The immune receptor signaling pathway is used by nonimmune cells, but the molecular adaptations that underlie its functional diversification are not known. Circulating platelets use the immune ...receptor homologue glycoprotein VI (GPVI) to respond to collagen exposed at sites of vessel injury. In contrast to immune cell responses, platelet activation must take place within seconds to successfully form thrombi in flowing blood. Here, we show that the GPVI receptor utilizes a unique intracellular proline-rich domain (PRD) to accelerate platelet activation, a requirement for efficient platelet adhesion to collagen under flow. The GPVI PRD specifically binds the Src-family kinase Lyn and directly activates it, presumably through SH3 displacement. In resting platelets, Lyn is constitutively bound to GPVI in an activated state and platelets lacking Lyn exhibit defective collagen adhesion like that of platelets with GPVI receptors lacking the PRD. These findings define a molecular priming mechanism that enables an immune-type receptor to adopt a hemostatic function. These studies also demonstrate that active kinases can constitutively associate with immune-type receptors without initiating signal transduction before receptor ligation, consistent with a recent molecular model of immune receptor signaling in which receptor ligation is required to bring active kinases to their receptor substrates.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Commensal butyrate-producing bacteria in the Firmicutes phylum are abundant in the human intestine and are important for maintaining health. However, understanding of the metabolism and host ...interaction of these bacteria is limited by the lack of genetic modification techniques. Here we establish a protocol enabling the transfer of autonomously-replicating shuttle vectors by conjugative plasmid transfer from an Escherichia coli donor into representatives of an important sub-group of strictly anaerobic human colonic Firmicutes. Five different plasmid shuttle vectors were tested, each carrying a different origin of replication from Gram-positive bacteria. Plasmid pMTL83151 (pCB102 replicon) were successfully transferred into two strains of Eubacterium rectale, while pMTL83151 and pMTL82151 (pBP1 replicon) were transferred into Roseburia inulinivorans A2-194. Plasmids that carried a Streptococcus bovis JB1 glycoside hydrolase family 16 β-(1,3-1,4)-glucanase gene were constructed and conjugated into Roseburia inulinivorans A2-194 and Eubacterium rectale T1-815, resulting in successful heterologous expression of this introduced enzymatic activity in these two strains of butyrate-producing Firmicutes.
•Butyrate producing human gut Firmicutes were genetically modified by gene transfer.•Shuttle vectors were transferred by conjugation from an Escherichia coli donor.•Transconjugants were obtained for Roseburia inulinivorans and Eubacterium rectale.•An enzymatically active glycoside hydrolase was heterologously expressed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We have analyzed the total metagenomic DNA from both human oral and fecal samples derived from healthy volunteers from six European countries to determine the molecular basis for tetracycline and ...erythromycin resistance. We have determined that tet(M) and tet(W) are the most prevalent tetracycline resistance genes assayed for in the oral and fecal metagenomes, respectively. Additionally, tet(Q), tet(O), and tet(O/32/O) have been shown to be common. We have also shown that erm(B), erm(V), and erm(E) are common erythromycin resistance genes present in these environments. Further, we have demonstrated the ubiquitous presence of the Tn916 integrase in the oral metagenomes and the Tn4451 and Tn1549 integrase genes within the fecal metagenomes.
Cardiac motion results in image artefacts and quantification errors in many cardiovascular magnetic resonance (CMR) techniques, including microstructural assessment using diffusion tensor ...cardiovascular magnetic resonance (DT‐CMR). Here, we develop a CMR‐compatible isolated perfused porcine heart model that allows comparison of data obtained in beating and arrested states. Ten porcine hearts (8/10 for protocol optimisation) were harvested using a donor heart retrieval protocol and transported to the remote CMR facility. Langendorff perfusion in a 3D‐printed chamber and perfusion circuit re‐established contraction. Hearts were imaged using cine, parametric mapping and STEAM DT‐CMR at cardiac phases with the minimum and maximum wall thickness. High potassium and lithium perfusates were then used to arrest the heart in a slack and contracted state, respectively. Imaging was repeated in both arrested states. After imaging, tissue was removed for subsequent histology in a location matched to the DT‐CMR data using fiducial markers. Regular sustained contraction was successfully established in six out of 10 hearts, including the final five hearts. Imaging was performed in four hearts and one underwent the full protocol, including colocalised histology. The image quality was good and there was good agreement between DT‐CMR data in equivalent beating and arrested states. Despite the use of autologous blood and dextran within the perfusate, T2 mapping results, DT‐CMR measures and an increase in mass were consistent with development of myocardial oedema, resulting in failure to achieve a true diastolic‐like state. A contiguous stack of 313 5‐μm histological sections at and a 100‐μm thick section showing cell morphology on 3D fluorescent confocal microscopy colocalised to DT‐CMR data were obtained. A CMR‐compatible isolated perfused beating heart setup for large animal hearts allows direct comparisons of beating and arrested heart data with subsequent colocalised histology, without the need for onsite preclinical facilities.
