1,721,028 research outputs found
Vannella pentlandii n. sp., (Amoebozoa, Discosea, Vannellida) a small, cyst-forming soil amoeba
Get PDFWe describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii, instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 μm in diameter, are the smallest so far described in genus Vannella. Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others.</p
Acanthamoeba programmed cell death
Get PDFAcanthamoeba is a free-living amoeba, ubiquitously distributed in the natural
environment including soil and a plethora of water habitats. It is characterised as an
opportunistic parasite that is able to cause several diseases, including life threating
granulomatous Acanthamoeba encephalitis and a painful vision-threating keratitis.
There is a clear and emerging need to understand how to treat infections caused by
this dangerous pathogen, since pharmaceutical approaches have been considered
insufficient. The presence and the importance of cell death pathways in unicellular
organisms including Acanthamoeba is not yet fully understood and its existence is still
debated. This research study presents a set of key characteristics and findings,
comprising morphological, biochemical and molecular evidence of Acanthamoeba
programmed cell death. Distinctive apoptotic features comprising cell shrinkage,
membrane vesiculation and granules appearance which could be easily described as
apoptotic like ‘bodies’ formation and nuclear shrinkage, accompanied by large scale
chromatin condensation in dense clusters, have been primarily observed.
Additionally, mitochondrial dysfunction, characterized by extended mitochondrial
outer membrane permeabilization and release of apoptotic factors including
cytochrome c, was also noted, indicating a mitochondrially mediated cell death
pathway. During the expansion of the aforementioned apoptotic characteristics
Acanthamoeba trophozoites were found to maintain their membrane integrity and
homeostasis, at least at the early stages of the process. In-depth transcriptomic
analysis based on RNA-sequence analysis revealed a plethora of differentially
expressed genes between Acanthamoeba undergoing cell death and control
trophozoites, indicating the correlation of a more defined and conserved signalling
self-destruct program. These discoveries suggest that Acanthamoeba could undergo
programmed cell death, which morphologically resembles apoptosis-like cell death,
under specific stress conditions. Furthermore, similar characteristics are also found in
cell death processes in higher eukaryotes and other unicellular organisms, indicating
that biological principles behind this behaviour are widespread and well conserved
among species. Identification of Acanthamoeba’s cell death signalling pathways might
provide alternatives not only to microorganism’s refractory infection treatment, but
also a more extended manipulation might become feasible across other species and
systems
Molecular identity of activity-dependent bulk endocytosis
Get PDFAt the neuronal synapse, neurotransmitter-filled synaptic vesicles (SVs) fuse
with the presynaptic plasma membrane during activity. Following exocytosis, SVs
must be retrieved for neurotransmission to be maintained. Several modes of SV
recycling have been identified. During mild neuronal activity, clathrin-mediated
endocytosis has been regarded as the dominant SV retrieval mode, however the
recently identified ultrafast endocytosis mode may also be important in this condition.
During elevated activity, activity-dependent bulk endocytosis (ADBE) is the dominant
SV retrieval pathway. In ADBE, large invaginations are formed from the plasma
membrane, which then undergo scission to create bulk endosomes. In a second distinct
step, SVs bud from these endosomes and specifically repopulate the reserve SV pool.
However, since its first identification, only few molecules have been shown to
participate in ADBE.
