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To understand the prevalence of depressive symptoms among foreign caregivers and the associated factors.A cross-sectional study.Data from 178 Indonesian foreign caregivers, selected based on convenience and snowball sampling in Taiwan, were collected between July 2019 and February 2020 using questionnaires. Stepwise multiple linear regression was used to identify the factors associated with depressive symptoms.Approximately 30.3% of the foreign caregivers displayed depressive symptoms. The symptoms were more prevalent among the participants who were younger; had more social support; shared a bed with others; and experienced higher work-related stress, more loneliness and physical discomfort. The findings suggest that nurses or nurse practitioners visiting patients at home should not only deliver care for them but also show concern for the psychological well-being of the foreign caregivers of these patients. Moreover, interventions should be developed to alleviate or prevent the emergence of depressive symptoms among foreign caregivers.

Summary Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general., Graphical Abstract, Highlights • TINC allows the isolation of a specific locus for molecular analyses • TINC identified hundreds of proteins at the Nanog promoter • RCOR2 is a component of the pluripotency network in embryonic stem cells • RCOR2 is required for efficient differentiation, Here, Knaupp and colleagues describe TINC, an epigenetic method that allows interrogation of mammalian regulatory complexes at a single-locus resolution. TINC was applied to dissect the transcriptional complex at the Nanog promoter in embryonic stem cells, revealing hundreds of interactors, including RCOR2, hence redefining how this gene is regulated in pluripotency.

This study aimed to examine the effectiveness of an educational board game in improving nursing students' medication knowledge.Maintaining patient safety is a core practice for nurses. Medication management is a central principle of patient safety. Nurses acquire pharmacology knowledge and medication safety skills in the classroom training. Thus, solidifying and strengthening nursing students' medication knowledge are crucial tasks for nursing faculty members. In recent years, board games, which offer both entertainment and competitive play, have been employed to educate students in a variety of disciplines and settings. Through board game play, students can learn in an enjoyable and fun atmosphere.A randomized controlled trial design.A convenience sample of 69 nursing students was obtained from a university in Taiwan. Participants were randomly assigned either to an experimental (board game) group (n = 35) or a comparison group (n = 34) using block randomization. The experimental group engaged in board game play to learn about medications, whereas the comparison group attended a one-hour didactic lecture. Using questionnaires, data were collected before the intervention, immediately post intervention and one month post intervention.Following the intervention, regardless of the learning method, both groups showed significant improvements in their immediate recall of medication information. However, when retested after one month, the experimental group obtained significantly higher scores than the comparison group. Moreover, students in the experimental group reported more satisfaction with the learning method than those in the comparison group.The study results suggest that learning through board games could enhance nursing students' retention of knowledge. Students reported favorable reactions to using a board game learning method for increasing knowledge of medication. With respect to this finding, faculty members may consider employing board games as teaching tools in nursing and other health science courses. Moreover, the findings of this study can also provide additional information for nursing managers in hospital wards or long-term care facilities where nurses are trained to familiarize themselves with frequently administered medications.Board game play can enhance nursing students' retention of knowledge; students reported positive reactions to game-based learning for medication training.

Methylation of DNA at cytosine bases is an important DNA modification underlying normal development and disease states. Despite decades of research into the biological function of DNA methylation, most of the observations so far have relied primarily on associative data between observed changes in DNA methylation states and local changes in transcriptional activity or chromatin state processes. This is primarily due to the lack of molecular tools to precisely modify DNA methylation in the genome. Recent advances in genome editing technologies have allowed repurposing the CRISPR-Cas9 system for epigenome editing by fusing the catalytically dead Cas9 (dCas9) to epigenome modifying enzymes. Moreover, methods of recruiting multiple protein domains, including the SunTag system, have increased the efficacy of epigenome editing at target sites. Here, we describe an end-to-end protocol for efficient targeted removal of DNA methylation by recruiting multiple catalytic domain of TET1 enzymes to the target sites with the dCas9-SunTag system, including sgRNA design, molecular cloning, delivery of plasmid into mammalian cells, and targeted DNA methylation analysis.

Purpose Due to the multifaceted chemotherapy workflow within the hospital, many patients often experience delays in receiving their treatment. This study aims to evaluate the causes for chemotherapy administration delays and implement new methods to reduce delays from order release to chemotherapy administration on an inpatient oncology unit at a community-focused academic medical center. Methods In this prospective quality improvement study, we developed a process map to track baseline time stamps and utilized performance improvement tools to identify causes for chemotherapy delays. Based on recognized areas for improvement, the Plan-Do-Study-Act (PDSA) model was used to implement one cycle of interventions. Chemotherapy orders were collected, and benchmark time stamps were documented from the electronic medical record. Results The primary outcome for the number of chemotherapy delays, based on compliance rate, was reduced from 63/100 (63.0%) to 48/100 (48.0%), a 15% reduction (p = 0.046). Our primary outcome of chemotherapy delays, based on our institutional benchmark of Conclusions This study allowed our institution to evaluate our current practice and reformulate the chemotherapy administration process. With the continuing education on the chemotherapy administration process and additional PDSA cycle interventions, it will help standardize our process and ultimately continue to reduce chemotherapy delays.

