Cholesterol Efflux Decreases TLR4-Target Gene Expression in Cultured Macrophages Exposed to T. brucei Ghosts.

Trypanosoma brucei causes African trypanosomiasis in humans. Infection with T. brucei elicits a potent pro-inflammatory immune response within infected human hosts, and this response is thought to at least be partially due to Toll-like receptor (TLR) activation. In response to stimulation by lipopolysaccharide and other pathogen antigens, TLR4 translocates to lipid rafts, which induces the expression of pro-inflammatory genes. However, cholesterol efflux is acknowledged as anti-inflammatory due to promoting lipid raft disruption. In this study, we wanted to assess the impact of T. brucei "ghosts", which are non-viable T. brucei essentially devoid of intracellular contents, in stimulating macrophage TLR4 translocation to lipid rafts, and whether promoting cholesterol efflux in macrophages incubated with T. brucei ghosts attenuates TLR4-target gene expression. When cultured macrophages were exposed to T. brucei ghosts, we observed an increase in lipid raft TLR4 protein content, which suggests certain surface molecules of T. brucei serve as ligands for TLR4. However, pretreating macrophages with cholesterol acceptors before T. brucei ghost exposure decreased lipid raft TLR4 protein content and the expression of pro-inflammatory TLR4-target genes. Taken together, these results imply that macrophage cholesterol efflux weakens pro-inflammatory responses which occur from T. brucei infection via increasing macrophage lipid raft disruption.

Inhibition of miR-33a-5p in Macrophage-like Cells In Vitro Promotes apoAI-Mediated Cholesterol Efflux.

Atherosclerosis is caused by cholesterol accumulation within arteries. The intima is where atherosclerotic plaque accumulates and where lipid-laden foam cells reside. Intimal foam cells comprise of both monocyte-derived macrophages and macrophage-like cells (MLC) of vascular smooth muscle cell (VSMC) origin. Foam cells can remove cholesterol via apoAI-mediated cholesterol efflux and this process is regulated by the transporter ABCA1. The microRNA miR-33a-5p is thought to be atherogenic via silencing ABCA1 which promotes cholesterol retention and data has shown inhibiting miR-33a-5p in macrophages may be atheroprotective via enhancing apoAI-mediated cholesterol efflux. However, it is not entirely elucidated whether precisely inhibiting miR-33a-5p in MLC also increases ABCA1-dependent cholesterol efflux. Therefore, the purpose of this work is to test the hypothesis that inhibition of miR-33a-5p in cultured MLC enhances apoAI-mediated cholesterol efflux. In our study, we utilized the VSMC line MOVAS cells in our experiments, and cholesterol-loaded MOVAS cells to convert this cell line into MLC. Inhibition of miR-33a-5p was accomplished by transducing cells with a lentivirus that expresses an antagomiR directed at miR-33a-5p. Expression of miR-33a-5p was analyzed by qRT-PCR, ABCA1 protein expression was assessed via immunoblotting, and apoAI-mediated cholesterol efflux was measured using cholesterol efflux assays. In our results, we demonstrated that lentiviral vector-mediated knockdown of miR-33a-5p resulted in decreasing expression of this microRNA in cultured MLC. Moreover, reduction of miR-33a-5p in cultured MLC resulted in de-repression of ABCA1 expression, which caused ABCA1 protein upregulation in cultured MLC. Additionally, this increase in ABCA1 protein expression resulted in enhancing ABCA1-dependent cholesterol efflux through increasing apoAI-mediated cholesterol efflux in cultured MLC. From these findings, we conclude that inhibiting miR-33a-5p in MLC may protect against atherosclerosis by promoting ABCA1-dependent cholesterol efflux.

Dissecting the Impact of Vascular Smooth Muscle Cell ABCA1 versus ABCG1 Expression on Cholesterol Efflux and Macrophage-like Cell Transdifferentiation: The Role of SR-BI.

