bluuebunny/crossref_metadata_2025 huggingface dataset:
Antimicrobial properties from lichens: An evaluation of the antimicrobial properties of English churchyard lichens
Authors: Taylor Judith , Fourie Toscane
Published: December 2019

The emergence of multidrug resistant bacteria has driven the need for novel antibiotics. Lichens naturally produce a wide range of unique defence chemicals and have already, historically shown medicinal efficacy. Antimicrobial properties of Lichens were investigated. Ten common churchyard species were sampled and extracted in acetone, namely Caloplaca flavescens, Diploicia canescens, Cladonia fimbriata, Psilolechia lucida, Lecanora campestris subsp. Campestris, Lecanora sulphurea, Pertusaria amara f.amara, Lepraria incana, Porpidia tuberculosa and Xanthoria calcicola. Extracts were impregnated into filter paper discs and tested against Gram positive and Gram negative bacteria and two fungi using the disc diffusion susceptibility test method. Extracts of Diploicia canescens, Psilolechia lucida, Lecanora sulphurea, Pertusaria amara f. amara and Lepraria incana showed inhibition against Gram positive bacteria. Diploicia canescens, Pertusaria amara and Lepraria incana also inhibited the dermatophyte fungi tested. Further studies should isolate their metabolites in order to find the agents responsible for the antimicrobial activity. These agents may or may not be novel to medicine but could be of current medicinal interest.
Deciphering the biological activity of propolis (bee glue) and its constituents; what is its potential as an antimicrobial?
Authors: Escalada Margarita Gomez
Published: December 2019

Area of expertise: I have a very strong background on the action of antimicrobials and their resistance, I originally trained on the study of disinfectant mechanisms of action and resistance with a number of publications on the topic. For some years I have investigated the antimicrobial properties of plant extracts such as tinctures and oils as well as specific phytochemicals. More recently, through a collaboration with a company in North Yorkshire I have become involved in the study of propolis, a product of the hive. Propolis is a resinous substance used by the bees for protection. It is highly complex, comprising in excess of 300 different chemicals; it is also very variable as its chemical composition depends on the time of year collected, the plants the bees are harvesting from and thus the geographical location.

In my group we are utilising our expertise to study the antimicrobial and antioxidant properties of propolis from different geographical locations in order to establish a link between the different aspects of biological activity with chemical composition. We are also investigating the effect of propolis on biofilm formation and quorum sensing, as well as its effects on antibiotic resistance.

Future goals: There is general awareness of the potential of propolis, from researchers and clinicians alike. However, understanding of this amazing product is relatively limited particularly regarding its biological activity as most of the research has been carried out by chemists analysing the different components. We would like to further understand the extent of the biological activity of propolis particularly regarding its antimicrobial and antioxidant activity. So that in the future we will be able to elucidate its mechanism (or most likely mechanisms) of action.

The main hurdles we currently face is regarding solubility which makes the use of established methodologies such as MICs very challenging. We are looking for researchers with experience in alternatives to these tests, particularly for test compounds with very limited solubility, as many phytochemicals are.

It has been suggested by colleagues that perhaps it would be necessary to investigate the effects of propolis on the whole organism at the molecular level, and thus carry out genomic and proteomic analysis following exposure to propolis. We would like to make links with researchers with experience of this kind of analysis to help us further understand the outcome of this kind of investigations. In the longer term in order to be able to work towards conducting clinical trials we would benefit from contacts with researchers that have taken work through this process.
A novel class of small molecule inhibitors targeting the chikungunya virus capping machinery with a high barrier to resistance
Authors: Abdelnabi Rana , Kovacikova Kristina , Kirchebner Julia , Leyssen Pieter , Marchand Arnaud , Chaltin Patrick , Puerstinger Gerhard , Langer Thierry , Quérat Gilles , Coutard Bruno , Hemert Martijn van , Neyts Johan , Delang Leen
Published: December 2019

Background: Despite the worldwide re-emergence of the chikungunya virus (CHIKV) and the high morbidity associated with CHIKV infections, there is no approved vaccine or antiviral treatment available. Here, we identified a novel class of CHIKV inhibitors i.e. the CHVB series.

Methods: CPE-reduction and virus yield assays were performed in Vero cells. Drug-resistant variants were selected using clonal resistance selection. The enzymatic assays for alphavirus capping were done using the non-structural protein 1 (nsP1) of Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEEV).

Results: CHVB compounds inhibited the in vitro replication of CHIKV isolates with EC50 values in the low μM range. In virus yield assays, the most potent analogues reduced the viral load with 4-5 log10. CHVB-resistant variants were selected and found to carry (i) two mutations in the gene encoding nsP1 (responsible for viral RNA capping), (ii) one mutation in nsP2 and (iii) one mutation in nsP3. Reverse-engineering suggested that nsP1 is the target of CHVB, since both nsP1 mutations were needed to achieve 10-fold resistance. Interestingly, the CHVBres virus proved cross-resistant to the MADTP series, a class of CHIKV capping inhibitors that we described earlier, suggesting a similar mode of action. In enzymatic assays, CHVB proved a potent inhibitor of the methyltransferase and guanylyltransferase activities of nsP1 of SFV and VEEV.

