Search

Quantitative micro-elastography (QME) is a compression-based optical coherence elastography technique enabling the estimation of tissue mechanical properties on the micro-scale. QME utilizes a compliant layer as an optical stress sensor, placed between an imaging window and tissue, providing quantitative estimation of elasticity. However, the implementation of the layer is challenging and introduces unpredictable friction conditions at the contact boundaries, deteriorating the accuracy and reliability of elasticity estimation. This has largely limited the use of QME to ex vivo studies and is a barrier to clinical translation. In this work, we present a novel implementation by affixing the stress sensing layer to the imaging window and optimizing the layer thickness, enhancing the practical use of QME for in vivo applications by eliminating the requirement for manual placement of the layer, and significantly reducing variations in the friction conditions, leading to substantial improvement in the accuracy and repeatability of elasticity estimation. We performed a systematic validation of the integrated layer, demonstrating >30% improvement in sensitivity and the ability to provide mechanical contrast in a mechanically heterogeneous phantom. In addition, we demonstrate the ability to obtain accurate estimation of elasticity (<6% error compared to <14% achieved using existing QME) in homogeneous phantoms with mechanical properties ranging from 40 to 130 kPa. Furthermore, we show the integrated layer to be more robust, exhibiting increased temporal stability, as well as improved conformity to variations in sample surface topography, allowing for accurate estimation of elasticity over acquisition times 3× longer than current methods. Finally, when applied to ex vivo human breast tissue, we demonstrate the ability to distinguish between healthy and diseased tissue features, such as stroma and cancer, confirmed by co-registered histology, showcasing the potential for routine use in biomedical applications., Competing Interests: B.F.K. reports funding from OncoRes Medical during the conduct of the study and other support from OncoRes Medical: ownership interest (including patents) outside the submitted work; in addition, B.F.K. is an inventor on a patent for a device and a method for evaluating a mechanical property of a material Intl. Pub. No. WO/2016/ 119011, Appl. No. PCT/AU2016/212695, Priority date: 30/01/2015 issued to OncoRes Medical. No disclosures were reported by the other authors., (© 2024 Author(s).)

Introduction: The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum)., Methods: Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 10 5 TCID 50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24 h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR)., Results: No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR., Discussion: This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yen, Nelli, Twu, Mora-Díaz, Castillo, Sitthicharoenchai and Giménez-Lirola.)

The ability to control gene expression in mammalian cells is crucial for safe and efficacious gene therapies and for elucidating gene functions. Current gene regulation systems have limitations such as harmful immune responses or low efficiency. We describe the pA regulator, an RNA-based switch that controls mammalian gene expression through modulation of a synthetic polyA signal (PAS) cleavage introduced into the 5' UTR of a transgene. The cleavage is modulated by a 'dual-mechanism'-(1) aptamer clamping to inhibit PAS cleavage and (2) drug-induced alternative splicing that removes the PAS, both activated by drug binding. This RNA-based methodology circumvents the immune responses observed in other systems and achieves a 900-fold induction with an EC 50 of 0.5 µg ml - 1 tetracycline (Tc), which is well within the FDA-approved dose range. The pA regulator effectively controls the luciferase transgene in live mice and the endogenous CD133 gene in human cells, in a dose-dependent and reversible manner with long-term stability., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The WHO currently recommends dolutegravir (DTG)-based ART for persons living with HIV infection in resource-limited-settings (RLS). To expand access to testing for HIV drug resistance (DR) to DTG in RLS, we developed probes for use in the oligonucleotide ligation assay (OLA)-Simple, a near-point of care HIV DR kit. Genotypic data from clinical trials and case reports were used to determine the mutations in HIV-1 integrase critical to identifying individuals with DTG-resistance at virologic failure of DTG-based ART. Probes to detect G118R, Q148H/K/R, N155H and R263K in HIV-1 subtypes A, B, C, D and CRF01&#95;AE were designed using sequence alignments from the Los Alamos database and validated using 61 clinical samples of HIV-1 subtypes A, B, C, D, CRF01&#95;AE genotyped by PacBio ( n = 15) or Sanger ( n = 46). Initial OLA probes failed to ligate for 16/244 (6.5%) codons (9 at G118R and 7 at Q148H/K/R). Probes revised to accommodate polymorphisms interfering with ligation at codons G118R and Q148R reduced indeterminates to 3.7% (5 at G118R and 4 at Q148H/K/R) and detected DTG-mutations with a sensitivity of 96.5% and 100% specificity. These OLA DTG resistance probes appear highly sensitive and specific across HIV-1 subtypes common in RLS with high burden of HIV infection.

In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world., (© 2024 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

The second decade of the twenty-first century witnessed a new challenge in the handling of microscopy data. Big data, data deluge, large data, data compliance, data analytics, data integrity, data interoperability, data retention and data lifecycle are terms that have introduced themselves to the electron microscopy sciences. This is largely attributed to the booming development of new microscopy hardware tools. As a result, large digital image files with an average size of one terabyte within one single acquisition session is not uncommon nowadays, especially in the field of cryogenic electron microscopy. This brings along numerous challenges in data transfer, compute and management. In this review, we will discuss in detail the current state of international knowledge on big data in contemporary electron microscopy and how big data can be transferred, computed and managed efficiently and sustainably. Workflows, solutions, approaches and suggestions will be provided, with the example of the latest experiences in Australia. Finally, important principles such as data integrity, data lifetime and the FAIR and CARE principles will be considered., (© 2023. The Author(s).)

Background and Aims: Fatty streaks initiating the formation of atheromatous plaque appear in the tunica intima. The tunica media is not known to be a nidus for lipid accumulation initiating atherogenesis. We assessed changes to the tunica media in response to a micro-injury produced in the pig aorta. In addition, we assessed human carotid endarterectomy plaques for indication of atheroma initiation in the tunica media., Methods: Three healthy landrace female pigs underwent laparotomy to inject autologous blood and create micro-hematomas at 6 sites within the tunica media of the infrarenal abdominal aorta. These pigs were fed a high-fat diet (HFD) for 4-12 weeks. Post-mortem aortas from all pigs, including a control group of healthy pigs, were serially stained to detect lipid deposits, vasa vasora (VV), immune cell infiltration and inflammatory markers, as well as changes to the vascular smooth muscle cell (vSMC) compartment. Moreover, 25 human carotid endarterectomy (CEA) specimens were evaluated for their lipid composition in the tunica media and intima., Results: High lipid clusters, VV density, and immune cell infiltrates were consistently observed at 5 out of 6 injection sites under prolonged hyperlipidemia. The hyperlipidemic diet also affected the vSMC compartment in the tunica media adjacent to the tunica adventitia, which correlated with VV invasion and immune cell infiltration. Analysis of human carotid specimens post-CEA indicated that 32% of patients had significantly greater atheroma in the tunica media than in the arterial intima., Conclusion: The arterial intima is not the only site for atherosclerosis initiation. We show that injury to the media can trigger atherogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Belhoul-Fakir, Wu, Yeow, Musk, Kershaw, Ingley, Zhao, Reid, Lagat, Evans, Thompson, Brown, Hamzah and Jansen.)

