bluuebunny/crossref_metadata_2025 huggingface dataset:
Concentration of Chondrogenic Soluble Factors in Freshly Harvested Lipoaspirate
Authors: Ruhl Tim , Stromps Jan-Philipp , Depenau Lisa-Marie Maxi , Kim Bong-Sung , Pallua Norbert
Published: April 2019

Background

Cartilage tissue has a limited capacity for healing with the consequence that patients are often treated symptomatically until they become candidates for osteotomy or total joint replacement. Alternative biological therapies, for example, application of platelet-rich plasma and implantation of chondrocytes and mesenchymal stem cells, have emerged as a new treatment modality to repair articular cartilage. In addition, autologous fat transfer is performed for treatment of cartilage defects, example given, in osteoarthrosis, but several questions regarding basic biochemical properties of the transplant remain unanswered. Bone morphogenetic protein 4 (BMP4), matrix metalloproteinase (MMP)-8, cartilage oligomeric matrix protein (COMP), and chitinase-3-like protein 1 (CHI3L1) have been shown to be involved in chondrogenic regeneration and represent potential therapeutic agents for cartilage repair. However, no study regarding naturally occurring levels of these soluble factors in transplanted adipose tissue has yet been performed.

Methods

To investigate the influence of age, body mass index, donor site, and sex on the concentration of BMP4, MMP-8, COMP, and CHI3L1 in freshly aspirated adipose tissue, their content was measured by means of enzyme-linked immunosorbent assay readings.

Results

There were significant quantities of BMP4, MMP-8, COMP, and CHI3L1 (23.6, 249.9, 298.0, and 540.6 pg/mg, respectively) in the lipoaspirate harvested for transplantation. There was no correlation between the content of soluble factors and the patients' age or body mass index. Furthermore, the sex did not affect the amount of the investigated factors. However, there were significantly lower contents of BMP4, COMP, and CHI3L1 found in lipoaspirates harvested from the abdomen compared with nonabdominal donor sites.

Conclusions

Naturally occurring differences in the concentrations of the investigated soluble factors will favor certain donor sites for autologous fat transfer in the field of cartilage repair. Thus, increasing knowledge will enable researchers and clinicians to make autologous fat transfer procedures more reliable and efficient for treatment of articular cartilage defects.
Buprenorphine Markedly Elevates a Panel of Surrogate Markers in a Murine Model of Sepsis
Authors: Chen Weiqiang , Brenner Max , Aziz Monowar , Chavan Sangeeta S. , Deutschman Clifford S. , Diamond Betty , Pavlov Valentin A. , Sherry Barbara , Wang Ping , Tracey Kevin J. , Wang Haichao
Published: April 2019

Sepsis can be simulated in animals by perforating the cecum via a surgical procedure termed “cecal ligation and puncture” (CLP), which induces similar inflammatory responses as observed during the clinical course of human sepsis. In addition to anesthetic agents, many Institutional Animal Care and Use Committees often recommend the use of additional analgesic agents (such as opioid) to further augment the initial anesthetic effects. However, emerging evidence suggest that a commonly recommended opioid, buprenorphine, dramatically elevated circulating interleukin (IL)-6 levels, and reduced animal survival in male C57BL/6 mice, but not in female mice possibly due to the complex interference of estrous cycles, fueling an ongoing debate regarding the possible impact of analgesic administration on the sepsis-induced systemic inflammation. As per the recommendation of a local government agency, we performed a pilot study and confirmed that repetitive administration of buprenorphine indeed markedly elevated circulating levels of four sepsis surrogate markers (e.g., IL-6, KC, monocyte chemoattractant protein-1, and granulocyte-colony stimulating factor) in 20% to 60% of septic animals. This complication may adversely jeopardize our ability to use the CLP model to reliably simulate human sepsis, and to understand the complex mechanism underlying the pathogenesis of lethal sepsis. Thus, for experimental sepsis studies set to survey systemic inflammation and animal lethality at relatively later stages (e.g., at 24 h post CLP and beyond), we strongly recommend not to repetitively administer buprenorphine to eliminate its potential complication to animal sepsis models.
Remote Ischemic Conditioning in Emergency Medicine—Clinical Frontiers and Research Opportunities
Authors: Ho Andrew Fu Wah , Chong Jun , Ong Marcus Eng Hock , Hausenloy Derek J.
Published: April 2019

Time-critical acute ischemic conditions such as ST-elevation myocardial infarction and acute ischemic stroke are staples in Emergency Medicine practice. While timely reperfusion therapy is a priority, the resultant acute ischemia/reperfusion injury contributes to significant mortality and morbidity. Among therapeutics targeting ischemia/reperfusion injury (IRI), remote ischemic conditioning (RIC) has emerged as the most promising.

RIC, which consists of repetitive inflation and deflation of a pneumatic cuff on a limb, was first demonstrated to have protective effect on IRI through various neural and humoral mechanisms. Its attractiveness stems from its simplicity, low-cost, safety, and efficacy, while at the same time it does not impede reperfusion treatment. There is now good evidence for RIC as an effective adjunct to reperfusion in ST-elevation myocardial infarction patients for improving clinical outcomes. For other applications such as acute ischemic stroke, subarachnoid hemorrhage, traumatic brain injury, cardiac arrest, and spinal injury, there is varying level of evidence.