Conclusion: We identified a class of CHIKV inhibitors that targets the viral capping machinery. The potent anti-CHIKV activity and the high barrier to resistance make this chemical scaffold a potential candidate for CHIKV drug development.
Establishment of a stable subgenomic dengue virus type 1 replicon system in Aedes albopictus mosquito cells for identification of DENV transmission blocking molecules
Authors: Patel Sameera , Besson Benoit , Rij Ronald van , Dechering Koen
Published: December 2019

To combat DENV transmission, we aim to screen for molecules that target mosquito host cell factors playing a role in the viral replication, consequently inhibiting the replication of virus in the mosquito. Use case scenarios for such compounds include human mass drug administration in endemic populations, application in sugar baits or bed-nets.

In order to accomplish this, we have set up high throughput screening (HTS) using viral replication inhibition assays. We describe here the generation of a novel stable Nanoluciferase-reporter based dengue replicon system in U4.4 mosquito cells. The U4.4-DENV1 replicon cell line has been stably and successfully maintained for over 30 passages without significant loss of reporter signal. In order to characterize the cell line further, the cells were subjected to treatment with antiviral compound and viral inhibition was observed with ribavirin (IC50 =1.69 × 10-6M) and siRNA against NS3. For the purpose of HTS, viral inhibition, cytotoxicity and luciferase interference assays were established on a 384-well plate using the cryopreserved U4.4_DENV1 replicon cells. The latter two were used as deselection assays to differentiate between false- and true- positives. To conclude, we have developed a robust screening cascade to identify small molecules that may act as transmission blocking compounds. Screening of a chemical diversity library is ongoing and the results will be presented at the meeting.
Glucose regulated protein 78 (GRP78) interacts with Zika virus envelope and is required for a productive infection
Authors: Royle Jamie , Ramirez-Santana Carolina , Donald Claire , Akpunarlieva Snezhana , Gestuveo Rommel , Anaya Juan-Manuel , Burchmore Richard , Kohl Alain , Varjak Margus
Published: December 2019

Zika virus (ZIKV) is a member of the Flaviviridae family and was until recently a relatively obscure tropical disease. Subsequently, ZIKV has been shown to be the causative agent of fetal abnormalities and Guillain-Barré syndrome in outbreaks across the Americas and so efforts towards delineating important factors in the viral lifecycle have increased. Combining protein pull-down with mass spectrometry, it was found that ZIKV envelope (Env) interacts with the endoplasmic reticulum (ER) resident chaperone, glucose regulated protein 78 (GRP78) in A549 cells. Flaviviruses such as Japanese encephalitis virus and dengue virus are known to co-opt ER resident proteins and members of the unfolded protein response, including GRP78, to enhance viral infectivity and propagation. The role these proteins play during the ZIKV lifecycle has yet to be elucidated.

To determine the importance of this interaction during ZIKV infection, A549 cells were treated with GRP78-specific siRNAs prior to infection with a NanoLuc expressing reporter virus or a wild-type virus. Depletion of GRP78 significantly reduced both virus luciferase readings and viral titres, indicating that GRP78 is necessary for efficient infection of mammalian cell culture. In contrast, inhibition of GRP78 with small molecule inhibitors did not reduce ZIKV infection. Interestingly, immunofluorescence of ZIKV infected cells reveal that GRP78 re-localises following infection and co-localises with Env. Depletion of GRP78 abrogated localisation of viral replication factories. Further experiments have shown that GRP78 is important for infection post entry and replication, and that putative GRP78 interactions partners are also required during infection.
Identification of candidate molecular determinants of the vector competence of Ixodes ricinus for members of the tick-borne encephalitis complex
Authors: Lemasson Manon , Caignard Grégory , Unterfinger Yves , Attoui Houssam , Bell-Sakyi Lesley , Moutailler Sara , Bonnet Sarah , Vitour Damien , Richardson Jennifer , Lacour Sandrine A.
Published: December 2019

In Europe, tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are two flaviviruses both transmitted by the tick Ixodes ricinus. While the mechanisms of viral replication and transmission in this vector are incompletely understood, they are presumed to be largely governed by protein-protein interactions established between viruses and cells.

To elucidate the molecular determinants involved in vector competence, we have mapped the network of protein-protein interactions established between viral proteins of both TBEV and LIV and tick proteins encoded by a cDNA library of I. ricinus, by using yeast two-hybrid methodology. Twenty-two cellular partners from I. ricinus have been identified and all evidenced to interact with both viruses. Upon functional annotation, some of these tick proteins seem to be involved in such biological processes as the immune response or ribosomal maturation. To gain insight into the role of each tick protein in viral replication, the impact of gene silencing will be assessed by dsRNA knockdown. In parallel, I. ricinus cell lines have been persistently infected with TBEV or LIV and the level of expression of selected antiviral effectors monitored over time, in an effort to characterize the antiviral response of this arthropod.

