High speed sCMOS-based oblique plane microscopy applied to the study of calcium dynamics in cardiac myocytes

blique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo-4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure

Non-Chlamydial Bacterial Infection and Progression of Conjunctival Scarring in Trachoma.

Purpose: The purpose of this study was to assess whether non-chlamydial bacterial infection is associated with progression of trachomatous scarring in adults. Methods: This was a cohort study involving 800 participants in northern Tanzania who underwent clinical examination, photography, and conjunctival swab collection for microbiology over a 24-month period. Samples for microbiology were inoculated onto blood and chocolate agar, and Chlamydia trachomatis was detected by PCR. Progression was determined by comparison of baseline to 24-month photographs. Results: C. trachomatis was detected in only four participants at baseline. At 24 months, 617 participants (77.1%) were followed up. Of those seen at 24 months, 452 could be reliably assessed. Definite scarring progression (progressors) was seen in 345 (55.9%); there was no progression (nonprogressors) in 107 (17.3%). Using combined baseline and 12-month microbiology results, progressors had significantly higher levels of commensal and pathogenic bacterial organisms detected compared with nonprogressors. After adjusting for age, baseline scarring, and ethnicity, there was weak evidence (P = 0.07) that the bacteria category was associated with scarring progression (commensal organisms only: odds ratio [OR] = 1.61; 95% confidence interval [CI]: 0.90 to 2.89; pathogenic organisms either with or without commensal: OR = 2.39; 95% CI: 1.10 to 5.16). Conclusion: The findings were consistent with the possibility that trachomatous scarring in adults is associated with the presence of non-chlamydial bacterial organisms, particularly pathogenic organisms. C. trachomatis was detected very infrequently and may not be an important factor in the pathogenesis of scarring progression in adults. This has implications for trachoma control programs, which largely concentrate on reducing C. trachomatis levels and transmission

Population genetics of trypanosoma brucei rhodesiense: clonality and diversity within and between foci

African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda) and Southern (Malawi) Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics

Low Prevalence of Ocular Chlamydia trachomatis Infection and Active Trachoma in the Western Division of Fiji.

BACKGROUND: Trachoma is the leading infectious cause of blindness and is caused by ocular infection with the bacterium Chlamydia trachomatis (Ct). While the majority of the global disease burden is found in sub-Saharan Africa, the Western Pacific Region has been identified as trachoma endemic. Population surveys carried out throughout Fiji have shown an abundance of both clinically active trachoma and trachomatous trichiasis in all divisions. This finding is at odds with the clinical experience of local healthcare workers who do not consider trachoma to be highly prevalent. We aimed to determine whether conjunctival infection with Ct could be detected in one administrative division of Fiji. METHODS: A population-based survey of 2306 individuals was conducted using the Global Trachoma Mapping Project methodology. Population prevalence of active trachoma in children and trichiasis in adults was estimated using the World Health Organization simplified grading system. Conjunctival swabs were collected from 1009 children aged 1-9 years. DNA from swabs was tested for the presence of the Ct plasmid and human endogenous control. RESULTS: The prevalence of active trachoma in 1-9 year olds was 3.4%. The age-adjusted prevalence was 2.8% (95% CI: 1.4-4.3%). The unadjusted prevalence of ocular Ct infection in 1-9 year-olds was 1.9% (19/1009), and the age-adjusted infection prevalence was 2.3% (95% CI: 0.4-2.5%). The median DNA load was 41 Ct plasmid copies per swab (min 20, first quartile 32, mean 6665, third quartile 161, max 86354). There was no association between current infection and follicular trachoma. No cases of trachomatous trichiasis were identified. DISCUSSION: The Western Division of Fiji has a low prevalence of clinical trachoma. Ocular Ct infections were observed, but they were predominantly low load infections and were not correlated with clinical signs. Our study data suggest that trachoma does not meet the WHO definition of a public health problem in this Division of Fiji, but the inconsistency with previous studies warrants further investigation

Immunopathogenesis of Progressive Scarring Trachoma: Results of a 4-Year Longitudinal Study in Tanzanian Children.