This review aims to describe the RIC phenomenon, briefly recount its historical development, and appraise the experimental and clinical evidence for RIC in selected emergency conditions. Finally, it describes the practical issues with RIC clinical application and research in Emergency Medicine.
Examining the Increased Rates of Deceased Donor Kidney Nonutilization in Australia: What Has Changed?
Authors: Sypek Matthew P. , Ullah Shahid , Hughes Peter D. , Clayton Philip A. , McDonald Stephen P.
Published: April 2019

Background.

From 2013, Australia has experienced a sustained increase in the proportion of deceased donor kidneys that are retrieved but not utilized for transplantation. We aimed to determine whether this could be explained by changes in donor characteristics over time.

Methods.

Registry data were used to examine predictors of kidney nonutilization over the period 2005–2017. Multilevel mixed effect logistic regression modeling and propensity score analysis were used to determine whether era of donation (2013–2017 versus 2005–2012) was an independent predictor of organ nonutilization after controlling for donor characteristics.

Results.

A total of 7810 kidneys were retrieved for the purpose of transplantation with 334 (4.3%) not utilized. The nonutilization rate was 5.8% in 2013–2017 compared to 2.7% in 2005–2012. Despite adjustment for donor characteristics, donation in the more recent era remained a significant predictor of kidney nonutilization (adjusted odds ratio, 1.98; 95% confidence interval, 1.54-2.54; P < 0.001). This finding was confirmed in the propensity score analysis.

Conclusions.

Kidneys retrieved in Australia since 2013 were more likely not to be utilized for transplantation even after adjusting for changes in donor characteristics. The abrupt increase may be explained by increased clinical risk aversion, changes in unmeasured donor factors or logistical issues. Although nonutilization rates in Australia remain low by international standards, further clinical auditing of the reasons for offer decline may help to optimize resource utilization and maximize transplant opportunities.
Mechanism and modelling of CO
2
corrosion on downhole tools
Authors: Wang Jigang , Meng Lihui , Fan Zhenzhong , Liu Qingwang , Tong Zhineng
Published: April 2019

The conditions surrounding oil and gas exploration are becoming more hazardous, especially in oil and gas fields with a high quantity of corrosive components such as CO
2
. CO
2
causes localized corrosion of tools made from metal, rubber and other materials in humid environments; this leads to corrosion failure in metal equipment and downhole tools such as drills pipes, casings and oil pipes, thereby reducing their service life. In this study, the composition, lattice and crystalline forms of corrosion products and corroded materials were analysed using scanning electron microscopy, energy spectrum analysis, X-ray diffraction and polarization curves, in order to investigate the corrosion mechanisms and influential factors for several common tool materials. A CO
2
corrosion model was established for two materials and the results were verified with optimal prediction values.
Facile synthesis of reduced graphene oxide by modified Hummer's method as anode material for Li-, Na- and K-ion secondary batteries
Authors: Jo Jeonggeun , Lee Seulgi , Gim Jihyeon , Song Jinju , Kim Sungjin , Mathew Vinod , Alfaruqi Muhammad Hilmy , Kim Seokhun , Lim Jinsub , Kim Jaekook
Published: April 2019

Reduced graphene oxide (rGO) sheets were synthesized by a modified Hummer's method without additional reducing procedures, such as chemical and thermal treatment, by appropriate drying of graphite oxide under ambient atmosphere. The use of a moderate drying temperature (250°C) led to mesoporous characteristics with enhanced electrochemical activity, as confirmed by electron microscopy and N
2
adsorption studies. The dimensions of the sheets ranged from nanometres to micrometres and these sheets were entangled with each other. These morphological features of rGO tend to facilitate the movement of guest ions larger than Li
+
. Impressive electrochemical properties were achieved with the rGO electrodes using various charge-transfer ions, such as Li
+
, Na
+
and K
+
, along with high porosity. Notably, the feasibility of this system as the carbonaceous anode material for sodium battery systems is demonstrated. Furthermore, the results also suggest that the high-rate capability of the present rGO electrode can pave the way for improving the full cell characteristics, especially for preventing the potential drop in sodium-ion batteries and potassium-ion batteries, which are expected to replace the lithium-ion battery system
Efficient production of α-keto acids by immobilized
E. coli
-pETduet-1-
Pmi
LAAO in a jacketed packed-bed reactor
Authors: Wu Licheng , Guo Xiaolei , Wu Gaobing , Liu Pengfu , Liu Ziduo
Published: April 2019

