting by selecting the beads that only capture the fluorescently-labeled target while excluding beads that carry competitor proteins labeled with a different color. Finally, the device can be operated in either bulk or single bead separation mode. In bulk separation mode all positive beads are directed to a common flask, whereas in single bead separation mode each positive bead is individually directed to a single well in a 96 well-plate. In this work, we validated the integrated bead-sorting device and code using a dual fluorescence library mimetic. This comprises a combination of ChemMatrix beads labeled either by covalent conjugation of fluorescent dyes (Texas Red and Fluorescein) or by adsorption of fluorescently-labeled, streptavidin-conjugated proteins of different molecular weight to biotinylated beads resulting in either a homogeneous or halo-like radial color distribution, respectively. The fluorescence intensity and radial pattern observed in each class of beads were used as metric to inform sorting parameters. Beads with different fluorescence patterns  were analyzed to quantify the accuracy of bead-sorting protocol. Our results indicate that the device was capable of sorting beads with a detection yield and accuracy of ~92% and 94% respectively. It should be noted that the system performs all sorting and quantitative image analysis without any user supervision, thereby enabling a fully automated library screening process. It is worth mentioning that high sensitivity and specificity were achieved while microscopy was performed in a non-confocal setup demonstrating the robustness of algorithm in handling images with suboptimal quality. The flexibility of quantitative image analysis algorithm in handling images with various qualities, enables the set up to be used with different instruments. In this work, we designed and fabricated an automated, low-cost, accessible, and portable microfluidic platform capable of sorting targets with various fluorescence patterns with high precision and accuracy.Microfluidic platforms have previously been used to sort particles, cells and dropletsets Fig. . This sy34. Streamlining their identification requires affordable, automated devices capable of sorting solid-phase peptide library beads with accurate and rigorous orthogonal selection logics. To meet these needs, we have developed a low-cost accessible automated microfluidic platform to handle, capture, image, analyze, and sort beads based on fluorescence patterns that directly translate into the binding affinity of the peptides carried by the selected library beads. We applied this technology for the isolation of positive  beads from a library mimetic consisting of a mixture of labeled ChemMatrix beads with different fluorescent color, intensity, and radial distribution.Peptides and peptide mimetics with high affinity and selectivity for biological targets will play a critical role in next generation medicinal chemistry, diagnostics, and downstream biomanufacturing technology35, PEG-based ChemMatrix resin has been successfully utilized as a substrate for the synthesis and screening of solid-phase peptide libraries against target proteins for the selection of peptide ligands. In this context, however, the small pore diameter of ChemMatrix resin has been shown to limit protein diffusion through the bead, resulting in a protein-rich corona, or “halo” whose width decreases with the molecular weight of the protein18. When fluorescently labeled targets are utilized, this translates in a halo-like fluorescence pattern; in theory, smaller proteins produce a diffuse halo, while larger ones produced a narrow halo. To replicate this effect, a number of fluorescent bead classes were prepared, either by conjugating different amine-reactive fluorophores  onto aminomethyl ChemMatrix beads or by adsorbing fluorescently labeled streptavidin or streptavidin-conjugated proteins of different molecular weight onto biotin-ChemMatrix beads ; accordingly, the selection of beads carrying only the desired label ensures capture specificity. To demonstrate the full potential of our technology, we sorted the beads from the library mimetic according to various fluorescence patterns and intensities, showing that the device is capable of unsupervised bead sorting with high accuracy in response to criteria set by the user.To enable automated bead selection and sorting, we developed customized algorithms to detect the presence of a bead, to extract image-based descriptive metrics required for classification, and to sort the bead according to the criteria provided by the operator into a positive line to the collection receptacle or a negative line to be discarded. Bead selection was based on (i) radial distribution of fluorescence, (ii) fluorescence intensity, and (iii) difference of color 1 Screening the peptide library using the microfluidic platform comprises a series of tasks performed in a loop. Throughout the process, on/off-chip valves are utilized to trap single beads in the imaging zone and sort them as positive or negative based on the selection criteria. The first step involves loading an individual bead to the imaging zone Fig. .Figure 3vs. positive beads fed and ratio of positive beads collected vs. total beads collected in the positive flask.Several tests were performed to evaluate the bead sorting accuracy and precision of the system using fluorescently labeled beads of Class 1–7. Library mimetics depicting different screening scenarios were prepared by mixing beads of one class, considered as positive, with a combination of beads from other classes. In every experiment, a different library mimetic was suspended in PBS at a density of ~2 beads/mL and maintained under gentle agitation to prevent aggregation. Beads were fed at a rate of ~2.5 beads/min. The beads conforming to the set selection criteria were sorted as positive, and subsequently analyzed to calculate the yield and accuracy of the sorting process, respectively defined as ratio of positive beads collected th percentile of the bead’s intensity in the red channel and the 90th percentile of the bead’s intensity in the green channel. Beads exhibiting values above 0.5 in normalized red channel 90th percentile and below 0.2 in normalized green channel 90th percentile were considered positive  or dual (red and green) color Fig. . In the ive Fig. . These tth percentile pixel intensity higher than 0.5 in the green channel and normalized 90th percentile pixel intensity lower than 0.2 in red channel were considered positive  from a mixture of Class 2, Class 1, and unlabeled beads. For this test, beads with normalized 90ive Fig. . Using tely Fig. . As a re18 on screening ChemMatrix-based peptide libraries indicates that, due to diffusion limitations, larger target proteins effectively penetrate only the outer corona of the beads. This translates into a “halo”-like fluorescence pattern, potentially accompanied by fluorescing spots randomly distributed throughout the bead  beads from a mixture of Class 6, Class 4 , Class 7 , and unlabeled beads. To distinguish beads with subtler fluorescence patterns, a more complex set of parameters was defined. Specifically, to be accepted as positive, a bead ought to meet three criteria:As a result, 21 positive beads and 3 false positives were retrieved out of 24 positive beads fed to the sorting device (~87.5% yield). Considering that 3 beads sorted as false positive, we obtained a ~87.5% accuracy Fig. . The preAs a final experiment, we aimed to sort Class 5 beads  from a mixture of Class 5, Class 4 , Class 7 , and unlabeled beads. Similar to the previous test, three criteria were defined to identify positive beads with non-uniform patterns:By applying these thresholds, we were able to retrieve 30 out of 33 positive beads fed to the sorting device (~91% yield), with 4 false positives and 2 false negatives (~88% accuracy) Fig. .th percentile pixel in the red channel and the normalized 90th percentile pixel of non-bright segment of bead in the red channel  using an equal ratio of alanine, aspartatic acid, tyrosine, arginine, glycine, glutamate, histidine, leucine, glutamine, and serine. The library was then spiked with ~5% in volume of ChemMatrix beads functionalized with the IgG-binding control peptide HWRGWV-GSG. This peptide ligand has been previously shown to selectively bind IgG in complex fluids, including human plasma and Chinese hamster ovary (CHO) cell culture fluids37. The procedures for the synthesis of the library and HWRGWV-GSG on ChemMatrix beads are detailed in the Supplemental Information. The HWRGWVGSG-spiked library was incubated with red-labeled IgG (tagged with Texas Red NHS ester) mixed with green-labeled CHO host cell proteins  and sorted using the proposed device to identify beads with “high” IgG binding and “low” HCP-binding (red only beads). In this context, we aimed to sort and sequence beads displaying selective halo-patterned binding of IgG to verify that the platform is capable of extracting IgG-binding sequences, including HWRGWVGSG. Nonetheless, positive sequences other than HWRGWVGSG were expected due to limited compositional bias in the combinatorial library. Prior to library screening, optimized hyperplanes for sorting positive beads were determined. In order to identify these hyperplanes, ~30–40 positive control beads were flown through the device and quantitative data was extracted. Based on the results acquired by running these beads through the device, the following thresholds were set for a bead to be considered positive: (i) αor − αog > 0, (ii) αor > 0.25, (iii) 90th percentile pixel intensity of entire bead in green channel <0.1, and (iv) 95th percentile pixel intensity of entire bead in red channel >0.08. Using the platform, we screened ~200 beads of this library and identified 12 beads as positive. To further verify the existence of fluorescence pattern of interest, the selected positive beads were imaged again individually