Neuroendocrine tumors are a rare type of cancer found in hormone-producing cells throughout the body. Research on disease-specific patient education assessments in this population is lacking. We previously demonstrated the feasibility and validity of NET VITALS, a patient-centered self-assessment designed to improve patients' knowledge of their neuroendocrine tumor diagnosis/treatment and facilitate communication with their physician. In this report, we provide a brief overview of patient assessments that have been used for patients with neuroendocrine tumors. We summarize NET VITALS and present a proposed infrastructure for its implementation into standard clinical care in both academic and community practice settings at City of Hope. Incorporating NET VITALS into standard of care treatment for patients with neuroendocrine tumors may improve patients' overall clinical care experience.

This study characterized the susceptibility and dynamic of porcine deltacoronavirus infection in grower pigs under experimental conditions using a combination of syndromic and laboratory assessments. Seven-week-old conventional pigs ( n = 24) were randomly distributed into PDCoV- ( n = 12) and mock-inoculated ( n = 12) groups. Serum was collected at -7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42 days post-inoculation (DPI) to evaluate viremia (RT-qPCR) and antibody response (S1-based ELISA). Viral shedding and potential infectivity were determined using pen-based oral fluids and feces collected every other day between DPI 0 and 42. Pigs showed no clinical signs or viremia throughout the study. Active virus shedding was detected in feces (6-22 DPI) and oral fluids (2-30 DPI), peaking at DPI 10. IgG was first detected at DPI 10, being statistically significant after DPI 14 and increasing thereafter, coinciding with the progressive resolution of the infection. Likewise, a significant increase in proinflammatory IL-12 was detected between DPI 10 and 21 in PDCoV-inoculated pigs, which could enhance innate resistance to PDCoV infection. This study demonstrated that active surveillance based on systematic sampling and laboratory testing combining molecular and serological tools is critical for the accurate detection of subclinical circulation of PDCoV in pigs after weaning.

The MyScope online training resource has been supporting the global microscopy community since 2011. Since then, it has expanded from six to twelve modules, growing in line with emerging and increasingly important areas of microscopy such as FIB and Cryo-EM. By sharing the expertise and commitment of Microscopy Australia staff to effective and efficient microscopy training, MyScope is being maintained as an up-to-date training tool. Online teaching and training resources such as MyScope have proven to be more important than ever in helping facilities and course leaders manage pandemic-impacted teaching, training and access regimes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae and genus Deltacoronavirus , is a major enteric pathogen in swine. Accurate PDCoV diagnosis relying on laboratory testing and antibody detection is an important approach. This study evaluated the potential of the receptor-binding subunit of the PDCoV spike protein (S1), generated using a mammalian expression system, for specific antibody detection via indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at day post-inoculation (DPI) -7 to 42, from pigs ( n = 83) experimentally inoculated with different porcine coronaviruses (PorCoV). The diagnostic sensitivity of the PDCoV S1-based ELISA was evaluated using serum samples ( n = 72) from PDCoV-inoculated animals. The diagnostic specificity and potential cross-reactivity of the assay was evaluated on PorCoV-negative samples ( n = 345) and samples collected from pigs experimentally inoculated with other PorCoVs ( n = 472). The overall diagnostic performance, time of detection, and detection rate over time varied across different S/P cut-offs, estimated by Receiver Operating Characteristic (ROC) curve analysis. The higher detection rate in the PDCoV group was observed after DPI 21. An S/P cut-off of 0.25 provided 100% specificity with no serological cross-reactivity against other PorCoV. These results support the use of S1 protein-based ELISA for accurate detection of PDCoV infections, transference of maternal antibodies, or active surveillance.

Introduction Although cancer most directly affects the patient, its impact is also widely recognized to extend to those who are caring for the patient. Cancer patient caregivers endure psychological distress, have high levels of depression, and report isolation and strain. Research on targeted caregiver interventions is limited. This case report examines the use of health and wellness coaching (HWC) with a caregiver of a patient with neuroendocrine tumors, a rare, insidious type of cancer. Case Presentation We present the first known case report on using HWC with a 44-year-old woman adult cancer patient caregiver who was caring for a patient with neuroendocrine tumors. The patient had a chronically elevated body mass index, cholesterol, and stage 2 hypertension. Her primary care physician had prescribed weight loss medication (naltrexone/bupropion), which the patient hesitated to take and wanted to try HWC instead. The 10-session intervention targeted multiple components of health, including blood pressure, body mass index, cholesterol, weight loss, stress management, relationship success, and vocational progress. Outcomes were followed over a 1-year period. Conclusion This case demonstrates multiple unique aspects of the HWC process that support successful, sustainable behavioral change. The case patient's success suggests HWC may be effective in supporting beneficial physical and psychosocial outcomes with an adult cancer caregiver and should be considered a viable option for promoting health in caregivers.

Background: An endotracheal tube cuff pressure between 20 and 30 cmH2O is recommended to prevent ventilator-associated respiratory infection (VARI). We aimed to evaluate whether continuous cuff pressure control (CPC) was associated with reduced VARI incidence compared with intermittent CPC., Methods: We conducted a multicenter open-label randomized controlled trial in intensive care unit (ICU) patients within 24 hours of intubation in Vietnam. Patients were randomly assigned 1:1 to receive either continuous CPC using an automated electronic device or intermittent CPC using a manually hand-held manometer. The primary endpoint was the occurrence of VARI, evaluated by an independent reviewer blinded to the CPC allocation., Results: We randomized 600 patients; 597 received the intervention or control and were included in the intention to treat analysis. Compared with intermittent CPC, continuous CPC did not reduce the proportion of patients with at least one episode of VARI (74/296 [25%] vs 69/301 [23%]; odds ratio [OR] 1.13; 95% confidence interval [CI] .77-1.67]. There were no significant differences between continuous and intermittent CPC concerning the proportion of microbiologically confirmed VARI (OR 1.40; 95% CI .94-2.10), the proportion of intubated days without antimicrobials (relative proportion [RP] 0.99; 95% CI .87-1.12), rate of ICU discharge (cause-specific hazard ratio [HR] 0.95; 95% CI .78-1.16), cost of ICU stay (difference in transformed mean [DTM] 0.02; 95% CI -.05 to .08], cost of ICU antimicrobials (DTM 0.02; 95% CI -.25 to .28), cost of hospital stay (DTM 0.02; 95% CI -.04 to .08), and ICU mortality risk (OR 0.96; 95% CI .67-1.38)., Conclusions: Maintaining CPC through an automated electronic device did not reduce VARI incidence., Clinical Trial Registration: NCT02966392., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objectives: There is a lack of effective patient education regarding diagnosis/treatment of neuroendocrine tumors (NETs), possibly related to their rare incidence., Methods: In this cross-sectional survey study, NET patients attending the 2019 Annual Los Angeles NET Education Conference were approached to complete NET VITALS, a self-assessment tool gauging patients' perception/awareness of their NET diagnosis/treatment, and a satisfaction survey. Feasibility of NET VITALS, patient satisfaction with NET VITALS, and patients' perception/awareness of their NET diagnosis/treatment were evaluated., Results: This analysis included 68 patients (median age, 63 years; 47.1% gastrointestinal NETs; 88.2% metastatic disease). Participation was 88.3% (68/77), with a median of 85.7% of items completed (range, 61.9%-100.0%). More than 30% of the patients answered "Don't know/Not familiar"/left blank questions related to tumor characteristics, years of symptoms, and liver-directed therapies. In addition, 69.5% of the patients did not feel sufficient information about NETs was provided at diagnosis. Overall, 67.8% of the patients felt that NET VITALS provides topics to discuss with providers and 76.3% would recommend NET VITALS to others., Conclusions: NET VITALS is a feasible and acceptable self-assessment tool to potentially help patients improve communication about their NET diagnosis/treatment with their physician. Further studies will examine NET VITALS' generalizability and discuss its incorporation into clinical care., Competing Interests: D.L. reports research funding to his institution from AstraZeneca and Brooklyn ImmunoTherapeutics. He serves as a consultant and has received honoraria from Adagene, Advanced Accelerator Applications, Bayer, Coherus, Eisai, Exelixis, Genentech, Ipsen Biopharmaceuticals, Lexicon, Merck, MiNA Therapeutics, QED, Servier, Sun Pharma, Taiho, and TerSera Therapeutics, all outside the submitted work. The remaining authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