α-keto acids are compounds of primary interest for the fine chemical, pharmaceutical and agrochemical sectors.
l
-amino acid oxidases as an efficient tool are used for α-keto acids preparation in this study. Firstly, an
l
-amino acid oxidase (
Pmi
LAAO) from
Proteus mirabilis
was discovered by data mining
.
Secondly, by gene expression vector screening, pETDuet-1-
Pmi
LAAO activity improved by 130%, as compared to the pET20b-
Pmi
LAAO.
Pmi
LAAO production was increased to 9.8 U ml
−1
by optimized expression condition (OD
600
= 0.65, 0.45 mmol l
−1
IPTG, 20 h of induction). Furthermore, The
Pmi
LAAO was stabile in the pH range of 4.0–9.0 and in the temperature range of 10–40°C; the optimal pH and temperature of recombinant
Pmi
LAAO were 6.5 and 37°C, respectively. Afterwards, in order to simplify product separation process,
E. coli
-pETduet-1-
Pmi
LAAO was immobilized in Ca-alginate beads. Continuous production of 2-oxo-3-phenylpropanoic acid was conducted in a packed-bed reactor via immobilized
E. coli
-pETduet-1-
Pmi
LAAO. Significantly, 29.66 g l
−1
2-oxo-3-phenylpropanoic acid with a substrate conversion rate of 99.5% was achieved by correspondingly increasing the residence time (25 h). This method holds the potential to be used for efficiently producing pure α-keto acids.
Effect of fractions of toxic cyanobacterium, Nodularia spumingena KAC 66 and its purified nodularin and nodulopeptin 901 on inhibition of colorimetric protein phosphatase 1 assay and lethality against two species of Daphnia
Authors: Hameed Shaista , Lawton Linda , Edwards Christine
Published: April 2019

The strains of Nodularia are known to produce potent hepatotoxic nodularins (NOD) and other bioactive metabolites i.e. nodulopeptins and spumigins. Contemporary, three new nodulopeptins were also isolated from N. spumigena KAC 66 collected from the Baltic Sea. The wide distribution and toxicological effects of N. spumigena on food chain, has caused significant attention towards the effects of the species. In the present investigation the toxicity of NOD and nodulopeptin 901, produced by N. spumigena, was fractionated by Reversed Phase Flash Chromatography (RPFC) and their toxicity was determined by their lethality to water fleas, Daphnia pulex and D. magna along with inhibition of protein phosphatase 1 assay (PP1; eukaryotic threonine phosphatase/protein serine). All fractions showed lethality to Daphnids and inhibitory activity against PP1, the toxicity was due to additional compounds as NOD and nodulopeptin 901 were only detected in 7 fractions. The pure NOD was lethal to D. pulex and D. magna LC50=8.4 µg. ml-1 and 5.0 µg .ml-1, respectively. The newly characterized nodulopeptin 901 was also tested against D. magna (LC50=>100 µg .ml-1). NOD and nodulopeptin 901 inhibited PP1 with weak IC500.038 μg .ml-1 and 25 µg .ml-1, respectively. The anabaenopeptin A (ANA) and anabaenopeptin B (ANAB) were used as reference peptides as they have similarity in structure with newly characterized nodulopeptin 901. The ANA, ANB and linear nodularin (LNOD) inhibited PP1 with IC5070μg .ml-1,100μg .ml-1 and20μg .ml-1, correspondingly. This is the first study that indicates the toxic and inhibitory activities of nodulopeptin 901 and fractions of N. spumigena KAC 66 against daphnids and PP1.
Sero-epidemiology of scrub typhus among suspected cases in selected areas of Nepal
Authors: Gautam Rajendra , Parajuli Keshab , Sherchand Jeevan Bahadur
Published: April 2019

Introduction

Scrub typhus is an acute, febrile, infectious disease which is caused by Orientia tsutsugamushi. In the current days of Nepal huge burden of scrub typhus have been found, mainly Southern Nepal.

Methodology

Blood samples were collected from the suspected patients of scrub typhus who is having acute febrile illness. Detection of Immunoglobulin M (IgM) antibody to Orientia tsutsugamushi was performed by using Scrub Typhus Detect™ kit, In Bios International USA.

Results

A total 1585 cases, 358 (22.58 %) were positive for IgM Antibodies to Orientia tsutsugamushi. Multivariate analysis demonstrated that the following factors were significantly associated with the scrub typhus. 1. Females (odd ratio [OR]= 2.037, P=< 0.001, confidence interval [CI]= 1.465 – 2.831) 2. Rural residential location (odd ratio [OR]= 0.431, P=0.001, confidence interval [CI]= 0.261 – 0.714), 3. House near grassland (odd ratio [OR]= 3.279, P=<0.001, confidence interval [CI]= 1.932 – 5.563), 4. Presence of mouse inside the house (odd ratio [OR]= 5.462, P=<0.001, confidence interval [CI]= 4.048 – 7.371), 5. Working in the field (odd ratio [OR]= 9.845, P=0.004, confidence interval [CI]= 2.068 – 46.954).

Conclusion

The study indicated that Scub typhus is a big burden of Nepal, where we have identified the prevalence rate was 23  % . Use of IgM ELISA test will help for early diagnosis and it is urgent to investigate to save the life of people who lives endemic areas of Scrub typhus in Nepal.
The HamE scaffold positively regulates MpkB phosphorylation to promote development and secondary metabolism in Aspergillus nidulans
Authors: Frawley Dean , Karahoda Betim , Bayram Ozlem Sarikaya , Bayram Ozgur
Published: April 2019

