title: Transcriptome and metabolome analyses reveal pathways associated with fruit color in plum (Prunus salicina Lindl.)

abstract:
BackgroundIn order to reveal the mechanism of fruit color changes in plum, two common plum cultivars Changli84 (Ch84, red fruit) and Dahuangganhe (D, yellow fruit) in Northeast China were selected as plant materials. Transcriptome sequencing and metabonomic analyzing were performed at three different developmental stages: young fruit stage, colour-change stage, and maturation stage.

Results"Flavonoid biosynthesis" was significantly enriched in the KEGG analysis. Some DEGs in "Flavonoid biosynthesis" pathway had an opposite trend between the two cultivars, such as CHS, DFR and FLS. Also, transcriptional control of MBW (MYB-bHLH-WD) protein complexes showed a close relationship with plum fruit color, especially the expression of MYBs and bHLHs. In the current study, procyanidin B1 and B2 had the highest level at young fruit stage in Ch84 and the content of procyanidin B2 decreased sharply at the color change stage. Conversely, the content of cyanidin increased with the growth of fruit and reached the peak at the maturation stage.

ConclusionThe content of procyanidin B1 and B2 in plums at young fruit stage might be the leading factors of the matured fruit color. At the maturation stage, the cyanidin produced by procyanidins keeps the color of the fruit red. Correspondingly, genes in "flavonoid biosynthesis" pathway play critical roles in regulating the accumulation of anthocyanin in plum.

category: plant biology


title: Automated identification of piglet brain tissue from MRI images using Region-Based Convolutional Neural Networks

abstract: Magnetic Resonance Imaging is an important tool for characterizing volumetric changes of the piglet brain during development. Typically, an early step of an imaging analysis pipeline is brain extraction, or skull stripping. Brain extractions are usually performed manually; however, this approach is time-intensive and can lead to variation between brain extractions when multiple raters are used. Automated brain extractions are important for reducing the time required for analyses and improving the uniformity of the extractions. Here we demonstrate the use of Mask R-CNN, a Region-based Convolutional Neural Network (R-CNN), for automated brain extractions of piglet brains. We validate our approach using Nested Cross-Validation on six sets of training/validation data drawn from 32 pigs. Visual inspection of the extractions shows acceptable accuracy, and Dice coefficients are in the range of 0.95-0.97. These results demonstrate that R-CNNs provide a viable tool for skull stripping of piglet brains.

category: neuroscience


title: Identification and Validation of N-Acetylputrescine in Combination With Non-Canonical Clinical Features As a Parkinsons Disease Biomarker Panel

abstract: Parkinsons disease is a progressive neurodegenerative disorder in which loss of dopaminergic neurons in the substantia nigra results in a clinically heterogeneous group with variable motor and non-motor symptoms with a degree of misdiagnosis. Only 3-5% of sporadic Parkinsons patients present with genetic abnormalities, thus environmental, metabolic, and other unknown causes contribute to the pathogenesis of Parkinsons disease, which highlights the critical need for biomarkers. There could be a significant clinical benefit to treating Parkinsons disease at the earliest stage and identify at-risk populations once disease-modifying treatments are available. In the present study, we prospectively collected and analyzed plasma samples from 201 Parkinsons disease patients and 199 age-matched non-diseased controls. Multiomic and Bayesian artificial intelligence analysis of molecular and clinical data identified the diagnostic utility of N-acetyl putrescine (NAP) in combination with smell (B-SIT), depression/anxiety (HADS), and acting out dreams (RBD1Q) clinical measurements. The clinical and biomarker panel demonstrated an area under the curve, AUC = 0.9, positive predictive value, PPV = 0.91, and negative predictive value, NPV = 0.66 utilizing all four variables. The assessed diagnostic panel demonstrates combinatorial utility in diagnosing Parkinsons disease, allowing for an integrated interpretation of disease pathophysiology and highlighting the use of multi-tiered panels in neurological disease diagnosis.


title: TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction

abstract: Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spikes polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

category: microbiology


title: Virulence and antimicrobial resistance profile of non-typhoidal Salmonella enterica serovars recovered from poultry processing environments at wet markets in Dhaka, Bangladesh

abstract: The rapid emergence of virulent and multidrug-resistant (MDR) non-typhoidal Salmonella (NTS) enterica serovars are a growing public health concern globally. The present study focused on the assessment of the pathogenicity and antimicrobial resistance (AMR) profiling of NTS enterica serovars isolated from chicken processing environments at wet markets in Dhaka, Bangladesh. A total number of 870 samples consisting of carcass dressing water (CDW), chopping board swabs (CBS), and knife swabs (KS) were collected from 29 wet markets. The prevalence of Salmonella was found to be 20% in CDW, 19.31% in CBS and 17.58% in KS, respectively. Meanwhile, the MDR Salmonella was found to be 72.41%, 73.21% and 68.62% in CDW, CBS, and KS, respectively. All isolates were screened by polymerase chain reaction (PCR) for eight virulence genes, namely invA, agfA, IpfA, hilA, sivH, sefA, sopE, and spvC. The S. Enteritidis and untyped Salmonella isolate harbored all virulence genes while S. Typhimurium isolates carried six virulence genes except sefA and spvC. Phenotypic resistance revealed decreased susceptibility to ciprofloxacin, streptomycin, ampicillin, tetracycline, gentamycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid and azithromycin. Genotypic resistance showed higher prevalence of plasmid mediated blaTEM followed by tetA, sul1, sul2, sul3, and strA/B genes. Harmonic and symmetrical trend was observed among the phenotypic and genotypic resistance patterns of the isolates. The research findings anticipate that MDR and virulent NTS enterica serovars are prevailing in the wet market environments which can easily enter into the human food chain. There was a resilient and significant correlation existent among the phenotypic and genotypic resistance patterns and virulence genes of Salmonella isolate recovered from carcass dressing water, chopping board swabs, and knife swabs (p < 0.05), respectively.


title: Evaluation of the sensitivity and specificity of three diagnostic tests for Coxiella burnetii infection in cattle and buffaloes in Punjab (India) using Bayesian latent class analysis

abstract: Q Fever is a zoonotic disease of significant animal and public health concern, caused by Coxiella burnetii (C. burnetii), an obligate intracellular bacterium. This study was done to evaluate the sensitivity (Se) and specificity (Sp) of three diagnostic methods to diagnose C. burnetii infection in cattle and buffaloes in Punjab, India: an indirect ELISA method applied in serum samples and a trans-Polymerase Chain Reaction (trans-PCR) technique applied in milk samples and genital swabs. Bayesian Latent Class Models were developed following the STARD-BLCM reporting guidelines. Conditional independence was assumed between the tests, given (i) the different biological principle of ELISA and trans-PCR and (ii) the fact that the trans-PCR was performed on different tissues. The ELISA method in the serum samples showed the highest Se of 0.97 (95% Probability Intervals (PIs): 0.93; 0.99) compared to the trans-PCR method applied in milk samples 0.76 (0.62; 0.87) and genital swabs 0.7 (0.55; 0.82). The Sps of all tests were high, with trans-PCR in genital swabs recording the highest Sp of 0.99 (0.98; 1), while the Sp of trans-PCR in milk samples and ELISA in serum samples were 0.98 (0.96; 0.99) and 0.95 (0.93; 0.97) respectively. The study results show that none of the applied tests are perfect, therefore, a testing regimen based on the diagnostic characteristic of the tests may be considered for diagnosis of C. burnetii.


title: Gene editing in bacteria by CRISPR-Cas9 via SpCas9/sgRNA ribonucleoprotein complexes

abstract: The authors have withdrawn this manuscript due to a duplicate posting of manuscript number 449263. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

category: molecular biology


title: Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant

abstract: The translocator protein (TSPO) has been implicated in mitochondrial transmembrane cholesterol transport, brain inflammation, and other mitochondrial functions. It is upregulated in glial cells during neuroinflammation in Alzheimers disease. High affinity TSPO imaging radioligands are utilized to visualize neuroinflammation. However, this is hampered by the common A147T polymorphism which compromises ligand binding. Furthermore, this polymorphism has been linked to increased risk of neuropsychiatric disorders, and possibly reduces TSPO protein stability. Here, we used immunoprecipitation coupled to mass-spectrometry (IP-MS) to establish a mitochondrial protein binding profile of wild-type (WT) TSPO and the A147T polymorphism variant. Using mitochondria from human glial cells expressing either WT or A147T TSPO, we identified 30 WT TSPO binding partners, yet only 23 for A147T TSPO. Confirming that A147T polymorphism of the TSPO might confer loss of function, we found that one of the identified interactors of WT TSPO, 14-3-3 theta (YWHAQ), a protein involved in regulating mitochondrial membrane proteins, interacts much less with A147T TSPO. Our data presents a network of mitochondrial interactions of TSPO and its A147T polymorphism variant in human glial cells and indicate functional relevance of A147T in mitochondrial protein networks.