A cardiovascular magnetic resonance (CMR)‐compatible isolated perfused beating porcine heart model was developed that allows direct comparison of data acquired in multiple states of contraction between the beating and arrested heart. We describe the development of this model, its successes and failures, including the presence of oedema in the hearts. In this study, microstructural measures were obtained from the porcine hearts using diffusion tensor CMR in the most and least contracted states with subsequent colocalised histology.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The Tor1p and Tor2p kinases, targets of the therapeutically important antibiotic rapamycin, function as components of two distinct protein complexes in yeast, termed TOR complex 1 (TORC1) and TORC2. ...TORC1 is responsible for a wide range of rapamycin-sensitive cellular activities and contains, in addition to Tor1p or Tor2p, two highly conserved proteins, Lst8p and Kog1p. By identifying proteins that co-purify with Tor1p, Tor2p, Lst8p, and Kog1p, we have characterized a comprehensive set of protein-protein interactions that define further the composition of TORC1 as well as TORC2. In particular, we have identified Tco89p (YPL180w) and Bit61p (YJL058c) as novel components of TORC1 and TORC2, respectively. Deletion of TOR1 or TCO89 results in two specific and distinct phenotypes, (i) rapamycin-hypersensitivity and (ii) decreased cellular integrity, both of which correlate with the presence of SSD1-d, an allele of SSD1 previously associated with defects in cellular integrity. Furthermore, we link Ssd1p to Tap42p, a component of the TOR pathway that is believed to act uniquely downstream of TORC1. Together, these results define a novel connection between TORC1 and Ssd1p-mediated maintenance of cellular integrity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Seven recently cultured bacterial isolates, although similar in their 16S rRNA gene sequences to Roseburia intestinalis L1-82T (DSM 14610T), were not sufficiently related for inclusion within ...existing species, forming three separate clusters in a 16S rRNA gene phylogenetic tree. The isolates, which were obtained from human stools, were Gram-variable or Gram-negative, strictly anaerobic, slightly curved rods; cells from all strains measured approximately 0.5x1.5-5.0 micrometer and were motile. Two strains belonging to one cluster (A2-181 and A2-183T) were the only strains that were able to grow on glycerol and that failed to grow on any of the complex substrates tested (inulin, xylan and amylopectin). Strains belonging to a second cluster (represented by M6/1 and M72/1T) differed from the other isolates in their ability to grow on sorbitol. Isolates belonging to a third cluster (L1-83 and A2-194T) were the only strains that failed to grow on xylose and that gave good growth on inulin (strains M6/1 and M72/1T gave weak growth). All strains were net acetate utilizers. The DNA G+C contents of representative Roseburia strains A2-183T, A2-194T, M72/1T and R. intestinalis L1-82T were 47.4, 41.4, 42.0 and 42.6 mol%, respectively. Based on 16S rRNA gene sequence similarity, three novel Roseburia species are proposed, with the names Roseburia hominis sp. nov. (type strain A2-183T=DSM 16839T=NCIMB 14029T), Roseburia inulinivorans sp. nov. (type strain A2-194T=DSM 16841T=NCIMB 14030T) and Roseburia faecis sp. nov. (type strain M72/1T=DSM 16840T=NCIMB 14031T).
•Following up clinicians who diagnosed individuals with hepatitis C virus through the department of health's surveillance systems has the potential of increasing the number of people living with the ...virus who initiate treatment.•This was the first prospective randomised study using existing HCV surveillance systems.•Directly engaging primary care practitioners has the potential of improving HCV care cascades.•The intervention increased HCV RNA testing.
Despite subsidised access to direct-acting antivirals (DAAs), hepatitis C (HCV) treatment uptake in Australia is declining. Interventions are needed to link people living with HCV to care and treatment. We implemented and measured effectiveness of a state-wide, health department-led, enhanced case management through the primary care practitioner for all HCV notifications, aiming to encourage and support treatment commencement.
A randomised controlled trial compared enhanced case management, delivered by the health department to diagnosing clinicians, with standard of care using notifiable disease systems in Tasmania, Australia (2020–21). The intervention involved a nurse specialist contacting and providing support by telephone to primary care practitioners making an HCV notification. The primary outcome was the proportion of cases notified with chronic hepatitis C who commenced treatment within 12 weeks of notification. We allowed a 12-week extended follow-up period at the end of the study for participants with no outcomes.