Mitogen-activated protein kinase (MAPK) pathways are conserved signalling cascades in eukaryotes which regulate a myriad of processes in fungi from sexual reproduction to stress responses. These pathways rely on recruitment of three kinases on a scaffold protein to facilitate efficient kinase phosphorylation and subsequent downstream signalling to the nucleus. The model filamentous fungus Aspergillus nidulans utilises a MAPK pathway termed the pheromone module to regulate both development and secondary metabolism. This complex consists of the MAP3K (SteC), MAP2K (MkkB), MAPK (MpkB) and adaptor protein SteD. To date, there has been no scaffold protein identified for this MAPK pathway. In this study, we characterised a protein termed HamE, which we propose as a scaffold that regulates kinase phosphorylation and signalling in the pheromone module. Mass spectrometry analysis and BIFC experiments revealed that HamE physically interacts with both MkkB and MpkB and transiently interacts with SteC. Deletion of hamE or any of the pheromone module kinases results in reduced sporulation and complete abolishment of cleistothecia production. Mutants also exhibited reductions in expression of secondary metabolite gene clusters, including the velvet complex and sterigmatocystin genes. HamE acts as a positive regulator of MpkB phosphorylation, allowing for HamE to subsequently regulate development and secondary metabolism.
Rabies virus: defining antigenic requirements for pan-lyssavirus neutralisation
Authors: Shipley Rebecca , Selden David , Guanghui Wu , Wright Edward , Fooks Anthony R , Banyard Ashley C
Published: April 2019

The lyssavirus genus is a diverse group of viruses all capable of causing an invariably fatal disease known as rabies, most commonly caused by the prototype species rabies virus (RABV). Alongside RABV the lyssavirus genus currently contains 15 other viruses capable of causing rabies. These viruses are broadly categorised into phylogroups according to the predicted level of vaccine protection, with protection from current vaccines and therapeutics afforded against phylogroup I but not II or III. Current evidence suggests that for a protective neutralising antibody response against RABV a neutralising antibody titre of 0.5 IU ml−1 is sufficient. This arbitrary value has been developed and promoted as a serological cut-off based on the reactivity of defined sera with a standardised dose of RABV. Studies using cross protection assays, have suggested that for protection against more divergent members of the genus, even those in phylogroup I, 10-fold or greater than the 0.5 IU ml−1 antibody titres are required. The continued discovery of novel lyssaviruses globally warrants an in-depth assessment of the protective titre required to protect against all the lyssaviruses to inform occupationally high-risk groups (e.g., scientists, bat workers and speleologists). Based on live virus neutralization assays, a minimum of 7 distinct lyssavirus glycoprotein antigens would have to be included in any pan-lyssavirus vaccine. Certainly, representative immunogens from all lyssavirus species characterized in phylogroups II and III are required to stimulate a pan-lyssavirus response.
The Sodalis system and flux balance analysis as a tool for investigating insect-microbe interactions and the evolution of symbioses
Authors: Hall Rebecca , Flanagan Lindsey , Bottery Michael , Springthorpe Vicki , Thorpe Stephen , Darby Alistair , Wood Jamie , Thomas Gavin
Published: April 2019

The development of new microbial growth and analytical techniques is becoming increasingly relevant in relation to ‘unculturable’ organisms. This may involve the modification of existing methods or the development of new, custom procedures. One important application of this is in the symbiotic bacteria of insects. Symbionts, due to adaptations to their host, are often difficult to culture in vitro. With the growing interest in the use of modified microbiomes to control vector-borne diseases, improved culture techniques that further the understanding of an insect’s microbiome are becoming increasingly important. The tsetse fly, genus Glossina, is the insect vector for Trypanosoma brucei. This parasite is responsible for human African trypanosomiasis (HAT), endemic in sub-Saharan Africa, as well as the wasting disease nagana in cattle. The tsetse’s secondary symbiont, Sodalis glossinidius, provides a unique potential target for reducing the spread of T. brucei. Here, we describe the use of metabolic modelling to design an entirely defined growth medium for S. glossinidius. This medium was used to verify predictions about carbon and nitrogen usage in the symbiont, including amino acid and vitamin auxotrophies. Furthermore, we discuss the use of multiobjective evolutionary algorithms combined with flux balance analysis to investigate computationally the evolution of symbioses, with S. glossinidius and its free-living relative S. praecaptivus as an exemplar. This work not only improves our understanding of the metabolic interactions within the tsetse microbiome, but serves also as a template for future investigations into symbiont evolution.
Prevalence, susceptibility patterns and virulence factors of bacterial isolates from neonate-mother pair samples in Benin City, Nigeria
Authors: Aziegbemhin Samuel
Published: April 2019

Neonatal sepsis remains a major cause of morbidity and mortality in neonates. The prevalence of bacterial isolates in neonates admitted due to sepsis together with mothers and their susceptibility to routinely used antibacterial agents were investigated. Ethical Approval was obtained from the Hospital Management Board while informed consents were obtained from their parents. Forty-five (45) saliva samples from neonates (with signs and symptoms of sepsis admitted at the neonatal unit of Central Hospital, Benin City, Nigeria) were obtained. Vaginal swab samples were also obtained from their mothers to attain a total of ninety samples comprising forty-five saliva-vaginal swab sample pairs. Samples were immediately transported to the Laboratory and processed using standard microbiological protocols. All samples showed significant bacterial growth. At least one similar bacterial isolate were recovered from each neonate-mother pair. Bacteria isolated include Staphylococcus aureus, Klebsiella oxytoca, Klebsiella spp., Escherichia coli, Enterobacter spp., Proteus miriabilis, Acinetobacter sp., Proteus vulgaris, Pseudomonas aeruginosa, Streptococcus agalaticeae, Citrobacter sp. and Streptococcus pneumoniae. Both neonatal and maternal isolates were sensitive to unacin, azithromycin, cefotaxime and cefuroxime. Bacterial isolates also showed varying degrees of resistance to bactericidal action of normal serum. Isolates also produced haemolysin. This study gives important insight to the role of saliva in bacteriological analysis of sepsis and has implications for neonatal survival.
Whole-genome sequence analyses of Fusobacterium necrophorum
Authors: Hoyles Lesley , Brimah Karima , Greenwell Pamela
Published: April 2019