in a single well. All 12 beads exhibited halo pattern in post-sorting microscopy, which is indicative of platform’s ability in identifying true positive. Finally, the peptides carried by the selected beads were sequenced by Edman degradation38 using a Shimadzu PPSQ 33A Protein Sequencer to verify the presence of the control sequence HWRGWV-GSG . In particular, we focused on the identification of immunoglobulin G (IgG)-binding ligands as a case study. Initially, a one-bead-one-component (OBOC) octameric peptide library X19. Despite its success, manual screening is extremely labor-intensive and commercial devices for automated screening are likely unaffordable to academic labs. In this work, we developed a low-cost accessible platform for automated screening of solid-phase peptide libraries that integrates lab-scale microfluidics and microscopy with user-friendly software that enables unsupervised bead imaging and sorting. The device, which can process 100–150 beads per hour, was tested to evaluate yield and accuracy of automated bead sorting. This setup was successfully able to handle beads of various size (~100–300) and flexible enough to detect and sort beads with different fluorescence pattern. To this end, we utilized seven classes of beads featuring different patterns of fluorescence labeling that mimic the appearance of library beads screened against protein targets with different size. The average yield and accuracy of positive beads recovered by the device from mixtures of different classes was found to be 92% and 94% respectively. Particularly encouraging was the recovery of beads with complex fluorescence patterns, which afforded ~88% yield and ~88% accuracy. Notably, the acquisition of the metrics needed to perform the bead sorting was unsupervised; specifically, two bead patterns  were produced using labeled proteins of different molecular weight, automatically acquired, and successfully utilized to recover positive beads from both classes. This demonstrates that the device provides a cost-efficient, accessible alternative for the automated sorting of bead-based combinatorial libraries with high sensitivity and specificity. As a demonstrative case study, we employed the device for selecting IgG-binding peptides by screening a combinatorial library spiked with IgG-binding HWRGWV-ChemMatrix beads upon incubation with IgG spiked in a complex protein mixture (CHO cell culture fluid). We finally confirmed the presence of IgG-capturing beads by Edman sequencing of the positive beads. This platform achieved high accuracy and yield in sorting beads via incorporation of fluorescence microscopy. In addition, as a microfluidic platform, multiple devices and setups can be assembled in a relatively short period of time.Screening combinatorial peptide libraries using fluorescence-based readouts is a powerful approach for the identification of protein-binding peptides. With solid-phase libraries, in particular, which feature peptides conjugated on porous beads, fluorescence detection of the beads following capture of the labeled protein target is a successful approach for high-throughput screening of combinatorial solid-phase librariesThe proposed device platform can also be integrated with other analytical instruments as well as systems that enhance the decision-making algorithms. Additionally, image processing and pattern recognition can be carried out using machine-learning algorithms that would improve the accuracy of the decisions made during sorting over the currently utilized statistical algorithm. Integrating these supervised machine-learning algorithms would lead to detecting more subtle and complicated patterns occurring throughout screening a library of peptides. The throughput of this platform can also be improved by implementing parallel devices simultaneously or by increasing the concentration of beads in the suspension flown through the device.The microfluidic platform was fabricated by traditional photolithography followed by soft lithography. Negative photoresist SU-8 2150 was spun at 1460 rpm to achieve a feature height of 400 μm Fig. . Soft baet al.18. Beads with homogeneous fluorescence were prepared by labeling ChemMatrix aminomethyl beads with Texas Red NHS ester, fluorescein isothiocyante (FITC), or both; to this end, 50 mg ChemMatrix dry resin was swollen in 1 ml 0.1 M sodium bicarbonate, pH 8.3, for 1 hour at room temperature and incubated with 50 µl of 2 mg/mL fluorescent dye solution in dimethyl sulfoxide (DMSO) in dark, for 1 hour at room temperature under gentle agitation. After incubation, the dye solution was removed and the beads were washed thoroughly with 0.1% Tween 20 in phosphate buffered saline (PBS), pH 7.4 (PBS-T) for storage and to remove unreacted fluorescent dye. Beads featuring halo-like beads were produced using proteins with a range of molecular weights. To this end, biotin was initially conjugated onto ChemMatrix aminomethyl beads by incubating the resin with a 1 mg/mL solution of biotin and 2 mg/mL 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in 0.1 M MES, pH 4.6, to achieve a ratio of 1 mg biotin:2 mg EDC:100 mg resin, for 2 hours under gentle agitation at room temperature. Meanwhile, high purity bovine serum albumin  and alcohol dehydrogenase  were dissolved at 2 mg/ml in phosphate buffered saline and conjugated to streptavidin (53 kDa) using a LYNX Rapid Streptavidin conjugation kit. The streptavidin-conjugated proteins were then fluorescently labeled with Texas Red NHS ester. Briefly, 10 µL of streptavidin-conjugated protein was dissolved at 2 mg/ml in 0.1 M sodium bicarbonate, pH 8.3, and mixed with 1 µL of 10 mg/mL solution of Texas Red NHS dye in DMSO under gentle agitation for 1 hour, light-protected at room temperature. The unreacted dye was removed by diafiltration against PBS-T. For labeled protein-bead interactions, 20 µL of the labeled streptavidin and protein conjugates was incubated with 2–5 µl settled volume of biotinylated beads for at least 1 hour at 2–8 °C.Beads were prepared to feature two different fluorescence profiles, namely homogeneous and “halo”-like distributions, as described by Marani 2 camera equipped with two charge-coupled devices (CCDs) enabling simultaneous microscopy in wavelengths of interest. The ChemMatrix beads used in this study tend to aggregate in solution, leading to clogging of the microfluidic platform and sorting errors . To prevent aggregation, beads were maintained in a diluted suspension in PBS buffer (150 beads per 80 ml) and gently stirred on an orbital shaker throughout the duration of the sorting cycle. On-chip valves were filled and degassed with a 50% glycerol solution with a similar refractive index as PDMS, which improves image quality in the vicinity of the valves. A custom-built pressure box equipped with pressure regulators was used to drive fluid flow in the tubing and device. Valve operations was controlled by a custom-developed MATLAB Graphical User Interface (GUI).In this study, beads are sorted based on their fluorescence intensity or pattern in both red and green channels. Simultaneous dual color fluorescence microscopy was performed using a LEICA DMi8 inverted microscope connected to a Hamamatsu Orca-Dth-percentile, and different combinations of these metrics are extracted. The values extracted using this algorithm are normalized by converting the 12-bit pixel value range from 0 (black)-4095(white) to 0 (black)-1 (white). Positive beads are detected and isolated based on the values of these metrics. In this study, more than 20 metrics were extracted for each bead. Depending on the pattern of interest, a combination of two or three metrics is used as criteria for the identification and sorting of positive beads.To automate the sorting process in an unbiased and quantitative manner, computer-vision algorithms were implemented to detect bead presence in the imaging zone and classify it as negative or positive based on the criteria provided by the operator. Bead detection was carried out by a custom developed MATLAB algorithm that identifies the presence of beads in the channel independently of bead size or fluorescence intensity. As shown in Supplemental Fig. Supplementary Information


Arracacha virus Y (ArVY) derived from an arracacha (Arracacia xanthorrhiza) sample originally collected in 1976 in Peru and compare it with other potyvirus genome sequences. It had a 79% nucleotide identity with a 2013 Brazilian Arracacha mottle virus (AMoV) sequence, suggesting that AMoV is ArVY.We describe here the first genome sequence of  Arracacha virus Y (ArVY) derived from an arracacha (Arracacia xanthorrhiza) sample originally collected in 1976 in Peru and compare it with other potyvirus genome sequences. It had a 79% nucleotide identity with a 2013 Brazilian Arracacha mottle virus (AMoV) sequence, suggesting that AMoV is ArVY.We describe here the first genome sequence of  Arracacha virus A (genus Nepovirus) and Arracacha virus B  were isolated from arracacha  plants showing pronounced yellow mosaic symptoms in young leaves . A fourth virus with 650-nm filamentous particles typical of the genus Carlavirus was also present. It was first named Arracacha latent virus , and a complete genomic sequence of it was obtained in 2013  was used in paired-end mode to 3′ trim the resulting 593,960 paired reads to a quality score of 20. The reads were assembled with Trinity v2, with the maximum memory allocation set to 99 gigabytes of RAM, and the process allocated 64 central processing units . Given that ArVY had a nucleotide identity of >75% with AMoV, both belong to the same potyvirus species (In this paper, the methods used were the same as those described previously –9. Brief–ng units . We usedng units  to compang units  was used species . As the MH716807. Raw data were deposited in the SRA under BioSample number SAMN10081144 and SRA run number SRR7873470, which are part of SRA study number SRP162046.The ArVY sequence described here was deposited in GenBank under the accession number