Trachoma is initiated during childhood following repeated conjunctival infection with Chlamydia trachomatis, which causes a chronic inflammatory response in some individuals that leads to scarring and in-turning of the eyelids in later life. There is currently no treatment to halt the progression of scarring trachoma due to an incomplete understanding of disease pathogenesis. A cohort study was performed in northern Tanzania in 616 children aged 6 to 10 years at enrollment. Every 3 months for 4 years, children were examined for clinical signs of trachoma, and conjunctival swabs were collected for C. trachomatis detection and to analyze the expression of 46 immunofibrogenic genes. Data were analyzed in relation to progressive scarring status between baseline and the final time point. Genes that were significantly associated with scarring progression included those encoding proinflammatory chemokines (CXCL5, CCL20, CXCL13, and CCL18), cytokines (IL23A, IL19, and IL1B), matrix modifiers (MMP12 and SPARCL1), immune regulators (IDO1, SOCS3, and IL10), and a proinflammatory antimicrobial peptide (S100A7). In response to C. trachomatis infection, IL23A and PDGF were significantly upregulated in scarring progressors relative to in nonprogressors. Our findings highlight the importance of innate proinflammatory signals from the epithelium and implicate interleukin 23A (IL-23A)-responsive cells in driving trachomatous scarring, with potential key mechanistic roles for PDGFB, MMP12, and SPARCL1 in orchestrating fibrosis

Oral doxycycline for the prevention of postoperative trachomatous trichiasis in Ethiopia: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Trachomatous trichiasis is treated surgically to prevent sight loss. Unfavourable surgical outcomes remain a major challenge. We investigated the hypothesis that doxycycline might reduce the risk of postoperative trichiasis following surgery in patients with trachomatous trichiasis through anti-matrix metalloproteinase and anti-inflammatory activity. METHODS: In this randomised, double-blind, placebo-controlled trial, adults (aged >18 years) with upper lid trachomatous trichiasis in association with tarsal conjunctive scarring were recruited through community-based screening and surgical outreach campaigns in Ethiopia. Individuals who had previously had eyelid surgery were excluded. Participants were randomly assigned (1:1), with random block sizes of four or six, to receive oral doxycycline (100 mg once a day) or placebo for 28 days immediately after trichiasis surgery. Randomisation was stratified by surgeon. Patients, investigators, surgeons, and all other study team members were masked to study group allocation and treatment. Participants were examined at 10 days, and 1, 6, and 12 months after surgery. The primary outcome was the cumulative proportion of individuals who developed postoperative trichiasis by 12 months. Primary analyses were done in all participants who attended at least one of the four follow-up assessments. Safety analyses were done in all participants who attended either the 10 day or 1 month follow-up assessments. This trial is registered with the Pan African Clinical Trials Registry, number PACTR201512001370307. FINDINGS: Between Dec 21, 2015, and April 6, 2016, 1000 patients with trichiasis were enrolled and randomly assigned to treatment (499 patients to doxycycline, 501 patients to placebo). All but one participant attended at least one follow-up assessment. Thus, 999 participants were assessed for the primary outcome: 498 in the doxycycline group and 501 in the placebo group. By month 12, 58 (12%) of 498 patients in the doxycycline group and 62 (12%) of 501 patients in the placebo group had developed postoperative trichiasis (adjusted odds ratio 0·91, 95% CI 0·61 to 1·34, p=0·63), with a risk difference of -0·5% (-4·5% to 3·5%). Significantly more patients in the doxycycline group had an adverse event than in the placebo group (18 [4%] of 498 vs six [1%] of 501; odds ratio 3·09, 95% CI 1·21-7·84; p=0·02). The most frequent adverse events in the doxycycline group were gastritis symptoms (n=9), constipation (n=4), and diarrhoea (n=4). INTERPRETATION: Doxycycline did not reduce the risk of postoperative trichiasis and is therefore not indicated for the improvement of outcomes following trachomatous trichiasis surgery. Surgical programmes should continue to make efforts to strengthen surgical training and supervision to improve outcomes. FUNDING: The Wellcome Trust