This work represents the first description of the protein-protein interaction network for TBEV, LIV and I. ricinus. Certain tick partners may well represent molecular determinants of vector competence of I. ricinus for TBEV and LIV and potentially other flaviviruses, which we will ascertain by in vivo silencing of selected tick partners.
The Cryopreservation of Cos Lettuce Callus by Using Liquid Nitrogen
Authors: Tangkananond Wipa
Published: December 2019

The cryopreservation of cos lettuce (CL) callus by using liquid nitrogen (LN) was further study from the previous research on callus induction and micropropagation of CL. That was studied on seed culture and micropropagation of CL and its embryogenic growth. The result was shown that the most suitable Murashing and Skoog (MS) medium concentration was in 1/2 MS. The medium supplemented with 0.1 mg/l of 6-benzylaminopurine (BAP) and 0.5 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D) gave the best for leaf inducing callus. Then it was brought to study the effects of cryoprotectants with the different percentages of dimethyl sulfoxide (DMSO). The cryopreserved callus by using LN was brought to subculture for the best inducing shoot in MS medium supplemented with 4.0 mg/l of BAP. The results that the best cryoprotectant at the percentage of DMSO at 7.5 gave the highest average number of shoots at 2.1 shoots/culture and the average shoot length was 1.56 cm. The MS medium supplemented with 0.5 mg/l of 1-Naphthaleneacetic acid was the best inducing root. The results that the best cryoprotectant at the percentage of DMSO at 7.5 gave the highest average number of roots at 9.7 roots/culture and the average root length was 1.59 cm. Plant differentiation from callus of CL leaf was transferred to grow in soil and had the same morphology as normal CL that was grown in soil by seed germination.

CL will be the host of Japanese encephalitis virus expression for the developmental swine vaccine.
The Tick Cell Biobank: new arthropod cell lines for arbovirus research
Authors: Bell-Sakyi Lesley , Hartley Catherine , Darby Alistair , Baylis Matthew , Makepeace Benjamin
Published: December 2019

The Tick Cell Biobank is the world’s only dedicated culture collection for cell lines derived from ticks and other arthropod vectors. As well as storing and distributing arthropod cell lines and training in their maintenance to UK and international researchers, the Tick Cell Biobank generates novel cell lines from arthropod species and geographic strains not already represented in the collection. Currently, efforts are focussed on European Argas, Dermacentor, Hyalomma, Rhipicephalus and Ixodes spp. ticks, Lutzomyia and Phlebotomus spp. sandflies, Culicoides spp. biting midges, Rhodnius prolixus kissing bugs and Glossina morsitans tsetse flies. Techniques used previously for ticks and insects are applied or adapted for use with embryonic or larval arthropods to generate primary cell and tissue cultures; these primary cultures are then maintained until significant cell multiplication commences and subculture can be attempted, which may take several years. This approach has yielded new cell lines from the soft tick Argas reflexus, the hard tick Hyalomma lusitanicum, the New World sand fly Lutzomyia longipalpis and the UK midge Culicoides nubeculosus. Most of these novel cell lines are now available for arbovirus research through the Tick Cell Biobank.
The effect of co-infecting Anaplasma phagocytophilum on replication of Langat Virus, a model for Louping ill virus, in Ixodes spp. tick cells
Authors: Luu Lisa , Bradley George , Al-Khafaji Alaa , Bell-Sakyi Lesley
Published: December 2019

Tick borne fever (TBF) caused by Anaplasma phagocytophilum (Ap), and louping ill caused by the flavivirus louping ill virus (LIV) are the two most economically important vector-borne diseases in UK sheep populations. Both pathogens are transmitted by the tick Ixodes ricinus, which also harbours protozoan parasites and Borrelia spp. spirochetes. I. ricinus ticks may be co-infected with multiple microorganisms and potentially transmit more than one pathogen to hosts during blood feeding.

Ap infection is not limited to sheep, causing pasture fever in cattle and granulocytic anaplasmosis in horses, dogs and humans. There is no vaccine available for TBF, and disease control relies on tick control and antibiotic treatment. LIV mainly infects ruminants, but can also infect other livestock including horses, pigs, alpacas and llamas. Since 2016, the LIV vaccine has been unavailable and there is no alternative prophylactic treatment for livestock. Both Ap and LIV are zoonotic diseases with occasional human cases reported in the UK.

Importantly, Ap infection leads to host immunosuppression and consequently increases vulnerability to secondary infections. Both Ap and LIV have been studied as single infections in tick and mammalian cells. However the dynamics and implications of co-infections within the arthropod vector or mammalian host have, to date, not been fully explored.

Using embryo-derived Ixodes spp. cell lines infected with Langat virus (a BSL-2 model for LIV) and Ap, we examined the effect of co-infection on viral RNA replication by qRT-PCR and bacterial growth by qPCR. The results of this study will be presented and discussed.
A Viral Metagenomic Analysis Reveals Rich Viral Abundance and Diversity in Mosquitoes from Pig Farms
Authors: Wahaab Abdul
Published: December 2019

Mosquitoes harbor a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns and significant economic losses across the globe. The viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms in the vicinity of Shanghai, China to explore the viral community carried by mosquitoes. The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms, which comprised of more than 48 viral taxonomic families, specific to vertebrates, invertebrates, plants, fungi, bacteria, and protozoa. The read sequences related to animal viruses included parvoviruses, anelloviruses, circoviruses, flavivirus, rhabdovirus, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, sample G1 contained the most abundant sequence related to Banna virus, which is of public health interest because it causes encephalitis in humans. Furthermore, non-classified viruses also shared considerable virus sequences in all the samples, presumably belonging to unexplored virus category. Overall, the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms, which is a potential source of diseases for mammals including humans and animals. These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.
The Expression of Japanese Encephalitis Virus Envelope Gene in Green Cos
Authors: Tangkananond Wipa , Lomonossoff George
Published: December 2019

The expression of Japanese encephalitis virus (JEV) envelope gene in green cos leaf was a project undertaken at the John Innes Centre during 6 September to 20 October 2018 and is one of the projects of the research training program at the John Innes Centre during long vacation every year since 2010 until now.