PLOS Pathogens intends to contribute to this ongoing debate with four balanced and differing perspectives from notable members of the scientific community.The implications of publishing dual use research of concern (DURC) continues to be a topic widely and deeply discussed in the scientific community. In a series of Opinion articles, PLOS ONE study on Horsepox virus [PLOS ONE paper as a springboard for their Opinions, have each provided insightful commentary related to DURC studies and the possible impact of these studies on infectious diseases research more generally.The four author groups represented in these Opinions include Drs. Ryan S. Noyce and David H. Evans, two authors of a recent ox virus , Dr. Volde novo approach to synthesize the complete sequence of a horsepox virus (HPVX), which has not been detected in the wild since the mid-1980s [de novo and a discussion of how these methods could be used to develop other live virus-based vaccines. The publishing of this study led to intense debate among the public and scientific community as to whether this type of study should be permissible given that the methods described could be used to synthesize any poxvirus, particularly variola virus, which was declared eradicated in 1980 and is the infectious agent responsible for smallpox.In early 2018, Ryan S. Noyce, Seth Lederman, and David H. Evans described a PLOS ONE article, the editors of PLOS Pathogens felt that providing a forum for individuals holding opinions on both sides of this debate to explain their rationale would be appropriate and interesting, particularly given the broad infectious diseases readership of the journal. As a PLOS Pathogens Opinions editor, I was eager to facilitate this process and guide the four papers through peer review and publication. The intent of publishing these Opinions together is to facilitate and encourage a healthy and balanced discussion of the implications of publishing DURC-related research and to provide a forum to consider the HPVX study as a model by which to consider future similar work. Together, and along with the original article in PLOS ONE, these four Opinions form a compendium of commentaries on this important topic. I hope that you find these articles as thought-provoking as I did and that they allow you to consider all sides of this debate as you form your own opinions.In light of the debate around the


Measurement of serum cobalamin concentration, often in combination with serum folate concentration, is routinely performed as a diagnostic test in clinical practice. While the detection of hypocobalaminemia has therapeutic implications, interpretation of cobalamin status in dogs can be challenging. The aim of this review is to define hypocobalaminemia and cobalamin deficiency, normocobalaminemia, and hypercobalaminemia in dogs, describe known cobalamin deficiency states, breed predispositions in dogs, discuss the different biomarkers of importance for evaluating cobalamin status in dogs, and discuss the management of dogs with hypocobalaminemia.Disorders of cobalamin (vitamin B AMNamnionlessCIEchronic inflammatory enteropathiesCUBNcubilinEPIexocrine pancreatic insufficiencyGC/MSgas chromatography‐mass spectrometryGIgastrointestinalHCYhomocysteineIBDinflammatory bowel diseaseIFintrinsic factorIGSImerslund‐Gräsbeck syndromeIREimmunosuppressant‐responsive enteropathyLC/MS‐MSliquid chromatography‐tandem mass spectrometryLoQlower limit of quantificationMMAmethylmalonic acidNREnonresponsive enteropathyPLEprotein‐losing enteropathyRIreference intervalSIsmall intestine112) metabolism are increasingly recognized in small animal medicine. Causes of cobalamin deficiency in dogs vary from chronic gastrointestinal (GI) disease to hereditary defects in cobalamin metabolism. Measurement of serum cobalamin concentration, often in combination with serum folate concentration, is routinely performed as a diagnostic test in small animal practice to detect dogs with vitamin B deficiency. The detection of hypocobalaminemia also has therapeutic implications. Because interpretation of the cobalamin status in dogs and the management of dogs with a suboptimal cobalamin status can be challenging, an update on this topic and a definition of the different cobalamin states in dogs are provided here.Disorders of cobalamin , describe causes of cobalamin deficiency states in dogs including breed predispositions, summarize breed‐specific changes in cobalamin metabolism, discuss biomarkers of importance for evaluating cobalamin status in dogs, and discuss the treatment options for dogs in which a suboptimal or deficient cobalamin status has been diagnosed.22.112. It is mainly ingested with food of animal origin with liver, kidney, meat, egg, milk products as well as fish having high cobalamin content, but smaller amounts can also be produced by the intestinal microbiota.Cobalamin is a water‐soluble vitamin, also referred to as vitamin BPlants and plant products contain virtually no cobalamin and, whereas ruminants and other herbivores can produce a sufficient amount of cobalamin in their intestines, omnivorous or carnivorous animals including dogs and cats are not able to produce cobalamin. The intestinal microbiota of dogs and cats can produce cobalamin in the presence of cobalt, but because this site of cobalamin production is distal to the site of its absorption, this ability appears to be of little benefit to the animal. Most commercial pet foods, including vegan or vegetarian diets, are supplemented with cobalamin,2.22.2.16)‐dependent enzyme, catalyzes the regeneration of methionine from homocysteine (HCY). This reaction links both cobalamin and folate metabolism , and a large amount of cobalamin is conserved by enterohepatic recirculation.Renal glomerular filtration of the transcobalamin II‐cobalamin complex is followed by tubular reabsorption to minimize urinary losses of cobalamin. Megalin, an endocytic receptor in the proximal renal tubulus that has a high affinity for the transcobalamin II‐cobalamin complex, mediates renal reabsorption and retention of cobalamin. Free cobalamin is excreted in the urine.3Subnormal serum cobalamin concentration or cellular cobalamin deficiency in dogs can be detected by measuring serum cobalamin, HCY, and MMA concentrations. Although serum cobalamin concentrations are measured routinely in dogs and cats and determination of serum cobalamin is offered by a number of veterinary diagnostic laboratories, quantification of serum HCY and MMA is not routinely available, although both are considered as useful markers to determine the cellular cobalamin status of companion animals.3.13.1.1There are several commercially available immunoassays for the measurement of serum cobalamin concentrations. An automated chemiluminescence assay is routinely used in North America and Europe, and the reference intervals (RI) for serum cobalamin concentrations in dogs that are used by the different veterinary diagnostic laboratories are similar Table . An immuThe main indication for measuring serum cobalamin is to identify a subnormal cobalamin status.3.1.2Serum HCY concentrations can be measured by gas chromatography‐mass spectrometry (GC/MS), with RIs of 5.0‐22.1 μmol/L12), folic acid (vitamin B9), and pyridoxine (vitamin B6) are essential cofactors for enzymes involved in the metabolism of HCY.Homocysteine is a sulfurated intermediate amino acid that is produced from dietary methionine and is then either remethylated to methionine or metabolized to cysteine.vitamin B are esseIn humans, HCY has direct toxic effects on neurons and endothelial cells, can induce DNA strand damage, oxidative stress, and apoptosisCirculating HCY is mostly bound to albumin. As a consequence, diseases associated with hypoalbuminemia, such as protein‐losing enteropathy (PLE), might be associated with a lower degree of hyperhomocysteinemia than expected or even with normohomocysteinemia.3.1.3http://www.vetmed.tamu.edu/gilab]) and 393‐1,476 nmol/L.Methylmalonic acid concentrations can be determined in serum and urine samples. Serum MMA concentrations are measured routinely in human medicine by either a GC/MS or liquid chromatography‐tandem mass spectrometry (LC/MS‐MS) assay. However, measurement of serum MMA concentrations is currently not routinely performed in companion animals. RIs for serum MMA concentrations in dogs are 415‐1,193 nmol/L , depletion of cobalamin stores in the liver and kidneys secondary to cobalamin malabsorption, or a combination of these, leading to insufficient amounts of cobalamin entering the cells. Tissues or cells with a high turnover rate such as enterocytes or blood cells are primarily affected. Dogs with a serum cobalamin concentration below RI have significantly higher serum MMA concentrations compared to dogs with normocobalaminemia, but serum MMA concentrations are increased in some (12%) normocobalaminemic dogs.Careful interpretation of serum MMA concentrations should be exercised in the presence of other conditions that can affect serum MMA concentrations, such as renal insufficiency causing increased serum MMA concentrations.Of healthy normocobalaminemic Border Collies, 38% have increased urine MMA concentrations.Urine MMA concentrations are also measured by GC/MS3.2The currently recommended interpretation of the cobalamin status in dogs is summarized in Table 3.2.1Normocobalaminemia refers to a serum cobalamin concentration within the RI for healthy dogs. However, serum cobalamin concentration does not accurately reflect the cobalamin status on the cellular level. If cobalamin transport into the cell is hindered, intracellular cobalamin deficiency might result while the cobalamin concentration in serum remains within normal limits.Twelve percent of dogs with chronic enteropathy and a serum cobalamin concentration within the normal RI have an increased serum MMA concentration.3.2.2Hypocobalaminemia is typically