Cholesterol-laden macrophages are recognized as a major contributor to atherosclerosis. However, recent evidence indicates that vascular smooth muscle cells (VSMC) that accumulate cholesterol and transdifferentiate into a macrophage-like cell (MLC) phenotype also play a role in atherosclerosis. Therefore, removing cholesterol from MLC may be a potential atheroprotective strategy. The two transporters which remove cholesterol from cells are ABCA1 and ABCG1, as they efflux cholesterol to apoAI and HDL, respectively. In this study, the well-characterized immortalized VSMC line MOVAS cells were edited to generate ABCA1- and ABCG1-knockout (KO) MOVAS cell lines. We cholesterol-loaded ABCA1-KO MOVAS cells, ABCG1-KO MOVAS cells, and wild-type MOVAS cells to convert cells into a MLC phenotype. When we measured apoAI- and HDL-mediated cholesterol efflux in these cells, we observed a drastic decrease in apoAI-mediated cholesterol efflux within ABCA1-KO MOVAS MLC, but HDL-mediated cholesterol efflux was only partially reduced in ABCG1-KO MOVAS cells. Since SR-BI also participates in HDL-mediated cholesterol efflux, we assessed SR-BI protein expression in ABCG1-KO MOVAS MLC and observed SR-BI upregulation, which offered a possible mechanism explaining why HDL-mediated cholesterol efflux remains maintained in ABCG1-KO MOVAS MLC. When we used lentivirus for shRNA-mediated knockdown of SR-BI in ABCG1-KO MOVAS MLC, this decreased HDL-mediated cholesterol efflux when compared to ABCG1-KO MOVAS MLC with unmanipulated SR-BI expression. Taken together, these major findings suggest that SR-BI expression in MLC of a VSMC origin plays a compensatory role in HDL-mediated cholesterol efflux when ABCG1 expression becomes impaired and provides insight on SR-BI demonstrating anti-atherogenic properties within VSMC/MLC.

miR-33a Expression Attenuates ABCA1-Dependent Cholesterol Efflux and Promotes Macrophage-Like Cell Transdifferentiation in Cultured Vascular Smooth Muscle Cells.

Recent evidence suggests that the majority of cholesterol-laden cells found in atherosclerotic lesions are vascular smooth muscle cells (VSMC) that have transdifferentiated into macrophage-like cells (MLC). Furthermore, cholesterol-laden MLC of VSMC origin have demonstrated impaired ABCA1-dependent cholesterol efflux, but it is poorly understood why this occurs. A possible mechanism which may at least partially be attributed to cholesterol-laden MLC demonstrating attenuated ABCA1-dependent cholesterol efflux is a miR-33a expression, as a primary function of this microRNA is to silence ABCA1 expression, but this has yet to be rigorously investigated. Therefore, the VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells, and we used KO and wild-type (WT) MOVAS cells to delineate any possible proatherogenic role of miR-33a expression in VSMC. When WT and KO MOVAS cells were cholesterol-loaded to convert into MLC, this resulted in the WT MOVAS cells to exhibit impaired ABCA1-dependent cholesterol efflux. In the cholesterol-loaded WT MOVAS MLC, we also observed a delayed restoration of the VSMC phenotype when these cells were exposed to the ABCA1 cholesterol acceptor, apoAI. These results imply that miR-33a expression in VSMC drives atherosclerosis by triggering MLC transdifferentiation via attenuated ABCA1-dependent cholesterol efflux.

Assessing Anti-Adipogenic Effects of Mango Leaf Tea and Mangiferin within Cultured Adipocytes.

Obesity is a condition caused by surplus adipose tissue and is a risk factor for several diet-related diseases. Obesity is a global epidemic that has also been challenging to treat effectively. However, one promoted therapy to safely treat obesity is anti-adipogenic therapeutics. Therefore, identifying potent anti-adipogenic bioactive compounds that can safely be used clinically may effectively treat obesity in humans. Mango leaf has potential medicinal properties due to its many bioactive compounds that may enhance human health. Mangiferin (MGF) is a primary constituent in mango plants, with many health-promoting qualities. Therefore, this study investigated the effect of MGF, and tea brewed with mango leaves in cultured adipocytes. The anti-adipogenic efficacy of mango leaf tea (MLT) and MGF in 3T3-L1 cells were assessed, along with cell viability, triglyceride levels, adiponectin secretion, and glucose uptake analyzed. In addition, changes in the mRNA expression of genes involved in lipid metabolism within 3T3-L1 cells were determined using quantitative real-time PCR. Our results showed while both MLT and MGF increased glucose uptake in adipocytes, only MLT appeared to inhibit adipogenesis, as determined by decreased triglyceride accumulation. We also observed increased secretory adiponectin levels, reduced ACC mRNA expression, and increased FOXO1 and ATGL gene expression in 3T3-L1 cells treated with MLT but not MGF. Together, these results suggest that MLT may exhibit anti-adipogenic properties independent of MGF content.

Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods.

Filamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they are not composed of discrete cells of similar size. This research focused on filamentous fungal biofilms that are representative of those in the built environment. The objective of this study was to develop a rapid method to examine biofilm structure and quantify live (metabolically active/ membrane undamaged) and dead (inactive/ membrane damaged) cells in Aspergillus niger biofilms utilizing a fluorescent probe staining method and confocal laser scanning microscopy (CLSM). For this, we compared two commercially available probe staining kits that have been developed for bacterial and yeast systems. One method utilized the classic cell stain FUN 1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells are fluoresced green. The second method utilized a combination of SYTO9 and propidium iodide (PI), and stains cells based on their membrane morphology. SYTO9 is a green fluorescent stain with the capacity to penetrate the living cell walls, and PI is a red fluorescent stain that can only penetrate dead or dying cells with damaged cell membranes. Following staining, the biofilms were imaged using CLSM and biofilm volumes and thickness were quantified using COMSTAT, a computer program that measures biofilm accumulation from digital image stacks. The results were compared to independent measurements of live-dead cell density, as well as a classic cell viability assay-XTT. The data showed that the combination of SYTO9 and PI is optimal for staining filamentous fungal biofilms.

Comparison of the capillary-channeled polymer (C-CP) fiber spin-down tip approach to traditional methods for the isolation of extracellular vesicles from human urine.

Capillary-channeled polymer fiber (C-CP) solid-phase extraction tips have demonstrated the ability to produce clean and concentrated extracellular vesicle (EV) recoveries from human urine samples in the small EV size range (< 200 nm). An organic modifier-assisted hydrophobic interaction chromatography (HIC) approach is applied in the spin-tip method under non-denaturing conditions-preserving the structure and bioactivity of the recovered vesicles. The C-CP tip method can employ either acetonitrile or glycerol as an elution modifier. The EV recoveries from the C-CP tip method (using both of these solvents) were compared to those obtained using the ultracentrifugation (UC) and polymer precipitation (exoEasy and ExoQuick) EV isolation methods for the same human urine specimen. The biophysical and quantitative characteristics of the recovered EVs using the five isolation methods were assessed based on concentration, size distribution, shape, tetraspanin surface marker protein content, and purity. In comparison to the traditionally used UC method and commercially available polymeric precipitation-based isolation kits, the C-CP tip introduces significant benefits with efficient (< 15 min processing of 12 samples here) and low-cost (< $1 per tip) EV isolations, employing sample volumes (10 µL-1 mL) and concentration (up to 4 × 1012 EVs mL-1) scales relevant for fundamental and clinical analyses. Recoveries of the target vesicles versus matrix proteins were far superior for the tip method versus the other approaches.

Sex ratios at birth in Australia according to mother's country of birth: A national study of all 5 614 847 reported live births 1997-2016.

OBJECTIVES: Son preference and sex selective practices have resulted in a deficit of girls in several countries, primarily across Asia. Emerging evidence indicates that son preference survives migration to Western high-income countries. The objective of this study was to assess male-to-female (M/F) ratios at birth per mother's country of birth in Australia 1997-2016, in total and by parity, and by states/territories and over time. METHODS: Data for this national population-based cross-sectional study were obtained from the National Perinatal Data Collection (NPDC) and included all live births in Australia 1997-2016 (N = 5 614 847). M/F ratios with 95% Confidence Intervals were estimated. RESULTS: The M/F ratio for births to Australian-born mothers was within the expected range (1.03-1.07) regardless of parity and time period. M/F ratios were elevated above the expected range for births to mothers born in China in the total sample (M/F ratio 1.084, 95% confidence interval 1.071-1.097) and at parity 2 (1.175, 1.120-1.231), and for births to mothers born in India at parity 2 (1.146, 1.090-1.204). Parity 2 births were the most consistently male-biased across time. Across states, elevated M/F ratios were identified for both groups in New South Wales (China parity 2: 1.182, 1.108-1.260; India parity 2: 1.182, 1.088-1.285), for births to Chinese-born mothers in Victoria (total births: 1.097, 1.072-1.123; parity 1: 1.115, 1.072-1.159) and Australian Capital Territory (total births: 1.189, 1.085-1.302) and births to Indian-born mothers Western Australia (parity 2: 1.307, 1.122-1.523). CONCLUSIONS: Son preference persists in some immigrant communities after migration to Australia. The consistent pattern of elevated M/F ratios across the larger states indicates that sex imbalances at birth are largely independent of restrictiveness of local abortion laws. Drivers and consequences of son preference in Western high-income settings should be explored to further promote gender equality, and to strengthen support for women who may be vulnerable to reproductive coercion.