Fusobacterium necrophorum is a strictly anaerobic, non-spore-forming, Gram-negative Bacillus encompassing two subspecies. Fusobacterium necrophorum subsp. necrophorum (FNN) is commonly found in animals, where it is the causative agent of foot rot and abscesses. Fusobacterium necrophorum subsp. fundiliforme (FNF) is found in humans and is the causative agent of Lemierre’s disease, a condition associated with throat infections. Little is currently known about the genomic diversity of the two subspecies. Whole-genome sequences were generated for 18 Fusobacterium necrophorum isolates recovered from recurrent and persistent sort throats in UK patients. Sixty-three whole genome sequences available for Fusobacterium spp. were downloaded from GenBank and compared with the newly sequenced isolates. All-against-all comparisons of average nucleotide identity (ANI) were done to confirm species affiliation. Strains of FNN (n=6) and FNF (n=43) shared ∼97 % ANI with each other, while strains of the same subspecies shared >98 % with one another. All the newly sequenced isolates belonged to FNF. All 49 Fusobacterium necrophorum sensu stricto genomes were subject to comparative and pangenome analyses. The strains fell into two groups, with the majority of strains clustering with the type strain of FNF. Three distinct clusters of strains were identified within the FNF group, and analyses are underway to determine their potential clinical significance. Analyses of virulence-associated leukotoxin genes are also being undertaken. It is envisaged that in-depth analyses of a large collection of Fusobacterium necrophorum genomes, particularly those of FNF, will provide novel insights into pathogenesis of these fastidious anaerobic bacteria.
Optimisation of an in vitro cell system using pseudoviruses to investigate HBV entry mechanisms
Authors: Goddard Chun , Irving William , Tarr Alexander
Published: April 2019

Aim

To explore the roles of large (L), middle (M) and small (S) surface antigens in an in vitro model of hepatitis B virus (HBV) entry, and thereby to achieve an optimal in vitro cell system using pseudoviruses to investigate HBV entry mechanisms.

Methods

RNA encoding sodium taurocholate co-transporting polypeptide (NTCP) was extracted from human hepatocytes and cloned into the pHIV-EGFP expression vector. The resulting pHIV-NTCP-EGFP construct was delivered into Huh7 hepatoma cells with the aid of pCMVR87.4 (packaging vector) and pCMV.VSV.G (glycoprotein), subsequently, a cell line over-expressing NTCP was generated. Meanwhile, by silencing start codons at L, M or S, seven constructs were obtained, i.e. L + M -S-, L-M + S -, l -M-S+, L + M + S-, L-M + S +, L + M -S+, l -M-S-. A matrix with various amounts of the seven constructs was used to generate HBV pseudoparticles (HBVpp) using a Luciferase-based HIV (pNL4.3.luc.R-E-) pseudotype entry model system. The infectivity of the HBVpp was tested in NTCP naïve and NTCP over-expressing Huh7 hepatoma cells.

Results

The relative amounts of L, M and S were critical in determining the efficiency of entry of HBVpp into NTCP + ve Huh7 cells.

Conclusions

Creation of NTCP-over expressing cells together with optimisation of conditions to maximise HBVpp entry provides an important tool to investigate the entry step in the HBV life cycle, and may allow identification of non-NTCP-dependent viral entry pathways.
Differential amplicons for the evaluation of RNA integrity extracted from complex environmental samples
Authors: Cholet Fabien
Published: April 2019

Reliability and reproducibility of transcriptomics-based studies are dependent on RNA integrity. Microfluidics-based techniques targeting rRNA are currently the only approaches to evaluate RNA integrity. However, the relationship between rRNA and mRNA integrity is unknown. Here we present a new integrity index, the Ratio amplicon, Ramp, to monitor mRNA integrity based on the differential amplification of RT-Q-PCR amplicons of the glutamine-synthetase A (glnA) transcript. We showed, in a suite of experimental degradations of RNA extracted from sediment, that while the RIN generally reflected the degradation status of RNA the Ramp mapped mRNA degradation better. Furthermore, we examined the effect of degradation on transcript community structure byamplicon sequencing of the 16S rRNA, amoA and glnA transcripts. We successful sequenced transcripts for all three targets even from highly-degraded RNA samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed for the 16S rRNA transcripts profiles. Since both RT-Q-PCR and sequencing results were obtained, even from highly degraded samples, we strongly recommend evaluating RNA integrity prior to downstream processing to ensure meaningful results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.
Identification of viral transcripts in RNA-seq datasets from bees, ants, wasps and mites
Authors: Brown Katherine , Olendraite Ingrida , Firth Andrew
Published: April 2019