The aim of this PhD was to identify novel molecules and elucidate the
molecular mechanism of ADBE. To achieve this, two independent biochemical
approaches were designed to purify and enrich bulk endosomes from primary neuronal
cultures. In the first approach, bulk endosomes and SVs were labelled with a dye,
FM1-43, using a strong stimulus. Cells were broken mechanically and the post nuclear
supernatant, that contains all intracellular organelles, was collected. The supernatant
was then subjected to subcellular fractionation using discontinuous Nycodenz
gradients. This stimulated sample was always processed in parallel with a basal
sample, where no neuronal stimulus was applied, in order to visualise activity
dependent FM loading. After different fractionation protocols were applied, bulk
endosomes were efficiently separated from SVs, as revealed by tracking fluorescence
in different fractions. The fractionation results were further validated by electron
microscopy, where bulk endosomes and SVs were labelled with horseradish
peroxidase and purified using the established protocol. Immunoblotting against
selected SV cargo proteins from stimulated bulk endosome and SV samples, indicated
the specific and preferential localisation of VAMP4 on bulk endosomes, in contrast to
other SV cargo. The molecular identity of bulk endosomes was also approached by
submitting the bulk endosome fractions to semi-quantitative mass spectrometry. This
analysis revealed many different proteins that were identified in bulk endosome
samples and quantification approaches further indicated proteins that can be localised
on bulk endosomes and have a potential role in ADBE.
A second magnetic isolation approach was designed, to purify bulk endosomes
using a completely different methodology. In this case, bulk endosomes were
specifically labelled with iron nanoparticles, which are preferentially taken up by bulk
endosomes since they are larger than SVs. The cells were broken as before and post
nuclear supernatant was acquired. In this case, the supernatant was submitted to
magnetic isolation that separated iron beads labelled structures from all other
intracellular organelles. An extensive immunoblotting analysis of magnetic bulk
endosomes validated that VAMP4 and syndapin I, two essential ADBE proteins, were
enriched in these purified samples. These magnetic bulk endosomes were also
analysed using semi-quantitative MS and revealed many proteins with a potential role
in ADBE. Significant overlap between the two independent methods was observed,
further validating these approaches. Combining these two methods with bioinformatics tools allowed the
identification of the molecular signature of ADBE as well as novel key candidates for
this process. Specific molecules were investigated for their role in ADBE and SV
recycling using a variety of different real-time fluorescent imaging assays. A major
focus was on rab small GTPases. High molecular weight dextran uptake was used to
specifically study the role of these proteins in ADBE, as it preferentially reports uptake
via larger bulk endosomes. A pH sensitive chimeric protein, synaptophysin-pHluorin,
was used to investigate the role of these proteins in CME. Additional imaging assays
were used to answer emerging questions regarding the function and localisation of
these targets in the presynapse. Using these approaches, rab11A and rab35 were found
to promote ADBE and accelerate clathrin-mediated endocytosis. This effect was
specific to high intensity stimulation, while SV exocytosis was not affected.
Further research on the role of both novel and established ADBE molecules
will provide key future insights into the mechanism of both bulk endosome
generation/scission and subsequent SV reformation. A very promising group is rab
proteins and now evidence for their implication in SV recycling is presented here.
Identification and characterisation of new targets will allow to investigate the role of
ADBE in neurotransmission in both physiology and pathophysiology
Characterising the function of a novel embryonic stem cell-associated signal transducer, Gab1β
Get PDFActivation of Ras/mitogen-activated protein kinase (ERK MAPK) signalling
controls the differentiation of mouse embryonic stem (ES) cells. An
established modulator of the ERK MAPK pathway is the IRS-1 (Insulin
Receptor Substrate 1) family adaptor protein Gab1 (Grb2-associated binder
1). Gab1 is ubiquitously expressed and is activated by a wide range of cell
surface receptors, mediating growth factor, cell-cell and cell-substratum
interactions. The N-terminal region of Gab1 contains a pleckstrin homology
(PH) domain required for membrane binding and a nuclear localisation
sequence (NLS) that facilitates nuclear translocation. Undifferentiated mouse