One common genetic alteration in cancer is gene fusion resulting from chromosomal translocations. The mechanisms that create such oncogenic fusion genes are not well understood. Previously, we provided the direct evidence that expression of a designed chimeric RNA can drive the formation of TMPRSS2-ERG gene fusion. Central to this RNA-mediated gene fusion mechanism is a proposed three-way junction formed by RNA/DNA hybrid and the intergenic DNA stem formed by target genes. In this study, we determined the important parameters for chimeric RNA-mediated gene fusion using TMPRSS2-ERG fusion gene as the model. Our results indicate that both the chimeric RNA lengths and the sizes of unpaired bulges play important roles in inducing TMPRSS2-ERG gene fusion. The optimal length of unpaired bulges was about 35 nt, while the optimal chimeric RNA length was about 50 nt for targeting. These observations were consistent regardless of the target locations within TMPRSS2 and ERG genes. These empirically determined parameters provide important insight for searching cellular RNAs that may initiate oncogenic fusion genes. The knowledge could also facilitate the development of useful genomic technology for manipulating mammalian genomes.

Oncogenic fusion genes as the result of chromosomal rearrangements are important for understanding genome instability in cancer cells and developing useful cancer therapies. To date, the mechanisms that create such oncogenic fusion genes are poorly understood. Previously we reported an unappreciated RNA-driven mechanism in human prostate cells in which the expression of chimeric RNA induces specified gene fusions in a sequence-dependent manner. One fundamental question yet to be addressed is whether such RNA-driven gene fusion mechanism is generalizable, or rather, a special case restricted to prostate cells. In this report, we demonstrated that the expression of designed chimeric RNAs in human endometrial stromal cells leads to the formation of JAZF1-SUZ12, a cancer fusion gene commonly found in low-grade endometrial stromal sarcomas. The process is specified by the sequence of chimeric RNA involved and inhibited by estrogen or progesterone. Furthermore, it is the antisense rather than sense chimeric RNAs that effectively drive JAZF1-SUZ12 gene fusion. The induced fusion gene is validated both at the RNA and the genomic DNA level. The ability of designed chimeric RNAs to drive and recapitulate the formation of JAZF1-SUZ12 gene fusion in endometrial cells represents another independent case of RNA-driven gene fusion, suggesting that RNA-driven genomic recombination is a permissible mechanism in mammalian cells. The results could have fundamental implications in the role of RNA in genome stability, and provide important insight in early disease mechanisms related to the formation of cancer fusion genes., Competing Interests: The authors have declared that no competing interests exist.

The ability of SARS-CoV to infect different species, including humans, dogs, cats, minks, ferrets, hamsters, tigers, and deer, pose a continuous threat to human and animal health. Pigs, though closely related to humans, seem to be less susceptible to SARS-CoV-2. Former in vivo studies failed to demonstrate clinical signs and transmission between pigs, while later attempts using a higher infectious dose reported viral shedding and seroconversion. This study investigated species-specific cell susceptibility, virus dose-dependent infectivity, and infection kinetics, using primary human (HRECs) and porcine (PRECs) respiratory epithelial cells. Despite higher ACE2 expression in HRECs compared to PRECs, SARS-CoV-2 infected, and replicated in both PRECs and HRECs in a dose-dependent manner. Cytopathic effect was particularly more evident in PRECs than HRECs, showing the hallmark morphological signs of apoptosis. Further analysis confirmed an early and enhanced apoptotic mechanism driven through caspase 3/7 activation, limiting SARS-CoV-2 propagation in PRECs compared to HRECs. Our findings shed light on a possible mechanism of resistance of pigs to SARS-CoV-2 infection, and it may hold therapeutic value for the treatment of COVID-19., (© 2021. The Author(s).)

AMPA receptors (AMPARs) mediate the majority of excitatory neurotransmission. Their surface expression, trafficking, gating, and pharmacology are regulated by auxiliary subunits. Of the two types of TARP auxiliary subunits, type I TARPs assume activating roles, while type II TARPs serve suppressive functions. We present cryo-EM structures of GluA2 AMPAR in complex with type II TARP γ5, which reduces steady-state currents, increases single-channel conductance, and slows recovery from desensitization. Regulation of AMPAR function depends on its ligand-binding domain (LBD) interaction with the γ5 head domain. GluA2-γ5 complex shows maximum stoichiometry of two TARPs per AMPAR tetramer, being different from type I TARPs but reminiscent of the auxiliary subunit GSG1L. Desensitization of both GluA2-GSG1L and GluA2-γ5 complexes is accompanied by rupture of LBD dimer interface, while GluA2-γ5 but not GluA2-GSG1L LBD dimers remain two-fold symmetric. Different structural architectures and desensitization mechanisms of complexes with auxiliary subunits endow AMPARs with broad functional capabilities., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

During surveys conducted on freshwater fungi from Vietnam, a new aquatic anamorphic fungus was isolated from decaying leaves in Vietnam. The fungus produced phialidic, swollen conidiophore and conidiogenous cells with one-celled conidia with four very tiny lobes. Based on its conidial development and other morphological characteristics, we confirmed that this is a novel fungus and described it as P. ambigua sp. nov. Phylogenetic analyses of P. ambigua based on the LSU nrDNA sequences showed that this fungus clusters in a single clade with the two known Polylobatispora spp. with 97% bootstrap value which is sister with Pseudoprobiscisporaceae . The Polylobatispora clade is nested in the Attracrosporales , Sordariomycetes . Furthermore, in this study two known species of Polylobatispora ( P. deltoidea and P. quinquecornuta ) were also isolated and illustrated., Competing Interests: The authors declare no conflicts of interest. All the experiments undertaken in this study complied with the current laws of the country where they were performed., (2021, by The Mycological Society of Japan, © 2021 Her Majesty the Queen in Right of Canada.)