Many honey bee colonies suffer large losses due to colony collapse disorder. This phenomenon, which has dramatically increased in frequency since 2006, has led to widespread efforts in sequencing honey bee pathogens, including RNA viruses such as deformed wing virus. However, honey bees coexist with a number of other arthropods, whose viruses are less thoroughly characterised. Many viruses currently classified as honey bee pathogens may therefore have a much wider host range. In particular, ants, which like bees are members of the Hymenoptera order, often coexist with bees and the two groups have previously been shown to exchange viruses. Parasitism by Varroamites, known to act as effective vectors for a number of RNA viruses, is also almost ubiquitous amongst honey bees, but little is known about viruses endemic to mites. We have previously demonstrated that it is possible to detect and characterise viral RNA in publicly available RNA-seq datasets. There are over 3000 such datasets for diverse Hymenoptera and mite species. We have developed a computational pipeline to identify viral transcripts in these datasets. This pipeline performs quality control, removes low complexity reads and reads generated from host RNA and various known contaminants, assembles the remaining reads into transcripts and detects the presence of regions with homology to known RNA viruses. Viral fragments identified with this pipeline will be examined phylogenetically to identify novel pathogens, clarify host range and specificity, and characterise transmission patterns.
Inflammation associated ethanolamine facilitates infection by Crohn’s disease-linked adherent-invasive Escherichia coli
Authors: Ormsby Michael , Logan Michael , Johnson Síle , McIntosh Anne , Hanson Richard , Ijaz Umar , Russell Richard , Gerasimidis Konstantinos , Wall Donal
Published: April 2019

The predominance of specific bacteria within the Crohn’s disease intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. Here we show that the intestinal short chain fatty acid propionic acid stimulates increased ethanolamine degradation by one such Crohn’s disease associated pathogen, adherent-invasive Escherichia coli (AIEC). This degradation occurs within bacterial microcompartments that are subsequently excreted in outer membrane vesicles. Additionally ethanolamine, added extracellularly at concentrations comparable to those in the human intestine, is accessible to intracellular AIEC and stimulates significant increases in growth within macrophages. Finally, expression of the operon for ethanolamine degradation (eut) is increased in children with active Crohn’s disease compared to healthy controls. After clinical remission was seen with exclusive enteral nutrition treatment, Crohn’s disease patient’s exhibit significantly reduced eutexpression. Our data indicates a role for ethanolamine metabolism in facilitating AIEC colonization of the Crohn’s disease intestine and warrants further study of its potential use as an indicator of inflammatory status in Crohn’s disease.
Blessing in disguise: a protocol to quantify biofouling effects on drinking water electrochemical sensors is a gateway to investigate biofilm using electroanalytival methods
Authors: Raditya Aldyla , O’Hare Danny
Published: April 2019

Provision of clean drinking water is regarded as being the most significant positive intervention in human health and it plays a significant role in supporting global health. Population growth, economic development and climate change all drive urbanisation and increase demand for clean water and the infrastructure for its delivery. As demand for clean water increases, so will the pressure to ensure its safety. Indwelling sensor networks offer real-time, long-term and intelligent monitoring system, and would enable optimisation of networks for quality. Electrochemical sensors are inexpensive and simple to construct and operate. However, prolonged exposure of the sensors to water causes biofouling which compromise their performance even in weeks or days, making early detection of performance failure critical. By using a combination of electroanalytical methods (cyclic voltammetry, chronoamperometry, electrochemical impedance spectroscopy and hydrodynamic voltammetry) we can quantify the mass transport and kinetics effects of the first six days old biofilm layer to electrode reactions. We used agarose hydrogels and cellulose acetate layer as model biofilms to provide rapid and simplified characterisation of early fouling effects which then compared with lab grown biofilm of Pseudomonas fluorescens. We present a protocol forin situdiagnosis of electrochemical sensors fouling during early stages of biofouling. We also demonstrate that the behaviour of lab grown biofilm can be approached by using a simple model made of hydrogels (i.e. agarose) or a dried layer of an organic solution (i.e. cellulose acetate) which provides a potential application in other fields beyond environmental sensors development such as infectious disease and novel antibiotic researches.
Use of nanosensor technology to investigate biofilm formation and resulting malodour in washing machines
Authors: Blunk Birte , Perkins Mark , Walsh Dean , Chauhan Veeren , Camara Miguel , Williams Paul , Aylott Jonathan , Hardie Kim
Published: April 2019

Biofilms are communities of microorganisms that attach to various surfaces and are widely associated with infection for animals and plants. Our investigation is focussed on a current and growing concern: the distribution and formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures around 30 to 40 °C degrees rather than at higher temperatures above 60 °C that would kill the bacteria. Survival of the bacteria is associated with biofouling, malodour and an increased infection risk due to the distribution of human pathogens such as Pseudomonas aeruginosa into the environment. P. aeruginosa is one of the predominant bacteria found in washing machines and is highly resistant to many antibiotics. Little is known about environmental microniches present in biofilms. In this work, we focus on the pH variation throughout P. aeruginosa biofilms knowing that the pH can influence biofilm formation and could be an important aspect for the prevention of biofilm formation. Here, we use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to variation in pH. Confocal laser scanning microscopy revealed that the nanosensors can penetrate biofilms within minutes and interact with the biofilm structure. Different washing detergents were tested resulting in altered biofilm formation and killing abilities. Using time lapse imaging, pH changes were tracked in real time at a microcolony and single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms, underpinning the development of more effective antimicrobials to limit the emergence of AMR.
Bactericidal fully-human monoclonal antibodies can be cloned from patients convalescing from invasive meningococcal disease
Authors: Bidmos Fadil , Nadel Simon , Screaton Gavin , Kroll Simon , Langford Paul
Published: April 2019

