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Abstract Background The rumen fluke, Calicophoron daubneyi, is the major paramphistome species infecting ruminants within Europe. Adult flukes reside within the rumen where they are in direct contact with a unique collection of microorganisms. Here, we report a 1.76-Gb draft genome for C. daubneyi, the first for any paramphistome species. Results Several gene families have undergone specific expansion in C. daubneyi, including the peptidoglycan-recognition proteins (PGRPs) and DM9 domain-containing proteins, which function as pattern-recognition receptors, as well as the saposin-like proteins with putative antibacterial properties, and are upregulated upon arrival of the fluke in the microbe-rich rumen. We describe the first characterisation of a helminth PGRP and show that a recombinant C. daubneyi PGRP binds to the surface of bacteria, including obligate anaerobes from the rumen, via specific interaction with cell wall peptidoglycan. We reveal that C. daubneyi eggshell proteins lack L-DOPA typically required for eggshell crosslinking in trematodes and propose that C. daubneyi employs atypical eggshell crosslinking chemistry that produces eggs with greater stability. Finally, although extracellular digestion of rumen ciliates occurs within the C. daubneyi gut, unique ultrastructural and biochemical adaptations of the gastrodermal cells suggest that adult flukes also acquire nutrients via uptake of volatile fatty acids from rumen fluid. Conclusions Our findings suggest that unique selective pressures, associated with inhabiting a host environment so rich in microbial diversity, have driven the evolution of molecular and morphological adaptations that enable C. daubneyi to defend itself against microorganisms, feed and reproduce within the rumen.

The most common equine tapeworm, Anoplocephala perfoliata, has often been neglected amongst molecular investigations and has been faced with limited treatment options. However, the recent release of a transcriptome dataset has now provided opportunities for in-depth analysis of A. perfoliata protein expression. Here, global, and sub-proteomic approaches were utilized to provide a comprehensive characterization of the A. perfoliata soluble glutathione transferases (GST) (ApGST). Utilizing both bioinformatics and gel-based proteomics, GeLC and 2D-SDS PAGE, the A. perfoliata ‘GST-ome’ was observed to be dominated with Mu class GST representatives. In addition, both Sigma and Omega class GSTs were identified, albeit to a lesser extent and absent from affinity chromatography approaches. Moreover, 51 ApGSTs were localized across somatic (47 GSTs), extracellular vesicles (EVs) (Whole: 1 GST, Surface: 2 GSTs) and EV depleted excretory secretory product (ESP) (9 GSTs) proteomes. In related helminths, GSTs have shown promise as novel anthelmintic or vaccine targets for improved helminth control. Thus, provides potential targets for understanding A. perfoliata novel infection mechanisms, host–parasite relationships and anthelmintic treatments.

Abstract Background Over the past decade, evidence has emerged of the ability of gastrointestinal (GI) helminth parasites to alter the composition of the host gut microbiome; however, the mechanism(s) underpinning such interactions remain unclear. In the current study, we (i) undertake proteomic analyses of the excretory-secretory products (ESPs), including secreted extracellular vesicles (EVs), of the ‘brown stomach worm’ Teladorsagia circumcincta, one of the major agents causing parasite gastroenteritis in temperate areas worldwide; (ii) conduct bioinformatic analyses to identify and characterise antimicrobial peptides (AMPs) with putative antimicrobial activity; and (iii) assess the bactericidal and/or bacteriostatic properties of T. circumcincta EVs, and whole and EV-depleted ESPs, using bacterial growth inhibition assays. Methods Size-exclusion chromatography was applied to the isolation of EVs from whole T. circumcincta ESPs, followed by EV characterisation via nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of EVs and EV-depleted ESPs was conducted using liquid chromatography-tandem mass spectrometry, and prediction of putative AMPs was performed using available online tools. The antimicrobial activities of T. circumcincta EVs and of whole and EV-depleted ESPs against Escherichia coli were evaluated using bacterial growth inhibition assays. Results Several molecules with putative antimicrobial activity were identified in both EVs and EV-depleted ESPs from adult T. circumcincta. Whilst exposure of E. coli to whole ESPs resulted in a significant reduction of colony-forming units over 3 h, bacterial growth was not reduced following exposure to worm EVs or EV-depleted ESPs. Conclusions Our data points towards a bactericidal and/or bacteriostatic function of T. circumcincta ESPs, likely mediated by molecules with antimicrobial activity. Graphical Abstract