referred to in dogs with a serum cobalamin concentration between the lower limit of quantification (LoQ) of the assay and the lower reference limit Fig. . HoweverThirty‐one percent of hypocobalaminemic dogs (as defined above) have serum MMA concentrations above the RI,After supplementation, cobalamin is rapidly extracted from plasma by tissues where it binds to cobalamin‐dependent enzymes with a relatively long half‐life.3.2.3The term cobalamin deficiency is not well‐defined in the veterinary literature. Most current cobalamin assays have an LoQ between 45 and 150 ng/L :1070‐1071 [abstract]) in a smaller number of dogs with clinical signs similar to those reported above (unpublished data), the upper reference limit in which was also much lower (590 ng/L) which might at least in part explain the discrepancy between the reported prevalence of hypercobalaminemia in dogs. Measurement of serum cobalamin concentrations at 2 different laboratories also needs to be considered when comparing the prevalence of hypercobalaminemia between these 2 studies. Serum MMA concentrations have not been evaluated in dogs with hypercobalaminemia.Hypercobalaminemia refers to a serum cobalamin concentration above the normal RI. Until recently, hypercobalaminemia was considered as a benign finding and was basically ignored in companion animals. However, recent data in people suggest that hypercobalaminemia is an underestimated finding that can reflect a number of serious underlying diseases such as solid neoplasms, hematological and other malignancies, and hepatic or renal diseases.4Hypocobalaminemia and cobalamin deficiency can have a variety of causes in dogs including exocrine pancreatic insufficiency (EPI), chronic severe ileal disease , SI dysbiosis , and inherited conditions Fig. B.49, 64,4.1Blood cell count abnormalities, typically nonregenerative anemia with megaloblastosis, neutropenia, and hypersegmented neutrophils, can be early signs of derangements in cobalamin metabolism.In addition to blood cell dyscrasias and organic acidemias associated with neurological signs, especially dogs with hereditary cobalamin deficiency can show severe clinical signs such as failure to thrive, anorexia, lethargy, vomiting, and diarrhea. The age of affected dogs at presentation with clinically apparent disease also varies, with the majority of dogs showing clinical signs early in life and a smaller proportion of dogs having a more delayed onset of clinical signs.4.2https://www.esvcp.org/index.php/docman/clin-path-cases/106-case-2-maguire-with-diagnosis/file.html); genetic defects and hypocobalaminemia has been confirmed for a few canine breeds. Certain dog breeds have a predisposition for hypocobalaminemia for which the underlying mechanism or cause has not been elucidated  depends on the normal structure of the other part of the receptor for epithelial brush border expression and function.AMN gene in Australian Shepherd dogs and Giant Schnauzers.CUBN mutation causes IGS in Border Collies.CUBN mutation is also responsible for canine IGS in the Beagle breed.CUBN mutation in juvenile Beagle dogs causes more severe cobalamin malabsorption than a different CUBN defect in Border ColliesAMN mutations in Giant Schnauzers and Australian Shepherd dogs. A residual CUBN expression, which does not exist in affected Beagle dogs, might explain why the diagnosis in some Border Collies can be delayed compared to affected dogs of other breeds.CUBN mutation was relatively low at 6.2%,CUBN splice‐site variant causes cobalamin deficiency.https://centerofanimalgenetics.com, https://animalabs.com, or http://www.laboklin.de). A large genomic study in Chinese Shar‐Peis revealed an association of 2 canine microsatellite markers, located on canine chromosome 13, with cobalamin deficiency in this breed.Cubam expression is disrupted by 2 independent mutations in the 4.34.3.1Cobalamin deficiency is common in dogs with EPI. A study showed 82% of dogs with EPI have decreased serum cobalamin concentrations, and 36% of these dogs had severe hypocobalaminemia .Enterococcus faecium SF68Exocrine pancreatic insufficiency is characterized by an inadequate production of digestive enzymes from pancreatic acinar cells, leading to the typical clinical signs of weight loss in the face of polyphagia and increased fecal volume.Because the exocrine pancreas is the major site of IF synthesis in dogs, cobalamin supplementation is often necessary in these dogs. However, cobalamin deficiency in some cases is not corrected by enzyme replacement alone likely because IF is not consistently a component of the exogenous enzyme mixture.4.3.2Chronic inflammatory enteropathies (CIE), further classified based on the response to treatment as either food‐responsive enteropathy, antibiotic‐responsive enteropathy, immunosuppressant‐responsive enteropathy (IRE), or nonresponsive enteropathy (NRE), are characterized by chronic persistent or recurrent clinical signs of GI disease  for longer than 3 weeks and are diagnosed retrospectively based on the response to treatment.It has been postulated that chronic mucosal disease affecting the ileum reduces the epithelial expression or function of the cubam receptor leading to a reduced mucosal uptake of cobalamin. However, currently there are no studies that have investigated the expression levels of the ileal cobalamin‐IF‐receptor in dogs with chronic enteropathies.In addition, cobalamin has been hypothesized to be decreased in dogs with CIE because of secondary SI dysbiosis. Once the body stores of cobalamin are depleted, cobalamin deficiency can occur.Reported prevalences of hypocobalaminemia in dogs with CIE ranged from 19% to 38%,Histological lesions in dogs with CIE are often multifocally distributed, and different degrees of disease severity might be found among the different segments of the intestinal tract and also among different regions within the same segment .Hypocobalaminemia correlates with an increased number of intraepithelial lymphocytes in the ileal mucosa,Several factors have been evaluated for their potential to predict outcome in dogs with CIE.1‐proteinase inhibitor concentrations (presumably due to PLE) in Yorkshire Terriers.Protein‐losing enteropathy is a syndrome associated with an abnormal loss of albumin into the intestinal lumen. Common causes for PLE are altered lymphatic drainage or increased mucosal permeability.Serum cobalamin concentrations should be evaluated in all dogs with PLE and, if needed, supplemental cobalamin be started early.4.3.3Sixteen percent of dogs with multicentric lymphoma have hypocobalaminemia, and this finding is associated with a poor outcome.4.3.4Small intestinal (SI) dysbiosis is defined as an altered composition, richness, or both of the intestinal microbiota.Bacteroides spp. are the principle competitors for cobalamin with the host because of the ability to utilize cobalamin‐IF complexes, in contrast to other bacteria that can only bind free cobalamin.Competition between bacteria and host cells for essential nutrients such as cobalamin can lead to malnutrition.5Cobalamin should be supplemented whenever serum cobalamin concentration is subnormal. There is a 12% probability that CIE dogs with low‐normal serum cobalamin concentration  might also benefit from supplementation.In dogs with CIE and suboptimal cobalamin status, cobalamin is recommended to be given at a dose of 50 μg/kg SC every 7 days or 50 μg/kg PO q24h.Dogs with selective cobalamin malabsorption—if left untreated—will die as a result of metabolic derangements and immunodeficiency.5.15.1.1Initially, weekly SC injections of 50 μg/kg SC cyanocobalamin , cobalamin supplementation was traditionally recommended to be continued every 2‐4 weeks. More than 90% of dogs with a suboptimal serum cobalamin status achieve hypercobalaminemia within 4 weeks of parenteral cobalamin supplementation, but serum cobalamin concentrations decreased within the RI towards the 12‐week mark of cobalamin supplementation in the majority of these dogs.A study investigating the effect of injectable hydroxocobalamin for the treatment of Beagle dogs with IGS showed a dose of 1 mg hydroxocobalamin administered IM to be adequate for maintaining a normal clinical status and normal urine MMA concentrations for up to 2 months.5.25.2.1Cobalamin (50 μg/kg PO) is administered once daily (250‐1000 μg in dogs, depending on the size of the dog; see 5.2.2Several studies in human patients with hypocobalaminemia suggest that supplementation with oral cobalamin might be as effective as parenteral administration.Romy Heilmann has received speaker honoraria from Dechra company for presentations at continuing education meetings. Dechra has an oral cyanocobalamin formulation commercially available.Authors declare no off‐label use of antimicrobials.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.Appendix S1: Supporting informationClick here for additional data file.


Aging is associated with normative declines in cognitive resources that increase the costs associated with mobilizing resources in cognitively demanding activities. Selective Engagement Theory  hypothesizes that changes in costs influence the motivation to engage in such activities in everyday life. We used an economic discounting task to examine the relationship between both objective estimates (systolic blood pressure responses) and subjective estimates (NASA Task Load Index) of cognitive costs in 78 older adults’ (ages 64-85) decisions to engage in more or less demanding activities. Perceptions of costs were meaningfully tied to actual costs (SBP), but further influenced by personal or primed attitudes about aging. Interestingly, decisions to engage in demanding activities—as reflected in discounting decisions—were less influenced by effort expenditure in the activity than by perceptions of difficulty. These results underscore the role that negative stereotypes play in undermining motivation to engage in potentially beneficial activities.