Reverse vaccinology 2.0 (RV 2.0), in which the cloning and recombinant expression of antigen-specific antibodies is followed by determination of their functional activity, is a valuable approach that can unravel novel vaccine antigens, or reinforce the vaccine candidacy of already known antigens. Invasive meningococcal disease (IMD) remains a serious source of concern even with the availability of vaccines. Incomplete strain coverage is a limitation of current vaccines, hence efforts to identify candidate antigens that will compose supplementary or replacement vaccines are necessitated. In this proof-of-principle study, we sought to assess the applicability of RV 2.0 to anti-meningococcal vaccine antigen discovery. Antibody-secreting cells (ASCs) obtained from a patient convalescing from serogroup B (MenB) IMD, were isolated and sorted singly using FACS. The specificity and functionality of each antibody produced by individual ASCs were assessed in ELISA and bactericidal assays, respectively. Eight cross-reactive anti-meningococcal antibodies were successfully cloned; three of these mediated complement-dependent killing of antigenically-heterologous MenB strains. Western blot data shows binding of these three bactericidal antibodies to a ∼35 kDa antigen. None of the three bactericidal antibodies were reactive with a target in current vaccine formulations strongly suggesting that the ∼35 kDa antigen does not compose available vaccines. Unequivocal determination of the identity of the ∼35 kDa antigen is ongoing. Given the need for antigens that would compose improved or novel anti-meningococcal vaccines, this study shows that the RV 2.0 approach has the potential to be a powerful tool in the identification of functionally-immunogenic anti-meningococcal antigens.
The unorthodox chromosomal organisation of the dinoflagellates
Authors: Hu I Ian , Waller Ross
Published: April 2019

Dinoflagellates nuclei are unlike any other. They have: 1) highly inflated genome sizes, 2) practically lack histones and nucleosomal organization; 3) have permanently condensed liquid crystalline chromosomes throughout the life cycle, and; 4) contain a novel a major new nuclear DNA-binding protein. This protein, called Dinoflagellate/Viral NucleoProtein (DVNP) is small in size (10–20 kDa), highly positively charged (30–40 % R+K), and its gene is one of the most highly transcribed in dinoflagellate cells. It has no homology to histone proteins, has no homologues in either eukaryotes or prokaryotes, but is found in a number of marine large DNA viruses. To understand the role of DVNP in the dinoflagellate nuclei we have expressed and purified DVNP and are studying the properties of this novel protein and its interaction with DNA. We show that DVNP is a monomer in solution, but upon exposure to DNA it rapidly binds to and compacts DNA into complexes micrometers in size. Using single-molecule imaging and optical tweezers, DVNP is seen to compact DNA a rates of over 50 µm/sec and change the mechanical properties of DNA. Most interestingly, the DVNP/DNA aggregates show a propensity to travel along the DNA strand en masse. Together, these observations suggest that DVNP plays a central role in the novel model for chromatin management found in dinoflagellates.
The modulation of autophagy by African swine fever virus
Authors: Shimmon Gareth , Dixon Linda , Wileman Tom , Netherton Chris
Published: April 2019

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of domestic pigs with mortality rates of up to 100 %. An outbreak in Russia in 2007 has since spread into Europe. There is currently no vaccine available, however infection with attenuated strains of ASFV can protect against infection with closely related virulent strains. Autophagy is a conserved, essential cell process that regulates multiple pathways that are critical for mounting an effective immune response. Experiments have shown that inhibiting the ability of viruses to regulate autophagy can lead to enhanced immune responses. We have shown that ASFV does not require autophagy for replication and that autophagosome formation is inhibited during infection. In addition, through analysis of key proteins in the upstream autophagy pathway, we describe a novel mechanism of ASFV inhibition of autophagy. This research will expand our understanding of the interaction between ASFV and the autophagy pathway with the potential that a low virulent ASFV strain with an altered ability to modulate autophagy will provide enhanced immunity against virulent isolates.
Transformation of the dinoflagellate chloroplast
Authors: Howe Christopher , Nisbet Ellen , Barbrook Adrian , Nimmo Isabel
Published: April 2019