Long non-coding (lnc)RNAs are a class of eukaryotic RNA that do not code for protein and are linked with transcriptional regulation, amongst a myriad of other functions. Using a custom in silico pipeline we have identified 6,436 putative lncRNA transcripts in the liver fluke parasite, Fasciola hepatica, none of which are conserved with those previously described from Schistosoma mansoni. F. hepatica lncRNAs were distinct from F. hepatica mRNAs in transcript length, coding probability, exon/intron composition, expression patterns, and genome distribution. RNA-Seq and digital droplet PCR measurements demonstrated developmentally regulated expression of lncRNAs between intra-mammalian life stages; a similar proportion of lncRNAs (14.2%) and mRNAs (12.8%) were differentially expressed (p

Abstract Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross‐species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well‐recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth‐derived EVs in these processes and highlights EVs as an important participant in cross‐phylum communication. While the mammalian EV field is guided by a community‐agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan‐helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth‐derived EVs to complement the MISEV guidelines. We summarise community‐agreed standards for studying EVs derived from this broad set of non‐model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.

Parasite derived extracellular vesicles (EVs) have been proposed to play key roles in the establishment and maintenance of infection. Calicophoron daubneyi is a newly emerging parasite of livestock with many aspects of its underpinning biology yet to be resolved. This research is the first in-depth investigation of EVs released by adult C. daubneyi. EVs were successfully isolated using both differential centrifugation and size exclusion chromatography (SEC), and morphologically characterized though transmission electron microscopy (TEM). EV protein components were characterized using a GeLC approach allowing the elucidation of comprehensive proteomic profiles for both their soluble protein cargo and surface membrane bound proteins yielding a total of 378 soluble proteins identified. Notably, EVs contained Sigma-class GST and cathepsin L and B proteases, which have previously been described in immune modulation and successful establishment of parasitic flatworm infections. SEC purified C. daubneyi EVs were observed to modulate rumen bacterial populations by likely increasing microbial species diversity via antimicrobial activity. This data indicates EVs released from adult C. daubneyi have a role in establishment within the rumen through the regulation of microbial populations offering new routes to control rumen fluke infection and to develop molecular strategies to improve rumen efficiency.

Abstract Background Diseases caused by parasitic flatworms of rumen tissues (paramphistomosis) are a significant threat to global food security as a cause of morbidity and mortality in ruminant livestock in subtropical and tropical climates. Calicophoron daubneyi is currently the only paramphistome species commonly infecting ruminant livestock in temperate European climates. However, recorded incidences of C. daubneyi infection in European livestock have been increasing over the last decade. Whilst clinical paramphistomosis caused by adult worms has not been confirmed in Europe, fatalities have been attributed to severe haemorrhagic enteritis of the small intestine resulting from the migration of immature paramphistomes. Large numbers of mature adults can reside in the rumen, yet to date, the impact on rumen fermentation, and consequently on productivity and economic management of infected livestock, have not been resolved. Limited publicly available nucleotide and protein sequences for C. daubneyi underpin this lack of biological and economic understanding. Here we present for the first time a de novo assembled transcriptome, with functional annotations, for adult C. daubneyi, which provides a reference database for protein and nucleotide sequence identification to facilitate fundamental biology, anthelmintic, vaccine and diagnostics discoveries. Results This dataset identifies a number of genes potentially unique to C. daubneyi and, by comparison to an existing transcriptome for the related Paramphistomum cervi, identifies novel genes which may be unique to the paramphistome group of platyhelminthes. Additionally, we present the first coverage of the excretory/secretory and soluble somatic proteome profiles for adult C. daubneyi and identify the release of extracellular vesicles from adult C. daubneyi parasites during in vitro, ex-host culture. Finally, we have performed the first analysis of rumen fluke impacting upon rumen fermentation parameters using an in vitro gas production study resulting in a significant increase in propionate production. Conclusions The resulting data provide a discovery platform (transcriptome, proteomes, EV isolation pipeline and in vitro fermentation system) to further study C. daubneyi-host interaction. In addition, the acetate: propionate ratio has been demonstrated to decrease with rumen fluke infection suggesting that acidotic conditions in the rumen may occur.

Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host–parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host–parasite interaction within the horse host.

Faecal egg counts (FECs) are the standard method of diagnosing the level of parasitic helminth egg shedding in horses and other grazing animals. Testing before treatment is an important factor in slowing the appearance of anthelmintic resistance in nematode parasites. The FECPAKG2, optimised for livestock, is reported to allow owners to perform FECs on their own animals without the need for a separate microscope or any specialist knowledge by tapping into remote expertise. However, the performance of the FECPAKG2 has yet to be assessed for equids. Therefore, a comparison of the FECPAKG2 (G2) method with an accepted equine FEC method (FECPAKG1(G1)) was performed, using faecal samples from 57 horses in Wales and 22 horses in New Zealand. There was a significant correlation between the FECs obtained by the two methods (p < 0.001) and no effect of the country of origin on the data (p = 0.157). The mean percentage accuracy compared to the control values (mean G2 count as a percentage of the mean G1 count, ±SStandard Error (SE)) was 101 ± 4%. There was no significant interaction between the method applied and the country of origin of the data (p = 0.814). The relative accuracy of the G2 method compared to the control method (FECPAKG1) was not affected by the level of infection (p = 0.124) and it was concluded that the FECPAKG2 method is a suitable method of performing FECs in horses. It is anticipated that the user-friendliness of the method will increase the uptake of FECs amongst horse owners, either by the direct use of the technology or through their veterinary practice, likely slowing the development of anthelmintic resistance.

Fasciola hepatica, the causative agent of fasciolosis, is a global threat to public health, animal welfare, agricultural productivity, and food security. In the ongoing absence of a commercial vaccine, independent emergences of anthelmintic-resistant parasite populations worldwide are threatening the sustainability of the few flukicides presently available, and particularly triclabendazole (TCBZ) as the drug of choice. Consequently, prognoses for future fasciolosis control and sustained TCBZ application necessitate improvements in diagnostic tools to identify anthelmintic efficacy. Previously, we have shown that proteomic fingerprinting of F. hepatica excretory/secretory (ES) products offered new biomarkers associated with in vitro TCBZ-sulfoxide (SO) recovery or death. In the current paper, two of these biomarkers (calreticulin (CRT) and triose phosphate isomerase (TPI)) were recombinantly expressed and evaluated to measure TCBZ efficacy via a novel approach to decipher fluke molecular phenotypes independently of molecular parasite resistance mechanism(s), which are still not fully characterised or understood. Our findings confirmed the immunoreactivity and diagnostic potential of the present target antigens by sera from TCBZ-susceptible (TCBZ-S) and TCBZ-resistant (TCBZ-R) F. hepatica experimentally infected sheep.

BACKGROUND:Robust protocols for the isolation of extracellular vesicles (EVs) from the rest of their excretory-secretory products are necessary for downstream studies and application development. The most widely used purification method of EVs for helminth pathogens is currently differential centrifugation (DC). In contrast, size exclusion chromatography (SEC) has been included in the purification pipeline for EVs from other pathogens, highlighting there is not an agreed research community 'gold standard' for EV isolation. In this case study, Fasciola hepatica from natural populations were cultured in order to collect EVs from culture media and evaluate a SEC or DC approach to pathogen helminth EV purification. METHODOLOGY/PRINCIPAL FINDINGS:Transmission electron and atomic force microscopy demonstrated that EVs prepared by SEC were both smaller in size and less diverse than EV resolved by DC. Protein quantification and Western blotting further demonstrated that SEC purification realised a higher EV purity to free excretory-secretory protein (ESP) yield ratio compared to DC approaches as evident by the reduction of soluble free cathepsin L proteases in SEC EV preparations. Proteomic analysis further highlighted DC contamination from ESP as shown by an increased diversity of protein identifications and unique peptide hits in DC EVs as compared to SEC EVs. In addition, SEC purified EVs contained less tegumental based proteins than DC purified EVs. CONCLUSIONS/SIGNIFICANCE:The data suggests that DC and SEC purification methods do not isolate equivalent EV population profiles and caution should be taken in the choice of EV purification utilised, with certain protocols for DC preparations including more free ES proteins and tegumental artefacts. We propose that SEC methods should be used for EV purification prior to downstream studies.