We report detection of Lassa virus and Crimean-Congo hemorrhagic fever virus infections in the area of Bamako, the capital of Mali. Our investigation found 2 cases of infection with each of these viruses. These results show the potential for both of these viruses to be endemic to Mali. Numerous viral hemorrhagic fevers are endemic to countries in Africa. Despite the underlying pathogens originating from diverse virus families, clinical features of hemorrhagic fevers are similar, including fever, malaise, abdominal pain, vomiting, headache, and myalgia  is the causative agent of Lassa fever. The multimammate rat, Mastomys spp., is the natural host of LASV and sheds the virus in urine and droppings. Transmission of LASV to humans usually occurs through contact with the excreta of infected rodents or with body fluids from persons with symptomatic illness.Lassa virus  is endemic to many regions, including Eurasia, Central America, and parts of Europe and Africa. To date, reports of acute CCHFV infections in West Africa are limited to Senegal . https://www.qiagen.com) and amplified viral RNA by using LASV reverse transcription PCR  for further investigation.Diagnostic amplicons of the LASV small (S) RNA segment, 278 nt from the boy and 276 nt from the girl, were sequenced by Sanger sequencing (GenBank accession nos. MH473586–7). Sequence analysis of the viral S segment showed that 1 patient was infected by a LASV strain that clusters genetically with viruses belonging to lineage IV . BecauseThe second identified Lassa fever case was caused by a LASV strain belonging to the Mano River clade with 91% similarity to Lassa Soromba R (GenBank accession no. KF478765) . This viWe also detected CCHFV, which previously was not known to circulate in the population of Mali. Using reverse transcription quantitative PCR, we detected acute CCHFV infection in 2 patients hospitalized at Hospital Gabriel Touré in April 2017, a 1-year-old boy (cycle threshold 32.74) and a 2-year-old boy (cycle threshold 36.95). We obtained sequence data from the viral S segment for the 1-year-old boy. Phylogenetic analysis showed that this virus is related to CCHFV strain ArD39554 from Mauritania and CCHFV sequence (GenBank accession no. KF793333) recently detected in ticks collected only 25 km from Bamako . We wereIn summary, our study detected 2 cases of infection with LASV in Bamako, Mali, indicating a broader distribution of LASV in Mali than previously known. This finding raises serious public health concerns for future LASV infection in cities in Mali. We also identified 2 human cases of infection with CCHFV in Mali, suggesting an extended CCHFV-endemic region in Africa. Our results underline the need for LASV and CCHFV surveillance programs in sub-Saharan regions of Mali, Burkina Faso, and Niger, which have similar ecology.


Highly variable bead stabilities with dissimilar sensitivities to pH were achieved using these methods. At low pH levels , chitosan/SDS beads were the most stable, requiring roughly 2 days to dissolve. In weakly acidic media (at pH 3.0–5.0), however, chitosan/TPP beads exhibited the highest stability, remaining intact throughout the entire experiment. Beads prepared using only NaOH solution  were the least stable, except at pH 5.0, where the NaC10 and NaC12-derived beads dissolved slightly faster. Collectively, these findings provide further guidelines for tailoring supramolecular chitosan bead stability in acidic media.Chitosan beads attract interest in diverse applications, including drug delivery, biocatalysis and water treatment. They can be formed through several supramolecular pathways, ranging from phase inversion in alkaline solutions, to the ionic crosslinking of chitosan with multivalent anions, to polyelectrolyte or surfactant/polyelectrolyte complexation. Many chitosan bead uses require control over their stability to dissolution. To help elucidate how this stability depends on the choice of supramolecular gelation chemistry, we present a comparative study of chitosan bead stability in acidic aqueous media using three common classes of supramolecular chitosan beads: (1) alkaline solution-derived beads, prepared through simple precipitation in NaOH solution; (2) ionically-crosslinked beads, prepared using tripolyphosphate (TPP); and (3) surfactant-crosslinked beads prepared via surfactant/polyelectrolyte complexation using sodium salts of dodecyl sulfate (SDS), caprate (NaC Due tion (DD) ,2. The i soluble ,16.When its amine groups are protonated, chitosan becomes a polyelectrolyte that can be assembled into diverse, gel-like soft materials ,6,8,9. TA key property that determines the performance of these chitosan-based materials is their degradation stability, which can be limited by either hydrolytic degradation of the chitosan chains ,28,29 orTo date, there have been many studies examining the stability of ionically crosslinked nanoparticles ,39, film10), sodium laurate (NaC12) or sodium dodecyl sulfate (SDS) solutions through a 20-gauge syringe needle and equilibrating the resulting mixtures  at 30 °C for at least 24 h. Here, the 1 wt% ionic crosslinker/surfactant concentrations were selected to remain fixed  and to safely not exceed the solubility limit of the least soluble surfactant, NaC12 [Supramolecular chitosan beads were generated via dropwise addition of a 3 wt% aqueous chitosan solution (pH. 5.0) into either 0.1 M NaOH or 1 wt% TPP, sodium caprate , the beads persisted throughout the entire 7-day quantitative dissolution experiment  and exhia,3K = 2.8, pa,4K = 6.5, and pa,5K = 9.2 [The bead stability was even more limited when the beads were placed in highly acidic pH 1.2–2.0 media. At pH 2.0, for instance, the opaque beads became both swollen and translucent after 12 h, and fully dissolved after roughly 18 h . Similar,5 = 9.2 ) and, at,5 = 9.2 , and by ,5 = 9.2 . Yet, Mi,5 = 9.2 ,40. This,5 = 9.2 . Thus, i,5 = 9.2 ,32,40 or,5 = 9.2 ) using c12 > NaC10 [10 beads only decreased from ~11–12 h to slightly over 3 h, while the stability of chitosan/NaC12 and chitosan/SDS beads decreased from roughly 1 day to about 4 h and from roughly 3.5 to 2 days, respectively. Throughout, the beads formed with stronger surfactants persisted longer, and the 1.8- to 5.9-fold reductions in bead stability as the dissolution medium pH was decreased from 5.0 to 1.2 were mild compared to the multiple-order-of-magnitude reduction exhibited by the alkaline solution-derived beads and chitosan/TPP beads . As PP beads . 12 and SDS) formed more stable complexes with chitosan than the shorter-tailed NaC10. This improved stability was qualitatively consistent with previous work on the stability of quaternized chitosan/fatty acid complexes [2 groups into the hydrophobic environment of the surfactant/polyelectrolyte complex [12 (23–24 mM), and more than ten times lower than that of NaC10 (94–96 mM) [Beads formed from surfactants with longer, dodecyl aliphatic tails  ,46. Unli4–96 mM) ), howeve4–96 mM) . This pr10 and NaC12 dissolved slightly faster at pH 5.0 than beads prepared in NaOH solution , whose binding to chitosan was intermediate to that of the strongly binding SDS and weakly binding NaC10.Interestingly, at lower pH levels, the outer layer delamination was less evident . Despitesolution . When thOverall, this bead dissolution study demonstrates that: (1) though each of the examined supramolecular chitosan beads ultimately dissolve (or at least shows signs of dissociation) in acidic media, some of these beads are more stable than others; and (2) the order of this stability may depend on whether the media is weakly or strongly acidic. In weakly acidic media , TPP-crosslinked beads are the most stable, remaining intact  for several weeks. In highly acidic environments , however, TPP becomes protonated and, due to its lower anionic charge and weaker binding, provides only a minor stabilizing effect under the conditions examined in this work. Further, though earlier publications by Mi et al. reported chitosan/TPP beads prepared using 10 wt% TPP at a low pH (which likely resulted in beads with a higher TPP content relative to those prepared in the present study) to remain stable even at pH 1.0 ,40, thesUnlike the chitosan/TPP gels, beads prepared through chitosan complexation with the strong anionic surfactant SDS are relatively insensitive to pH and require 2 days to fully dissolve at the gastric pH of 1.2. Thus, in the limit of low pH, chitosan/SDS beads are the most stable of the beads in our study  also generate beads whose dissolution stability is less sensitive to pH than that of beads formed in NaOH and TPP solutions. Due to their weaker binding to chitosan, however, they do not provide as strong of a stabilizing effect as SDS. Indeed, as discussed in Weak anionic surfactants  are highly sensitive to pH, dissolving within an hour at low pH levels , but persisting over at least weeks at the mildly acidic pH of 5.0. Conversely, anionic surfactant-associated chitosan beads are the least sensitive to pH, remain stable in acidic media for up to days at all of the examined pH levels, and have dissolution properties that can be extensively tuned by varying the surfactant/polyelectrolyte binding strength . Thus, the type of bead that provides the highest stability may depend on whether the dissolution media is weakly acidic  or strongly acidic . Overall, these findings extend existing guidelines on tuning the stability of supramolecular chitosan gel beads to their diverse technological applications.12 were purchased from Sigma-Aldrich . SDS was obtained from MP Biomedicals , HCl was bought from both Sigma-Aldrich  and VWR International, LLC , and NaC10 was purchased from Spectrum . NaCl and NaOH were obtained from Fisher Scientific . All materials were used as received.All experiments were performed using Millipore Direct-Q 3 deionized water (18.2 MΩ·m resistivity). Chitosan , TPP (sChitosan solution (3 wt%) at pH 5.0 was prepared by dissolving chitosan in water, while adding HCl until the pH was 5.00 ± 0.05. The solution was then equilibrated for 12–16 h at 30 °C, whereupon the pH was checked one final time to ensure that it remained stable. The chitosan solution was then loaded into 3 mL BD Luer-Lok tip syringes, discarding approximately 10% of the solution at the bottom of the bottle, to avoid loading any undissolved impurities. 10, 1 wt% NaC12 or 1 wt% SDS. All solutions were stored at 30 °C prior to bead preparation to maintain their temperature above the Krafft point of the least soluble surfactant  solutions and the chitosan solution was maintained above 10:1 throughout the dropwise addition process, which in the case of ionically associating TPP, NaC10, NaC12 and SDS, kept the gelling agent:chitosan charge ratios above 1.7:1, 3.1:1, 2.7:1 and 2.1:1, respectively . Once the beads were formed, the resulting mixtures were stored in a 30 °C water bath for at least 24 h .To form the beads, the 3 wt% chitosan solution was added dropwise (at a rate of roughly 30 drops/min) through a 20-gauge syringe needle into gently agitated solutions of either 0.1 M of NaOH, 1 wt% TPP, 1 wt% NaCt (NaC12 ) and ensTo test their dissolution stability, chitosan beads  were equilibrated at room temperature in an excess of 10 mM NaCl solution pH adjusted to 1.2, 2.0, 3.0, 4.0 or 5.0 using HCl. Dissolution was considered complete when no visible material remained in the dissolution medium. To prepare the beads for dissolution, they were removed from the solutions in which they were formed and rinsed with 5–8 mL of water before being placed into 1 L of dissolution medium. Dissolution experiments were then performed by allowing one bead to dissolve in a 1 L Pyrex Vista beaker, agitated with a 5/16” octagonal stir bar at 500 rpm. Besides recording the times required for the beads to fully dissolve, evolutions in their size and morphology during dissolution were tracked by periodically imaging them with a Leica EZ4 D  digital stereomicroscope. During each imaging step, the beads were removed from the dissolution medium using a wire mesh, and imaged after removing the surface water with a VWR  light duty tissue wiper. The imaged beads were then returned to the dissolution media. For experiments lasting longer than 24 h, the dissolution medium was replaced daily. Each dissolution experiment was reproduced six times.