title: Highly selective Cooperative Primers for multiplex detection of rare mutations

abstract: We describe a real time PCR-based technique capable of detecting and quantifying rare somatic mutations in circulating tumor DNA reference materials. Our approach utilizes previously described Cooperative Primers, structurally modified to exhibit high allele-specificity and one-copy target sensitivity. Cooperative Primers are bi-functional molecules, consisting of a high affinity probe fragment that guarantees sensitivity, and a covalently attached lower affinity primer providing specificity. Additional optimization of Cooperative Primer structure generated molecules capable of reliable detection of allele changes as small as a single nucleotide. These highly selective Cooperative Primers maintain excellent discrimination properties in rare mutant allele scenarios, in both monoplex and multiplex assays. With synthetic DNA samples, Cooperative Primers can detect as little as 100 copies of mutant template amongst 1 000 000 copies of wild-type template (minor allele fraction of 0.01 %). Multiplex Cooperative Primer assay was validated with cell-free DNA reference materials and consistently detected the lowest minor allele fraction available (0.1 %) for EGFR L858R, G719S and V769-D770insASV mutations, while simultaneously providing qualitative and quantitative assessment of cell-free DNA with integrated {beta}-Actin assay. Easy to design, rapid and inexpensive, Cooperative Primer - based real time PCR assays are a promising tool for evaluation of cancer therapy response, occurrence of resistance mutations and relapse monitoring.


title: Buenos Aires, June 2021Inhibition of Mitochondrial Fission by Drp-1 Blockade by Short-Term Leptin and Mdvi-1 Treatment Improves White Adipose Tissue Abnormalities in Obesity and Diabetes

abstract:
BackgroundObesity and type 2 diabetes are chronic diseases characterized by insulin resistance, mitochondrial dysfunction and morphology abnormalities.

ObjectiveHerein, we investigated if dysregulation of mitochondrial dynamics and biogenesis is involved in an animal model of obesity and diabetes.

MethodsThe effect of short-term leptin and mdivi-1 -a selective inhibitor of Drp-1 fission-protein- treatment on mitochondrial dynamics and biogenesis was evaluated in epididymal white adipose tissue (WAT) from male ob/ob mice.

ResultsAn increase in Drp-1 protein levels and a decrease in Mfn2 and OPA-1 protein expression were observed with enhanced and sustained mitochondrial fragmentation in ob/ob mice compared to wt C57BL/6 animals (p<0.05). The content of mitochondrial DNA and mRNA expression of PGC-1 -both parameters of mitochondrial biogenesis- were reduced in ob/ob mice (p<0.05). Leptin and mdivi-1 treatment significantly increased mitochondrial biogenesis, improved fusion-to-fission balance and attenuated mitochondrial dysfunction, thus inducing white-to-beige adipocyte transdifferentiation. Measurements of glucose and lipid oxidation in adipocytes revealed that both leptin and mdivi-1 increase substrates oxidation while in vivo determination of blood glucose concentration showed decreased levels by 50% in ob/ob mice, almost to the wt level.

ConclusionsPharmacological targeting of Drp-1 fission protein may be a potential novel therapeutic tool for obesity and type 2 diabetes.


title: A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

abstract: Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called  endosomal escape problem, i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, "TAT-CaM," evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

category: cell biology


title: Community health and human-animal contacts on the edges of Bwindi Impenetrable National Park, Uganda

abstract: Cross-species transmission of pathogens is intimately linked to human and environmental health. With limited healthcare and challenging living conditions, people living in poverty may be particularly susceptible to endemic and emerging diseases. Similarly, wildlife is impacted by human influences, including pathogen sharing, especially for species in close contact with people and domesticated animals. Here we investigate human and animal contacts and human health in a community living around the Bwindi Impenetrable National Park (BINP), Uganda. We used contact and health survey data to identify opportunities for cross-species pathogen transmission, focusing mostly on people and the endangered mountain gorilla. We conducted a survey with background questions and self-reported diaries to investigate 100 participants health, such as symptoms and behaviours, and contact patterns, including direct contacts and sightings over a week. Contacts were revealed through networks, including humans, domestic, peri-domestic, and wild animals for 1) network of contacts seen in the week of background questionnaire completion, 2) network of contacts seen during the diary week. Participants frequently felt unwell during the study, reporting from one to 10 disease symptoms at different intensity levels (maximum of seven symptoms in one day), with severe symptoms comprising 6.4% of the diary records and tiredness and headaches the most common symptoms. Besides human-human contacts, direct contacts with livestock and peri-domestic animals were the most common. Wildlife contacts were the rarest, including one direct contact with gorilla with a concerning timeline of reported symptoms. The contact networks were moderately connected and revealing a preference in contacts within the same species or taxon and within their groups. Despite sightings of wildlife being much more common than touching, one participant declared direct contact with a mountain gorilla during the week. Gorillas were seen very close to six animal taxa (including themselves) considering all interaction types, mostly seen closer to other gorillas, but also people and domestic animals. Our findings reveal a local human population with recurrent symptoms of illness in a location with intense exposure to factors that can increase pathogen transmission, such as direct contact with domestic and wild animals and proximity among animal species. Despite significant biases and study limitations, the information generated here can guide future studies, such as models for disease spread and One Health interventions.

category: ecology


title: Long-term changes in populations of rainforest birds in the Australia Wet Tropics bioregion: a climate-driven biodiversity emergency

abstract: Many authors have suggested that the vulnerability of montane biodiversity to climate change worldwide is significantly higher than in most other ecosystems. Despite the extensive variety of studies predicting severe impacts of climate change globally, few studies have empirically validated the predicted changes in distribution and population density. Here, we used 17 years of bird monitoring across latitudinal/elevational gradients in the rainforest of the Australian Wet Tropics World Heritage Area to assess changes in local abundance and distribution. We used relative abundance in 1977 surveys across 114 sites ranging from 0-1500m above sea level and utilised a trend analysis approach (TRIM) to investigate elevational shifts in abundance of 42 species between 2000 - 2016. The local abundance of most mid and high elevation species has declined at the lower edges of their distribution by >40% while lowland species increased by up to 190% into higher elevation areas. Upland-specialised species and regional endemics have undergone dramatic population declines of almost 50%. The "Outstanding Universal Value" of the Australian Wet Tropics World Heritage Area, one of the most irreplaceable biodiversity hotspots on Earth, is rapidly degrading. These observed impacts are likely to be similar in many tropical montane ecosystems globally.


title: Verification of our empirical understanding of the physiology and ecology of two contrasting plantation species using a trait database

abstract: The effects of climate change on forest ecosystems take on increasing importance more than ever. Information on plant traits is a powerful predictor of ecosystem dynamics and functioning. We reviewed the major ecological traits, such as foliar gas exchange and nutrients, xylem morphology and drought tolerance, of Cryptomeria japonica and Chamaecyparis obtusa, which are major timber species in East Asia, especially in Japan, by using a recently developed functional trait database for both species (SugiHinokiDB). Empirically, C. obtusa has been planted under drier conditions, whereas C. japonica has been planted under wetter conditions. Our analyses revealed followings: The maximum photosynthetic rate, stomatal conductance, foliar nutrient content and soil-to-foliage hydraulic conductance were higher in C. japonica than in C. obtusa and were consistent with the higher growth rate of C. japonica. In contrast, the foliar turgor loss point and xylem pressure corresponding to 50% conductivity, which indicate drought tolerance, were lower in C. obtusa than in C. japonica and are consistent with the drier habitat of C. obtusa. Ontogenetic shifts were also observed; as the age and height of the trees increased, many foliar nutrient concentrations decreased, and the foliar minimum midday water potential and specific leaf area also decreased. This suggests that an ontogenetic reduction in photosynthesis occurred due to an increase in drought stress with tree height and age. However, among the Cupressaceae worldwide, the drought tolerance of C. japonica and C. obtusa is not as high. This may be related to the fact that the Japanese archipelago has historically not been subjected to strong dryness. The maximum photosynthetic rate showed intermediate values within the family, indicating that C. japonica and C. obtusa exhibit relatively high growth rates in the Cupressaceae family, and this is thought to be the reason why they have been selected as economically suitable timber species in Japanese forestry. This study clearly demonstrated that the plant trait database provides us a promising opportunity to verify out empirical knowledge of plantation management and helps us to understand effect of climate change on plantation forests by using trait-based modelling.