Corallococcus is an abundant genus of predatory soil myxobacteria, containing two species, C. coralloides (for which a genome sequence is available) and C. exiguus. To investigate the genomic basis of predation, we genome-sequenced 23 Corallococcus strains. Genomic similarity metrics grouped the sequenced strains into at least nine distinct genomospecies, divided between two major sub-divisions of the genus, encompassing previously described diversity. The Corallococcus pan-genome was found to be open, with strains exhibiting highly individual gene sets. On average, only 30.5% of each strain’s gene set belonged to the core pan-genome, while more than 75% of the accessory pan-genome genes were present in less than four of the 24 genomes. The Corallococcus accessory pan-proteome was enriched for the COG functional category “Secondary metabolism,” with each genome containing on average 55 biosynthetic gene clusters (BGCs), of which only 20 belonged to the core pan-genome. Predatory activity was assayed against ten prey microbes and found to be mostly incongruent with phylogeny or BGC complement. Thus, predation seems multifactorial, depending partially on BGC complement, but also on the accessory pan-genome – genes most likely acquired horizontally. These observations encourage further exploration of Corallococcus as a source for novel bioactive secondary metabolites and predatory proteins.

Myxobacteria are natural predators of microorganisms and the subjects of concerted efforts to identify novel antimicrobial compounds. Myxobacterial predatory activity seems to require more than just the possession of specific antimicrobial metabolites. Thus a holistic approach to studying predation promises novel insights into antimicrobial action. Here, we report the isolation of 113 myxobacteria from samples of soil taken from a range of habitats in mid Wales. Predatory activity of each isolate was quantified against a panel of clinically important prey organisms, including Klebsiella pneumoniae, Proteus mirabilis, Candida albicans, Enterococcus faecalis, and three species of Staphylococcus. Myxobacterial isolates exhibited a wide range of predation activity profiles against the panel of prey. Efficient predation of all prey by isolates within the collection was observed, with K. pneumoniae and C. albicans proving particularly susceptible to myxobacterial predation. Notably efficient predators tended to be proficient at predating multiple prey organisms, suggesting they possess gene(s) encoding a broad range killing activity. However, predatory activity was not congruent with phylogeny, suggesting prey range is subject to relatively rapid specialization, potentially involving lateral gene transfer. The broad but patchy prey ranges observed for natural myxobacterial isolates also implies multiple (potentially overlapping) genetic determinants are responsible for dictating predatory activity.

The parasitic flatworm Fasciola hepatica is a global food security risk. With no vaccines, the sustainability of triclabendazole (TCBZ) is threatened by emerging resistance. F. hepatica excretory/secretory (ES) products can be detected in host faeces and used to estimate TCBZ success and failure. However, there are no faecal based molecular diagnostics dedicated to assessing drug failure or resistance to TCBZ in the field. Utilising in vitro maintenance and sub-proteomic approaches two TCBZ stress ES protein response fingerprints were identified: markers of non-killing and lethal doses. This study provides candidate protein/peptide biomarkers to validate for detection of TCBZ failure and resistance.

Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200-320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised.In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection.We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity.

fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES) products are also the most promising vaccine candidates, the cathepsin L (Cat L) protease family.the sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples.we have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase.

The article discusses the study on the control of cell cycle and mitotic spindle. Cyclin-dependent kinases (CDK) inhibitors downstream targets of various signal transduction cascades that regulate cell proliferation. Studying CDK multiple downstream events provides further explanation on the roles played by various CDK complexes. CDK regulates pathways resulted in the formation of mitotic spindle which is responsible for controlling high fidelity of chromosome transmission during cell division.