ES cells preferentially express high levels of a novel form of Gab1 (Gab1β)
lacking the N-terminal region. Based on its novel structure and abundance,
Gab1β may act in a dominant negative manner by binding and mislocalising
downstream effectors. Alternatively, it may have a deregulated function
unrestrained by the PH or NLS domains. Data presented here shows that
Gab1β is tyrosine phosphorylated in response to the self-renewal factor
Leukemia Inhibitory Factor (LIF) and/or Foetal Bovine Serum (FBS)
stimulation. This then leads to the formation of complexes with Shp2 and the
p85 subunit of PI3K. Experiments comparing the responses of wild-type and
Gab1β knock-out ES cells indicate that Gab1β enhances ERK and potentially
AKT phosphorylation in response to LIF. In contrast, Gab1β has a negative
effect on ERK and AKT phosphorylation in response to IGF-1 (Insulin Growth Factor 1). These results suggest that the contribution of Gab1β to
signalling activity is receptor specific and may imply that the response of ES
cells to ERK activation is context specific. By reintroducing fluorescently
tagged Gab1 proteins into Gab1β knockout ES cells, I investigated the
localisation of Gab1β in ES cells. Gab1β localised at the cell membrane as
well as in a perinuclear body. I next investigated the potential role of Gab1β
in the differentiation of ES cells into neural precursors. A monolayer
differentiation protocol was used to differentiate Gab1β wild-type and
knock-out cells into neural precursors. Furthermore, the effect of insulin on
the emergence of neural precursors from Gab1β-targeted cells was also
explored
Morphological properties of articular chondrocytes in various experimental and clinical conditions
Get PDFPrevious work has suggested that there exists a relationship between chondrocyte
morphology and matrix metabolism. Changes to chondrocyte morphology have been
reported in human cartilage however it is unclear if these are involved in the
degenerative process associated with osteoarthritis (OA). In this work, the
morphology of human and bovine chondrocytes has been characterised under a range
of conditions. Bovine chondrocytes have been utilised in these experiments as bovine
cartilage is non-degenerate and the chondrocytes have ‘normal’ morphology.
However, if human cartilage have been used instead then there is possibility of
having chondrocytes of mixed shapes i.e. both ‘normal’ and ‘abnormal’ cells. The
thesis aimed at experimentally inducing morphological changes to chondrocytes to
determine whether these changes resemble those observed in human cartilage. The
ultimate aim is to model these changes to clarify the link between morphology and
matrix metabolism by determining how morphological changes influence matrix
metabolism.
A classification system was developed for chondrocyte morphology allowing the
quantification of chondrocyte shapes under different conditions permitting statistical
comparisons. The different conditions utilised were (1) non-degenerate and mildly-degenerate
human articular cartilage and (2) two in vitro models (a) weak 3D agarose
gels to study the effect of gel strength and increasing concentrations of foetal calf
serum (FCS) on morphology of bovine chondrocytes and (b) scalpel induced
mechanically-injured bovine cartilage model to study in situ chondrocyte viability
and morphology at the injured site in various culture conditions. Additionally, the
effect of raised medium osmolarity on the response of chondrocytes to injury was
studied to determine if the abnormal morphology could be reversed.
Using fluorescence-mode confocal laser scanning microscopy (CLSM), chondrocyte
viability, volume and morphology were determined and quantified by using
VolocityTM 3D image analysis software. Histological evaluation of matrix by using
Haematoxylin and eosin, Alcian blue and Masson’s trichrome staining of matrix
produced by chondrocytes cultured in strong or weak agarose gels and in injured
cartilage was determined. Additionally, immunohistochemical evaluation of matrix
(collagen Types I & II) produced by chondrocytes was also performed.
Results demonstrated that in non-degenerate human femoral head cartilage, ~83%
chondrocytes were normal in morphology and 17±2% chondrocytes had cytoplasmic
processes as compared to mildly-degenerate cartilage where 35±5% abnormal
chondrocytes with cytoplasmic processes were present. In non-degenerate cartilage,
11±3% chondrocytes formed small sized clusters however clustering was quite
evident in the superficial zone of mildly-degenerate human femoral head cartilage
where 43±16% chondrocytes had formed large clusters. In mildly-degenerate
cartilage the number of abnormal chondrocytes with processes, length of processes
and number of processes per cell were greater in the superficial as compared to mid
and deep zones.