title: Population size, density, and ranging behaviour in a key leopard population in the Western Cape, South Africa

abstract: Globally, leopards are the most widespread large felid. However, mounting anthropogenic threats are rapidly reducing viable leopard populations and their range. Despite the clear pressures facing this species, there is a dearth of robust and reliable population and density estimates for leopards across their range, which is particularly important in landscapes that consist of protected and non-protected areas. We conducted a camera trapping survey between 2017 and 2018 in the Western Cape, South Africa to estimate the occupancy, density, and population size of a leopard population. Leopards were recorded at 95% of camera trapping sites, which resulted in a high occupancy that showed no significant variation between seasons, habitat types, or along an altitudinal gradient. Our results indicated a low leopard density in the study area, with an estimated 1.53 leopards/100 km2 in summer and 1.62 leopards/100 km2 in winter. Mean leopard population size was therefore estimated at 107 and 113 individuals in the winter and summer respectively. Leopard activity centres for female ranges were centred in the core study area and could be predicted with good certainty, while males appeared to move out of the study area during winter which resulted in a higher uncertainty in locations of activity centres. Interestingly, livestock depredation events in the surrounding farmlands were significantly higher in winter, which coincides with male leopards moving outside the core protected area into the surrounding farmlands. To reduce livestock losses and retaliatory leopard killings, we suggest that human-carnivore conflict mitigation measures be intensely monitored during the winter months in the study area. We also suggest that future leopard conservation efforts should focus on privately-owned land as these non-protected areas contain the majority of the remaining suitable leopard habitat and may provide important dispersal corridors and buffer zones on which the long-term sustainability of leopard populations depends.


title: IDENTIFICATION OF HIGH SEED OIL YIELD AND HIGH OLEIC ACID CONTENT IN BRAZILIAN GERMPLASM OF WINTER SQUASH (Cucurbita moschata D.)

abstract: Cucurbita moschata D. seed oil contains approximately 75% unsaturated fatty acids, with high levels of monounsaturated fatty acids and antioxidant compounds such as vitamin E and carotenoid, constituting a promising food in nutritional terms. Associated to this, the Brazilian germplasm of C. moschata exhibits remarkable variability, representing an important source for the genetic breeding of this vegetable and other cucurbits. In this context, the present study evaluated the productivity and profile of the seed oil of 91 C. moschata accessions from different regions of Brazil and maintained in the Vegetable Germplasm Bank of the Federal University of Vicosa (BGH-UFV). A field experiment was conducted between January and July 2016. The tested C. moschata accessions showed high genetic variability in terms of characteristics related to seed oil productivity (SOP), such as the mass of seeds per fruit and productivity of seeds, providing predicted selection gains of 29.39 g and 0.26 t ha-1, respectively. Based on the phenotypic and genotypic correlations, greater SOP can be achieved while maintaining high oleic acid content and low linoleic acid content, providing oil of better nutritional and chemical quality. In variability analysis, the accessions were clustered into five groups, which presented different averages for SOP and fatty acid content of seed oil; approach that will guide the use of appropriate germplasm in programs aimed at genetic breeding for SOP and seed oil profile. Per se analysis identified BGH-4610, BGH-5485A, BGH-6590, BGH-5556A, BGH-5472A, and BGH-5544A as the most promising accessions in terms of SOP, with average (+g) of approximately 0.20 t ha-1. The most promising accessions for higher oleic acid content of seed oil were BGH-5456A, BGH-3333A, BGH-5361A, BGH-5472A, BGH-5544A, BGH-5453A, and BGH-1749, with average (+g) of approximately 30%, and almost all of these accessions were also the most promising in terms of lower linoleic acid content of seed oil, with average (+g) of approximately 45%. Overall, part of the C. moschata accessions evaluated in the present study can serve as a promising resource in genetic breeding programs for SOP and fatty acid profile, aiming at the production of oil with better nutritional and physicochemical quality.

category: genetics


title: Effects of pen enrichment on leg health of fast and slower-growing broiler chickens

abstract: Pen enrichment for broiler (meat-type) chickens is one of the potential strategies to stimulate locomotion and consequently contribute to leg health and welfare. This study was designed to evaluate effects of using a plethora of pen enrichments (barrier perches, angular ramps, horizontal platforms, large distance between feed and water and providing live Black Soldier fly larvae in a dustbathing area) on tibia characteristics, locomotion, leg health and home pen behaviour of fast and slower-growing broiler chickens. The experiment was set up as a 2 x 2 factorial arrangement with a total of 840 male broiler chickens in a complete randomized design (7 replicates per treatment and 30 chickens per replicate) with the following treatments: 1) pen enrichment (enriched pen or non-enriched pen); 2) broiler strain (fast-growing Ross 308 or slower-growing Hubbard JA 757). Home pen behaviour and use of enrichment were observed. At approximately 1400 and 2200 gram body weight, two chickens per pen were randomly selected and slaughtered, to investigate tibia morphological, biophysical and mechanical characteristics and leg health. Pen enrichment positively affected tibia biophysical characteristics, e.g., osseous volume ({Delta}=1.8 cm3, P=0.003), total volume ({Delta}=1.4 cm3, P=0.03) and volume fraction ({Delta}=0.02 %, P=0.002), in both fast and slower-growing chickens, suggesting that pen enrichment particularly affects ossification and mineralization mechanisms. Accordingly, locomotion and active behaviours were positively influenced by pen enrichment. However, pen enrichment resulted in lower body weight gain in both strains, which might be due to higher activity or lower feed intake as a result of difficulties of crossing the barrier perches. Regarding the strain, slower-growing chickens showed consistently more advanced tibia characteristics and more active behaviour than fast-growing chickens. It can be concluded that pen enrichment may lead to more activity and better bone development in both fast and slower-growing chickens.

category: animal behavior and cognition


title: Decoding the Neural Signatures of Valence and Arousal From Portable EEG Headset

abstract: This paper focuses on classifying emotions on the valence-arousal plane using various feature extraction, feature selection and machine learning techniques. Emotion classification using EEG data and machine learning techniques has been on the rise in the recent past. We evaluate different feature extraction techniques, feature selection techniques and propose the optimal set of features and electrodes for emotion recognition. The images from the OASIS image dataset were used for eliciting the valence and arousal emotions, and the EEG data was recorded using the Emotiv Epoc X mobile EEG headset. The analysis is additionally carried out on publicly available datasets: DEAP and DREAMER. We propose a novel feature ranking technique and incremental learning approach to analyze the dependence of performance on the number of participants. Leave-one-subject-out cross-validation was carried out to identify subject bias in emotion elicitation patterns. The importance of different electrode locations was calculated, which could be used for designing a headset for emotion recognition. Our study achieved root mean square errors (RMSE) of less than 0.75 on DREAMER, 1.76 on DEAP, and 2.39 on our dataset.


title: Waves and currents decrease the available space in a salmon cage

abstract: Due to increasing demand for salmon and environmental barriers preventing expansion in established sites, salmon farmers seek to move or expand their production to more exposed sites. In this study we investigate the effects of strong currents and waves on the behaviour of salmon and how they choose to use the space available to them. Observations are carried out in a site with strong tidal currents and well mixed water. Using video cameras and echo sounders, we show that salmon prefer to use the entire water column, narrowing their range only as a response to cage deformation, waves, or daylight. Conversely, salmon show strong horizontal preference, mostly occupying the portions of the cage exposed to currents. Additionally, waves cause salmon to disperse from the exposed side of the cage to the more sheltered side. Even when strong currents decrease the amount of available space, salmon choose to occupy the more exposed part of the cage. This indicates that at least with good water exchange, the high density caused by limited vertical space is not so aversive that salmon choose to move to less desirable areas of the cage. However, the dispersal throughout the entire available water column indicates that ensuring enough vertical space, even in strong currents, would be beneficial to salmon welfare.


title: TELSAM polymers accelerate crystallization of fused target proteins by stabilizing minimal crystal contacts and in the absence of direct inter-TELSAM contacts

abstract: We extend investigation into the usefulness of genetic fusion to TELSAM polymers as an effective protein crystallization strategy. We tested various numbers of the target protein fused per turn of the TELSAM helical polymer and various TELSAM-target connection strategies. We provide definitive evidence that: 1. A TELSAM-target protein fusion can crystallize more rapidly than the same target protein alone, 2. TELSAM-target protein fusions can form well-ordered, diffracting crystals using either flexible or rigid TELSAM-target linkers, 3. Well-ordered crystals can be obtained when either 2 or 6 copies of the target protein are presented per turn of the TELSAM helical polymer, 4. The TELSAM polymers themselves need not directly contact one another in the crystal lattice, and 5. Fusion to TELSAM polymer confers immense avidity to stabilize exquisitely weak inter-target protein crystal contacts. We report features of TELSAM-target protein crystals and outline future work needed to define the requirements for reliably obtaining optimal crystals of TELSAM-target protein fusions.

category: biochemistry


title: Synthesis and characterization of peptide conjugated human serum albumin nanoparticles for targeted cardiac uptake and drug delivery.