Rapid isolation of extracellular vesicles from diverse biofluid matrices via capillary-channeled polymer fiber solid-phase extraction micropipette tips.

Extracellular vesicles (EVs) play essential roles in biological systems based on their ability to carry genetic and protein cargos, intercede in cellular communication and serve as vectors in intercellular transport. As such, EVs are species of increasing focus from the points of view of fundamental biochemistry, clinical diagnostics, and therapeutics delivery. Of particular interest are 30-200 nm EVs called exosomes, which have demonstrated high potential for use in diagnostic and targeted delivery applications. The ability to collect exosomes from patient biofluid samples would allow for comprehensive yet remote diagnoses to be performed. While several exosome isolation methods are in common use, they generally produce low recoveries, whose purities are compromised by concomitant inclusion of lipoproteins, host cell proteins, and protein aggregates. Those methods often work on lengthy timescales (multiple hours) and result in very low throughput. In this study, capillary-channeled polymer (C-CP) fiber micropipette tips were employed in a hydrophobic interaction chromatography (HIC) solid-phase extraction (SPE) workflow. Demonstrated is the isolation of exosomes from human urine, saliva, cervical mucus, serum, and goat milk matrices. This method allows for quick (<15 min) and low-cost (<$1 per tip) isolations at sample volume and time scales relevant for clinical applications. The tip isolation was evaluated using absorbance (scattering) detection, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Exosome purity was assessed by Bradford assay, based on the removal of free proteins. An enzyme-linked immunosorbent assay (ELISA) to the CD81 tetraspanin protein was used to confirm the presence of the known exosomal-biomarker on the vesicles.

MOVAS Cells: A Versatile Cell Line for Studying Vascular Smooth Muscle Cell Cholesterol Metabolism.

Cholesterol metabolism is paramount to cells. Aberrations to cholesterol metabolism affects cholesterol homeostasis, which may impact the risk of several diseases. Recent evidence has suggested that vascular smooth muscle cell (VSMC) cholesterol metabolism may play a role in atherosclerosis. However, there is scant in vitro mechanistic data involving primary VSMC that directly tests how VSMC cholesterol metabolism may impact atherosclerosis. One reason for this lack of data is due to the impracticality of gene manipulation studies in primary VSMC, as cultured primary VSMC become senescent and lose their morphology rapidly. However, there are no immortalized VSMC lines known to be suitable for studying VSMC cholesterol metabolism. The purpose of this study was to determine whether MOVAS cells, a commercially available VSMC line, are suitable to use for studying VSMC cholesterol metabolism. Using immunoblotting and immunofluorescence, we showed that MOVAS cells express ABCA1, ABCG1, and SREBP-2. We also determined that MOVAS cells efflux cholesterol to apoAI and HDL, which indicates functionality of ABCA1/ABCG1. In serum-starved MOVAS cells, SREBP-2 target gene expression was increased, confirming SREBP-2 functionality. We detected miR-33a expression in MOVAS cells and determined this microRNA can silence ABCA1 and ABCG1 via identifying conserved miR-33a binding sites within ABCA1/ABCG1 3'UTR in MOVAS cells. We showed that cholesterol-loading MOVAS cells results in this cell line to transdifferentiate into a macrophage-like cell, which also occurs when VSMC accumulate cholesterol. Our characterization of MOVAS cells sufficiently demonstrates that they are suitable to use for studying VSMC cholesterol metabolism in the context of atherosclerosis.

Role of strengthening during nonoperative treatment of lateral epicondyle tendinopathy.