Dinoflagellate algae are ecologically and environmentally important, as symbionts of corals and many other aquatic organisms, and the causative agents of red tides. However, attempts over the last twenty years to establish genetic manipulation systems for dinoflagellates have met with little success. We have exploited the unusual chloroplast genome of dinoflagellates to establish a system for transformation of this organelle. The chloroplast genome of peridinin-containing (the ancestral state) dinoflagellates is highly reduced and composed of a number of small, plasmid-like molecules, referred to as ‘minicircles’. We have constructed shuttle vectors that are fusions of minicircles and Escherichia coli plasmids and carry selectable markers. We used biolistic transformation to introduce these into the model dinoflagellate Amphidinium carterae. We found that the plasmids confer the expected phenotype on the dinoflagellate cells, and we can detect the plasmid DNA and associated transcripts following selection, indicating successful transformation. This opens up the possibility of studying many aspects of dinoflagellate chloroplast biology, including the maintenance and expression of the minicircles, and the role of the chloroplast in phenomena such as coral bleaching.
Coupling of subunit availability to activation of PMF-driven flagellar type III secretion
Authors: Bryant Owain , Chung Betty , Fraser Gillian
Published: April 2019

Bacterial flagella are assembled from thousands of protein subunits that are unfolded and exported via a specialized type III secretion system. Subunit export is fuelled by the proton motive force (PMF) facilitated by a cytoplasmic ATPase complex comprising FliH, FliI and FliJ, which are evolutionarily related to components of the F1 ATPase. The FliJ stalk component of the ATPase binds the export gate protein FlhA, allowing it to utilise ΔΨ to drive highly efficient subunit export. What is unclear is how FliJ activation of FlhA is regulated to prevent constitutive proton influx when there are no subunits available. FliJ-mediated export gate activation could be regulated by other proteins that bind FliJ. We have shown that FliJ recruits unladen export chaperones, transferring them to their cognate subunits to create a local cycle of chaperone-subunit binding. To investigate whether chaperones also regulate FliJ activation of FlhA, we sought to isolate chaperone variants that were defective in FliJ binding but retained their ability to bind subunits and other export components. Disruption of chaperone-FliJ binding attenuated motility and cognate subunit export. To test whether chaperones blocked the FlhA-FliJ interaction, we developed in vitro and in vivo competition assays. Our data showed that chaperones and FlhA compete for a common binding site on FliJ, and that unladen chaperones, which would be present in the cell when subunit levels are low, disrupt the FliJ-FlhA interaction, preventing activation of the export gate. This provides a mechanism whereby the export gate is only activated when subunits are available.
Engineered extremes: microbial interactions with steel and bentonite in a geological disposal facility
Authors: Barnett Megan , Green Kay , Kilpatrick Andy , Field Lorraine , Kemp Simon , Gregory Simon
Published: April 2019

The conditions of the deep subsurface, combined by perturbations caused by geological disposal of radioactive waste create multiple extreme conditions (limited space availability, high pH, high temperature etc.) for microbial communities. Microbial activity has the potential to cause corrosion of steel and alteration of bentonite clays used in geological disposal facilities. To understand the limits on microbial growth, and the potential for microbial activity to affect the swelling behaviour of the bentonite and metal corrosion, a suite of laboratory experiments is being conducted. In situ repository conditions have been replicated in the MIND (Horizon2020) project. Preliminary results show evidence of corrosion in all experiments, an increase in the basal spacings of smectites in the zone immediately surrounding the steel and inoculated samples had evidence of calcite crystal formation, accompanied by differences in the iron phases. These experiments simulate the in situ conditions well, but the complex nature of this experimental design (high pressure and flow) reduces the practicality of varying the environmental conditions. To complement these investigations, a low-tech solution has been implemented with unpressurised, hydrated bentonite batch experiments. The simpler nature of this set-up allows for investigation of more parameters. Microcosms with artificial groundwater used in the MIND set-up are being compared to the MIND groundwater composition, modelled to represent permafrost conditions. The effect of incubation temperature is also being investigated. Combined, these experiments will help to understand the influence of microbes under the extremes of geological disposal facilities and how their behaviour may change by external parameters.
Development of serologic tools for diagnosis and surveillance of Chikungunya
Authors: Fonseca Bagno Flávia , Marta Figueiredo Maria , Carvalho Godoi Lara , Guimarães da Fonseca Flávio
Published: April 2019

Chikungunya (CHIKV), an arbovirus that belongs to the Alphavirus genus of the Togaviridae family, causes a disease characterized by acute onset of fever accompanied by arthralgia. CHIKV also has been associated with cases of meningoencephalitis (primarily in neonates), Guillain–Barré syndrome and hemorrhagic disease. The clinical similarities, cross-reactivity and cocirculation of arboviruses in Brazil have complicated their differentiation, highlighting the need for new diagnostic tools. A serologic test can be useful for acute detections as well as for surveillance and epidemiological studies. Point of care tests currently available to detect CHIKV infections have been associated with low accuracy. The aim of this work was to design a synthetic gene and generate a recombinant protein to be used as antigens in diagnostic assays. Computational methods were used to predict its structure and antigenic potential. To confirm predictions, the gene coding for the recombinant CHIKV protein (rCHIKV) was synthetized and the protein was purified by affinity chromatography. Antigenicity of the protein was initially confirmed by western-blot using sera from CHIKV infected mice. Additionally, the seroreactivity of r-CHIKV protein was evaluated using a panel of sera samples from human patients, CHIKV seropositive or not, by indirect IgG ELISA. The r-CHIKV protein showed sensitivity of 95 % and specificity of 96 %. No cross-reactivity was found against sera of Zika and Dengue positive patients. These results indicate that this proteins maybe useful antigen to detect CHIKV infections in ELISA assays. Our next step includes the development of a rapid test kit.