Introduction: Antibiotic use drives antibiotic resistance. The UK antimicrobial resistance (AMR) strategy aims to reduce antibiotic use. We aimed to quantify excess antibiotic use in a district general hospital in south-west England., Methods: Medical patients discharged in August 2020 who had received antibiotics were included. An audit tool of antibiotic prescribing appropriateness was used to collect relevant clinical information regarding each patient case. The appropriateness of antibiotic use was then determined by two infection specialists and excess days of therapy (DOTs) calculated., Results: 647 patients were discharged in August 2020. Of the 1658 antibiotic DOTs for the 184 patients reviewed, 403 (24%) were excess DOTs. The excess antibiotic DOTs were prescribed in 92 patients (50%); 112/403 (27.8%) excess DOTs originated at the initiation of antibiotic therapy (time point A); 184/403 (45.7%) of excess DOTs occurred at the antibiotic review pre-72 hours (time point B); and 107/403 (26.6%) of excess DOTs were due to protracted antibiotic courses (time point C)., Conclusion: 24% of antibiotic DOTs were deemed unnecessary. The greatest opportunity to reduce antibiotic use safely was the pre-72 hours antibiotic review, which may provide a target for reducing excess antimicrobial therapy in line with the national AMR strategy., Competing Interests: Competing interests: None declared., (© European Association of Hospital Pharmacists 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei , the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events., Competing Interests: FL, AB, OK, MJ, TM, EM, TK, SS, NJ, MM, MS, CS, JM, ME No competing interests declared, (© 2023, Link et al.)

Rationale: The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates., Objective: We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid., Methods and Results: Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics., Conclusions: Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.

Introduction: Antibiotic use drives antibiotic resistance. Reducing antibiotic use through reducing antibiotic course lengths could contribute to the UK national ambition to reduce total antibiotic use., Methods: Medical notes were reviewed for patients who had received at least 5 days of antibiotic therapy; had been discharged in January 2019; and were from a 750-bed acute secondary care hospital in England. UK national guidelines were used to determine the excess antibiotic use in common medical infections: community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP), pyelonephritis, cellulitis, infective exacerbation of chronic obstructive pulmonary disease (IECOPD) and cholangitis., Results: Four-hundred and twenty-three patients met the inclusion criteria. Of these, 307 (73%) patient notes were retrieved and reviewed. One-hundred and seventy-three patients met the study case definitions, of which, 137 met short course criteria.Potential antibiotic reductions (measured in defined daily doses) were identified for five of the six infections: 32% in CAP, 20% in HAP, 14% in IECOPD, 11% in cellulitis and 10% in pyelonephritis. These reductions were estimated to reduce total antibiotic use in medical specialties by 12.4%, which equates to 3.6% of the hospital's total antibiotic use., Conclusion: Clinical application of the evidence-based guidance for shorter antibiotic course lengths appears to be a valid strategy for reducing total antibiotic consumption., (© Royal College of Physicians 2021. All rights reserved.)

Metaphase spindles exert pole-directed forces on still-connected sister kinetochores. The spindle must counter these forces with extensive forces to prevent spindle collapse. In small spindles, kinetochore microtubules (KMTs) connect directly with the poles, and countering forces are supplied either by interdigitating MTs that form interpolar bundles or by astral MTs connected to the cell cortex. In bigger spindles, particularly those without structured poles, the origin of extensive forces is less obvious. We have used electron tomography of well-preserved metaphase cells to obtain structural evidence about interactions among different classes of MTs in metaphase spindles from Chlamydomonas rheinhardti and two strains of cultured mammalian cells. In all these spindles, KMTs approach close to and cross-bridge with the minus ends of non-KMTs, which form a framework that interdigitates near the spindle equator. Although this structure is not pole-connected, its organization suggests that it can support kinetochore tension. Analogous arrangements of MTs have been seen in even bigger spindles, such as metaphase spindles in Haemanthus endosperm and frog egg extracts. We present and discuss a hypothesis that rationalizes changes in spindle design with spindle size based on the negative exponential distribution of MT lengths in dynamically unstable populations of tubulin polymers.