INTRODUCTION: Lateral epicondyle tendinopathy (LET) is the most common cause of lateral elbow pain. The literature on rehabilitation of the condition encompasses a plethora of interventions with most current evidence indicating that stretches and some form of strengthening are vital components. However, patient outcomes are infrequently reported further than 12 weeks from the start of therapy and it is unclear which components of a home exercise program are necessary to alleviate symptoms up to one year from the initiation of a therapy program. PURPOSE OF THE STUDY: The purpose of the study is to determine if a therapy program with 4 to 6 visits spaced out over 12 weeks focusing on self-management and strengthening is more effective in reducing pain and improving function long term than the same program without strengthening, for individuals with LET. STUDY DESIGN: This is a randomized controlled trial. METHODS: Ninety-four patients were randomly allocated into two groups: both groups received the interventions of education in pertinent pathoanatomy, stretching, pain management through rest and icing, and activity modification. Group 1 (n = 38) was also provided with a strengthening component to the home exercise program, whereas group 2 did not (n = 21). Our primary outcome measure was pain at rest and pain with activity; our secondary measure was the level of functional impairment as measured by the quick disabilities of arm shoulder and hand. Outcome measurements were assessed at baseline, 6, 12, 24, and 52 weeks after initiation of therapy. RESULTS: Both groups demonstrated statistically significant improvement with a moderate to large effect size in pain and function scores when compared with previous time point at 6, 12, and 24 weeks. Pain continued to decrease for both groups from 24 weeks to 52 weeks, but interestingly, there was a significant increase with moderate effect size in the quick disabilities of arm shoulder and hand score at 52 weeks when compared with week 24. No statistically significant difference was found between the two groups at any time point up to 52 weeks from the start of therapy. CONCLUSIONS: This study demonstrates that a therapy program consisting of a low number of visits spaced out over 12 weeks based on education, stretches, activity modification, and pain management techniques is effective at reducing pain and increasing function in patients with LET. The addition of strengthening to this program did not improve outcomes. The therapy approach used in this study is consistent with the International Classification of Function guidelines and focuses on engaging patients in self-management of the condition through patient education and self-empowerment.

Solid-phase extraction of exosomes from diverse matrices via a polyester capillary-channeled polymer (C-CP) fiber stationary phase in a spin-down tip format.

Exosomes, a subset of the extracellular vesicle (EV) group of organelles, hold great potential for biomarker detection, therapeutics, disease diagnosis, and personalized medicine applications. The promise and potential of these applications are hindered by the lack of an efficient means of isolation, characterization, and quantitation. Current methods for exosome and EV isolation (including ultracentrifugation, microfiltration, and affinity-based techniques) result in impure recoveries with regard to remnant matrix species (e.g., proteins, genetic material) and are performed on clinically irrelevant time and volume scales. To address these issues, a polyethylene terephthalate (PET) capillary-channeled polymer (C-CP) fiber stationary phase is employed for the solid-phase extraction (SPE) of EVs from various matrices using a micropipette tip-based format. The hydrophobic interaction chromatography (HIC) processing and a spin-down workflow are carried out using a table-top centrifuge. Capture and subsequent elution of intact, biologically active exosomes are verified via electron microscopy and bioassays. The performance of this method was evaluated by capture and elution of exosome standards from buffer solution and three biologically relevant matrices: mock urine, reconstituted non-fat milk, and exosome-depleted fetal bovine serum (FBS). Recoveries were evaluated using UV-Vis absorbance spectrophotometry and ELISA assay. The dynamic binding capacity (50%) for the 1-cm-long (~ 5 µL bed volume) tips was determined using a commercial exosome product, yielding a value of ~ 7 × 1011 particles. The novel C-CP fiber spin-down tip approach holds promise for the isolation of exosomes and other EVs from various matrices with high throughput, low cost, and high efficiency. Graphical abstract.

Improving Patient Throughput With an Electronic Nursing Handoff Process in an Academic Medical Center: A Rapid Improvement Event Approach.