The summary of this research is as follows. The production of the envelope protein of the JEV in green cos by using the envelope gene expression by 8 epitopes (MEP: multiepitope) of Japanese encephalitis virus vaccine strain SA14-14-2 that provides the highest immune response against JEV. After incorporated the envelope gene into pEAQ-HT and pEAQ-HT-HBcAg-tEL vectors, we cloned genes in Escherichia coli and Agrobacterium tumefaciens, respectively, then expressed the envelope protein of JEV in green cos leaves. The result can be seen in photographs of green cos leaves by both visible light camera and ultraviolet light camera.

Further research should include the analysis and identification by chemiluminescence immunoassay and by the Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF). Further purification of the envelope protein expression of JEV in green cos that will be benefited for the production of the envelope protein of the JEV plant-based vaccine.

Key words: Japanese encephalitis virus, plant-based vaccine, chemiluminescence immunoassay, MALDI-TOF
Stage specific transcriptome profiling of castor bean tick Ixodes ricinus
Authors: Vechtova Pavlina , Füssy Zoltan , Sterba Jan , Cegan Radim , Benes Vladimir , Grubhoffer Libor
Published: December 2019

Arthropod borne diseases are ubiquitously discussed topic whose relevance increases with ongoing changing climate, which extends the area of their incidence and affects profoundly the size of vector population, as well as its reproductive capacity, the abundance and spread of reservoir hosts and other variables that are generally tightly correlated with the spread of zoonoses.

The life cycle of arthropod borne pathogens are tightly bound to the life cycle of their host as well as to their vector organism. Therefore, the description of the vector life cycle should elucidate some questions related to the vector-pathogen dynamics including the factors important for successful disease transmission.

Tick has complex life cycle and for its completion it requires feeding on several host organisms, which is abused by the pathogen for its spreading within reservoir and host organisms.

Thus, more thorough description of factors driving tick developmental processes controlling its life cycle will be instrumental in understanding the nature of I. ricinus and its pathogens interactions and may also shed light on the process of blood feeding as an integral event in tick development as well as in potential pathogen transmission.

In our study we perform transcriptional profiling of all life stages of I. ricinus and provide a list of genes associated with particular life stages of I. ricinus and hence extend our knowledge in pursuit of potential acaricidal strategies.
Lumpy skin disease virus: transmission to dipteran vectors using animal and ex-vivo models
Authors: Bernardo Beatriz Sanz , Suckoo Rey , Darpel Karin , Hawes Pippa , Basu Sanjay , Langlands Zoe , Gubbins Simon , Beard Pip
Published: December 2019

Lumpy skin disease (LSD) is a tropical neglected viral disease of cattle, characterised by numerous cutaneous lesions disseminated throughout the body. Historically endemic to the African continent, it has become a threat to Europe following the outbreaks of LSD in the Middle East and Eastern Europe.

LSD virus (LSDV) is a Capripoxvirus transmitted by insect vectors. Experimental and epidemiological studies have indicated a role for the stable fly (Stomoxys calcintrans) and the mosquito Aedes aegypti. Nevertheless the relative importance of these vector species and others is unclear. A study was designed to explore the risk of transmission of LSDV from cattle to different vector species including Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus. Cattle was challenged with LSDV to produce a bovine experimental model used as a natural source of LSDV to the potential vectors. Cattle samples were taken to quantify LSDV in different tissues and characterise the disease. All insect species were allowed to feed on LSDV-challenged cattle at regular intervals and incubated for up to eight days. This data was then used to model the dynamics of LSDV infection and transmission. All four species were able to acquire and maintain LSDV for up to eight days post feeding, and the risk of transmission from bovine donor to insect was dependent on the severity of the disease. A model was then generated using ex-vivo skin lesions and infectious blood that will allow further studies of the role of these vectors.
Antagonistic action of blood-feeding and mating on the gut immunity in the female mosquito Aedes aegypti
Authors: Almire Floriane , Terry Sandra , Kean Joy , Donald Claire , Varjak Margus , McFarlane Melanie , Kohl Alain , Pondeville Emilie
Published: December 2019