abstract: Congestive heart failure, a prominent cardiovascular disease results primarily from myocardial infarction or ischemia. Milrinone (MRN), a widely used clinical drug for heart failure, improves myocardial contractility and cardiac function through its inotropic and vasodilatory effects. However, lacking target specificty, it exhibits low bioavailability and lower body retention time. Therefore, in this study, angiotensin II (AT1) peptide conjugated human serum albumin nanoparticles (AT1-HSA-MRN-NPs) have been synthesized for targeted delivery of MRN to the myocardium, overexpressing AT1 receptors under heart failure. The NPs were surface functionalized through a covalent conjugation reaction between HSA and AT1. Nanoparticle size was 215.2{+/-}4.7 nm and zeta potential -28.8{+/-}2.7 mV and cumulative release of MRN was [~]72% over 24 hrs. The intracellular uptake of nanoparticles and cell viability was studied in H9c2 cells treated with AT1-MRN-HSA-NPs vs the control non-targeted drug, MRN Lactate under normal, hypoxic and hypertrophic conditions. The uptake of AT1-HSA-MRN-NPs in H9c2 cells was significantly higher as compared to non-targeted nanoparticles, and the viability of H9c2 cells treated with AT1-MRN-HSA-NPs vs MRN Lactate was 73.4{+/-}1.4% vs 44.9{+/-}1.4%, respectively. Therefore, AT1-HSA-MRN-NPs are safe for in vivo use and exhibit superior targeting and drug delivery characteristics for treatment of heart failure.

category: bioengineering


title: Performance of Deep Convolutional Neural Network Approaches and Human Level in Detecting Mosquito Species

abstract: Recently, mosquito-borne diseases have been a significant problem for public health worldwide. These diseases include dengue, ZIKA and malaria. Reducing disease spread stimulates researchers to develop automatic methods beyond traditional surveillance Well-known Deep Convolutional Neural Network, YOLO v3 algorithm, was applied to classify mosquito vector species and showed a high average accuracy of 97.7 per cent. While one-stage learning methods have provided impressive output in Aedes albopictus, Anopheles sinensis and Culex pipiens, the use of image annotation functions may help boost model capability in the identification of other low-sensitivity (< 60 per cent) mosquito images for Cu. tritaeniorhynchus and low-precision Ae. vexans (< 80 per cent). The optimal condition of the data increase (rotation, contrast and blurredness and Gaussian noise) was investigated within the limited amount of biological samples to increase the selected model efficiency. As a result, it produced a higher potential of 96.6 percent for sensitivity, 99.6 percent for specificity, 99.1 percent for accuracy, and 98.1 percent for precision. The ROC Curve Area (AUC) endorsed the ability of the model to differentiate between groups at a value of 0.985. Inter-and intra-rater heterogeneity between ground realities (entomological labeling) with the highest model was studied and compared to research by other independent entomologists. A substantial degree of near-perfect compatibility between the ground truth label and the proposed model (k = 0.950 {+/-} 0.035) was examined in both examinations. In comparison, a high degree of consensus was assessed for entomologists with greater experience than 5-10 years (k = 0.875 {+/-} 0.053 and 0.900 {+/-} 0.048). The proposed YOLO v3 network algorithm has the largest capacity for support-devices used by entomological technicians during local area detection. In the future, introducing the appropriate network model based methods to find qualitative and quantitative information will help to make local workers work quicker. It may also assist in the preparation of strategies to help deter the transmission of arthropod-transmitted diseases.

category: bioinformatics


title: Gastric cancer derived exosomes induce peritoneal mesothelial cell EMT through TGF-β1/Smads pathway to promote peritoneal metastasis

abstract: Epithelial-mesenchymal transition (EMT) plays an important role in peritoneal metastasis of Gastric cancer (GC). Tumor exosomes can mediate tumor directed metastasis, and TGF-{beta}1 is an important factor in inducing tumor Epithelial mesenchymal transition. However, it is not clear whether GC derived exosomes can induce peritoneal mesothelial cells through the TGF-{beta}1/ Smads pathway and the effect of injured peritoneal mesothelial cells on the biological characteristics of GC cells. In this study, we demonstrated that GC-derived exosomes can activate the TGF-{beta}1/Smads pathway in peritoneal mesothelial cells and induce the corresponding EMT process, and that the injured peritoneal mesothelial cells can improve the migration and adhesion of GC cells. Taken together, these data further support the critical role of exosomes in the remodeling of the pre-metastatic microenvironment.

category: cancer biology


title: US Dog Importations during the COVID-19 Pandemic: Do we have an erupting problem?

abstract: Dog importation data from 2018-2020 were evaluated to ascertain whether the dog importation patterns in the United States changed during the COVID-19 pandemic, specifically with regard to denial of entry. Dog denial of entry reports from January 1, 2018, to December 31, 2020, stored within the Centers for Disease Control and Prevention (CDC) Quarantine Activity Reporting System, were reviewed. Basic descriptive statistics were used to analyze the data. Reason for denial, country of origin, and month of importation were all examined to determine which countries of origin resulted in the largest number of denials, and whether there was a seasonal change in importations during the COVID-19 pandemic (2020), compared to previous years (2018 and 2019). During 2020, CDC denied entry to 458 dogs. This represents a 52% increase in dogs denied entry compared to the averages in 2018 and 2019. Dogs were primarily denied entry for falsified rabies vaccination certificates (56%). Three countries exported 74% of all dogs denied entry into the United States, suggesting that targeted interventions may be needed for certain countries. Increased attempts to import inadequately vaccinated dogs from countries with canine rabies in 2020 may have been due to the increased demand for domestic pets during the COVID-19 pandemic. Educational messaging should highlight the risk of rabies and the importance of making informed pet purchases from foreign entities to protect pet owners, their families, and the public.

category: scientific communication and education


title: Nano-Insecticides Against the Black Cutworm Agrotis ipsilon (Lepidoptera: Noctuidae): Toxicity, Development, Enzyme activity, and DNA Mutagenicity

abstract: High-frequency doses of chemical pesticides cause environmental pollution with high pesticide residues. In addition, increasing insecticide resistance in many insect pests requires novel pest control methods. Nanotechnology could be a promising field of modern agriculture, and is receiving considerable attention in the development of novel nano-agrochemicals, such as nanoinsectticides and nanofertilizers. This study assessed the effects of the lethal and sublethal concentrations of chlorantraniliprole, thiocyclam, and their nano-forms on the development, reproductive activity, oxidative stress enzyme activity, and DNA changes at the molecular level of the polyphagous species of black cutworm Agrotis ipsilon. The results revealed that A. ipsilon larvae were more susceptible to the nano-formsthan the regular forms of both nano chlorine and sulfur within the chlorantraniliprole and thiocyclam insecticides, respectively, with higher toxicities than the regular forms (ca. 3.86, and ca.2.06-fold, respectively). Significant differences in biological parameters, including developmental time and reproductive activity (fecundity and hatchability percent) were also observed. Correspondingly, increases in oxidative stress enzyme activities were observed, as were mutagenic effects on the genomic DNA of A. ipsilon after application of the LC50 of the nano-forms of both insecticides compared to the control. The positive results obtained here have led us to apply these nano-forms indifferent insect models in additional studies.

category: zoology


title: {-}CardiOvascular examination in awake Orangutans (Pongo pygmaeus pygmaeus): Low-stress Echocardiography including Speckle Tracking imaging (the COOLEST method)

abstract:
IntroductionCardiovascular diseases have been identified as a major cause of mortality and morbidity in Borneo orangutans (Pongo pygmaeus pygmaeus). Transthoracic echocardiography is usually performed under anesthesia in great apes, which may be stressful and risky in cardiac animals. The aim of the present pilot study was hence to develop a quick and non-stressful echocardiographic method (i.e., the COOLEST method) in awake Borneo orangutans (CardiOvascular examination in awake Orangutans: Low-stress Echocardiography including Speckle Tracking imaging) and assess the variability of corresponding variables.

Materials and MethodsFour adult Borneo orangutans trained to present their chest to the trainers were involved. A total of 96 TTE examinations were performed on 4 different days by a trained observer examining each orangutans 6 times per day. Each examination included four two-dimensional views, with offline assessment of 28 variables (i.e., two-dimensional (n=12), M-mode and anatomic M-mode (n=6), Doppler (n=7), and speckle tracking imaging (n=3)), representing a total of 2,688 measurements. A general linear model was used to determine the within-day and between-day coefficients of variation.

ResultsMean{+/-}SD (minimum-maximum) images acquisition duration was 3.8{+/-}1.6 minutes (1.3-6.3). All within-day and between-day coefficients of variation but one (n=55/56, 98%) were <15%, and most (51/56, 91%) were <10% including those of speckle tracking systolic strain variables (2.7% to 5.4%).

DiscussionHeart morphology as well as global and regional myocardial function can be assessed in awake orangutans with good to excellent repeatability and reproducibility.