OBJECTIVE: Rush University Medical Center nursing leadership undertook a process improvement project to revamp nursing handoff during unit transfer with the goal of improving patient throughput. The aim was to decrease assign-to-occupy time, the duration from bed assignment to bed occupancy. BACKGROUND: There was a lengthy lag time in admitting/transferring patients, leading to delays in patient throughput and potential threats to patient safety. In fiscal year 2016, assign-to-occupy time averaged 97 minutes. The goal was to decrease that time to 60 minutes or less. METHODS: Process improvement leaders held a rapid improvement event to determine viable solutions. A team then standardized handoff workflow; created an electronic tool, virtually eliminating verbal report; and implemented a new handoff process. RESULTS: Assign-to-occupy time at 1 year after go-live averaged 55 minutes, and it has been staying less than 60 minutes since the implementation. CONCLUSIONS: Key success strategies included engaging stakeholders during the rapid improvement event, imploring frontline nurses to create and promote the revised process to facilitate staff engagement, and leveraging electronic health records.

Ergot alkaloid exposure during gestation alters: 3. Fetal growth, muscle fiber development, and miRNA transcriptome1.

The objective of this study was to assess how exposure to ergot alkaloids during 2 stages of gestation alters fetal growth, muscle fiber formation, and miRNA expression. Pregnant ewes (n = 36; BW = 83.26 ± 8.14 kg; 4/group; 9 groups) were used in a 2 × 2 factorial arrangement with 2 tall fescue seed treatments [endophyte-infected (E+) vs. endophyte-free (E-)] fed during 2 stages of gestation (MID, days 35 to 85 vs. LATE, days 86 to 133), which created 4 possible treatments (E-/E-, E+/E-, E-/E+, or E+/E+). Ewes were individually fed a total mixed ration containing E+ or E- fescue seed according to treatment assignment. Terminal surgeries were conducted on day 133 of gestation for the collection of fetal measurements and muscle samples. Data were analyzed as a 2 × 2 factorial with fescue treatment, stage of gestation, and 2-way interaction as fixed effects. Fetuses exposed to E+ seed during LATE gestation had reduced (P = 0.0020) fetal BW by 10% compared with E- fetuses; however, fetal body weight did not differ (P = 0.41) with E+ exposure during MID gestation. Fetuses from ewes fed E+ seed during MID and LATE gestation tended to have smaller (P = 0.058) kidney weights compared with E- fetuses. Liver weight was larger (P = 0.0069) in fetuses fed E- during LATE gestation compared with E+. Fetal brain weight did not differ by fescue treatment fed during MID (P = 0.36) or LATE (P = 0.40) gestation. The percentage of brain to empty body weight (EBW) was greater (P = 0.0048) in fetuses from ewes fed E+ fescue seed during LATE gestation, which is indicative of intrauterine growth restriction (IUGR). Primary muscle fiber number was lower (P = 0.0005) in semitendinosus (STN) of fetuses exposed to E+ during MID and/or LATE gestation compared with E-/E-. miRNA sequencing showed differential expression (P < 0.010) of 6 novel miRNAs including bta-miR-652_R+1, mdo-miR-22-3p, bta-miR-1277_R-1, ppy-miR-133a_L+1_1ss5TG, hsa-miR-129-1-3p, and ssc-miR-615 in fetal STN muscle. These miRNA are associated with glucose transport, insulin signaling, intracellular ATP, hypertension, or adipogenesis. This work supports the hypothesis that E+ tall fescue seed fed during late gestation reduces fetal weight and causes asymmetrical growth, which is indicative of IUGR. Changes in primary fiber number and miRNA of STN indicate that exposure to E+ fescue fed during MID and LATE gestation alters fetal muscle development that may affect postnatal muscle growth and meat quality.

Exposure to arsenic during embryogenesis impairs olfactory sensory neuron differentiation and function into adulthood.