The female mosquito Aedes aegypti needs a blood meal to reproduce and is therefore a vector of arthropod borne viruses (arboviruses) such as Zika (ZIKV) or chikungunya (CHIKV) viruses. Even though mosquitoes transmit viruses, they possess different immune pathways to fight against pathogens. Surprisingly, the analysis of immune gene expression in different tissues from non blood fed (NBF) or blood fed (BF) females revealed that these genes were repressed in the digestive tract from 2 to 30 h following blood meal. As the digestive tract is the first barrier to be overcome by an arbovirus during the first few hours/days after blood meal, our findings could explain the high susceptibility of Ae. aegypti mosquitoes to arboviruses. To investigate this hypothesis, it is required to identify the mechanism by which gut immunodeficiency is triggered. We would then be able to inhibit it and analyse the impact on mosquito infection/transmission. In addition to the acquisition of a blood meal, the female must mate before laying fertilized eggs. Mating has been shown to cause changes in the female physiology and behaviour such as refractoriness to further mating in many insects but also, mating has been shown to influence immunity in Drosophila. We found that mating transiently antagonizes the immune gene repression in the digestive tract of BF females compared to virgin BF females. The mechanisms by which blood-feeding and mating modulate gut immunity are under investigation and will be presented during the conference.
ARIPOR2in-frame deletion is a frequent and highly penetrant cause of adult-onset hearing loss
Authors: de Bruijn Suzanne E. , Smits Jeroen J. , Liu Chang , Lanting Cornelis P. , Beynon Andy J. , Blankevoort Joëlle , Oostrik Jaap , Koole Wouter , de Vrieze Erik ,   DOOFNL Consortium, Cremers Cor W.R.J. , Cremers Frans P. M. , Roosing Susanne , Yntema Helger G. , Kunst Henricus P.M. , Zhao Bo , Pennings Ronald J.E. , Kremer Hannie
Published: December 2019

Hearing loss is one of the most prevalent disabilities worldwide, and has a significant impact on quality of life. The adult-onset type of the condition is highly heritable but the genetic causes are largely unknown, which is in contrast to childhood-onset hearing loss. We identified an in-frame deletion of 12 nucleotides inRIPOR2as a highly penetrant cause of adult-onset progressive hearing loss that segregated as an autosomal dominant trait in 12 families from the Netherlands. Hearing loss associated with the deletion in 63 subjects displayed variable audiometric characteristics and an average age of onset of 30.6 years (SD 14.9 years, range 0-70 years). A functional effect of theRIPOR2variant was demonstrated by aberrant localization of the mutant RIPOR2 in the stereocilia of cochlear hair cells and failure to rescue morphological defects in RIPOR2-deficient hair cells, in contrast to the wildtype protein. Strikingly, theRIPOR2variant is present in 18 of 22,952 individuals not selected for hearing loss in the Southeast Netherlands. Collectively, these data demonstrate that an inherited form of adult-onset hearing loss is relatively common, with potentially thousands of individuals at risk in the Netherlands and beyond, which makes it an attractive target for developing a (genetic) therapy.
Loss ofprdm1aaccelerates melanoma onset and progression
Authors: Iwanaga Ritsuko , Truong Brittany T. , Hsu Jessica Y. , Lambert Karoline A. , Vyas Rajesh , Orlicky David , Shellman Yiqun G. , Tan Aik-Choon , Ceol Craig , Artinger Kristin Bruk
Published: December 2019

Melanoma is an aggressive, deadly skin cancer derived from melanocytes, a neural crest cell derivative. Melanoma cells mirror the developmental program of neural crest cells in that they exhibit the same gene expression patterns and utilize similar cellular mechanisms, including increased cell proliferation, EMT and migration. Here we studied the role of neural crest regulator PRDM1 in melanoma onset and progression. In development, Prdm1a functions to promote neural crest progenitor fate, and in melanoma, we found thatPRDM1has reduced copy number and is recurrently deleted in both zebrafish and humans. When examining expression of neural crest and melanocyte development genes, we show thatsox10progenitor expression is high inprdm1a-/-mutants, while more differentiated melanocyte markers are reduced, suggesting that normally Prdm1a is required for differentiation. Data mining of human melanoma datasets indicate that highPRDM1expression in human melanoma is correlated with better patient survival and decreasedPRDM1expression is common in metastatic tumors. When one copy ofprdm1ais lost in the zebrafish melanoma model (Tg[mitfa:BRAFV600E];p53-/-;prdm1a+ /-), melanoma onset occurs more quickly, and the tumors that form have a larger area with increased expression ofsox10.These data demonstrate a novel role for PRDM1 as a tumor suppressor in melanoma.
Contrast-enhanced ultrasound with sub-micron sized contrast agents detects insulitis and β-cell mass decline in mouse models of type 1 diabetes
Authors: Ramirez David G. , Abenojar Eric , Hernandez Christopher , Papazian Lucine A. , Passman Samantha , Pham Vinh , Exner Agata A. , Benninger Richard K.P.
Published: December 2019

