A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

MTHFR Glu429Ala and ERCC5 His46His Polymorphisms Are Associated with Prognosis in Colorectal Cancer Patients: Analysis of Two Independent Cohorts from Newfoundland

Introduction

In this study, 27 genetic polymorphisms that were previously reported to be associated with clinical outcomes in colorectal cancer patients were investigated in relation to overall survival (OS) and disease free survival (DFS) in colorectal cancer patients from Newfoundland.

Methods

The discovery and validation cohorts comprised of 532 and 252 patients, respectively. Genotypes of 27 polymorphisms were first obtained in the discovery cohort and survival analyses were performed assuming the co-dominant genetic model. Polymorphisms associated with disease outcomes in the discovery cohort were then investigated in the validation cohort.

Results

When adjusted for sex, age, tumor stage and microsatellite instability (MSI) status, four polymorphisms were independent predictors of OS in the discovery cohort MTHFR Glu429Ala (HR: 1.72, 95%CI: 1.04–2.84, p = 0.036), ERCC5 His46His (HR: 1.78, 95%CI: 1.15–2.76, p = 0.01), SERPINE1 −675indelG (HR: 0.52, 95%CI: 0.32–0.84, p = 0.008), and the homozygous deletion of GSTM1 gene (HR: 1.4, 95%CI: 1.03–1.92, p = 0.033). In the validation cohort, the MTHFR Glu429Ala polymorphism was associated with shorter OS (HR: 1.71, 95%CI: 1.18–2.49, p = 0.005), although with a different genotype than the discovery cohort (CC genotype in the discovery cohort and AC genotype in the validation cohort). When stratified based on treatment with 5-Fluorouracil (5-FU)-based regimens, this polymorphism was associated with reduced OS only in patients not treated with 5-FU. In the DFS analysis, when adjusted for other variables, the TT genotype of the ERCC5 His46His polymorphism was associated with shorter DFS in both cohorts (discovery cohort: HR: 1.54, 95%CI: 1.04–2.29, p = 0.032 and replication cohort: HR: 1.81, 95%CI: 1.11–2.94, p = 0.018).

Conclusions

In this study, associations of the MTHFR Glu429Ala polymorphism with OS and the ERCC5 His46His polymorphism with DFS were identified in two colorectal cancer patient cohorts. Our results also suggest that the MTHFR Glu429Ala polymorphism may be an adverse prognostic marker in patients not treated with 5-FU.     

Epidemiology,Diagnosis and Management of Extra-Pulmonary Tuberculosis in a Low-Prevalence Country: A Four Year Retrospective Study in an Australian Tertiary Infectious Diseases Unit

Objectives

Extra-pulmonary tuberculosis (EPTB) is relatively neglected and increasing in incidence, in comparison to pulmonary tuberculosis (PTB) in low-burden settings. It poses particular diagnostic and management challenges. We aimed to determine the characteristics of EPTB in Western Sydney, Australia, and to conduct a quality assurance investigation of adherence to guidelines among Infectious Diseases (ID) practitioners managing EPTB cases.

Methods

All adult EPTB cases managed by a large ID service during 01/01/2008–31/12/2011 were eligible for inclusion in the retrospective review. Data were extracted from patient medical records on demographic, diagnostic, clinical and management details, and on clinician adherence to local and international TB guidelines.

Results

129 cases managed by the ID service were identified, with files available for 117. 98 cases were managed by the Respiratory service and were excluded. 98.2%(112/114) had been born in a country other than Australia. HIV status was tested or previously known in 97 people, and positive in 4 (4%). Microbiological confirmation was obtained in 68/117 (58.1%), an additional 24 had histopathological findings considered confirmatory (92/117, 78.6%), with the remainder diagnosed on clinical and/or radiological grounds. Median time to diagnosis post-migration from a high TB-burden country was 5 years (range 0–41). 95 cases were successfully treated, 11 cases defaulted, refused therapy or transferred, 2 cases relapsed and outcomes unknown or pending in 9 cases. No deaths occurred in the sample analysed. Clinician adherence to guidelines was high, but with scope for improvement in offering testing for co-infections, performing eye checks, monitoring blood glucose in patients receiving adjunctive corticosteroids, and considering drug interactions.

Conclusions

Despite excellent TB outcomes in this setting, the low proportion of cases with susceptibility data is worrying in this era of increasing drug resistance, and illustrates the diagnostic difficulties faced even in a well-resourced setting. Vigilance for EPTB needs to remain high in those moving from high prevalence countries to Australia, even decades after immigration.     