The eradication of smallpox is one of the greatest medical successes in history. Vaccinia virus was made famous by being the virus used in the live vaccine that enabled this feat. Nearly 40 years on from that success, this prototypical poxvirus continues to empower the exploration of fundamental biology and the potential to develop therapeutics against some of the major causes of death and disease in the modern world. The eradication of smallpox was one of the greatest medical successes in history. In this Research Matters article, Susanna Bidgood explores the future of using poxviruses as versatile tools for cell biology research. In 1980, the World Health Organisation announced an extraordinary scientific achievement that would change the course of human history: their 14-year international vaccination campaign had finally and completely eradicated smallpox. This disease, caused by the poxvirus variola, first appeared around 10,000 B.C. and was the most deadly human disease to ever exist. With a devastating 20% to 60% mortality rate, smallpox killed an estimated 300 million people in the 20th century alone and left its few survivors severely disfigured, with a third of them blind [The practice of vaccination had begun much earlier, in the 18th century. It was commonly recognised at the time that milkmaids, who would catch cowpox from their cattle, never died of smallpox. The English physician Edward Jenner then systematically infected individuals with this nonlethal yet close relative of variola virus and showed that cowpox infection protected a person from smallpox. However, it took the World Health Organisation vaccination campaign, now using a vaccine containing the poxvirus vaccinia, to finally rid the world of the devastating killer .Health officials subsequently stopped routine smallpox vaccinations because the risk of using a live virus vaccine against a disease that no longer posed a threat far outweighed the benefits. Due to its bioterrorism potential and likely lethality upon accidental exposure, scientific research on variola virus is now restricted exclusively to government secure facilities where the few remaining stocks of variola virus are kept safely under lock and key. Smallpox has been defeated.This extraordinary victory was achieved before I was born, so I never received a shot of vaccinia to protect me from smallpox. In fact, for most people alive today, vaccinia virus is simply the vaccine once used to protect people from a disease that no longer exists. Despite this, just four years ago, I established a research project to study the fundamental biology of vaccinia virus.I was excited to study vaccinia because, independent of its disease-eradicating heroism, this virus is a powerful tool for cell biology research. Vaccinia virus lends itself to experimental manipulation by being both relatively safe, and quick and easy to perform experiments with. It can be handled at room temperature, maintains infectivity after multiple rounds of freeze-thawing, and is able to infect a broad range of species and cell types. The virus naturally undergoes homologous recombination during viral replication, which enables the easy addition or deletion of genes from its genome, and its complete replication cycle only takes 8 to 12 hours. Moreover, as it has been evolving alongside animals for thousands of years and is highly adapted to control the host cell, establish its replicative niche inside the cell, and down-regulate the host’s immune surveillance systems, I can learn a lot of cell biology by watching how it does this.In recent years, three specific vaccinia features have proved hugely valuable both for the development of therapeutics to treat major public health threats as well as aiding the development of cutting-edge imaging technologies that will be applicable to all areas of cell biology.Firstly, vaccinia encodes almost all of its own replication machinery despite being an obligate intracellular parasite. Many of these viral proteins are homologues of cellular proteins, giving scientists like me a simpler system in which to begin to understand the function of the closely related cellular proteins. For example, human cells encode over 600 kinase enzymes. As a result, it is sometimes hard to define the role of an individual human kinase within the cell. Vaccinia virus by contrast encodes just two kinases. One of these kinases, called B1, was first identified in the 1980s. The kinase activity of B1 was extensively studied and shown to be essential for viral DNA replication. A decade later, a screen for novel human proteins identified two proteins whose sequences were more than 40% identical to vaccinia B1, suggesting that these proteins would also be active kinases . Today, The second key vaccinia feature is its size. Boasting dimensions of 350 nm × 250 nm × 250 nm, vaccinia virus is large (for a virus!) and can be easily tracked by live cell microscopy. In 2008, the power of this feature was exemplified when a novel virus entry pathway into cells was discovered using vaccinia [This ‘apoptotic mimicry’ entry pathway is now known to be employed by 20 clinically relevant viruses, including chikungunya virus, dengue virus, hepatitis A virus, Lassa virus, Marburg virus, and Ebola virus . During The final key feature of vaccinia is its unusual subviral shape, which can clearly be seen by electron microscopy . PoxviruTwo years ago, Rob Gray, a PhD student in London, combined all these features to develop a piece of software that enhances the precision of the super-resolution images. His software automatically detects individual viruses within the image, aligns them, and then averages them together to produce high-confidence models with a 2-fold improvement in resolution. This is a general software that can be applied to a wide array of other data sets independent of the microscope used to collect the images .Vaccinia has also proved to be a powerful tool to enhance the accuracy of super-resolution microscopes. In her paper last year, Siân Culley developed software to automatically detect artifacts sometimes generated during the post-acquisition processing of super-resolution microscopy images. Siân used vaccinia images as test data sets during the development of the software . Their cThe super-resolution applications to date have employed isolated virions, but it seems likely that soon, similar approaches will be applied to the context of the infected cells. Therefore, vaccinia is proving an excellent tool to aid the improvement of light microscopy approaches. It follows that the better our microscopes, the more new biology we will be able to see and understand.The eradication of smallpox was undeniably a world-changing accomplishment, but vaccinia virus is more than simply the vaccine that enabled this feat. Vaccinia has a lot more biology to teach us. With vaccinia virus as a tool, poxvirologists all over the world are pushing the boundaries of technology, discovering fundamental biology, and developing therapeutics to combat some of the major causes of death and disease in the modern world.