Background: Platelet transfusion is required to treat haemo-oncology or trauma patients. Platelet apheresis (PA) performed with apheresis equipment has increased rapidly in recent years. Leucocyte-reduced platelet apheresis (LRPA) can reduce the risk of platelet refractoriness and febrile nonhemolytic transfusion reactions (FNHTRs) for transfusion. Accordingly, this study aimed to investigate and compare the platelet metabolic and functional responses between PA performed with Haemonetics and LRPA performed with Trima Accel cell separator., Methods: The qualities of platelets collected through PA and LRPA were evaluated in terms of visual appearance, morphology, platelet-aggregation changes, metabolic activities, and bacterium-screening test during 5-day storage. Statistical analyses included two-sample t-test and generalised estimating equation(GEE) method., Results: During 5-day storage in LRPA, residual leucocytes were all <1.0×10 6 , and the parameters of platelet function were as follows: platelet aggregated to agonists such as adenosine 5'-diphosphate (ADP) and collagen, and the extent of shape change and pO 2 showed no statistically significant difference between PA and LRPA. The hypotonic shock reaction (HSR) on days 0, 1, and 3 were significantly higher in LRPA than in PA (71.78±6.92 vs. 64.10±7.42; P=0.002; 71.53±8.98 vs. 62.96±9.84; P=0.007; 68.05±7.28 vs. 57.76±6.80; P<0.0001, respectively). Values of mean platelet volume (MPV) were statistically larger in PA than in LRPA on days 0, 1, and 3. On day 5, the swirling score was higher in LRPA than in PA. The mean lactate levels had no statistically significant difference between PA and LRPA. Moreover, no growth was observed through bacterium-screening test conducted on 40 samples., Conclusion: Comparison of LRPA and PA products collected from the Trima Accel and Haemonetics automated blood-collection systems, respectively, revealed that both products possessed good platelet qualities even though additional processes are needed to reduce leucocytes. Furthermore, investigating the outcomes of other apheresis instruments with focus on the safety of donors, products, and recipients is necessary., (Copyright © 2020 Société française de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved.)

Small-scale mangroves serve ecological functions similar to large-scale mangroves regarding biological conservation, environmental purification, and supporting biogeochemical processes. The rising aquaculture neighboring mangroves results in their serving as an important sink for massive nutrients and pollutants from aquaculture effluent. We assessed how long-term aquaculture effluent discharge influenced the soil properties of a mangrove-tidal flat continuum using field survey and geostatistics. Common soil physical-chemical properties presented significant spatial variability. Continued aquaculture effluent discharge caused a significant cumulation of soil total organic carbon (SOC) (64.13 g·kg -1 ), total nitrogen (TN) (2.44 g·kg -1 ) and total phosphorus (TP) (1.12 g·kg -1 ) in the mangrove soil, which were as 2-3 times as those on the mudflat. Most of the soil properties changed significantly with increasing distance from the effluent outlet along a tidal channel, and the maximum concentrations of SOC, TN and TP all occurred at 50 m away from the outlet. The results of principal component analysis indicated that aquaculture effluent significantly affected the spatial pattern of soil properties along the mangrove-tidal flat continuum. Continued aquaculture effluent input rendered extensive accumulation of SOC, TN and TP in the mangroves. The spatial heterogeneity of mangrove is the key driver to process the nutrient input spatially differently., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu 2+ ) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu 2+ and catalyzes its reduction to Cu 1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu 1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu 1+ ions in eukaryotes., (Copyright © 2020, American Association for the Advancement of Science.)

Background: Dementia is under-diagnosed in primary care. Timely diagnosis and care management improve outcomes for patients and caregivers. This research evaluated the effectiveness of a nationwide Continuing Medical Education (CME) program to enhance dementia-related awareness, practice, knowledge and confidence of general practitioners (GPs) in Australia., Methods: Data were collected from self-report surveys by GPs who participated in an accredited CME program face-to-face or online; program evaluations from GPs; and process evaluations from workshop facilitators. CME participants completed surveys at one or more time-points (pre-, post-program, six to 9 months follow-up) between 2015 and 2017. Paired samples t-test was used to determine difference in mean outcome scores (self-reported change in awareness, knowledge, confidence, practice) between time-points. Multivariable regression analyses were used to investigate associations between respondent characteristics and key variables. Qualitative feedback was analysed thematically., Results: Of 1352 GPs who completed a survey at one or more time-points (pre: 1303; post: 1017; follow-up: 138), mean scores increased between pre-CME and post-program for awareness (M post-pre  = 0.9, p <  0.0005), practice-related items (M post-pre  = 1.3, p <  0.0005), knowledge (M post-pre  = 2.2, p <  0.0005), confidence (M post-pre  = 2.1, p <  0.0005). Significant increases were seen in all four outcomes for GPs who completed these surveys at both pre- and follow-up time-points. Male participants and those who had practised for five or more years showed greater change in knowledge and confidence. Age, years in practice, and education delivery method significantly predicted post-program knowledge and confidence. Most respondents who completed additional program evaluations (> 90%) rated the training as relevant to their practice. These participants, and facilitators who completed process evaluations, suggested adding more content addressing patient capacity and legal issues, locality-specific specialist and support services, case studies and videos to illustrate concepts., Conclusions: The sustainability of change in key elements relating to health professionals' dementia awareness, knowledge and confidence indicated that dementia CME programs may contribute to improving capacity to provide timely dementia diagnosis and management in general practice. Low follow-up response rates warrant cautious interpretation of results. Dementia CME should be adopted in other contexts and updated as more research becomes available.