A model was developed to study the effect of external supporting agarose gel on
chondrocyte morphology and also to determine the influence of FCS. Bovine
chondrocytes cultured in weak gels after 7 days developed similar morphological
changes as those observed in degenerate human cartilage. However, in the strong
gels only few chondrocytes with morphological changes were present i.e. similar to
non-degenerate cartilage. These morphological changes (development of clusters and
processes) occurred more rapidly with increasing concentrations of FCS. Histology
revealed less Alcian blue staining intensity around chondrocytes cultured in weak
gels as compared to strong gels suggesting altered matrix produced by abnormal
chondrocytes. FCS and gel strength were therefore proposed as related factors in
regulating chondrocyte morphology.
In the bovine injured cartilage explant model, after 14 days chondrocytes at the
injury in the presence of FCS or synovial fluid (SF) produced morphological
changes. These changes comprised cell enlargement, flattening, elongation and
production of cytoplasmic processes. In the absence of FCS or SF, chondrocytes at
the injury remained unaffected and were morphologically ‘normal’. Throughout the
cartilage and even in the absence of subchondral bone, chondrocytes displayed
morphological abnormalities in the presence of FCS or SF. These findings suggested
that this is not the property of chondrocytes in the superficial layers alone rather it is
due to the extent of penetration of the ‘factors’ into the matrix and there is no
possibility of interference of injured site with osteocytes or bone factors. Histology
revealed that these abnormal chondrocytes showed less staining with Alcian blue at
the injury suggesting that these morphological changes might play a role in the
changes to matrix metabolism. By raising the osmolarity of the culture medium these
changes were inhibited and chondrocytes maintained their normal morphology.
The results suggest that morphogenic/proliferative factors in FCS or SF and
strength/damage to the matrix may be inter-related and act as potent controllers of
chondrocyte morphology. Raised osmolarity was found to inhibit the morphological
changes suggesting the possibly that hyperosmolarity can antagonise the effects of
these factors. The key conclusions from the thesis were (a) in non-degenerate human
femoral cartilage a large percentage of chondrocytes ~83% were normal in
morphology and the rest were abnormal however in mildly-degenerate cartilage
35±5% abnormal chondrocytes with processes were present (b) the changes to
chondrocyte morphology (development of clusters and processes) were exacerbated
with cartilage degeneration (c) chondrocytes cultured in the weak gels produced
morphological changes as compared to strong gels (d) chondrocytes at the injury
displayed marked morphological changes in the presence of FCS or SF (e) by raising
the medium osmolarity these morphological changes to chondrocytes at the injury
were inhibited. These results show that chondrocyte morphology is complex and
strongly dependent on the environmental settings. Experimental conditions were
therefore identified which showed increased chondrocyte volume, abnormal
morphology with cytoplasmic processes, enhanced proliferation/cluster formation
and matrix changes. These changes to volume and morphology of chondrocytes in
the models studied in this work had certain similarities to the changes observed in
human cartilage suggesting that these shape changes may play a role in the changes
to matrix metabolism occurring in OA.
These findings may be of translational relevance in clinical and experimental
research into cartilage injury and degeneration by providing new insights in
understanding the role played by chondrocyte morphology in cartilage degeneration
and injury
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Iron and copper transporters in amphizoic amoebae "Naegleria fowleri" and "Acanthamoeba castellanii"
No full textAmphizoic amoebae Naegleria fowleri and Acanthamoeba castellanii are distributed worldwide in diverse natural and anthropogenic environments. N. fowleri can infect healthy individuals, causing a rare but deadly brain disease called primary amoebic meningoencephalitis with a mortality rate of over 95%. Infection develops if amebae from contaminated water get into the nose. A. castellanii can infect the central nervous system of immunocompromised patients, causing granulomatous amoebic encephalitis with a survival rate of 2-3%, and the eyes of healthy people, leading to a severe sight-threatening infection called Acanthamoeba keratitis. N. fowleri and A. castellanii are aerobic organisms and require iron and copper cofactors. Both metals are crucial for almost all known organisms and toxic in excess, so the pathogens tightly control the homeostasis of iron and copper, which is critical for their virulence. However, the information about metal homeostasis in amphizoic amoeba is scarce. This work aimed to characterize some of the mechanisms employed by N. fowleri and A. castellanii for iron and copper acquisition and detoxification. Despite the similar morphology and lifestyle of studied amoebae, their mechanisms of iron homeostasis under iron-limiting conditions are entirely different. Both amoebae..