Arsenic is a contaminant of food and drinking water. Epidemiological studies have reported correlations between arsenic exposure and neurodevelopmental abnormalities, such as reduced sensory functioning, while in vitro studies have shown that arsenic reduces neurogenesis and alters stem cell differentiation. The goal of this study was assess whether arsenic exposure during embryogenesis reduced olfactory stem cell function and/or numbers, and if so, whether those changes persist into adulthood. Killifish (Fundulus heteroclitus) embryos were exposed to 0, 10, 50 or 200 ppb arsenite (AsIII) until hatching, and juvenile fish were raised in clean water. At 0, 2, 4, 8, 16, 28 and 40 weeks of age, odorant response tests were performed to assess specific olfactory sensory neuron (OSN) function. Olfactory epithelia were then collected for immunohistochemical analysis of stem cell (Sox2) and proliferating cell numbers (PCNA), as well as the number and expression of ciliated (calretinin) and microvillus OSNs (Gαi3) at 0, 4, 16 and 28 weeks. Odorant tests indicated that arsenic exposure during embryogenesis increased the start time of killifish responding to pheromones, and this altered start time persisted to 40 weeks post-exposure. Response to the odorant taurocholic acid (TCA) was also reduced through week 28, while responses to amino acids were not consistently altered. Immunohistochemistry was used to determine whether changes in odorant responses were correlated to altered cell numbers in the olfactory epithelium, using markers of proliferating cells, progenitor cells, and specific OSNs. Comparisons between response to pheromones and PCNA + cells indicated that, at week 0, both parameters in exposed fish were significantly reduced from the control group. At week 28, all exposure are still significantly different than control fish, but now with higher PCNA expression coupled with reduced pheromone responses. A similar trend was seen in the comparisons between Sox2-expressing progenitor cells and response to pheromones, although Sox2 expression in the 28 week-old fish only recovers back to the level of control fish rather than being significantly higher. Comparisons between calretinin expression (ciliated OSNs) and response to TCA demonstrated that both parameters were reduced in the 200 ppb arsenic-exposed fish in at weeks 4, 16, and 28. Correlations between TCA response and the number of PCNA + cells revealed that, at 28 weeks of age, all arsenic exposure groups had reductions in response to TCA, but higher PCNA expression, similar to that seen with the pheromones. Few changes in Gαi3 (microvillus OSNs) were seen. Thus, it appears that embryonic-only exposure to arsenic has long-term reductions in proliferation and differentiation of olfactory sensory neurons, leading to persistent effects in their function.

Factors associated with induced abortion over time: secondary data analysis of five waves of the Australian Longitudinal Study on Women's Health.

OBJECTIVE: A trend analysis of associations with induced abortion. METHODS: Secondary analysis of the 1973/78 cohort of the Australian Longitudinal Study of Women's Health of women responding to two or more consecutive surveys out of five (N=9,042), using generalised estimating equations. RESULTS: New abortions dropped from 7% to 2% at surveys 4 and 5. By survey 5, 16% of respondents reported abortions, only 2% of them new. Women aged in their twenties were more likely to terminate a pregnancy if they reported less-effective contraceptives (aOR2.18 CI 1.65-2.89); increased risky drinking (aOR1.65 CI 1.14-2.38); illicit drugs ≤12 months (aOR3.09 CI 2.28-4.19); or recent partner violence (aOR2.42 CI 1.61-3.64). By their thirties, women were more likely to terminate if they reported violence (aOR2.16 CI 1.31-3.56) or illicit drugs <12 months (aOR2.69 CI 1.77-4.09). Women aspiring to be fully- (OR1.58 CI 1.37-1.83) or self-employed (OR1.28 CI 1.04-1.57), with no children (OR1.41 CI 1.14-1.75) or further educated (OR 2.08 CI 1.68-2.57) were more likely to terminate than other women. CONCLUSIONS: Abortion remains strongly associated with factors affecting women's control over reproductive health such as partner violence and illicit drug use. Implications for public health: Healthcare providers should inquire about partner violence and illicit drug use among women seeking abortion, support women experiencing harm and promote effective contraception.

Exosome isolation and purification via hydrophobic interaction chromatography using a polyester, capillary-channeled polymer fiber phase.

Extracellular vesicles, including microvesicles and exosomes, are lipidic membrane-derived vesicles that are secreted by most cell types. Exosomes, one class of these vesicles that are 30-100 nm in diameter, hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. For exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Most current exosome isolation methods have practical problems including being too time-consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from diverse matrices of practical concern. Initial results demonstrate the ability to isolate extracellular vesicles enriched in exosomes with comparable yields and size distributions on a much faster time scale when compared to traditional isolation methods. As a demonstration of the potential analytical utility of the approach, extracellular vesicle recoveries from cell culture milieu and a mock urine matrix are presented. The potential for scalable separations covering submilliliter spin-down columns to the preparative scale is anticipated.

Flexor tendon rehabilitation in the 21st century: A systematic review.