Type 1 diabetes (T1D) is characterized by the infiltration of autoreactive T-cells into the islet of Langerhans, and depletion of insulin-secreting β-cells. This immune cell infiltration (insulitis) first occurs during an asymptomatic phase of T1D that can take place many years prior to clinical diagnosis. Methods to diagnose insulitis and changes in β-cell mass during this asymptomatic phase are limited, thus precluding early therapeutic intervention. While therapeutic treatments can delay T1D progression, treatment efficacy is limited and widely varying, and a method to track this efficacy is also lacking. During T1D progression, the islet microvasculature increases permeability as a result of insulitis, in both mouse models of T1D and humans with T1D. This increased permeability can allow nanoparticles, such as contrast agents for diagnostic imaging, to access the islet microenvironment. Contrast enhanced ultrasound (CEUS) uses shell-stabilized gas bubbles to provide high acoustic backscatter in vasculature and tissue and is clinically approved. A novel, sub-micron sized ‘nanobubble’ (NB) ultrasound contrast agent has been developed and shown to extravasate and accumulate in tumors, where microvascular permeability is high. To test whether CEUS can be used to measure increased islet microvasculature permeability and indicate the asymptomatic phase of T1D, we applied CEUS measurements with NBs in pre-clinical T1D models. NOD mice and mice receiving an adoptive-transfer of diabetogenic splenocytes showed accumulation of NBs specifically within the pancreatic islets, and only in the presence of insulitis. This accumulation was measured by both ultrasound contrast and histological analysis, and accumulation only occurred for sub-micron sized bubbles. Importantly, accumulation was detected as early as 4w in NOD mice. Thus, CEUS with sub-micron sized NB contrast agent may provide a predicative marker for disease progression early in asymptomatic T1D, as well as monitoring of disease prevention or reversal.
A switch in cilia-mediated Hedgehog signaling controls muscle stem cell quiescence and cell cycle progression
Authors: Cruz-Migoni Sara Betania , Imran Kamalliawati Mohd , Wahid Aysha , Rahman Oisharja , Briscoe James , Borycki Anne-Gaëlle
Published: December 2019

Tissue homeostasis requires a tight control of stem cells to maintain quiescence in normal conditions, and ensure a balance between progenitor cell production and the need to preserve a stem cell pool in repair conditions. Using ex-vivo and in-vivo genetic approaches, we provide evidence that primary cilium-mediated repressive Hedgehog (Hh) signalling is required to maintain skeletal muscle stem cells (MuSCs) in a quiescent state. De-repression and further activation of Hh signalling initiates MuSC entry and progression through the cell cycle, and controls self-renewal to ensure efficient repair of injured muscles. We propose a model whereby disassembly of primary cilia upon MuSC activation induces a switch in Hh signalling from a repressive to active state that controls exit from quiescence. Positive Hh response in bi-potential muscle progenitor cells regulates also cell cycle progression and drives MuSC self-renewal. These findings identify Hh signalling as a major regulator of MuSC activity.

Highlights

Cilia-containing quiescent MuSCs are Hh signalling suppressed

MuSC activation coincides with a switch to active Hh signalling

Smomutation delays cell cycle entry and progression, and causes impaired self-renewal

Ptch1mutation promotes exit from quiescence, rapid cell cycle and increased self-renewal

Graphical abstract
The DNA Methylation Landscape of Giant Viruses
Authors: Jeudy Sandra , Rigou Sofia , Alempic Jean-Marie , Claverie Jean-Michel , Abergel Chantal , Legendre Matthieu
Published: December 2019

DNA methylation is an important epigenetic mark that contributes to various regulations in all domains of life. Prokaryotes use it through Restriction-Modification (R-M) systems as a host-defense mechanism against viruses. The recently discovered giant viruses are widespread dsDNA viruses infecting eukaryotes with gene contents overlapping the cellular world. While they are predicted to encode DNA methyltransferases (MTases), virtually nothing is known about the DNA methylation status of their genomes. Using single-molecule real-time sequencing we studied the complete methylome of a large spectrum of families: the Marseilleviridae, the Pandoraviruses, the Molliviruses, the Mimiviridae along with their associated virophages and transpoviron, the Pithoviruses and the Cedratviruses (of which we report a new strain). Here we show that DNA methylation is widespread in giant viruses although unevenly distributed. We then identified the corresponding viral MTases, all of which are of bacterial origins and subject to intricate gene transfers between bacteria, viruses and their eukaryotic host. If some viral MTases undergo pseudogenization, most are conserved, functional and under purifying selection, suggesting that they increase the viruses’ fitness. While the Marseilleviridae, Pithoviruses and Cedratviruses DNA MTases catalyze N6-methyl-adenine modifications, some MTases of Molliviruses and Pandoraviruses unexpectedly catalyze the formation of N4-methyl-cytosine modifications. In Marseilleviridae, encoded MTases are paired with cognate restriction endonucleases (REases) forming complete R-M systems. Our data suggest that giant viruses MTases could be involved in different kind of virus-virus interactions during coinfections.
PSF broadening due to fluorescence emission
Authors: Becker Jan , Heintzmann Rainer
Published: December 2019