Background: Primary health care nurses can play an important role in assisting the diagnosis and management of dementia. This study describes the evaluation outcome of a training program developed on the 'Four Steps to Building Dementia Practice in Primary Care'., Objective: To evaluate a training program for primary health care nurses by assessing change in current practice and future intention; and their knowledge, confidence, and perceived importance about dementia diagnosis and management., Design: A longitudinal survey. Participants were surveyed at three time points: pre-training, immediately post-training and six months (+/- 3 months) following their training., Setting: All states and territories in Australia., Participants: Primary health care nurses (n = 1,290)., Methods: A face-to-face and online training program on timely diagnosis and management of dementia was offered to primary health care nurses. A questionnaire was administered face-to-face and online to assess whether certain processes and services were 'currently in practice', 'working towards', or 'not in current practice' in their primary care facility. Three 10-point Likert scales were created to assess self-perceived levels of importance, knowledge and confidence about the diagnosis and management of dementia. A paired t-test was used to examine the differences between (a) post and pre-scores, and (b) follow-up and post scores. Linear regressions were used to identify the significant factors associated with pre-training scores for importance, confidence and knowledge., Results: Of 1290 primary health care nurses who participated in the training, 471 attended face-to-face and 819 participated online. Participants demonstrated improvements in all items in all four steps of the survey, with considerably higher improvement in the face-to-face mode. The average post-training score was significantly higher than the pre-training score for perceived importance, knowledge and confidence. The average follow-up score was significantly higher than the post-training score for perceived knowledge and confidence but not for perceived importance. Primary health care nurses who had 20 or more years of experience reported significantly more knowledge in attending patients with dementia than those with less than five years of experience (0.56, 95% CI: 0.11-1.01)., Conclusions: With a growing ageing population, the demand for dementia care is rising. Primary health care nurses can lead practice change and promote the timely diagnosis and management of dementia in general practice. Training programs of this kind that build knowledge, confidence, awareness and skills should be made available to the primary care nursing workforce. Further research is recommended to examine the translation of this training outcome into practice., Competing Interests: Conflict of Interest None., (Copyright © 2020. Published by Elsevier Ltd.)

Objective Medication adherence is a significant public health concern. Australian studies of statins show patients facing the highest copayments are the least likely to be adherent. This study examined whether the association identified between adherence and costs for statins also applies to a wider group of medications prescribed for Australian patients with chronic conditions. Methods Data from 267086 participants in the Sax Institute's 45 and Up Study linked to data from the Pharmaceutical Benefits Scheme (PBS) provided by the Department of Human Services were used. Patients using angiotensin II receptor blockers, angiotensin-converting enzyme inhibitors, glitazones and bisphosphonates were identified and classified according to concessional status and whether they had access to the PBS 'safety net'. Data were analysed using mainly descriptive methods to investigate the association of adherence with cost and other selected covariates. Results Across medications, the group facing the highest copayment was least adherent. Speaking a language other than English at home and facing high levels of psychological distress were also associated with lower levels of adherence. Conclusions As for statins, the main financial determinant of adherence is cost in the form of prescribed copayments, suggesting that this may apply across many medications. What is known about the topic? Previous studies have shown patients' concern about the costs of pharmaceuticals, and more detailed studies of statins show that the lowest adherence relates to patients facing the highest copayments. What does this paper add? This paper provides support for the contention that the results found for statins broadly apply across more medications used by people with chronic conditions. What are the implications for practitioners? Although practitioners cannot affect legislated copayments, they can consider the costs of options for medications for patients with chronic conditions, especially those general patients who have not reached the safety net, and they can be aware that patients from homes where English is not spoken and patients with high levels of psychological distress are also likely to have low adherence without intervention.

Anti-MDA5-associated dermatomyositis (MDA5-associated DM) is an uncommon presentation of idiopathic inflammatory myositis, typically amyopathic, associated with rapidly progressive, treatment refractory interstitial lung disease and poor prognosis, particularly in patients with concomitant rapidly progressive interstitial lung disease (RP-ILD). We report two cases of MDA5-associated DM with fatal outcome in one of the patients, despite 'aggressive triple therapy' for RP-ILD., (© 2020 Royal Australasian College of Physicians.)

Objective: To describe the epidemiology of and risk factors associated with acute kidney injury (AKI) during acyclovir treatment in neonates and infants., Study Design: We conducted a multicenter (n = 4), retrospective cohort study of all hospitalized infants age <60 days treated with intravenous acyclovir (≥1 dose) for suspected or confirmed neonatal herpes simplex virus disease from January 2011 to December 2015. Infants with serum creatinine measured both before acyclovir (baseline) and during treatment were included. We classified AKI based on changes in creatinine according to published neonatal AKI criteria and performed Cox regression analysis to evaluate risk factors for AKI during acyclovir treatment., Results: We included 1017 infants. The majority received short courses of acyclovir (median, 5 doses). Fifty-seven infants (5.6%) developed AKI during acyclovir treatment, with an incidence rate of AKI at 11.6 per 1000 acyclovir days. Cox regression analysis identified having confirmed herpes simplex virus disease (OR, 4.35; P = .002), receipt of ≥2 concomitant nephrotoxic medications (OR, 3.07; P = .004), receipt of mechanical ventilation (OR, 5.97; P = .001), and admission to an intensive care unit (OR, 6.02; P = .006) as risk factors for AKI during acyclovir treatment., Conclusions: Among our cohort of infants exposed to acyclovir, the rate of AKI was low. Sicker infants and those exposed to additional nephrotoxic medications seem to be at greater risk for acyclovir-induced toxicity and warrant closer monitoring., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs), a class of long noncoding RNAs, are known to be enriched in mammalian neural tissues. Although a wide range of dysregulation of gene expression in autism spectrum disorder (ASD) have been reported, the role of circRNAs in ASD remains largely unknown. Here, we performed genome-wide circRNA expression profiling in postmortem brains from individuals with ASD and controls and identified 60 circRNAs and three coregulated modules that were perturbed in ASD. By integrating circRNA, microRNA, and mRNA dysregulation data derived from the same cortex samples, we identified 8170 ASD-associated circRNA-microRNA-mRNA interactions. Putative targets of the axes were enriched for ASD risk genes and genes encoding inhibitory postsynaptic density (PSD) proteins, but not for genes implicated in monogenetic forms of other brain disorders or genes encoding excitatory PSD proteins. This reflects the previous observation that ASD-derived organoids show overproduction of inhibitory neurons. We further confirmed that some ASD risk genes ( NLGN1 , STAG1 , HSD11B1 , VIP , and UBA6 ) were regulated by an up-regulated circRNA (circARID1A) via sponging a down-regulated microRNA (miR-204-3p) in human neuronal cells. Particularly, alteration of NLGN1 expression is known to affect the dynamic processes of memory consolidation and strengthening. To the best of our knowledge, this is the first systems-level view of circRNA regulatory networks in ASD cortex samples. We provided a rich set of ASD-associated circRNA candidates and the corresponding circRNA-microRNA-mRNA axes, particularly those involving ASD risk genes. Our findings thus support a role for circRNA dysregulation and the corresponding circRNA-microRNA-mRNA axes in ASD pathophysiology., (© 2020 Chen et al.; Published by Cold Spring Harbor Laboratory Press.)

Autonomic nervous system dysfunction (ANSD) is a significant cause of mortality in tetanus. Currently, diagnosis relies on nonspecific clinical signs. Heart rate variability (HRV) may indicate underlying autonomic nervous system activity and represents a potentially valuable noninvasive tool for ANSD diagnosis in tetanus. HRV was measured from three 5-minute electrocardiogram recordings during a 24-hour period in a cohort of patients with severe tetanus, all receiving mechanical ventilation. HRV measurements from all subjects-five with ANSD (Ablett Grade 4) and four patients without ANSD (Ablett Grade 3)-showed HRV was lower than reported ranges for healthy individuals. Comparing different severities of tetanus, raw data for both time and frequency measurements of HRV were reduced in those with ANSD compared with those without. Differences were statistically significant in all except root mean square SD, indicating HRV may be a valuable tool in ANSD diagnosis.