Sw-5 gene cluster encodes protein receptors that are potentially able to recognize microbial products and activate signaling pathways that lead to plant cell immunity. Although there are several Sw-5 homologs in the tomato genome, only one of them, named Sw-5b, has been extensively studied due to its functionality against a wide range of (thrips-transmitted) orthotospoviruses. The Sw-5b gene is a dominant resistance gene originally from a wild Peruvian tomato that has been used in tomato breeding programs aiming to develop cultivars with resistance to these viruses. Here, we provide an overview starting from the first reports of Sw-5 resistance, positional cloning and the sequencing of the Sw-5 gene cluster from resistant tomatoes and the validation of Sw-5b as the functional protein that triggers resistance against orthotospoviruses. Moreover, molecular details of this plant–virus interaction are also described, especially concerning the roles of Sw-5b domains in the sensing of orthotospoviruses and in the signaling cascade leading to resistance and hypersensitive response.The  Tospoviridae) cause substantial losses in crop production worldwide (Thysanoptera) in nature, replicating in both plant and invertebrate hosts  together with vertebrate-infecting viruses due to their similar virion shape, genomic organization and, of course, phylogenetic relationship (Orthotospoviruses (family orldwide . They cate hosts . Until rtionship .Solanum lycopersicum L.) is the subject of this mini-review. Initially, this resistance source used to be referred to as Sw-5, being utilized in tomato breeding programs. Sequencing of the resistance gene locus, originally from Solanum peruvianum Mill. (a wild Peruvian tomato), revealed five paralogs, the so-called Sw-5 gene cluster  date from the end of the 1930s (Stevens (Sw-5) was responsible for resistance that was initially found to be effective against a wide array of orthotospovirus isolates from United States  and leucine-rich repeat (LRR) domains, which are often observed in other plant resistance proteins  which provides resistance to TSWV isolates only  and impatiens necrotic spot virus (INSV) as well , groundnut ringspot virus (GRSV), and chrysanthemum stem necrosis virus (CSNV) . This brtes only . More re as well . Thus, try clade . Conversa plants .Sw-5b-transformed N. tabacum plants do not show any HR after mechanical inoculation with TSWV . This phenotype is usually observed as local necrotic lesions/spots and often appears  on leaves upon activation of immune protein receptors containing the NB and LRR domains . During ith TSWV , and strith TSWV , the Sw-itant HR . The maiequences  while inic virus . Whethern vector  or in thn vector .The NB-LRR proteins are also known as NLR proteins, an acronym that also stands to nucleotide-binding (NB) and LRR domains (L)-containing (R) receptors . Many clIn order to identify the avr-determinant of Sw-5b, indirect approaches have been performed due to the lack of an orthotospovirus infectious clone . The botSw-5b gene copy  TSWV isolate and the M segment of an RB TSWV isolate overcame the Sw-5 resistance in tomato plants . Furtherene copy . Moreoveene copy . All theN. benthamiana leaves -terminus either a Toll and interleukin-1 receptor (TIR) domain or a coiled-coil (CC) domain, which is then followed by the central NB- and carboxy (C)-terminal LRR domains . Therefoa leaves .Sw-5b gene lacking these domains are not resistant to orthotospoviruses  responsible to deliver the protein to the cell nucleus . This nuoviruses . Altogetoviruses . The SD oviruses . Interesoviruses .The NB domain, which also includes the subdomains ARC 1 and ARC 2, is solely responsible for the signaling pathway that leads to HR triggering . This doin vitro and in planta  and NSm triggers a strong HR. If only NB is co-expressed with NSm, no HR is seen . Thus, Ln planta . A consen planta . Within n planta . As suchSw-5a-transformed tobacco plants are susceptible to TSWV , it is likely that some point mutations within the LRR domain disrupted the avr-determinant sensing of Sw-5a. Proof for this could come from testing a chimeric Sw-5a NB-LRR construct, in which its LRR domain has been swapped for the functional one from Sw-5b. So far, no studies have yet been performed with the proteins Sw-5c, Sw-5d, and Sw-5e.In contrast to the findings with Sw-5b, co-expression of Sw-5a and TSWV NSm does not result in HR triggering , which c to TSWV . Dissect to TSWV . ConsideSw-5a and Sw-5b, the protein product of the most conserved ortholog from susceptible tomato isolines (not bred with S. peruvianum), named Sw-5aS, is not able to trigger auto-HR at all, even though these three proteins share about 94% aa identity  abolished the ability for HR-induction. A reversion of this single amino acid into the one found in the NB domains of Sw-5a and Sw-5b, restored the ability of triggering HR by the Sw-5aS NB domain  and a truncated gene are found in chromosome 9 of tomato cultivar “Heinz 1706”  upstream of the coding sequence of Sw-5aS . Apart fsequence , all Sw-nz 1706” . One of chiensis . This efchiensis . Figure Few plant immune receptors against viruses have been deeply studied to date. Apart from tomato Sw-5b, most of the efforts concentrated on the resistance mechanisms orchestrated by potato Rx against potato virus X (PVX) and tobacco N against tobacco mosaic virus (TMV) . The finG. pallida, respectively  modifications that have to be implemented to turn non-functional copies into one that resembles the functional Sw-5b. The insertion of these modifications in the tomato genome, especially in those containing only SSw-5a, could be done by genome editing tools such as CRISPR and TALENs (We do not rule out that in addition to Sw-5b, the other Sw-5-related proteins trigger resistance to different pathogens. This has been observed for NLRs from potato, in which the paralogs Rx and GPa2 trigger resistance to PVX and nematode ectively . Taking d TALENs .AdO organized and wrote most of this manuscript. LB, RK, and RR revised and added complementary information.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Yellow fever was transported during the slave trade in the 15th and 16th centuries from Africa to the Americas where the virus encountered favorable ecological conditions that allowed creation of a sustainable sylvatic cycle. Despite effective vector control and immunization programs for nearly a century, yellow fever epidemics reemerged in many Latin American countries, particularly Brazil. The emergence or reemergence of vector-borne diseases encompasses many intricate factors. Yellow fever outbreaks occur if at least three conditions are fulfilled: the introduction of the virus into a non-immune human community, presence of competent and anthropophilic vectors and insufficiency of prevention and/or adequate management of the growing outbreak. On the other hand, two weapons are available to constrain yellow fever: vector control and immunization. In contrast, yellow fever is absent from Asia and the Pacific despite the presence of the vector and the susceptibility of human populations to the virus. Based on a review of the global history of yellow fever and its epidemiology, the authors deliver some recommendations for improving the prevention of epidemics. Aedes aegypti in the 1970s, after an attempt to eliminate the vector [Haemagogus sp. and Sabethes sp., involved in the sylvatic cycle, while Aedes aegypti and A. albopictus, which induce epidemics in urban and peri-urban areas, respectively [Brazil has experienced an exceptional yellow fever (YF) outbreak since December 2016  or to the genera Haemagogus and Sabethes (in America). Epidemics are more frequent and important in Africa than in the Americas  was not the cause of YF but a secondary contaminant [A detailed review of potential candidates as etiological YF agents concluded that it was filterable, i.e., a pathogen not yet called a virus: “It is more than likely that the germs of yellow fever, as well as those of small pox, measles, hydrophobia, etc. belong to a group of organisms, smaller than our bacteria and as yet unknown, awaiting discovery” . The US taminant . The Fretaminant .The YF virus was isolated June 30, 1927, from the blood of a Ghanaian patient – who recovered – by Adrian Stokes, who contracted YF and died . The 17DYF has been known in Africa since the end of the fifteenth century through sporadic cases and/or limited small epidemics observed in the colonial counters of the West African coast. In East and South Africa, YF was probably rare or absent from coastal areas because of the lack of an appropriate vector.The epidemiology of YF in West and Central Africa was well described in the 1960s to 1980s –58 folloAedes species, notably A. africanus and A. furcifer, and non-human primates, especially arboreal monkeys [Aedes species, particularly A. furcifer, leave the plant coverage, penetrate the inhabited areas and bite humans that they infect [Endemic areas are those where the virus circulates between some  monkeys , 57. In  monkeys , 59. Humy infect .The endemic zone tends to spread, partly because of the transovarial transmission of YF in mosquitos –41 and, Aedes, thereby ensuring infection of the adults at their hatching at the beginning of the following season. On the other hand, monkeys – and possibly other small mammals –, especially the young still immunologically naïve ones, amplify, reveal and propagate the virus from the first rains, thus perpetuating the sylvatic cycle [In-depth studies of viral infection in wild mammals and specific antibodies showed that vertebrates in general, including monkeys, play only a secondary role in the persistence and resilience of the virus in the sylvatic cycle, because of the short duration of viremia and the resulting definitive immunity, which reflects an effective adaptation of the virus to its transient host , 60–62. ic cycle .Aedes species, first of all A. aegypti. The virus is spread in the municipality, amplified by the proliferation of A. aegypti, where there are favorable conditions, lack of immunity among a large proportion of the inhabitants and, even today, health-system insufficiency based on the tripartite approach: mass vaccination, vector control, screening and treatment of cases placed under bed nets.In the epidemic zone, the virus does not circulate and the wild vector does not occur. The population shows a low immune status, except in immunized individuals, which in case of low immunization coverage explains the intensity of the epidemic . In urbaYF vaccination was made mandatory in 1941 in all French-controlled colonies. Facilitated by its stability and administration by scarification associated with the smallpox vaccine, mass immunization led to remarkably effective control of YF , 53. ItsIn South America, YF was mentioned at the same time as in Africa and in similar circumstances - during the slave trade – first in the Caribbean and in Central America (Yucatan). In Brazil, the first indisputable