ConclusionsThis non-stressful method may be used for longitudinal cardiac follow-up in awake orangutans.


title: Insights on the historical biogeography of Philippine native pigs and its relationship with Continental domestic and wild boars

abstract: The Philippine archipelago was believed to have never been connected to the Asian continent even during the severe Quaternary sea-level drops. As a result, the history of pig dispersal in the Philippines remains controversial and must have some anthropogenic origin associated with some human migration events. In this study, the context of origin, dispersal, and the level of genetic introgression in Philippine native pigs were deduced using mitochondrial DNA D-loop analysis altogether with domestic pigs and wild boars corresponding to their geographic origin. Results revealed a considerable genetic diversity (0.900{+/-}0.016), and a widespread Asian pig-ancestry (94.60%) were revealed in the phylogenetic analysis with admixed European pig-origin (5.10%) harboring various fractions of ancestry from Berkshire and Landrace. The close genetic connection between the continental wild boars and domestic pigs present in the Philippine pigs corroborates our hypothesis of a genetic signal that could potentially be associated with the recently reported multiple waves of human migrations to the Philippines during the last 50,000 years. The high frequency of haplotypes (54.08%) that collapsed in the D7 haplogroup represent an interesting challenge as its distribution does not coincide with the hypothesized migratory route of the Neolithic Austronesian-speaking populations. We detected the first Pacific Clade signature and ubiquitously distributed D2 haplotypes which postulate the legitimate dispersal of pigs associated with the multiple waves of human migrations involving the Philippines. The multimodal mismatch and neutrality test statistics both Fus Fs and Tajimas D correlates the long stationary period of effective population size revealed in the Bayesian skyline plot. While the sudden decrease in population was consistent with the pronounced population bottleneck of Asian and European pigs during the interglacial periods of the Pleistocene.


title: Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1

abstract:
Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxins antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts.

We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs.

In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.


title: Fine-tuned method to extract high purified proteins from the seagrass Halophila stipulacea to be used for proteome analyses

abstract: The non-indigenous to the Mediterranean tropical seagrass Halophila stipulacea has the possibility to become more prevalent in the Mediterranean basin, exacerbated by the rapid increase of water temperature. Molecular profiling appears a promising tool to study the traits that render H. stipulacea tolerant and resilient and facilitate its rapid and vast geographical spread. Taking advantage from recent seagrass genomes sequencing, proteomics specialty has been applied to several seagrasses giving new insight on the biology and physiology of this group of angiosperms. Thus, it could be of interest to apply proteomics to H. stipulacea that it could be considered as a possible plant model species to study marine biological invasion. The first step to achieve this goal is to obtain high quality proteins from plant tissue. Tissue fixation and protein extraction protocol are the most challenging steps in proteomics. Here we report a fine-tuned procedure obtained by comparing protein yield from H. stipulacea plants frozen in liquid nitrogen or preserved in RNAlater and processed following two different extraction protocols. Higher protein yield have been extracted from the procedure that use the RNAlater preserved plants, extracted with trichloroacetic acid in water followed by trichloroacetic acid in acetone, compared to those obtained from all other procedures. Protein purity of these samples have been tested by the separation in SDS-PAGE comfirming a better resolved profile of peptide bands suitable for a gel-based proteomics. Then, to assess the quality of proteins the mHPLC-ESI-MS/MS mass spectrometry analyses and bioinformatics have been performed. Hundreds proteins have been identified against several seagrass genomic resources available at UniProt, NCBI, SeagrassDB and transcriptomic datasets, which were merged to form the first customized dataset useful for H. stipulacea proteomic investigations.


title: Improving RT-LAMP Detection of SARS-CoV-2 RNA through Primer Set Selection and Combination

abstract: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a viable molecular diagnostic method to expand the breadth and reach of nucleic acid testing, particularly for SARS-CoV-2 detection and surveillance. While rapidly growing in prominence, RT-LAMP remains a relatively new method compared to the standard RT-qPCR, and contribution to our body of knowledge on designing LAMP primer sets and assays can have significant impact on its utility and adoption. Here we evaluate 18 LAMP primer sets for SARS-CoV-2, comparing speed and sensitivity with different LAMP formulations and conditions across more than 5,000 RT-LAMP reactions and identifying several primer sets with similar high sensitivity for different SARS-CoV-2 gene targets. Significantly we observe a consistent sensitivity enhancement by combining primer sets for different targets, confirming and building on earlier work to create a simple, general approach to building better and more sensitive RT-LAMP assays.


title: Comparative analysis of transcriptomic profiles among ascidians, zebrafish, and mice: insights from tissue-specific gene expression

abstract: Tissue/organ-specific genes (TSGs) are important not only for understanding organ development and function, but also for investigating the evolutionary lineages of organs in animals. Here, we investigate the TSGs of 9 adult tissues of an ascidian, Ciona intestinalis Type A (Ciona robusta), which lies in the important position of being the sister group of vertebrates. RNA-seq and qRT-PCR identified the Ciona TSGs in each tissue, and BLAST searches identified their homologs in zebrafish and mice. Tissue distributions of the vertebrate homologs were analyzed and clustered using public RNA-seq data for 12 zebrafish and 30 mouse tissues. Among the vertebrate homologs of the Ciona TSGs in the neural complex, 48% and 63% showed high expression in the zebrafish and mouse brain, respectively, suggesting that the central nervous system is evolutionarily conserved in chordates. In contrast, vertebrate homologs of Ciona TSGs in the ovary, pharynx, and intestine were not consistently highly expressed in the corresponding tissues of vertebrates, suggesting that these organs have evolved in Ciona-specific lineages. Intriguingly, more TSG homologs of the Ciona stomach were highly expressed in the vertebrate liver (17-29%) and intestine (22-33%) than in the mouse stomach (5%). Expression profiles for these gene suggest that the biological roles of the Ciona stomach are distinct from those of their vertebrate counterparts. Collectively, Ciona tissues were categorized into 3 groups: i) high similarity to the corresponding vertebrate tissues (neural complex and heart), ii) low similarity to the corresponding vertebrate tissues (ovary, pharynx, and intestine), and iii) low similarity to the corresponding vertebrate tissues, but high similarity to other vertebrate tissues (stomach, endostyle, and siphons). The present study provides transcriptomic catalogs of adult ascidian tissues and significant insights into the evolutionary lineages of the brain, heart, and digestive tract of chordates.

category: evolutionary biology


title: Deficiency in Lyst function leads to accumulation of secreted proteases and predisposition to mechanic stress-induced retinal detachment

abstract: Chediak-Higashi syndrome, caused by mutations in the Lysosome Trafficking Regulator (Lyst) gene, is a recessive hypopigmentation disorder characterized by albinism, neuropathies, neurodegeneration, and defective immune responses, with enlargement of lysosomes and lysosome-related organelles. Although recent studies have suggested that Lyst mutations impair the regulation of sizes of lysosome and lysosome-related organelle, the underlying pathogenic mechanism of Chediak-Higashi syndrome is still unclear. Here we show striking evidence that deficiency in LYST protein function leads to accumulation of photoreceptor outer segment phagosomes in retinal pigment epithelial cells, and reduces adhesion between photoreceptor outer segment and retinal pigment epithelial cells in a mouse model of Chediak-Higashi syndrome. In addition, we observe elevated levels of cathepsins, matrix metallopeptidase (MMP) 3 and oxidative stress markers in the retinal pigment epithelium of Lyst mutants. Previous reports showed that impaired degradation of photoreceptor outer segment phagosomes causes elevated oxidative stress, which could consequently lead to increases of cysteine cathepsins and MMPs in the extracellular matrix. Taken together, we conclude that the loss of LYST function causes accumulation of phagosomes in the retinal pigment epithelium and elevation of several extracellular matrix-remodeling proteases through oxidative stress, which may, in turn, reduce retinal adhesion. Our work reveals previously unreported pathogenic events in the retinal pigment epithelium caused by Lyst deficiency, which may place Chediak-Higashi syndrome patients at increased risk for retinal detachment. The same pathogenic events may be conserved in other professional phagocytic cells, such as macrophages in the immune system, contributing to overall Chediak-Higashi syndrome pathology.


title: Vision-based monitoring and measurement of bottlenose dolphins' daily habitat use and kinematics

abstract: This research presents a framework to enable computer-automated observation and monitoring of bottlenose dolphins (Tursiops truncatus) in a professionally managed environment. Results from this work provide insight into the dolphins movement patterns, kinematic diversity, and how changes in the environment affect their dynamics. Fixed overhead cameras were used to collect [~]100 hours of observations, recorded over multiple days including time both during and outside of formal training sessions. Animal locations were estimated using convolutional neural network (CNN) object detectors and Kalman filter post-processing. The resulting animal tracks were used to quantify habitat use and animal dynamics. Additionally, Kolmogorov-Smirnov analyses of the swimming kinematics were used for high-level behavioral mode classification. The detectors achieved a minimum Average Precision of 0.76. Performing detections and post-processing yielded 1.24x107 estimated dolphin locations. Animal kinematic diversity was found to be lowest in the morning and peaked immediately before noon. Regions of the habitat displaying the highest activity levels correlated to locations associated with animal care specialists, conspecifics, or enrichment. The work presented here demonstrates that CNN object detection is not only viable for large-scale marine mammal tracking, it also enables automated analyses of dynamics that provide new insight into animal movement and behavior.


title: miR-206 inhibits estrogen-induced proliferation and invasion of ER-α36 positive gastric cancer cells

abstract:
ObjectiveTo explore the biological role of miR-206 in inhibiting the proliferation and invasion of estrogen-induced ER-36 positive gastric cancer cells and its related mechanisms.