Faithful genome propagation requires coordination between nuclear envelope (NE) breakdown, spindle formation, and chromosomal events. The conserved linker of nucleoskeleton and cytoskeleton (LINC) complex connects fission yeast centromeres and the centrosome, across the NE, during interphase. During meiosis, LINC connects the centrosome with telomeres rather than centromeres. We previously showed that loss of telomere-LINC contacts compromises meiotic spindle formation. Here, we define the precise events regulated by telomere-LINC contacts and address the analogous possibility that centromeres regulate mitotic spindle formation. We develop conditionally inactivated LINC complexes in which the conserved SUN-domain protein Sad1 remains stable but severs interphase centromere-LINC contacts. Strikingly, the loss of such contacts abolishes spindle formation. We pinpoint the defect to a failure in the partial NE breakdown required for centrosome insertion into the NE, a step analogous to mammalian NE breakdown. Thus, interphase chromosome-LINC contacts constitute a cell-cycle control device linking nucleoplasmic and cytoplasmic events., (Published by Elsevier Inc.)

The field of cardiovascular research has benefitted from rapid developments in imaging technology over the last few decades. Accordingly, an ever growing number of large, multidimensional data sets have begun to appear, often challenging existing pre-conceptions about structure and function of biological systems. For tissue and cell structure imaging, the move from 2D section-based microscopy to true 3D data collection has been a major driver of new insight. In the sub-cellular domain, electron tomography is a powerful technique for exploration of cellular structures in 3D with unparalleled fidelity at nanometer resolution. Electron tomography is particularly advantageous for studying highly compartmentalised cells such as cardiomyocytes, where elaborate sub-cellular structures play crucial roles in electrophysiology and mechanics. Although the anatomy of specific ultra-structures, such as dyadic couplons, has been extensively explored using 2D electron microscopy of thin sections, we still lack accurate, quantitative knowledge of true individual shape, volume and surface area of sub-cellular domains, as well as their 3D spatial interrelations; let alone of how these are reshaped during the cycle of contraction and relaxation. Here we discuss and illustrate the utility of ET for identification, visualisation, and analysis of 3D cardiomyocyte ultrastructures such as the T-tubular system, sarcoplasmic reticulum, mitochondria and microtubules., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Interfaces between spindle microtubules and kinetochores were examined in diverse species by electron tomography and image analysis. Overall structures were conserved in a mammal, an alga, a nematode, and two kinds of yeasts; all lacked dense outer plates, and most kinetochore microtubule ends flared into curved protofilaments that were connected to chromatin by slender fibrils. Analyses of curvature on >8,500 protofilaments showed that all classes of spindle microtubules displayed some flaring protofilaments, including those growing in the anaphase interzone. Curved protofilaments on anaphase kinetochore microtubules were no more flared than their metaphase counterparts, but they were longer. Flaring protofilaments in budding yeasts were linked by fibrils to densities that resembled nucleosomes; these are probably the yeast kinetochores. Analogous densities in fission yeast were larger and less well-defined, but both yeasts showed ring- or partial ring-shaped structures girding their kinetochore microtubules. Flaring protofilaments linked to chromatin are well placed to exert force on chromosomes, assuring stable attachment and reliable anaphase segregation.

High-pressure freezing (HPF) has been around since the mid-1980s as a cryopreparation technique for biological electron microscopy. It has taken quite some time to "catch on" but with the recent interest in cellular tomography and electron microscopy of vitreous cryosections it has been used more frequently. While HPF is relatively easy to do, there are a number of steps, such as loading the sample into the specimen carrier correctly, that are critical to the success of this method. In this chapter we discuss some of the "little" things that can make the difference between successful or unsuccessful freezing. We cover all aspects of HPF, from specimen loading to removing your sample from the carriers in polymerized resin. Our goal is to make it easier and more reliable for HPF users to get well-frozen samples for their research., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A key feature of immune evasion for African trypanosomes is the functional specialization of their surface membrane in an invagination known as the flagellar pocket (FP), the cell's sole site of endocytosis and exocytosis. The FP membrane is biochemically distinct yet continuous with those of the cell body and the flagellum. The structural features maintaining this individuality are not known, and we lack a clear understanding of how extracellular components gain access to the FP. Here, we have defined domains and boundaries on these surface membranes and identified their association with internal cytoskeletal features. The FP membrane appears largely homogeneous and uniformly involved in endocytosis. However, when endocytosis is blocked, receptor-mediated and fluid-phase endocytic markers accumulate specifically on membrane associated with four specialized microtubules in the FP region. These microtubules traverse a distinct boundary and associate with a channel that connects the FP lumen to the extracellular space, suggesting that the channel is the major transport route into the FP.