description of YF was made after the Pernambuco epidemic in Recife and Olinda in 1685 . Legend A. aegypti ensuring the perpetuation and dispersal of the virus [Anyway, the penetration of YF in America via Brazil is not excluded because of the considerable economic development of Brazil starting from 1554. The expansion of sugar plantations was favored by the large spaces available and the tax benefits granted by Portugal. On the one hand, the need for labor not met by indigenous natives was assumed by the massive influx of African slaves . On the other hand, the crossing from Africa to Brazil was shorter than that to the West Indies or North America due to the proximity of both coasts and favorable winds, resulting in the multiplication of YF transport opportunities , 70. In he virus . YF was he virus , 66. It Haemagogus janthinomys and H. leucocelaenus play an essential role while other species (including H. capricornii) or genera, notably Aedes, are little involved in the transmission [A. aegypti and A. albopictus represent a major risk of spreading YF in urban and peri-urban areas [The endemic zone of YF is located in the Amazon and encompasses, to the west, Peru and Colombia, to the north the Guyana plateau , and to the northeast, Brazil. The description of the sylvatic cycle was made on the occasion of the great 1942 epidemic in Brazil , 72. Thesmission –83. On tan areas .Despite common ecological conditions in the Amazonian region, there are two lineages of the YF virus: genotype 1 from Brazil (or possibly from the West Indies) and genotype 2 from Peru , 85–87. The reemergence of YF in Brazil benefits from particularly favorable environmental conditions: high deforestation increasing the contact of human populations with the sylvatic virus, substantial migration between endemic and epidemic regions, poorly controlled urbanization favoring the multiplication of vectors, low immunization coverage in epidemic risk areas . FinallyOn the occasion of the Rio de Janeiro epidemic of 1900, the French scientific mission demonstrated the vertical transmission of the pathogen in mosquitos, and identified the former as small filtering organism. The first vaccine trials were undertaken , 39, 40.A. aegypti started in the 1920s in most Latin American countries, reinforced by the use of DDT from 1947 [A. aegypti from jungle foci were unexpected [The control of rom 1947 . Mass vaexpected , 92.A. aegypti and A. albopictus and, on the other hand, the human population lacks herd immunity.The YF virus and its vector live in close symbiosis in which the evolution of the former seems irremediably linked to that of the latter. The virus-vector coadaptation is essential for maintaining the sylvatic cycle. This is the case in Africa and South America where the virus adapted to various species of Culicidae in forest areas that have become endemic. Humans become infected and spread the epidemic all the more rapidly so that, on the one hand, the mosquito is both receptive to the virus and anthropophilic, which is the case for A. aegypti, favor the transport of the virus because of the resistance of their eggs to the desiccation [A. aegypti. The short flying distance of Aedes, especially A. aegypti and A. albopictus, leaves no alternative other than passive transport, especially by the transportation of humans or goods [These two species, in particular iccation  that leaiccation , 95. Theor goods –102.A. aegypti strains confirmed the African origin of the species although genetic variability reflected massive mixings due to multiple importations over time from all continents, and human intervention for controlling mosquitoes [A. aegypti formosus as opposed to the subspecies A. aegypti aegypti, which is the domestic and urban form of the species – is still present in African jungle where the larvae grow in tree trunks, and whose females are not anthropophilic [A. aegypti formosus is involved in the transmission of YF in nonhuman primates within the sylvatic cycle [A. aegypti, which led to its diffusion in America, was earlier or contemporaneous with its transport during the slave trade [A. aegypti adaptation to human populations in West Africa was early, which explains the many large urban epidemics in this part of the continent at the end of the eighteenth century [A. aegypti does not seem to have adapted to the anthropization of the environment, as evidenced by the scarcity of urban epidemics up to the present day [The molecular phylogeny of squitoes , 104. Insquitoes –108. Thepophilic –111. A. ic cycle . It is n century . On the sent day .A. aegypti was later, probably at the end of the nineteenth century when it was revealed as an urban vector of dengue fever [A. aegypti suggests that the introduction was recent and accomplished by a limited number of entries [A. aegypti aegypti is monophyletic [The colonization of Asia by ue fever . Phylogeue fever , 111. In entries . Howeverphyletic , it has phyletic , 117 thaphyletic , 118.A. aegypti, although a recent arrival, is present in many places in Asia and Oceania. In addition, the abundant circulation of other arboviruses, dengue, chikungunya and Zika, the latter two coming from Africa like YF, indicates the possibility of YF extension in Asia. Recently, several human cases of YF introduced from Angola have been diagnosed in China [A. aegypti and A. albopictus as well as on their respective competencies in the transmission of dengue and YF to explain the absence of the latter in Asia and the rarity of the former in Africa [The absence of YF from Asia and Oceania remains an enigma. The population is susceptible to the virus as shown by Asian patients living in endemic areas (Africa or South America). in China , increasin China , 118, b)in China ), c) comin China , 122. Rein China . The firin China , 122. Thn Africa . Anothern Africa . MosquitHaemagogus and Sabethes – playing the role of virus reservoir, especially in the Amazon that is the refuge zone of the virus [A. albopictus and A. aegypti respectively, and follows the routes of human migrations, particularly forest galleries [Since its introduction in the seventeenth century, YF remained in the Amazonian region in the form of a jungle cycle that causes rural, peri-urban or urban epidemics at regular intervals. The sylvatic cycle is maintained by wild endemic vectors present before the introduction of YF in Brazil – belonging to the genera he virus , 79–83. alleries .Clinically, the Brazilian YF does not differ from the forms described in Africa or other American countries. The high case fatality rate (up to 50%) is likely not to be the manifestation of a particular virulence but resulted from underreporting of benign cases .A. aegypti, confirmed between 1971 and 1999 [A. aegypti from isolated and poorly controlled Amazonian and Southern Brazilian forest localities, as a result of a relaxation of vector control and vaccination campaigns, as well as the deterioration of health systems in some remote areas [A. aegypti and A. albopictus reinforce the risk of YF extension in cities, especially in the overcrowded suburbs of major Brazilian cities [The vector control carried out at the beginning of the twentieth century during the Rio de Janeiro epidemic was organized nationwide in 1928–29. It was reinforced by YF immunization in 1937 and DDT sprayings initiated 10 years later, which significantly reduced the burden of YF, that was considered as having been eliminated from Brazil in the 60s , 126. Hoand 1999 , which rte areas , 128. Ante areas , 129. Nete areas , 128. Thn cities .Fig. 6MuHaemagogus and Sabethes, as well as non-anthropophilic Aedes, i.e. other species than A. aegypti and A. albopictus) and spared densely populated areas. So far, humans have been infected outside cities and their suburbs from multiple sylvatic foci [The attempt to eliminate the YF during the twenty-first century, despite a relative failure, probably contained the risk of urban and peri-urban epidemics. For more than 50 years, Brazilian YF epidemics – and the most recent one was no exception – were sylvatic and involved wild vectors . In addition to vaccine hypersensitivity and anaphylactic shock, SAEs were categorized into two groups: YFV-associated neurotropic disease (YFV-AND), including encephalitis, myelitis, acute disseminated encephalo-myelitis, and Guillain-Barré syndrome, and YF-vaccine-associated viscerotropic disease (YDV-AVD) responsible for multi-organ failure . Risk faThe physiognomy of YF has reemerged abruptly over the last 50 years while the disease was likely to be controlled. Consequently, it is no longer a question of its eradication, if only because of the sylvatic foci present on two continents, but rather of limiting the epidemic risk .The reservoir of YF, both in Africa and the Americas, is the mosquito, especially the sylvatic species that maintain the jungle cycle. The short viremia and definitive immunity – or death – that follow the viral infection in mammals do not allow their consideration as a proper virus reservoir. However, they ensure amplification of the latter and, in the Americas in particular, are an alarm signal of viral reemergence that can result in human outbreaks able to extend into urban areas.The history of YF, especially its installation in the Americas five centuries ago, taught us that the virus and its vector presented a remarkable capacity for environmental adaptation. The pair seem to react very quickly to the conditions that they face. While until 1960, the virus was spread by boat – limited to transport through mosquitos – which required time and allowed the implementation of preventive or remedial measures, it is now rapidly spread via aircraft carrying infected humans, a scenario that is more difficult to control.However, there are flaws in the transmission of the YF, as evidenced by its absence from Asia. The emergence or re-emergence of vector-borne diseases depends on complex, entangled biological, climatic, ecological, socio-economic and political factors . Three cAedes larvae and adults, and preventive mass immunization [The objectives of the WHO are therefore to protect the at-risk population, prevent international spread and rapidly contain epidemics. These objectives include a prevention component through appropriate urban development and an immediate response in the event of an YF outbreak by combining vector control against nization . In Braznization , 150 andnization .Public health strategies must combine both scientific and political criteria. The history of YF stressed, as many authors have already noted, that the opinions of scientists – sometimes giving rise to expertise followed by the authorities – often came from assertions based on subjective arguments rather than evidence based on observations validated experimentally. Controversies conceal ideological cleavages, even political ones, which divert the public debate from its objectives of prevention and management, make them lose their effectiveness, and involve useless and considerable expenses , 16.