Fluorescent structures are nowadays commonly used in the field of biological imaging. However the emission of fluorescence is always given within a finite spectrum. When the imaging system’s point spread function is measured, this can be described as an incoherent summation of individual single-wavelength PSF’s, weighted by the emission spectrum. As the full-width-half-maximum of the PSF scales inversely with the wavelength and most emission spectra exhibit a fluorescence tail at longer wave-lengths, this contributes significantly to lateral broadening. In our work we theoretically quantify this effect by deriving an analytic expression, which has been verified against some numerical simulations (rel. error on average 3 %). We report a FWHM-broadening on the order of 10 nm and additionally propose a way to overcome this broadening by splitting the emission spectrum into multiple wavelength segments. The corresponding image data is recombined in Fourier space by weighted averaging, leading to an improved signal-to-noise ratio at high spatial frequencies and a reduction of the FWHM of up to 8 nm (relative reduction of the broadening by ≈ 70 %). We also introduce acorrectedwavelength, which in combination with already existing PSF calculation tools, describes a theoretical PSF which incorporates the aforementioned broadening effect.
Cross-species analysis of melanoma enhancer logic using deep learning
Authors: Minnoye Liesbeth , Taskiran Ibrahim Ihsan , Mauduit David , Fazio Maurizio , Van Aerschot Linde , Hulsemans Gert , Christiaens Valerie , Makhzami Samira , Seltenhammer Monika , Karras Panagiotis , Primot Aline , Cadieu Edouard , van Rooijen Ellen , Marine Jean-Christophe , Maskos Giorgia Egidy , Ghanem Ghanem-Elias , Zon Leonard , Wouters Jasper , Aerts Stein
Published: December 2019

Genomic enhancers form the central nodes of gene regulatory networks by harbouring combinations of transcription factor binding sites. Deciphering the combinatorial code by which these binding sites are assembled within enhancers is indispensable to understand their regulatory involvement in establishing a cell’s phenotype, especially within biological systems with dysregulated gene regulatory networks, such as melanoma. In order to unravel the enhancer logic of the two most common melanoma cell states, namely the melanocytic and mesenchymal-like state, we combined comparative epigenomics with machine learning. By profiling chromatin accessibility using ATAC-seq on a cohort of 27 melanoma cell lines across six different species, we demonstrate the conservation of the two main melanoma states and their underlying master regulators. To perform an in-depth analysis of the enhancer architecture, we trained a deep neural network, called DeepMEL, to classify melanoma enhancers not only in the human genome, but also in other species. DeepMEL revealed the presence, organisation and positional specificity of important transcription factor binding sites. Together, this extensive analysis of the melanoma enhancer code allowed us to propose the concept of a core regulatory complex binding to melanocytic enhancers, consisting of SOX10, TFAP2A, MITF and RUNX, and to disentangle their individual roles in regulating enhancer accessibility and activity.
Learning immune cell differentiation
Authors: Maslova Alexandra , Ramirez Ricardo N. , Ma Ke , Schmutz Hugo , Wang Chendi , Fox Curtis , Ng Bernard , Benoist Christophe , Mostafavi Sara ,   the Immunological Genome Project
Published: December 2019

The mammalian genome contains several million cis-regulatory elements, whose differential activity marked by open chromatin determines organogenesis and differentiation. This activity is itself embedded in the DNA sequence, decoded by sequence-specific transcription factors. Leveraging a granular ATAC-seq atlas of chromatin activity across 81 immune cell-types we show that a convolutional neural network (“AI-TAC”) can learn to infer cell-type-specific chromatin activity solely from the DNA sequence. AI-TAC does so by rediscovering, with astonishing precision, binding motifs for known regulators, and some unknown ones, mapping them with high concordance to positions validated by ChIP-seq data. AI-TAC also uncovers combinatorial influences, establishing a hierarchy of transcription factors (TFs) and their interactions involved in immunocyte specification, with intriguingly different strategies between lineages. Mouse-trained AI-TAC can parse human DNA, revealing a strikingly similar ranking of influential TFs. Thus, Deep Learning can reveal the regulatory syntax that drives the full differentiative complexity of the immune system.
NADPH diaphorase neuronal dystrophy in gracile nucleus, cuneatus nucleus and spinal trigeminal nucleus in aged rat
Authors: Hou Wei , Jia Yunge , Li Yinhua , Wei Zichun , Wen Xiaoxin , Rao Chenxu , Xu Ximeng , Li Fuhong , Wu Xianhui , Sun Haoran , Li Hang , Huang Yizhe , Sun Jingjing , Shu Gege , Wang Xinghang , Zhang Tianyi , Shi Geming , Guo Anchen , Xu Shengfei , Du Guanghui , Tan Huibing
Published: December 2019

NADPH-diaphorase (N-d) activity is commonly used to identify NOS-ergic neurons. In our previous study, N-d positive neuritic dystrophy and spheroid termed aging-related N-d Body is discovered in the lumbosacral spinal cord in the normal aging rats. Histological studies also reveal that N-d positive neurodegenerative changes occur in the gracile nucleus. We re-examined N-d activity in gracile nucleus in aged rat. We found N-d positive neuritic dystrophy and spheroid also occurred in the cuneatus nucleus and spinal trigeminal nucleus. Besides regular coronal section, longitudinal oriented dystrophic neurites were detected in the sagittal and horizontal section in gracile nucleus and dorsal column. We fziurther examined the medullary oblongata with regular classical histology including Golgi staining, immunocytochemistry of NOS and phosphorylated tau protein, neuronal tracing method with wheat germ agglutinin conjugated alexa-fluor-488 through sciatic nerve, and spinal cord transection at thoracic level. Most of N-d positive neuritic dystrophy and spheroid did not showed colocalization with NOS or phosphorylated tau protein. Neuronal tracing and spinal cord transection revealed that N-d dystrophic neurites in gracile nucleus originated from terminal of sensory projection from spinal cord and peripheral somatic input. The results suggested that aging-related N-d dystrophy in the gracile nucleus was unique morphological feature. In conclusion, it was postulated that the N-d dystrophy as a morphological marker of aging degenerative damage in normal aged organisms.
Pattern regulation in a regenerating jellyfish
Authors: Sinigaglia Chiara , Peron Sophie , Eichelbrenner Jeanne , Chevalier Sandra , Steger Julia , Barreau Carine , Houliston Evelyn , Leclère Lucas
Published: December 2019