While chimeric RNAs may be generated by transcription-mediated mechanisms such as "trans-splicing" and "read-through/splicing" (Zhang et al., Cancer Discov 2:598-607, 2012; Zaphiropoulos, Front Genet 2:92, 2011; Li et al., Cell Cycle 8:218-222, 2009; Jividen and Li, Genes Chromosomes Cancer 53:963-971, 2014; Jia et al., Trends Cancer 2:475-484, 2016), most highly expressed chimeric RNA species identified so far are usually transcribed directly from fusion genes. Fusion genes, formed by joining two parental genes as a result of chromosomal rearrangements, are hallmarks of many types of cancer. Various methods can be deployed for confirming a particular fusion gene as the original source of transcribed chimeric RNA. In this chapter, we discuss commonly used methodologies such as genomic DNA breakpoint mapping and fluorescent in situ hybridization useful for confirming gene fusion events. In addition, we highlight the development of new technologies such as de novo whole-genome optical mapping suitable for global analysis of genomic arrangements. The advantages and disadvantages of each of these technologies are presented and compared.

Polo-like kinase-1 (PLK1) regulates the MYC-dependent kinome in aggressive B-cell lymphoma. However, the role of PLK1 and MYC toward proliferation in diffuse large B-cell lymphoma (DLBCL) is unknown. We use multiplexed fluorescent immunohistochemistry (fIHC) to evaluate the co-localization of MYC, PLK1 and Ki67 to study their association with proliferation in DLBCL. The majority (98%, 95% CI 95-100%) of MYC/PLK1-double positive tumor cells expressed Ki67, underscoring the key role of the MYC/PLK1 circuit in proliferation. However, only 38% (95% CI 23-40%) and 51% (95% CI 46-51%) of Ki67-positive cells expressed MYC and PLK1, respectively. Notably, 40% (95% CI 26-43%) of Ki67-positive cells are MYC- and PLK-negative. A stronger correlation exists between PLK1 and Ki67 expression ( R  = 0.74, p  < .001) than with MYC and Ki67 expression ( R  = 0.52, p  < .001). Overall, the results indicate that PLK1 has a higher association than MYC in DLBCL proliferation and there are mechanisms besides MYC and PLK1 influencing DLBCL proliferation.

Transcription by RNA polymerase V (Pol V) in plants is required for RNA-directed DNA methylation, leading to transcriptional gene silencing. Global chromatin association of Pol V requires components of the DDR complex DRD1, DMS3 and RDM1, but the assembly process of this complex and the underlying mechanism for Pol V recruitment remain unknown. Here we show that all DDR complex components co-localize with Pol V, and we report the cryoEM structures of two complexes associated with Pol V recruitment-DR (DMS3-RDM1) and DDR' (DMS3-RDM1-DRD1 peptide), at 3.6 Å and 3.5 Å resolution, respectively. RDM1 dimerization at the center frames the assembly of the entire complex and mediates interactions between DMS3 and DRD1 with a stoichiometry of 1 DRD1:4 DMS3:2 RDM1. DRD1 binding to the DR complex induces a drastic movement of a DMS3 coiled-coil helix bundle. We hypothesize that both complexes are functional intermediates that mediate Pol V recruitment.

Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Debilitating heart conditions, notably dilated and hypertrophic cardiomyopathies (CMs), are associated with point mutations in metavinculin, a larger isoform of the essential cytoskeletal protein vinculin. Metavinculin is co-expressed with vinculin at sub-stoichiometric ratios in cardiac tissues. CM mutations in the metavinculin tail domain (MVt) occur within the extra 68-residue insert that differentiates it from the vinculin tail domain (Vt). Vt binds actin filaments (F-actin) and promotes vinculin dimerization to bundle F-actin into thick fibers. While MVt binds to F-actin in a similar manner to Vt, MVt is incapable of F-actin bundling and inhibits Vt-mediated F-actin bundling. We performed F-actin co-sedimentation and negative-stain EM experiments to dissect the coordinated roles of metavinculin and vinculin in actin fiber assembly and the effects of three known metavinculin CM mutations. These CM mutants were found to weakly induce the formation of disordered F-actin assemblies. Notably, they fail to inhibit Vt-mediated F-actin bundling and instead promote formation of large assemblies embedded with linear bundles. Computational models of MVt bound to F-actin suggest that MVt undergoes a conformational change licensing the formation of a protruding sub-domain incorporating the insert, which sterically prevents dimerization and bundling of F-actin by Vt. Sub-domain formation is destabilized by CM mutations, disrupting this inhibitory mechanism. These findings provide new mechanistic insights into the ability of metavinculin to tune actin organization by vinculin and suggest that dysregulation of this process by CM mutants could underlie their malfunction in disease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: Half of prescription drugs commonly given to children lack product labeling on pediatric safety, efficacy, and dosing. Two drugs most widely used off-label in pediatrics are azithromycin and fentanyl. We sought to determine the risk of serious adverse events (SAEs) when oral azithromycin or intravenous/intramuscular fentanyl are used off-label compared to on-label in pediatric intensive care units (ICUs)., Study Design: Six pediatric hospitals participated in a retrospective chart review of patients administered oral azithromycin (n = 241) or intravenous/intramuscular fentanyl (n = 367) between January 5, 2013 and December 26, 2014. Outcomes were SAEs by drug and labeling status: off-label compared to on-label by Food and Drug Administration (FDA)-approved age and/or indication. Statistical analysis was performed using logistic regression to estimate odds ratios (ORs) and Cox regression to estimate hazard ratios (HRs)., Results: Twenty-one (9%) children receiving azithromycin experienced SAEs. Off-label use of azithromycin was not associated with a higher risk of SAE (OR 0.87, 95% CI 0.27-2.71, p = 0.81). Ninety-five (26%) children receiving fentanyl experienced SAEs. Fentanyl off-label use by both age and indication was not associated with a higher risk of overall SAEs compared to on-label use (OR 1.99, 95% CI 0.94-4.19, p = 0.07). However, the risk of the SAE respiratory depression was significantly greater when fentanyl was used off-label by both age and indication (OR 5.05, 95% CI 1.08-23.56, p = 0.044). Results based on HRs were similar., Conclusions: Azithromycin off-label use in pediatric ICUs does not appear to be associated with an increased risk of SAEs. Off-label use of fentanyl appears to be more frequently associated with respiratory depression when used off-label by both age and indication in pediatric ICUs. Prospective studies should be undertaken to assess the safety and efficacy of fentanyl in the pediatric population so that data can be added to the FDA labeling.