The seroepidemiology of dengue in a US military population based in Puerto Rico during the early phase of the Zika pandemic

Understanding the burden and risk factors of dengue virus (DENV) infection in Puerto Rico is important for the prevention of dengue in local, traveler and military populations. Using sera from the Department of Defense Serum Repository, we estimated the prevalence and predictors of DENV seropositivity in those who had served in Puerto Rico, stratified by birth or prior residence (“birth/residence”) in dengue-endemic versus non-endemic regions. We selected sera collected in early 2015 from 500 U.S. military members, a time-point also permitting detection of early cryptic Zika virus (ZIKV) circulation. 87.2% were born or resided in a DENV-endemic area before their military service in Puerto Rico. A high-throughput, flow-cytometry-based neutralization assay was employed to screen sera for ZIKV and DENV neutralizing antibodies, and confirmatory testing was done by plaque-reduction neutralization test (PRNT). We identified one Puerto Rico resident who seroconverted to ZIKV by June 2015, suggesting cryptic ZIKV circulation in Puerto Rico at least 4 months before the first reported cases. A further six PRNT-positive presumptive ZIKV infections which were resolved as DENV infections only by the use of paired sera. We noted 66.8% of the total study sample was DENV seropositive by early 2015. Logistic regression analysis indicated that birth/residence in a dengue non-endemic region (before military service in Puerto Rico) was associated with a lower odds of DENV exposure by January—June 2015 (aOR = 0.28, p = 0.001). Among those with birth/residence in a non-endemic country, we noted moderate evidence to support increase in odds of DENV exposure for each year of military service in Puerto Rico (aOR = 1.58, p = 0.06), but no association with age. In those with birth/residence in dengue-endemic regions (before military service in Puerto Rico), we noted that age (aOR = 1.04, p = 0.02), rather than duration of Puerto Rico service, was associated with dengue seropositivity, suggesting earlier lifetime DENV exposure. Our findings provide insights into the burden and predictors of DENV infection in local, traveler and military populations in Puerto Rico. Our study also highlights substantial PRNT ZIKV false-positivity when paired sera are not available, even during periods of very low ZIKV prevalence.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Assessment of thin-layer breast aspirates for immunocytochemical evaluation of HER2 status

OBJECTIVE: To determine whether immunocytochemistry (ICC) for HER2 on ThinPrep (TP)-processed breast fine needle aspiration biopsies (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) is comparable to the findings of immunohistochemistry on corresponding surgically removed tissue. STUDY DESIGN: Immunostaining was performed on 63 malignant breast fine needle aspirates and compared to immunostaining on paraffin sections (PSs) from the subsequent biopsies. The HercepTest (Dako, Carpinteria, California, U.S.A.) and TAB250 antibodies were utilized. Cases in which the TP and paraffin HER2 results did not correlate were further assessed for gene amplification by differential polymerase chain reaction (dPCR). RESULTS: HER2 overexpression was found in 9 of the 63 cases (14%). TAB250 had higher specificity on PS versus TP (P = .008), and TAB250 had higher specificity on PS versus the HercepTest on PS and TP (P = .004 and .0001, respectively). CONCLUSION: HER2 immunostaining with both the HercepTest and TAB250 on TP is unreliable due to low specificity (72% and 83% for HercepTest and TAB250, respectively). However, both antibodies have high sensitivity (89% and 100%, respectively); suggesting that this method may have some utility as a preliminary screening test for HER2 status. Negative HER2 staining by ICC is highly predictive of the absence of HER2 overexpression, whereas positive HER2 staining on TP would require further validation by either dPCR of fluorescence in situ hybridization.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Prospective identification of myogenic endothelial cells in human skeletal muscle

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.     

Exacerbated tissue destruction in DSS-induced acute colitis of OPN-null mice is associated with downregulation of TNF-alpha expression and non-programmed cell death

Osteopontin (OPN), a pro-inflammatory mediator, is constitutively expressed in normal gut and is upregulated in inflammatory colitis. To determine the significance of OPN in inflammatory bowel disease, we studied the development of acute, experimental colitis induced by dextran sulfate sodium (DSS) in OPN-null and wild-type (WT) mice. OPN expression was markedly increased in WT diseased colons, while a higher disease activity index, including spleen enlargement, bowel shortening, and mucosal destruction, was observed in OPN-null mice. Although peripheral blood neutrophil numbers were lower in DSS-treated OPN-null mice, tissue myeloperoxidase levels, reflecting enhanced neutrophil activity, were increased in the diseased colons. In comparison, lymphocyte numbers in peripheral blood were increased earlier than in DSS-treated WT mice. Despite a significantly greater spleen enlargement, flow cytometric analysis of splenocytes from the DSS-treated OPN-null mice revealed lower numbers of differentiated macrophages and (CD4+ and CD8alpha+) lymphocytes. Whereas pro-inflammatory cytokines, including G-CSF, RANTES, MIP1alpha, and TNF-alpha, were increased < 10-fold in DSS-treated WT splenocytes, expression of these cytokines was dramatically suppressed in the DSS-treated OPN-null splenocytes as well as gut tissues. The suppressed TNF-alpha response in OPN-null mice was reflected in a marked increase in non-apoptotic cell death in diseased colons. Collectively, these studies demonstrate that OPN is required for mucosal protection in acute inflammatory colitis.     

On parameter estimation in population models

We describe methods for estimating the parameters of Markovian population processes in continuous time, thus increasing their utility in modelling real biological systems. A general approach, applicable to any finite-state continuous-time Markovian model, is presented, and this is specialised to a computationally more efficient method applicable to a class of models called density-dependent Markov population processes. We illustrate the versatility of both approaches by estimating the parameters of the stochastic SIS logistic model from simulated data. This model is also fitted to data from a population of Bay checkerspot butterfly (Euphydryas editha bayensis), allowing us to assess the viability of this population.