MethodsMTT and tranwell assays were used to detect the proliferation and invasion of BGC-823 cells under different concentrations and time of ER stimulation. RT-PCR was used to detect the effect of ER stimulation on the content of miR-206 in gastric cancer cells. miR-206 mimics were transfected into BGC-823 cells to detect cell proliferation and invasion; luciferase reporter assay was used to determine whether miR-206 targets the 3-untranslated region of the CDK14-encoding gene (3 -UTR).

ResultsWe confirmed that estrogen promoted the proliferation and invasion of BGC-823 cells; As the concentration of estrogen increases, the level of miR206 in the cell gradually rises.Overexpression of miR-206 significantly decreased the estrogen-induced BGC-823 cells. Proliferative and invasive ability; miR-206 inhibits the expression of CDK14 by directly binding to the 3-UTR of CDK14 mRNA, thereby inhibiting the proliferation and invasion of gastric cancer cells.

ConclusionsOur results suggest that miR-206 inhibits estrogen-induced proliferation and invasion of ER-36-positive gastric cancer cells by inhibiting CDK14 and may be a promising therapeutic option for anticancer therapy.


title: Bioerosion traces and borings in the Upper Devonian vertebrate remains from the Holy Cross Mountains, Poland Bioerosion in fish remains

abstract: Among the hundreds of collected Devonian vertebrate macrofossils in the Holy Cross Mountains, placoderms dominate and provide data on their morphology, distribution and taphonomy. So far 17 out of more than 500 studied specimens have revealed bones with surfaces covered by sediment-filled trace fossils. The traces have been made on the vertebrate remains before their final burial. The borings, oval in cross-section, include dendroidal networks of shallow tunnels or short, straight or curved individual scratches and grooves, which frequently create groups on the both sides of the bones. ?Karethraichnus isp. from Kowala and ?Osteocallis isp. from Wietrznia are the oldest record of these ichnogenera. Sedimentological clues indicate a shallow water environment, probably from the slope below the storm wave base.

category: paleontology


title: Targeting "Immunogenic Hotspots" in Dengue and Zika Virus:  A Novel Approach to a Common Vaccine

abstract: Diseases caused by Dengue (DENV) and Zika (ZIKV) viruses cause significant mortality and illness globally. Due to the high sequence similarity of the viral proteins and the purported cross-reactive immune responses against the viruses, we envisioned a common multi-epitope vaccine (MEV) against both viruses by adopting a novel approach of identifying "immunogenic hotspots". These stretches of the structural and non-structural proteins are enriched with MHC class I and class II supertype-restricted T cell epitopes, and B cell epitopes, in addition to being highly conserved between different DENV serotypes and ZIKV. Such an approach ensures inclusion of multiple overlapping T and B cell epitopes common to both viruses, and also warrants high population coverage. Importantly, epitopes known to cause antibody-dependent-enhancement of infection have been excluded. These immunogenic hotspots have then been stitched together with linkers in-silico along with an adjuvant, CTxB to develop the MEV candidate. Four structural models of the MEV were selected on the basis of conformational preservation of CTxB, and their biophysical parameters, which also conserved the immunogenicity of the multiple epitopes. Importantly, each of the MEV candidates were found to interact with TLR4-MD2 complex by molecular docking studies, indicative of their ability to induce TLR-mediated immune responses.

category: immunology


title: Identifying cell-state associated alternative splicing events and their co-regulation

abstract: Alternative splicing shapes the transcriptome and contributes to each cells unique identity, but single-cell RNA sequencing has struggled to capture the impact of alternative splicing. We previously showed that low recovery of mRNAs from single cells led to erroneous conclusions about the cell-to-cell variability of alternative splicing (1). Here, we present a method, Psix, to confidently identify splicing that changes across a landscape of single cells, using a probabilistic model that is robust against the data limitations of scRNA-seq. Its autocorrelation-inspired approach finds patterns of alternative splicing that correspond to patterns of cell identity, such as cell type or developmental stage, without the need for explicit cell clustering, labeling, or trajectory inference. Applying Psix to data that follow the trajectory of mouse brain development, we identify exons whose alternative splicing patterns cluster into modules of co-regulation. We show that the exons in these modules are enriched for binding by distinct neuronal splicing factors, and that their changes in splicing correspond to changes in expression of these splicing factors. Thus, Psix reveals cell-type-dependent splicing patterns and the wiring of the splicing regulatory networks that control them. Our new method will enable scRNA-seq analysis to go beyond transcription to understand the roles of post-transcriptional regulation in determining cell identity.

category: genomics


title: Fecal Microbiota Transplantation Drives Colonic Expression of Immune Activation Genes in a Mouse Model of Antibiotic Use

abstract: Clostridioides difficile infection (CDI) is the leading hospital acquired infection in North America. While the standard treatment for CDI remains antibiotics, fecal microbiota transplantation (FMT) has gained attention as an effective therapy to prevent relapse. Previous work has focused on colonization resistance mounted against C. difficile by FMT-delivered commensals, but the effects of FMT on the gut mucosal immune response are poorly understood. Better understanding of the molecular mechanisms driven by FMT would allow for more targeted therapy against CDI. To address this important gap in knowledge, microbial community structure and host gene expression were assessed after FMT in a mouse model of antibiotic use. Administration of FMT led to a significant increase in microbial diversity and partial restoration of community structure within 48 hours of treatment. RNA sequencing of cecal tissue identified large changes in gene expression between FMT recipient and vehicle control groups. Strikingly, genes upregulated after FMT treatment were enriched in immune activation pathways, many of which were associated with pro-inflammatory immune responses. FMT also upregulated several genes associated with type 2 immunity while repressing several associated with type 3 immunity, trends that are associated with improved response to CDI. These results highlight the interplay between the intestinal microbiota and host transcriptome and identify pathways of interest for exploring the role of FMT on treatment of recurring CDI.


title: Targeted drug delivery using Beeswax derived Albendazole-Loaded Solid Lipid Nanoparticles in Haemonchus contortus, an albendazole tolerant nematode.

abstract:
Targeted delivery has not been achieved for anthelmintic treatment, resulting in the requirement of excess drugs dose leading to side effects and therapeutic resistance. Gastrointestinal helminths ingest lipid droplets from digestive fluid for energy production, development, and defense. Worms habit inspired us to develop biocompatible, oral administrable, bee-wax derived solid lipid nanoparticles (SLN) with excellent drug (albendazole) loading efficiency of 83.3 {+/-} 6.5 mg/g and sustained-release properties (86.4 {+/-} 3.9 % of drug released within 24 h). Rhodamine B-loaded SLN showed time-dependent release and distribution of dye in vivo in Haemonchous contortus. The intestinal sustained-release property was shown by the particles that caused enhancement of albendazole potency for up to 50 folds. Therefore, this formulation has immense potential as an anthelminthic drug delivery vehicle that will not only be able to reduce the dose but will also reduce the drug-induced side effects by enhancing the bioavailability of the drug.

HighlightsO_LIAlbendazole-loaded Solid Lipid Nanoparticles (SLN-A) were formulated using Beeswax as stating material showing high drug loading capacity of 83 mg/g with sustained-release properties and 84 {+/-} 3 % of drug release within 24 h.
C_LIO_LISLN-A particles showed 50 fold enhancement of Albendazole activity against Haemonchous contortus worm.
C_LIO_LIRhodamine B-loaded SLN particles showed the specific uptake and in-vivo sustained release of dye in the worm.
C_LI


title: Bioinspired Dual-Functional Solid Lipid Nanoformulations for Targeted Drug Delivery and Sustained Release for Enhancement of Potency of Albendazole, an Antihelminthic Drug


title: Orfamide-A-mediated bacterial-algal interactions involve specific Ca2+ signalling pathways

abstract: The antagonistic bacterium Pseudomonas protegens secretes the cyclic lipopeptide orfamide A, which triggers a Ca2+ signal, causing the deflagellation of the green microalga Chlamydomonas reinhardtii. By investigating targeted synthetic orfamide A variants and inhibitors, we found that at least two Ca2+-signalling pathways and TRP channels are involved in this response.


title: Determination of the Two-Component Systems regulatory network reveals core and accessory regulations across Pseudomonas aeruginosa lineages

abstract: Pseudomonas aeruginosa possesses one of the most complex bacterial regulatory networks, which largely contributes to its success as a human opportunistic pathogen. However, most of its transcription factors (TFs) are still uncharacterized and the potential intra-species variability in regulatory networks has been mostly ignored so far. Here, to provide a first global view of the two-component systems (TCSs) regulatory network in P. aeruginosa, we produced and purified all DNA-binding TCS response regulators (RRs) and used DAP-seq to map the genome-wide binding sites of these 55 TFs across the three major P. aeruginosa lineages. The resulting networks encompass about 40% of all genes in each strain and contain numerous new important regulatory interactions across most major physiological processes, including virulence and antibiotic resistance. Strikingly, the comparison between the three representative strains shows that about half of the detected targets are specific to only one or two of the tested strains, revealing a previously unknown large functional diversity of TFs within a single species. Three main mechanisms were found to drive this diversity, including differences in accessory genome content, as exemplified by the strain-specific plasmid in the IHMA87 outlier strain which harbors numerous binding sites of chromosomally-encoded RRs. Additionally, most RRs display potential auto-regulation or RR-RR cross-regulation, bringing to light the vast complexity of this network. Overall, we provide the first complete delineation of the TCS regulatory network in P. aeruginosa that will represent an important resource for future studies on this pathogen.