Acanthamoeba and the bacterial pathogen interactions
Get PDFThe present study investigates Acanthamoeba-bacteria interaction and how this relation can
influence human health aiming at the influence of bacteria on Acanthamoeba in terms of their
isolation and diversity, and the effect of Acanthamoeba on bacteria focusing on two emerging
human bacterial pathogens Arcobacter butzleri and Rhodococcus equi. To first objective was
investigated by the test question “can the presence of a particular type of bacteria play role in
the diversity of Acanthamoeba by masking and/or favouring certain genotypes of
Acanthamoeba?”
To answer this, two different bacteria the Gram+ve Enterococcus the Gram-ve Arcobacter
were used as food source for isolation of Acanthamoeba from 102 soil samples while E. coli
was used as control. It was found that the presence of different bacteria could affect the
isolation of genotypes specially the subgroups and subtypes of Acanthamoeba as manifested
by greater diversity of 18S rRNA sequences of Acanthamoeba isolated from environmental
samples on Arcobacter (Arc) and Enterococcus (Ent) than those isolated on E. coli (Eco). The
Eco isolates consisted of only T4>T11=T13 compared to Ent isolates with T4>T16>T13/16
and the Arc isolates which comprised of T4>T2>T2/6=T13>T13/16. The T13/16 were the
intermediate sequence types with no match to any T types. There were also considerable
differences among the T4 subgroups; the Eco isolates consisted of T4-A>T4-B>T4-N>T4-
E>T4-D>T4-C while Ent isolates comprised of T4-A>T4-C=T4-D=T4-E=T4-N>T4-B and
the Arc isolates had only T4-E>T4-A>T4-B>T4-N. In both Eco and Ent isolates 11 subtypes
were recovered with T4-36 being the most abundant, however, in Arc isolates eight subtypes
were recovered with T4-12 as the most abundant. The non-Eco isolates were also different in
their bacterial endosymiotic profile from Eco isolates with Arc isolates having the greatest
proportion of bacterial endosymbionts (15.7%) as compared to 7.8% of Eco and 12.9% of Ent
isolates. Together these results indicate a prominent role of prey bacteria on favouring certain
genotypes and thus compelling consideration for use of different types of bacteria for isolation
of Acanthamoeba to help surface the masked populations as well for more realistic prevalence
that will help in better designing of prevention and control strategies.
The influence of Acanthamoeba on bacteria was investigated for A. butzleri and R. equi both
of which appeared to exploit the former as an environmental reservoir and for modulation of
their pathogenic potential. A. butzleri which are closely related to Campylobacter, appeared to
have a smooth interaction with Acanthamoeba. They were shown to be easily located through
chemotaxis, readily attached and internalized using monosaccharide receptors and a complex
phagocytic process, and could survive/proliferate in Acanthamoeba by defying the intra-vacuolar
killing processes. Intracellular survival in Acanthamoeba did play a role in promoting
the pathogenicity of these bacteria enabling them to survive more than three times longer. Co-culturing
of the two organisms also seemed to benefit the bacteria but not Acanthamoeba. A.
butzleri were found to be able to sense the environmental changes and thus modulate their
virulence, a feature that together with selection pressure for intracellular survival in
Acanthamoeba can cause rapid adaptation to intra-amoebal environment and enhance the
pathogenic potential of these bacteria for humans and animals.