One of the hallmarks of cancer is the formation of oncogenic fusion genes as a result of chromosomal translocations. Fusion genes are presumed to form before fusion RNA expression. However, studies have reported the presence of fusion RNAs in individuals who were negative for chromosomal translocations. These observations give rise to "the cart before the horse" hypothesis, in which the genesis of a fusion RNA precedes the fusion gene. The fusion RNA then guides the genomic rearrangements that ultimately result in a gene fusion. However, RNA-mediated genomic rearrangements in mammalian cells have never been demonstrated. Here we provide evidence that expression of a chimeric RNA drives formation of a specified gene fusion via genomic rearrangement in mammalian cells. The process is: ( i ) specified by the sequence of chimeric RNA involved, ( ii ) facilitated by physiological hormone levels, ( iii ) permissible regardless of intrachromosomal ( TMPRSS2-ERG ) or interchromosomal ( TMPRSS2-ETV1 ) fusion, and ( iv ) can occur in normal cells before malignant transformation. We demonstrate that, contrary to "the cart before the horse" model, it is the antisense rather than sense chimeric RNAs that effectively drive gene fusion, and that this disparity can be explained by transcriptional conflict. Furthermore, we identified an endogenous RNA AZI1 that functions as the "initiator" RNA to induce TMPRSS2-ERG fusion. RNA-driven gene fusion demonstrated in this report provides important insight in early disease mechanisms, and could have fundamental implications in the biology of mammalian genome stability, as well as gene-editing technology via mechanisms native to mammalian cells., Competing Interests: The authors declare no conflict of interest.

DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objective: To examine and describe the use of Western and Chinese Medicine for pregnancy preparation among women with breast cancer after cancer-related treatment in Taiwan., Methods: Women of reproductive age (20-49 years) women who were treated for breast cancer from January 2011 through June 2015 in a Taiwanese city participated in the study. Of 306 eligible study participants, interviews were completed in 197 (64%). Participants were asked about sociodemographic variables, disease and treatment characteristics, their desire for fertility, and their use of Western or Chinese Medicine., Results: The proportions of women who planned to use Western or Chinese Medicine to help achieve pregnancy were 17.3% and 14.7%, respectively. The result of binary logistic regression showed that current employment and lack of children were factors predictive of the use of Western Medicine. Younger age and the use of Chinese Medicine before treatment were predictors of using Chinese Medicine. While the desire for fertility preservation was related to the use of Western Medicine, the desire for pregnancy was related to the use of Chinese Medicine., Conclusion: Various characteristics guided the women's decisions to use Western or Chinese Medicine. Health care providers should learn patients' preferences for fertility preservation and provide appropriate advice, referring them to safe providers of their desired method of fertility treatment.

Background: Epithelial-to-mesenchymal transition (EMT) has, in recent years, emerged as an important tumor cell behavior associated with high metastatic potential and drug resistance. Interestingly, protein SUMOylation and hepatocyte growth factor could respectively reduce the effect of small molecule inhibitors on tyrosine kinase activity of mutated epidermal growth factor receptor of lung adenocarcinomas (LADC). The actual mechanism is yet to be resolved., Methods: Immunohistochemistry was used to stain proteins in LADC specimens. Protein expression was confirmed by Western blotting. In vitro, expression of proteins was determined by Western blotting and immunocytochemistry. Levels of circular RNA were determined by reverse transcription-polymerase chain reaction., Results: SAE2 and cirRNA CCDC66 were highly expressed in LADC. Expression of SAE2 was mainly regulated by EGFR; however, expression of cirRNA CCDC66 was positively regulated by FAK and c-Met but negatively modulated by nAchR7α. EGFR-resistant H1975 also highly expressed cirRNA CCDC66. Immediate response of hypoxia increased phosphorylated c-Met, SAE2, and epithelial-to-mesenchymal transition. Either activation of FAK or silencing of nAchR7α increased cirRNA CCDC66., Conclusions: HGF/c-Met regulates expression of SAE2 and cirRNA CCDC66 to increase EMT and drug resistance of LADC cells. Multimodality drugs concurrently aiming at these targets would probably provide more benefits for cancer patients.

Background: Despite recognition of the link between body mass index (BMI) and depression in adolescence, the underlying mechanisms behind this association remain understudied. This study aims to examine three mediational pathways from BMI to depressive symptoms through peer victimization and sleep problems. Sex differences in the mediating effects were also explored., Methods: Data came from 1893 adolescents participating in a multi-wave longitudinal study from grade 9 to 12 in northern Taiwan were analyzed. Measures included BMI in 2009, peer victimization in 2010, sleep problems in 2011, depressive symptoms in 2012 and other covariates (sex, age, parental education, family structure, family economic stress, stressful life events, pubertal development and previous scores of focal study variables). A series of multiple regression models were conducted to test mediation hypotheses. A bootstrapping approach was applied to obtain confidence intervals for determining the significance of indirect effects., Results: The association between BMI and depressive symptoms was significantly mediated by peer victimization and sleep problems. Higher BMI predicted more peer victimization and sleep problems, each of which led to higher levels of depressive symptoms. Our results further showed that higher BMI was associated with more peer victimization, which led to greater sleep problems and in turn resulted in increased depressive symptoms. No sex differences was found for the indirect effects of BMI on depressive symptoms through either peer victimization or sleep problems., Conclusions: Peer victimization and sleep problems partly explain the link between BMI and depressive symptoms. Interventions to prevent or manage depressive symptoms may yield better results if they consider the effects of these two psychosocial factors rather than targeting BMI alone.

Background: Recent studies indicate that recombinant human bone morphogenetic protein-2 (rhBMP-2) in a demineralized bone matrix scaffold is a comparable alternative to iliac bone autograft in the setting of secondary alveolar cleft repair. Postreconstruction occlusal radiographs demonstrate improved bone stock when rhBMP-2/demineralized bone matrix (DBM) scaffold is used but lack the capacity to evaluate bone growth in three dimensions. This study uses cone beam computed tomography to provide the first clinical evaluation of volumetric and density comparisons between these two treatment modalities., Methods: A prospective study was conducted with 31 patients and 36 repairs of the alveolar cleft over a 2-year period. Twenty-one repairs used rhBMP-2/DBM scaffold and 14 repairs used iliac bone grafting. Postoperatively, occlusal radiographs were obtained at 3 months to evaluate bone fill; cone beam computed tomographic images were obtained at 6 to 9 months to compare volumetric and density data., Results: At 3 months, postoperative occlusal radiographs demonstrated that 67 percent of patients receiving rhBMP-2/DBM scaffold had complete bone fill of the alveolus, versus 56 percent of patients in the autologous group. In contrast, cone beam computed tomographic data showed 31.6 percent (95 percent CI, 24.2 to 38.5 percent) fill in the rhBMP-2 group compared with 32.5 percent (95 percent CI, 22.1 to 42.9 percent) in the autologous population. Density analysis demonstrated identical average values between the groups (1.38 g/cc)., Conclusions: These data demonstrate comparable bone regrowth and density values following secondary alveolar cleft repair using rhBMP-2/DBM scaffold versus autologous iliac bone graft. Cone beam computed tomography provides a more nuanced understanding of true bone regeneration within the alveolar cleft that may contribute to the information provided by occlusal radiographs alone., Clinical Question/level of Evidence: Therapeutic, II.