STUDY DESIGN: Systematic review. INTRODUCTION: The rehabilitation of patients following flexor tendon injury has progressed from immobilization to true active flexion with the addition of wrist motion over the last 75 years. PURPOSE OF THE STUDY: This review specifically intended to determine whether there is evidence to support one type of exercise regimen, early passive, place and hold, or true active, as superior for producing safe and maximal range of motion following flexor tendon repair. METHODS: The preferred reporting items for systematic review and meta-analysis (PRISMA-P 2015) checklist was utilized to format the review. Both reviewers collaborated on all aspects of the research, including identifying inclusion/exclusion factors, search terms, reading and scoring articles, and authoring the paper. Articles were independently scored by each reviewer using the Structured Effectiveness Quality Evaluation Scale (SEQES). RESULTS: A total of nine intervention studies that included a rehabilitative comparison group were systematically reviewed: one pediatric, four comparing passive flexion protocols to place and hold flexion, and four comparing true active flexion to passive and/or place and hold flexion. DISCUSSION: This review provides moderate to strong evidence that place and hold exercises provide better outcomes than passive flexion protocols for patients with two to six-strand repairs. The studies included in this review suffered from methodological limitations including short timeframes for follow-up, unequal group distribution, and limited attention to repair site strength. CONCLUSIONS: Based on a lack of superior benefits following true active motion regimens, there is not sufficient evidence to support true active motion as an effective or preferable choice for flexor tendon rehabilitation at this time.

Women's views and experiences of publicly-funded homebirth programs in Victoria, Australia: A cross-sectional survey.

BACKGROUND: It is critical women's voices are heard if there is to be more widespread implementation of midwifery-led continuity models. Publicly-funded homebirth is one such model, yet there has been limited systematic evaluation from the women's perspective. AIM: Examine women's experiences of and views about the two publicly-funded homebirth programs in Victoria, Australia. METHODS: A cross-sectional design was used. All eligible women enrolled in the two pilot homebirth programs in metropolitan Melbourne whose infants were eight weeks of age or more during the evaluation period were invited to participate in a postal survey. A structured questionnaire was used, with some open-ended questions to enable extra comments. We explored women's reasons for choosing homebirth; views of care; experience of labour and birth; views on transfer; and overall experience of the homebirth program. Data were analysed using descriptive statistics. Simple thematic analysis was used for open-ended questions. FINDINGS: The survey response rate was 71% (96/136). A high percentage of women rated their care as 'Very good': pregnancy 81%; labour and birth 90%; and the early postpartum period 83%. Women reported low levels of anxiety during labour and birth, were able to express their feelings, felt in control, and coped physically and emotionally better than they had expected. They felt well supported by midwives and overall reported very positive experiences of the homebirth programs. CONCLUSIONS: These two publicly-funded homebirth pilot programs demonstrated very positive care ratings by women. These findings, along with the clinical outcomes (reported separately), support the continuation and expansion of the program.

Male-biased sex ratios in Australian migrant populations: a population-based study of 1 191 250 births 1999-2015.

Background: The naturally occurring male-to-female (M/F) ratio at birth is 1.05. Higher ratios found primarily in countries across Asia have been attributed to prenatal sex selection due to son preference. There is growing evidence that sex-selective practices continue following migration; however, little is known about these practices following migration to Australia. Methods: In this population-based study we assessed M/F ratios at birth per mother's country of birth for all registered births 1999-2015 in Victoria, Australia (n = 1 191 250). We also compared the M/F ratio among births to mothers born elsewhere to that of mothers born in Australia, stratified by time period and parity. Results: Compared with the naturally occurring M/F ratio as well as to the M/F ratio among births to mothers born in Australia, there was an increased ratio of male births to mothers born in India, China and South-East Asia, particularly at higher parities and in more recent time periods (elevated M/F ratios ranged from 1·079 to 1·248, relative risks of male birth ranged from 1·012 to 1·084 with confidence intervals between 1·001 and 1·160 and P-values between 0·005 and 0·039). The most male-biased sex ratios were found among multiple births to Indian-born mothers, and parity of two or more births to Indian and Chinese-born mothers in 2011-15. Conclusions: The male-biased sex ratios observed in this study indicate that prenatal sex selection may be continuing following migration to Australia from countries where these practices have been documented. The excess of males among multiple births raises the question as to what role assisted reproduction plays. Findings also suggest that systematic discrimination against females starts in the womb.