Exploitation of Acanthamoeba for survival was also found to be exhibited by the
Mycobacterium-resembling Gram+ve R. equi by utilizing similar strategies for
survival/proliferation as used for macrophages, which involved the definite presence of
virulence plasmid and its activation at higher temperatures. Moreover, similar genes (vapA,
vapC and vapF) were found to play role in intracellular survival in both the macrophages and
amoeba cells. The intra-amoebal survival/proliferation capabilities of A. butzleri and R. equi
appear to support the notion that free living protists like Acanthamoeba act as environmental
reservoirs/virulence trait selectors and are strong candidates for the “missing link” between
the ecology and pathology of these emerging pathogenic pathogens.
Overall, the observations made in this study explore the vital role of Acanthamoeba-bacteria
interaction not only mutually on each other but as a consequence the impact on human health
either as a result of masked genotypes in clinical diagnosis of Acanthamoeba or due to
environmental reservoir role of Acanthamoeba in selecting virulence traits of bacteria, can
pose serious challenges leaving ample opportunities for more emerging bacterial pathogens.
These observations call for revising the protocols for Acanthamoeba prevalence, eradication
and control strategies
The dance and diabetes project: an evolving model for engagement with underserved communities
Get PDFThe Dance and Diabetes project took place between 2017 and 2022, and utilised dance to engage South Asian women with diabetes research in Leicester. The rate of diabetes in the British South Asian population is about 20% higher than in White British population and manifests itself between 10-15 years earlier (Gholap et al 2011, Goff 2019). Reducing diabetes is a global priority (WHO 2016) and a particular concern for health and research services in Leicester where there is a large South Asian population. As such a project delivering engagement around diabetes was a strategic priority. The team assembled to deliver the project was multidisciplinary and included: Leicester Diabetes Centre (LDC), NIHR Leicester Biomedical Research Centre (BRC), University of Leicester (UoL), University Hospitals of Leicester NHS Trust (UHL), proximal research and NIHR infrastructure, Aakash Odedra Dance Company, Shiamak Midlands, Moving Together and the participating women.
The project was developed over multiple cycles using Sagor’s (2011) model of action research. This entailed, for each stage of the project, clarifying our vision and targets, articulating a theory of action, implementing action and collecting data, and reflecting on the data to plan informed action (Sagor 2011). Whilst every stage featured assessment of impact, in the final cycle focus groups confirmed that participant perspective aligned with our assessment. Transcriptions were analysed using content analysis. Each cycle of action research served to establish credibility and trustworthiness of themes by referring back to existing theory and research, and incorporating new theory and research as it became relevant.
The project rapidly and radically transformed. Early success encouraged the project team to greater aspiration. Ongoing impact assessment associated with the action research cycle indicated that the model had potential to impact health inequalities resulting in a refocus of the project on broader impacts. Situational factors, notably inability to source funds and restrictions associated with the COVID-19 pandemic also had a considerable impact on the direction and implementation of the project.
The project saw participating women empowered to assume responsibility and authority within the project, taking on roles as organisers and influencers, running discussion sessions around areas of interest, conducting formal community consultations themselves as Community Researchers and even running the project in the absence of funding during the summer of 2019.
The different cultures, skills and expertise of project partners established different ways of working, which necessitated consistent renegotiation and communication. Communication supported delivery; it also constructed and reconstructed our understanding of the project and its impacts, and supported the development of cultural competence between project partners.
The project found consistent support for O’Mara Eves et als (2013) recommendations for conduct of community engagement, particularly regards a culturally specific approach and relationship building. Dance, owing to its culturally specific nature and potential to create feelings of cohesion (Lakens and Shel 2011, Hove and Risen 2009, Miles Lind and McCrae 2009), was an effective tool to achieve this. The themes identified also reflected the domains identified by Sung et al (2013) as determinants of effective community engagement (flexibility, a sense of belonging, commitment, communication, being genuine, relevance, sustainability).
The impact assessment for the project supports assertions that high-quality community engagement can impact health inequalities and provides examples of social justice in action
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