Background: In Australia, as in many other Western countries, patient surveys suggest the costs of medicines lead to deferring or avoiding filling of prescriptions. The Australian Pharmaceutical Benefits Scheme provides approved prescription medicines at subsidised prices with relatively low patient co-payments. The Pharmaceutical Benefits Scheme defines patient co-payment levels per script depending on whether patients are "concessional" (holding prescribed pension or other government concession cards) or "general", and whether they have reached a safety net defined by total out-of-pocket costs for Pharmaceutical Benefits Scheme-approved medicines., Objective: The purpose of this study was to explore the impact of costs on adherence to statins in this relatively low-cost environment., Methods: Using data from a large-scale survey of older Australians in the state of New South Wales linked to administrative data from the national medical and pharmaceutical insurance schemes, we explore the relationships between adherence to medication regimes for statins and out-of-pocket costs of prescribed pharmaceuticals, income, other health costs, and a wide set of demographic and socio-economic control variables using both descriptive analysis and logistic regressions., Results: Within the general non-safety net group, which has the highest co-payment, those with lowest income have the lowest adherence, suggesting that the general safety threshold may be set at a level that forms a major barrier to statin adherence. This is reinforced by over 75% of those who were not adherent before reaching the safety net threshold becoming adherent after reaching the safety net with its lower co-payments., Conclusion: The main financial determinant of adherence is the concessional/general and safety net category of the patient, which means the main determinant is the level of co-payment.

Sensitization of allergic patients normally takes place over several years and is the result of repeated exposure to low levels of allergen. Most mouse asthma models use a high dose of allergen administered over a short period. We have investigated the role of dose in the immune response to an inhaled respiratory allergen (Blomia tropicalis). We observed the effect of priming dose on the allergic response in mice intranasally immunized with low (0·5 μg) and high (50 μg) doses of B. tropicalis extract and killed 1 day after the last challenge. For both doses of allergen, T helper type 2 (Th2) cells and Th2 cytokines were evident as well as eosinophilic inflammation accompanied by mucus hyper-secretion. By contrast, IgE and IgG1 antibody responses were normally only detected at high-dose priming. To investigate the mechanism for these effects, we found group 2 innate lymphoid cells (ILC2s) were increased 48 hr after challenge in the low-dose-treated but not the high-dose-treated mice. Furthermore, we determined whether repeated low-dose exposure with different priming protocols could induce an antibody response. Repeated low-dose exposure to 0·5 μg three times weekly for 4 weeks (cumulative 6 μg) had the same effect as a shorter high-dose exposure (cumulative 80 μg) and increasing cumulative dose induced antibody responses. These data indicate that low doses of allergen are sufficient to prime Th2 cells and ILC2s, but insufficient to induce antibody responses. Cumulative exposure to small amounts of allergen induces both Th2 and antibody responses and may better reflect natural sensitization., (© 2017 John Wiley & Sons Ltd.)

Circular RNA (circRNA) is a class of noncoding RNA whose functions remain mostly unknown. Recent studies indicate circRNA may be involved in disease pathogenesis, but direct evidence is scarce. Here, we characterize the functional role of a novel circRNA, circCCDC66, in colorectal cancer. RNA-Seq data from matched normal and tumor colon tissue samples identified numerous circRNAs specifically elevated in cancer cells, several of which were verified by quantitative RT-PCR. CircCCDC66 expression was elevated in polyps and colon cancer and was associated with poor prognosis. Gain-of-function and loss-of-function studies in colorectal cancer cell lines demonstrated that circCCDC66 controlled multiple pathological processes, including cell proliferation, migration, invasion, and anchorage-independent growth. In-depth characterization revealed that circCCDC66 exerts its function via regulation of a subset of oncogenes, and knockdown of circCCDC66 inhibited tumor growth and cancer invasion in xenograft and orthotopic mouse models, respectively. Taken together, these findings highlight a novel oncogenic function of circRNA in cancer progression and metastasis. Cancer Res; 77(9); 2339-50. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The purpose of this study was to explore domain-general cognitive skills, domain-specific academic skills, and demographic characteristics that are associated with calculation development from first grade to third grade among young children with learning difficulties. Participants were 176 children identified with reading and mathematics difficulties at the beginning of first grade. Data were collected on working memory, language, nonverbal reasoning, processing speed, decoding, numerical competence, incoming calculations, socioeconomic status, and gender at the beginning of first grade and on calculation performance at four time points: the beginning of first grade, the end of first grade, the end of second grade, and the end of third grade. Latent growth modeling analysis showed that numerical competence, incoming calculation, processing speed, and decoding skills significantly explained the variance in calculation performance at the beginning of first grade. Numerical competence and processing speed significantly explained the variance in calculation performance at the end of third grade. However, numerical competence was the only significant predictor of calculation development from the beginning of first grade to the end of third grade. Implications of these findings for early calculation instructions among young at-risk children are discussed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: The objective of this study is to examine the experiences of informal carers in terms of how their time spent caring is related to worry. Is worry about a care recipient a care practice, and if so, to what extent it can be understood temporally?, Methods: Classical phenomenology underpinned this study. Three qualitative studies of people living with chronic illness in Australia were conducted between 2009 and 2013. Semi-structured interviews were conducted. Interviews were audio recorded and transcribed verbatim. Phenomenological thematic analysis of the data was undertaken., Results: Twenty-five informal carers participated. The findings pivot around three interconnected themes: time, worry and cost. Worry was identified as a temporally bound unseen cost to carers that informs carer identity irrespective of culture, ethnicity, or social status., Discussion: Worry is a practice that most carers report engaging in and it is one that comes with a temporal cost-it keeps people busy looking after the needs of others during the day and it keeps some people awake when they would rather be sleeping. Worry takes time and effort, it informs people's construction of their own sense of self, motivates acts of care, and informs carers' imaginings of what their future and that of their loved one(s) may entail., (© The Author(s) 2016.)

Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3., Competing Interests: The authors declare no conflict of interest.

Through iterative cycles of selection, amplification, and mutagenesis, in vitro selection provides the ability to isolate molecules of desired properties and function from large pools (libraries) of random molecules with as many as 10(16) distinct species. This review, in recognition of a quarter of century of scientific discoveries made through in vitro selection, starts with a brief overview of the method and its history. It further covers recent developments in in vitro selection with a focus on tools that enhance the capabilities of in vitro selection and its expansion from being purely a nucleic acids selection to that of polypeptides and proteins. In addition, we cover how next generation sequencing and modern biological computational tools are being used to complement in vitro selection experiments. On the very least, sequencing and computational tools can translate the large volume of information associated with in vitro selection experiments to manageable, analyzable, and exploitable information. Finally, in vivo selection is briefly compared and contrasted to in vitro selection to highlight the unique capabilities of each method., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)