Eighty-five primary care practitioners randomised to the intervention and 86 to standard of care arms notified 111 and 115 HCV cases, respectively. The proportion of cases notified with chronic hepatitis (HCV RNA detected) commencing treatment within 12 weeks was similar between study arms (41% vs 33%; p=0·51) and after extended study follow-up (65% vs 48%; p=0·18). RNA test completion was higher in the intervention than in standard of care arm (89% vs. 78%; p=0·03), while completing pre-treatment workup for chronic patients (65% vs. 64%; p=0·93) was similar.
This was the first prospective randomised study of the utility of immediate HCV notification follow-up of primary care practitioners to enhance treatment uptake using disease notification surveillance data. We demonstrated improvement in HCV RNA testing and trend toward better engagement in care, but no significant increase in treatment uptake.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recent years have seen the development of high-accuracy and high-throughput genetic manipulation techniques, which have greatly improved our understanding of genetically tractable microbes. However, ...challenges remain in establishing genetic manipulation techniques in novel organisms, owing largely to exogenous DNA defence mechanisms, lack of selectable markers, lack of efficient methods to introduce exogenous DNA and an inability of genetic vectors to replicate in their new host. In this review, we describe some of the techniques that are available for genetic manipulation of novel microorganisms. While many reviews exist that focus on the final step in genetic manipulation, the editing of recipient DNA, we particularly focus on the first step in this process, the transfer of exogenous DNA into a strain of interest. Examples illustrating the use of these techniques are provided for a selection of human gut bacteria in which genetic tractability has been established, such as
,
and
. Ultimately, this review aims to provide an information source for researchers interested in developing genetic manipulation techniques for novel bacterial strains, particularly those of the human gut microbiota.
Aim
The aim of this study was to investigate changes in bowel function and anorectal physiology (ARP) after anterior resection for colorectal cancer.
Method
Patients were recruited from November 2006 ...to September 2008. Cleveland Clinic Incontinence (CCI) scores and stool frequency were determined by patient questionnaires before surgery (t0) and at three (t3), six (t6), nine (t9) and 12 (t12) months after restoration of intestinal continuity. ARP measurements were recorded at T0, T3 and T12. Endoanal ultrasound was performed at T0 and T12.
Results
Eighty‐nine patients were included. CCI score increased postoperatively then normalized, whereas stool frequency did not change. Patients who had neoadjuvant radiotherapy or a lower anastomosis had increased incontinence and stool frequency in the postoperative period, whereas those with defunctioning stomas or open surgery had increased stool frequency alone. Maximum resting pressure, volume at first urge and maximum rectal tolerance were reduced throughout the postoperative period. Radiotherapy, lower anastomosis and defunctioning stoma (but not operative approach) altered manometric parameters postoperatively. Maximum rectal tolerance correlated with incontinence and first urge with stool frequency. The length of the anterior internal anal sphincter decreased postoperatively.
Conclusions
Incontinence recovers in the first year after anterior resection. Radiotherapy, lower anastomosis, defunctioning stoma and open surgery have a negative influence on bowel function. ARP may be useful if bowel dysfunction persists beyond 12 months.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
By subsidising access to direct acting antivirals (DAAs) for all people living with hepatitis C (HCV) in 2016, Australia is positioned to eliminate HCV as a public health threat. However, uptake of ...DAAs has declined over recent years and new initiatives are needed to engage people living with HCV in care. Active follow-up of HCV notifications by the health department to the notifying general practitioner (GP) may increase treatment uptake. In this study, we explore the impact of using hepatitis C notifications systems to engage diagnosing GPs and improve patient access to treatment.
This study is a randomised controlled trial comparing enhanced case management of HCV notifications with standard of care. The intervention includes phone calls from a department of health (DoH) specialist HCV nurse to notifying GPs and offering HCV management support. The level of support requested by the GP was graded in complexity: level 1: HCV information only; level 2: follow-up testing advice; level 3: prescription support including linkage to specialist clinicians and level 4: direct patient contact. The study population includes all GPs in Tasmania who notified HCV diagnosis to the DoH between September 2020 and December 2021. The primary outcome is proportion of HCV cases who initiate DAAs after 12 weeks of HCV notification to the health department. Secondary outcomes are proportion of HCV notifications that complete HCV RNA testing, treatment workup and treatment completion. Multiple logistic regression modelling will explore factors associated with the primary and secondary outcomes. The sample size required to detect a significant difference for the primary outcome is 85 GPs in each arm with a two-sided alpha of 0.05% and 80% power.
The study was approved by University of Tasmania's Human Research Ethics Committee (Protocol ID: 18418) on 17 December 2019. Results of the project will be presented in scientific meetings and published in peer-reviewed journals.
NCT04510246.
The study commenced recruitment in September 2020 and end of study expected December 2021.
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