Recent advances in rapid freezing and fixation by freeze substitution have allowed structural cell biologists to apply these reliable modes of sample preparation to a wide range of specimens and scientific problems. Progress in electron tomography has produced cellular images with resolution approaching 4 nm in 3D, but our ability to localize macromolecules in these well-fixed, well-resolved samples has remained limited. When light fixation and low temperature embedding are employed with appropriate resins, immuno-localizations can recognize antigens at a section's surface, but labelling is therefore confined, not throughout the section's depth. Small, electron-dense markers, like Nanogold(R), will often enter a living cell, serving as reliable tracers for endocytic activity, but these markers are usually too small to be visible in the context of a cell. We have developed a method for the silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature. Here, we describe the development of this method, based on in vitro tests of reagents and conditions. We then show results from application of the method to an in vivo system, using Nanogold to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor-mediated membrane traffic.

This chapter is an update of material first published by McDonald in the first volume of this book. Here, we discuss the improvements in the technology and the methodology of high-pressure freezing (HPF) since that article was published. First, we cover the latest innovation in HPF, the Leica EM PACT2. This machine differs significantly from the BAL-TEC HPM 010 high-pressure freezer, which was the main subject of the former chapter. The EM PACT2 is a smaller, portable machine and has an optional attachment, the Rapid Transfer System (RTS). This RTS permits easy and reproducible loading of the sample and allows one to do correlative light and electron microscopy with high time resolution. We also place more emphasis in this article on the details of specimen loading for HPF, which is considered the most critical phase of the whole process. Detailed procedures are described for how to high-pressure freeze cells in suspension, cells attached to substrates, tissue samples, or whole organisms smaller than 300 microm, and tissues or organisms greater than 300 microm in size. We finish the article with a brief discussion of freeze substitution and recommend some sample protocols for this procedure.

The homeobox-containing aristaless-related protein ARX has been directly linked to the development of a number of human disorders involving mental retardation and epilepsy and clearly plays a critical role in development of the vertebrate central nervous system. In this work, we investigate the role of ALR-1, the Caenorhabditis elegans aristaless orthologue, in amphid sensory function. Our studies indicate that ALR-1 is required for maintenance of the amphid organ structure throughout larval development. Mutant analysis indicates a progressive loss in the amphid neurons' ability to fill with lipophilic dyes as well as a declining chemotactic response. The degeneration in amphid function corresponds with a failure of the glial-like amphid socket cell to maintain its specific cell shape and cell-cell contacts. Consistent with ALR-1 expression within the amphid socket cell, our results indicate a cell autonomous role for ALR-1 in maintaining cell shape. Furthermore, we demonstrate a role for ALR-1 in the proper morphogenesis of the anterior hypodermis. Genetic interaction tests also suggest that ALR-1 may function cooperatively with the cell adhesion processes in maintaining the amphid sensory organs.

In the budding yeast Saccharomyces cerevisiae, the calmodulin-binding protein Spc110p/Nuf1p facilitates mitotic spindle formation from the fungal centrosome or spindle pole body (SPB). The human Spc110p orthologue kendrin is a centrosomal, calmodulin-binding pericentrin isoform that is specifically overexpressed in carcinoma cells. Here we establish an evolutionary and functional link between Spc110p and kendrin through identification and analysis of similar calmodulin-binding proteins in the fission yeast Schizosaccharomyces pombe (Pcp1p, pole target of calmodulin in S. pombe) and the filamentous fungus Aspergillus nidulans. Like Spc110p and kendrin, Pcp1p and the A. nidulans protein contain predicted coiled-coil secondary structure and a COOH-terminal calmodulin-binding region. Green fluorescent protein fusions of Pcp1p localize to the SPB as analyzed by fluorescence and immunoelectron microscopy. Pcp1p overexpression causes chromosome missegregation, multiple mitotic spindle fragments, and multiple abnormal SPB-like structures, a phenotype remarkably similar to that of many human carcinoma lines, which exhibit chromosome and spindle defects, and supernumerary centrosomes.