Clytia hemisphaericajellyfish, with their tetraradial symmetry, offer a novel paradigm for addressing patterning mechanisms during regeneration. Here we show that an interplay between mechanical forces, cell migration and proliferation allows jellyfish fragments to regain shape and functionality rapidly, notably by efficient restoration of the central feeding organ (manubrium). Fragmentation first triggers actomyosin-powered remodeling that restores body umbrella shape, causing radial smooth muscle fibers to converge around “hubs” which serve as positional landmarks. Stabilization of these hubs, and associated expression ofWnt6, depends on the configuration of the adjoining muscle fiber “spokes”. Stabilized hubs presage the site of the manubrium blastema, whose growth is Wnt/β-catenin dependent and fueled by both cell proliferation and long-range cell recruitment. Manubrium morphogenesis is modulated by its connections with the gastrovascular canal system. We conclude that body patterning in regenerating jellyfish emerges mainly from local interactions, triggered and directed by the remodeling process.
Modeling acute kidney injury in kidney organoids with cisplatin
Authors: Digby Jenny L. M. , Przepiorski Aneta , Davidson Alan J. , Sander Veronika
Published: December 2019

Acute kidney injury (AKI) remains a major global healthcare problem and there is a need to develop human-based models to study AKIin vitro. Towards this goal, we have characterized induced pluripotent stem cell-derived human kidney organoids and their response to cisplatin, a chemotherapeutic drug that induces AKI and preferentially damages the proximal tubule. We found that a single treatment with 50 µM cisplatin inducesHAVCR1andCXCL8expression, DNA damage (γH2AX) and cell death in the organoids in a dose-dependent manner but greatly impairs organoid viability. DNA damage was not specific to the proximal tubule but also affected the distal tubule and interstitial populations. This lack of specificity correlated with low expression of the proximal tubule-specificSLC22A2/OCT2transporter for cisplatin. To improve viability, we developed a repeated low-dose regimen of 4x 5 µM cisplatin over 7 days and found this causing less toxicity while still inducing a robust AKI response that included secretion of known AKI biomarkers and inflammatory cytokines. This work validates the use of human kidney organoids to model aspects of AKIin vitro, with the potential to identify new AKI biomarkers and develop better therapies.
Skip segment Hirschsprung disease: modelling the trans-mesenteric origin of the enteric nervous system in the human colon
Authors: Newgreen Donald F , Osborne James M , Zhang Dongcheng
Published: December 2019

Skip segment Hirschsprung disease is a difficult to explain human enteric neuropathy where a ganglionated region lies within a region of total colonic aganglionosis.. Recently, trans-mesenteric migration was described in the mouse intestine whereby neural crest cells migrate via the mesentery across a U-shape gut loop from the midgut to the hindgut: this could explain skip segment Hirschsprung disease. To investigate this, human intestinal growth parameters were derived from published sources and correlated with enteric neural crest cell migration. These processes were then simulated using agent based mathematical models scaled to human intestinal growth. A Hirschsprung-associated slowing of migration was imposed and trans-mesenteric migration was allowed. From the developmental anatomy we conclude that trans-mesenteric migration is unlikely in normal human embryogenesis, but with a Hirschsprung-associated slowing of enteric neural crest cell migration it could occur at Carnegie stages 17 and 18. By varying the division rate of enteric neural crest agents we could reproduce full colonisation, short segment, long segment and skip segment Hirschsprung and hypoganglionic segments.

Summary Statement

Skip segment Hirschsprung disease in humans challenges current explanations. Mathematical modelling shows how this birth defect could develop.
Mutualistic cross-feeding in microbial systems generates bistability via an Allee effect
Authors: Vet Stefan , Gelens Lendert , Gonze Didier
Published: December 2019

Mutualistic interactions are characterized by the positive influence that two species exert on each other. Such mutualism can lead to bistability. Depending on the initial population size species will either survive or go extinct. Various phenomenological models have been suggested to describe bistability in mutualistic systems. However, these models do not account for interaction mediators such as nutrients. In contrast, nutrient-explicit models do not provide an intuitive understanding of what causes bistability. Here, we reduce a theoretical nutrient-explicit model of two mutualistic cross-feeders in a chemostat, uncovering an explicit relation to a growth model with an Allee effect. We show that the dilution rate in the chemostat leads to bistability by turning a weak Allee effect into a strong Allee effect. This happens as long as there is more production than consumption of cross-fed nutrients. Thanks to the explicit relationship of the reduced model with the underlying experimental parameters, these results allow to predict the biological conditions that sustain or prevent the survival of mutualistic species.