The conjunctival transcriptome in Ethiopians after trichiasis surgery: associations with the development of eyelid contour abnormalities and the effect of oral doxycycline treatment

Background: Surgery to correct trichiasis is a key component of the World Health Organisation trachoma control strategy, however unfavourable outcomes such as eyelid contour abnormalities (ECA) following surgery are relatively common. This study aimed to understand the transcriptional changes associated with the early development of ECA and the impact of doxycycline, which has anti-inflammatory and anti-fibrotic properties, upon these transcription patterns. Methods: One thousand Ethiopians undergoing trichiasis surgery were enrolled in a randomised controlled trial following informed consent. Equal groups of randomly assigned individuals were orally administered with 100mg/day of doxycycline (n=499) or placebo (n=501) for 28 days. Conjunctival swabs were collected immediately prior to surgery and at one- and six-months post-surgery. 3’ mRNA sequencing was performed on paired baseline and one-month samples from 48 individuals; 12 in each treatment/ECA outcome group. qPCR validation was then performed for 46 genes of interest in 145 individuals who developed ECA at one month and 145 matched controls, using samples from baseline, one and six months. Results: All treatment/outcome groups upregulated genes associated with wound healing pathways at one month relative to baseline, however no individual differences were detected between groups. The summed expression of a highly coexpressed cluster of pro-fibrotic genes was higher in patients that developed ECA in the placebo group relative to controls. qPCR validation revealed that all genes in this cluster and a number of other pro-inflammatory genes were strongly associated with ECA, however these associations were not modulated by trial arm. Conclusions: The development of post-operative ECA is associated with overexpression of pro-inflammatory and pro-fibrotic genes including growth factors, matrix metalloproteinases, collagens and extracellular matrix proteins. There was no evidence that doxycycline modulated the association between gene expression and ECA.</ns4:p

The conjunctival transcriptome in Ethiopians after trichiasis surgery: associations with the development of eyelid contour abnormalities and the effect of oral doxycycline treatment

Background: Surgery to correct trichiasis is a key component of the World Health Organisation trachoma control strategy, however unfavourable outcomes such as eyelid contour abnormalities (ECA) following surgery are relatively common. This study aimed to understand the transcriptional changes associated with the early development of ECA and the impact of doxycycline, which has anti-inflammatory and anti-fibrotic properties, upon these transcription patterns. Methods: One thousand Ethiopians undergoing trichiasis surgery were enrolled in a randomised controlled trial following informed consent. Equal groups of randomly assigned individuals were orally administered with 100mg/day of doxycycline (n=499) or placebo (n=501) for 28 days. Conjunctival swabs were collected immediately prior to surgery and at one- and six-months post-surgery. 3’ mRNA sequencing was performed on paired baseline and one-month samples from 48 individuals; 12 in each treatment/outcome group (Placebo-Good outcome, Placebo-Poor outcome, Doxycycline-Good outcome, Doxycycline-Poor outcome). qPCR validation was then performed for 46 genes of interest in 145 individuals who developed ECA at one month and 145 matched controls, using samples from baseline, one and six months. Results: All treatment/outcome groups upregulated genes associated with wound healing pathways at one month relative to baseline, however no individual differences were detected between groups. The summed expression of a highly coexpressed cluster of pro-fibrotic genes was higher in patients that developed ECA in the placebo group relative to controls. qPCR validation revealed that all genes in this cluster and a number of other pro-inflammatory genes were strongly associated with ECA, however these associations were not modulated by trial arm. Conclusions: The development of post-operative ECA is associated with overexpression of pro-inflammatory and pro-fibrotic genes including growth factors, matrix metalloproteinases, collagens and extracellular matrix proteins. There was no evidence that doxycycline modulated the association between gene expression and ECA.</ns4:p