title: Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

abstract: Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One virulence factor is the sortase-assembled endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that the pilus sortase covalently links pilus monomers prior to recognition, while the housekeeping sortase cleaves at the LPXTG motif within the terminal pilin subunit, and subsequently attaches assembled pilus fiber to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) throughout the E. faecalis cell cycle via immunofluorescence microscopy and fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of the E. faecalis diplococcus were completely saturated with Ebp, while Ebp appeared as two foci directly adjacent to the nascent septum in the newer hemisphere. A similar localization pattern was observed for another cell wall anchored substrate by sortase A, aggregation substance (AS), suggesting that this may be a general rule for all SrtA substrates in E. faecalis. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that binds and sequesters lipid II cell wall precursors, new Ebp deposition at the cell surface was not disrupted. These data suggest an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto un-crosslinked cell wall, independent of new PG synthesis.


title: SRS-FISH: High-Throughput Platform Linking Microbiome Function to Identity at the Single Cell Level

abstract: One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single cell level. Single cell isotope probing has become a key tool for this purpose, but the currently applied detection methods for measuring isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering - two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput structure-function analyses of microbial communities with single cell resolution. SRS-FISH has an imaging speed of 10 to 100 milliseconds per cell, which is two to three orders of magnitude faster than spontaneous Raman-FISH. Using this technique, we delineated metabolic responses of thirty thousand individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose.


title: The dual nature of bacteriophage: growth-dependent predation and generalised transduction of antimicrobial resistance

abstract: Bacteriophage ("phage") are both predators and evolutionary drivers for bacteria, notably contributing to the spread of antimicrobial resistance (AMR) genes by generalised transduction. Our current understanding of the dual nature of this relationship is limited. We used an interdisciplinary approach to quantify how these interacting dynamics can lead to the evolution of multi-drug resistant bacteria. We co-cultured two strains of Methicillin-resistant Staphylococcus aureus, each harbouring a different antibiotic resistance gene, with 80 generalized transducing phage. After a growth phase of 8h, bacteria and phage surprisingly coexisted at a stable equilibrium in our culture, the level of which was dependent on the starting concentration of phage. We detected double-resistant bacteria as early as 7h, indicating that transduction of AMR genes had occurred. We developed multiple mathematical models of the bacteria and phage relationship, and found that phage-bacteria dynamics were best captured by a model in which the phage burst size decreases as the bacteria population reaches stationary phase, and where phage predation is frequency-dependent. We estimated that one in every 108 new phage generated was a transducing phage carrying an AMR gene, and that double-resistant bacteria were always predominantly generated by transduction rather than by growth. Our results suggest a shift in how we understand and model phage-bacteria dynamics. Although rates of generalised transduction could be interpreted as too rare to be significant, they are sufficient to consistently lead to the evolution of multi-drug resistant bacteria. Currently, the potential of phage to contribute to the growing burden of AMR is likely underestimated.


title: The human neuronal receptor NgR1 bridges reovirus capsid proteins to initiate infection

abstract: Human Nogo-66 receptor 1 (NgR1) is a receptor for mammalian orthoreoviruses (reoviruses), but the mechanism of virus-receptor engagement is unknown. NgR1 binds a variety of structurally dissimilar ligands in the adult central nervous system (CNS) to inhibit axon outgrowth. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron regrowth, making NgR1 an important therapeutic target for diverse conditions such as spinal crush injuries and Alzheimer disease. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that NgR1 sequences in a central concave region of the molecule establish a bridge between two copies of the viral capsid protein, {sigma}3. This unusual binding interface produces high-avidity interactions between virus and receptor and likely primes early entry steps. NgR1 sequences engaged by reovirus also are required for NgR1 binding to ligands expressed by neurons and oligodendrocytes. These studies redefine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.


title: Malaria parasite evades mosquito immunity by glutaminyl cyclase mediated protein modification

abstract: Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that Plasmodium sporozoites of QC-null mutants are recognized by the mosquito immune system and melanized when they reach the hemocoel. Sporozoite numbers in salivary glands are also reduced in mosquitoes infected with QC-null or QC catalytically-dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito hemocytes or melanization immune responses. Mutation of a single QC-target glutamine of the major sporozoite surface protein (CSP) also results in immune recognition of sporozoites. These findings reveal QC-mediated post-translational modification of surface proteins as a major mechanism of mosquito immune evasion by Plasmodium sporozoites.


title: Sulfated host glycan recognition by carbohydrate sulfatases of the human gut microbiota

abstract: The vast microbial community that resides in the human colon, termed the human gut microbiota, performs important roles in maintaining host health. Sulfated host glycans comprise both a major nutrient source and important colonisation factors for this community. Carbohydrate sulfatases remove sulfate groups from glycans and are essential in many bacteria for the utilisation of sulfated host glycans. Additionally, carbohydrate sulfatases are also implicated in numerous host diseases, but remain some of the most understudied carbohydrate active enzymes to date, especially at the structural and molecular level. In this work, we analyse 7 carbohydrate sulfatases, spanning 4 subfamilies, from the human gut symbiont Bacteroides thetaiotaomicron, a major utiliser of sulfated host glycans, correlating structural and functional data with phylogenetic and environmental analyses. Together, these data begin to fill the knowledge gaps in how carbohydrate sulfatases orchestrate sulfated glycan metabolism within their environment.


title: DNA mismatch and damage detection using a FRET-based assay for monitoring the loading of multiple MutS sliding clamps

abstract: We developed a sensitive, homogeneous fluorescence assay for the detection of DNA mismatches and DNA damage based on the mismatch repair (MMR) protein MutS. The assay is based on Forster resonance energy transfer (FRET) between SYBR Green I (SG), non-covalently bound to DNA, and Alexa Fluor 647 (AF647) conjugated to MutS. In contrast to previous assays using only the mismatch binding activity of MutS, we exploited the ATP-dependent loading of multiple MutS sliding clamps provoked by mismatch/damage to the DNA, which increases the overall sensitivity of the assay. The assay was validated using a well-characterized 3 kb circular DNA containing a single G/T mismatch. We also demonstrate that treatment of long (multiple kb) DNA with various chemical or physical agents including non-denaturing bisulfite conversion of cytosine to uracil, cisplatin modification or ultraviolet light (UVC) results in changes in the DNA that can be detected by the FRET-based MutS biosensor.


title: Characterization of glucosyltransferase and glucuronosyltransferase family members reveals how major flavone glycoside accumulate in the root of Scutellaria baicalensis

abstract: Flavonoid glycosides extracted from roots of Scutellaria baicalensis exhibit strong pharmaceutical effect in antitumor, antioxidative, anti-inflammatory, and antiviral activity. UDP glycosyltransferase family members are responsible for the transfer of a glycosyl moiety from UDP sugars to a wide range of acceptor flavonoids. Here, we report the phylogenetic analysis, tissue-specific expression and biochemical characterization of 10 glucosyltrasferases (SbUGTs) and 6 glucuronosyltransferases (SbUGATs) based on the recently released genome of S. baicalensis. These results reveal that the high expression level and affinity to substrate of SbUGAT4 make baicalin become the richest flavonoid glycoside in the root of S. baicalensis.

category: plant biology


title: The transcription factor Zeb2 drives differentiation of age-associated B cells

abstract: Age-associated B cells (ABCs) accumulate after infection, during aging, and in systemic autoimmunity, contributing to pathogenesis in the latter1. To date, their precise ontogeny and whether they represent a B cell subset distinct from the other major effector subsets - plasma cells and memory B cells - remain unknown. We asked whether ABC formation requires the expression of a unique transcription factor. Here, we used single-cell RNA sequencing to identify differentially-expressed transcription factors shared by ABCs from both lupus patient and lupus-prone mice. Using an arrayed CRISPR screen, we show Zeb2 is essential for ABC differentiation in vitro. Selective deficiency of Zeb2 in B cells also leads to impaired ABC formation in vivo. Zeb2 drove ABC specification by directly binding the promoters of ABC signature genes including Itgax and Itgam encoding for CD11c and CD11b respectively. Zeb2 also targeted molecules involved in cytokine receptor signaling via the JAK/STAT pathway including Il21r and Socs5. Administration of the approved JAK/STAT inhibitor tofacitinib effectively ameliorated ABC accumulation in mice and patients with autoimmunity. This study reveals ABCs represent a distinct B cell subset dependent on Zeb2 expression, and provides a rationale for the use of JAK/STAT inhibitors to treat ABC-mediated autoimmune diseases.


title: FTO suppresses STAT3 activation and modulates proinflammatory interferon-stimulated gene expression

abstract: Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m6A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase FTO in the type I IFN response. FTO, which can remove either m6A or the cap-adjacent m6Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation is not through the actions of FTO on m6Am. Further, we found that depletion of FTO led to activation of STAT3, a transcription factor that mediates responses to various cytokines, but whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes.


title: Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters

abstract:
Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters.