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2(+) gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the localization of this complex in the fission yeast, Schizosaccharomyces pombe. Immunofluorescence microscopy shows a striking localization pattern whereby the proteasome is found predominantly at the nuclear periphery, both in interphase and throughout mitosis. Electron microscopic analysis revealed a concentration of label near the inner side of the nuclear envelope. The localization of green fluorescent protein (GFP)-tagged 26S proteasomes was analyzed in live cells during mitosis and meiosis. Throughout mitosis the proteasome remained predominantly at the nuclear periphery. During meiosis the proteasome was found to undergo dramatic changes in its localization. Throughout the first meiotic division, the signal is more dispersed over the nucleus. During meiosis II, there was a dramatic re-localization, and the signal became restricted to the area between the separating DNA until the end of meiosis when the signal dispersed before returning to the nuclear periphery during spore formation. These findings strongly imply that the nuclear periphery is a major site of protein degradation in fission yeast both in interphase and throughout mitosis. Furthermore they raise interesting questions as to the spatial organization of protein degradation during meiosis.

During fission yeast mitosis, the duplicated spindle pole bodies (SPBs) nucleate microtubule arrays that interdigitate to form the mitotic spindle. cut12.1 mutants form a monopolar mitotic spindle, chromosome segregation fails, and the mutant undergoes a lethal cytokinesis. The cut12(+) gene encodes a novel 62-kD protein with two predicted coiled coil regions, and one consensus phosphorylation site for p34(cdc2) and two for MAP kinase. Cut12 is localized to the SPB throughout the cell cycle, predominantly around the inner face of the interphase SPB, adjacent to the nucleus. cut12(+) is allelic to stf1(+); stf1.1 is a gain-of-function mutation bypassing the requirement for the Cdc25 tyrosine phosphatase, which normally dephosphorylates and activates the p34(cdc2)/cyclin B kinase to promote the onset of mitosis. Expressing a cut12(+) cDNA carrying the stf1.1 mutation also suppressed cdc25.22. The spindle defect in cut12.1 is exacerbated by the cdc25.22 mutation, and stf1.1 cells formed defective spindles in a cdc25.22 background at high temperatures. We propose that Cut12 may be a regulator or substrate of the p34(cdc2) mitotic kinase.

The gene encoding the actin-related protein Arp3 was first identified in the fission yeast Schizosaccharomyces pombe and is a member of an evolutionarily conserved family of actin-related proteins. Here we present several key findings that define an essential role for Arp3p in the functioning of the cortical actin cytoskeleton. First, mutants in arp3 interact specifically with profilin and actin mutants. Second, Arp3 localizes to cortical actin patches which are required for polarized cell growth. Third, the arp3 gene is required for the reorganization of the actin cytoskeleton during the cell cycle. Finally, the Arp3 protein is present in a large protein complex. We believe that this complex may mediate the cortical functions of profilin at actin patches in S. pombe.

Members of the RCC1 protein family are chromatin-associated guanine nucleotide exchange factors that have been implicated in diverse cellular processes in various organisms, yet no consensus has been reached as to their primary biological role. The fission yeast Schizosaccharomyces pombe, a single-celled eukaryote, provides an in vivo system in which to study the RCC1/Ran switch by using a temperature-sensitive mutant in the RCC1-related protein pim1. Mitotic entry in the pim1-d1ts mutant is normal, but mitotic exit leads to the accumulation of cells arrested with a medial septum and condensed chromosomes. Although the yeast nuclear envelope normally remains intact throughout the cell cycle, we found a striking fragmentation of the nuclear envelope in the pim1-d1ts mutant following mitosis. This resulted in chromatin that was no longer compartmentalized and an accumulation of pore-containing membranes in the cytoplasm. The development of this terminal phenotype was dependent on the passage of cells through mitosis and was coincident with the loss of viability. We propose that pim1 is required for the reestablishment of nuclear structure following mitosis in fission yeast.