IMPORTANCEMicrobial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals. We validate the approach by synthesizing and testing an array of bioreporters, including arsenic and mercury biosensors, that produce signal outputs in environments ranging from aerobic to highly reduced anaerobic growth conditions.


title: Cell-state specific aggresome formation in fibroblasts is driven by stress-activated MAPK signaling

abstract: The aggresome is a protein turnover system in which proteins are trafficked along microtubules to the centrosome for degradation. Despite extensive focus on aggresomes in immortalized cell lines, it remains unclear if the aggresome is conserved in all primary cells and all cell-states. Here we examined the aggresome in primary adult mouse dermal fibroblasts shifted into four distinct cell-states. We found that in response to proteasome inhibition, quiescent and immortalized fibroblasts formed aggresomes whereas proliferating and senescent fibroblasts did not. Using this model, we generated a resource to better understand the aggresome, providing a characterization of the proteostasis networks in which the aggresome is used and transcriptomic features associated with the presence or absence of aggresome formation. Using this resource, we validate a previously reported role for p38 MAPK signaling in aggresome formation and identify TAK1 as a novel driver of aggresome formation upstream of p38 MAPKs. Together, our data demonstrate that the aggresome is a non-universal protein degradation system which can be used cell-state specifically and provide a resource for better understanding aggresome formation and function.


title: Adult fibroblasts use aggresomes only in distinct cell-states


title: Blood flow coordinates collective endothelial cell migration during vascular plexus formation and promotes angiogenic sprout regression via vegfr3/flt4

abstract: Nascent vascular networks adapt to the increasing metabolic demands of growing tissues by expanding via angiogenesis. As vascular networks expand, blood vessels remodel, progressively refining vascular connectivity to generate a more haemodynamically efficient network. This process is driven by interplay between endothelial cell (EC) signalling and blood flow. While much is known about angiogenesis, considerably less is understood of the mechanisms underlying vessel remodelling by blood flow. Here we employ the zebrafish sub-intestinal venous plexus (SIVP) to characterise the mechanisms underlying blood flow-dependent remodelling. Using live imaging to track ECs we show that blood flow controls SIVP remodelling by coordinating collective migration of ECs within the developing plexus. Blood flow opposes continuous ventral EC migration within the SIVP and is required for regression of angiogenic sprouts to support plexus growth. Sprout regression occurs by coordinated polarisation and migration of ECs from non-perfused leading sprouts, which migrate in opposition to blood flow and incorporate into the SIV. Sprout regression is compatible with low blood flow and is dependent upon vegfr3/flt4 function under these conditions. Blood flow limits expansive venous remodelling promoted by vegfr3/flt4. Collectively, these studies reveal how blood flow sculpts a developing vascular plexus by coordinating EC migration and balancing vascular remodelling via vegfr3/flt4.

category: developmental biology


title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro/adipogenic progenitors in fibrodysplasia ossificans progressiva mice

abstract: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by progressive and catastrophic heterotopic ossification (HO) of skeletal muscle and associated soft tissues. FOP is caused by dominantly acting mutations in the bone morphogenetic protein (BMP) type I receptor, ACVR1 (also known as ALK2), the most prevalent of which is an arginine to histidine substitution [ACVR1(R206H)] in the glycine-serine rich intracellular domain of the receptor. A fundamental pathological consequence of FOP-causing ACVR1 receptor mutations is to enable activin A to initiate canonical BMP signaling in responsive progenitors, which drives skeletogenic commitment and HO. With the clear targets of activin A and ACVR1 identified, development of antibody therapeutics to prevent ligand-receptor interactions is an interventional approach currently being explored. Here, we developed a monoclonal blocking antibody (JAB0505) to the extracellular domain of ACVR1 and tested its ability to inhibit HO in established FOP mouse models. JAB0505 inhibited BMP-dependent gene expression in wild-type and ACVR1(R206H)-overexpressing cell lines. Strikingly, however, JAB0505 treatment markedly exacerbated injury-induced HO in two independent FOP mouse models in which ACVR1(R206H) was either broadly expressed, or more selectively expressed in fibro/adipogenic progenitors (FAPs). JAB0505 drove HO even under conditions of activin A inhibition, indicating that JAB0505 has receptor agonist activity. JAB0505-treated mice exhibited multiple, distinct foci of heterotopic lesions, suggesting an atypically broad anatomical domain of FAP recruitment to endochondral ossification. In addition, skeletogenic differentiation was both delayed and prolonged, and this was accompanied by dysregulation of FAP population growth. Collectively, alterations in the growth and differentiative properties of FAPs and FAP-derived skeletal cells are implicated in the aggravated HO phenotype. These data raise serious safety and efficacy concerns for the use of anti-ACVR1 antibodies to treat FOP patients.


title: Mapping drug-microenvironment-genetic interplay in CLL reveals trisomy 12 as a modulator of microenvironmental signals

abstract:
The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited to small scale studies and systematic analyses are lacking. We chose Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in adults, as a model disease to study this complex interplay systematically. We performed a combinatorial assay using 12 drugs individually co-applied with each of 17 microenvironmental stimuli in 192 primary CLL samples, generating a comprehensive map of drug-microenvironment interactions in CLL. This data was combined with whole-exome sequencing, DNA-methylation, RNA-sequencing and copy number variant annotation. Our assay identified four distinct CLL subgroups that differed in their responses to the panel of microenvironmental stimuli. These subgroups were characterized by distinct clinical outcomes independently of known prognostic markers. We investigated the effect of CLL- specific recurrent genetic alterations on microenvironmental responses and identified trisomy 12 as an amplifier of multiple microenvironmental stimuli. We further quantified the impact of microenvironmental stimuli on drug response, confirmed known interactions such as Interleukin (IL) 4 mediated resistance to B cell receptor (BCR) inhibitors, and identified new interactions such as Interferon-{gamma} induced resistance to BCR inhibitors. Finally, we identified interactions which were limited to genetic subgroups. Resistance to chemotherapeutics, such as Fludarabine, induced by Toll-Like Receptor (TLR) agonists could be observed in IGHV unmutated patient samples and IGHV mutated samples with trisomy 12. In-vivo relevance was investigated in CLL-infiltrated lymph nodes, which showed increased IL4 and TLR signalling activity compared to healthy samples (p<0.001). High IL4 activity in lymph nodes correlated with faster disease progression (p=0.038).

We provide a publicly available resource (www.dietrichlab.de/CLL_Microenvironment/) which uncovers tumour cell extrinsic influences on drug response and disease progression in CLL, and how these interactions are modulated by cell intrinsic molecular features.


title: Combinatorial drug-microenvironment interaction mapping reveals cell-extrinsic drug resistance mechanisms and clinically relevant patient subgroups in CLL


title: A ubiquitously expressed UDP-glucosyltransferase, UGT74J1, controls basal salicylic acid levels in rice

abstract: Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.


title: Genomic rearrangements have consequences for introgression breeding as revealed by genome assemblies of wild and cultivated lentil species

abstract: Understanding the genomic relationship between wild and cultivated genomes would facilitate access to the untapped variability found in crop wild relatives. We developed genome assemblies of a cultivated lentil (Lens culinaris) as well as a wild relative (L. ervoides). Comparative analyses revealed large-scale structural rearrangements and additional repetitive DNA in the cultivated genome, resulting in regions of reduced recombination, segregation distortion and permanent heterozygosity in the offspring of a cross between the two species. These novel findings provide plant breeders with better insight into how best to approach accessing the novel variability available in wild relatives.


title: A BioBricks toolbox for multiplexed metabolic engineering of central carbon metabolism in the tetracenomycin pathway

abstract: The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. We developed a BioBricks(R) genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: 1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a "metabolic regulatory switch" responsible for mobilizing triacylglycerols to {beta}-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. Altogether, this system is compatible with the BioBricks(R) [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE.

category: synthetic biology


title: IFITM3 regulates virus-induced inflammatory cytokine production by titrating Nogo-B orchestration of TLR responses

abstract: Interferon induced transmembrane protein 3 (IFITM3) is an important viral restriction factor in viral pathogenesis that also exhibits poorly understood immune regulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 regulates MyD88-dependent TLR-mediated cytokine production following dendritic cell exposure to cytomegalovirus (CMV), and this process limits viral pathogenesis in vivo. IFITM3 also restricted pro-inflammatory (IL-6) cytokine production in response to influenza. IFITM3 bound to and promoted ubiquitination and proteasomal degradation of the reticulon 4 isoform Nogo-B. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and this process involved alteration of TLR dynamics. The anti-inflammatory function of IFITM3 was intrinsically linked to its ability to regulate Nogo-B. Thus, we uncover Nogo-B as an unappreciated driver of viral pathogenesis and highlight a novel immune regulatory pathway where IFITM3 fine-tunes TLR responsiveness of myeloid cells to viral stimulation.