Hearing loss is the most common sensory deficit worldwide with no approved therapeutics for treatment. Local neurotrophin delivery into the cochlea has shown great potential in protecting and repairing the sensory cells important for hearing. However, delivery of these factors into the inner ear at therapeutic levels over a sustained period of time has remained a challenge restricting clinical translation. We have developed a method to test the pharmacokinetics of neurotrophin released from porous silica particles called ‘supraparticles’ that can provide sustained release of neurotrophins to the inner ear.•This report describes a radiolabeling method to examine neurotrophin retention and distribution in the cochlea. The neurotrophin was labeled with a radioactive tracer (iodine 125: 125I) and delivered into the cochlea via the supraparticle system.•Gamma counts reveal drug levels and clearance in the intact cochlea, as well as accumulation in off-target organs (safety test). Autoradiography analyses using film and emulsion permit quantification and visualization of drug distribution at the cellular level. The method has a detection limit of 0.8 pg of radiolabeled neurotrophin-3 in cochlear sections exposed to film.•The tracer 125I with a half-life of 59.4 days can be used to label other drugs/substances with a tyrosine residue and therefore be broadly applicable for long-term pharmacokinetic studies in other systems., Graphical abstract Image, graphical abstract

Aim: To determine whether epigenetic modifications of DNA regulate gene expression and alternative splicing during terminal granulopoiesis. Materials & methods: Using whole genome bisulfite sequencing, reduced representation hydroxymethylation profiling and mRNA sequencing, we compare changes in DNA methylation, DNA hydroxymethylation, gene expression and alternative splicing in mouse promyelocytes and granulocytes. Results & conclusion: We show reduced DNA methylation at the promoters and enhancers of key granulopoiesis genes, indicating a regulatory role in the activation of lineage-specific genes during differentiation. Notably, increased DNA hydroxymethylation in exons is associated with preferential inclusion of specific exons in granulocytes. Overall, DNA methylation and hydroxymethylation changes at particular genomic loci may play specific roles in gene regulation or alternative splicing during terminal granulopoiesis. Data deposition: Whole genome bisulfite sequencing of mouse promyelocytes and granulocytes: Gene Expression Omnibus (GSE85517); mRNA sequencing of mouse promyelocytes and granulocytes: Gene Expression Omnibus (GSE48307); reduced representation 5-hydroxymethylation profiling of mouse promyelocytes and granulocytes: Bioproject (PRJNA495696).

Introduction: DAWN and DEFUSE 3 trials utilized advanced imaging to prove the benefit of endovascular treatment (EVT) in patients within 6-24 hours. There is increasing evidence to suggest the limitations of advanced imaging in real-world practice. Non-contrast-enhanced computed tomography (NCCT) has proved with good sensitivity and specificity in the definition of infarct core. It is still unknown if a simpler and faster protocol may adequately select patients within 6-24 hours for EVT. Hypothesis: To compare outcomes of patients submitted to EVT presenting within 6 hours or 6-24 hours, selected using simple imaging protocol. Methods: An observational study was performed, which included consecutive patients with anterior circulation ischemic stroke eligible for EVT within 6 hours or 6-24 hours. Patients within early window received routine treatment, while patients presenting within late window were treated if they had a mismatch between the clinical deficit, the infarct volume, and collateral blood blow: (NIHSS score was ≥10 and ASPECTS was ≥7) or (ASPECTS was =6, and the collateral score was ≥2). ASPECTS of NCCT/DWI-MRI and collateral status on CTA/DSA were assessed by a blinded neuroradiologist. The collateral grading system was scored on a scale of 0-3 as in the ESCAPE trial. Results: Of the 184 patients were included, 77 (41,8%) received thrombectomy in late window, 107 (58,2%) received acute treatment in early window. Baseline characteristics were similar between groups, except for longer onset to groin puncture time (median, 300 vs 705 min; P Conclusions: This real-world observational study suggests that EVT may be safe and effective in patients presenting within 6-24 hours selected using clinical-core mismatch and collateral blood blow.

DNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of these tools, revealing consistent off-target binding events and DNA methylation deposition in the genome, limiting the capacity of these tools to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA-methylation. dC9Sun-D3A is tunable, specific and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

Objective. Established guidelines for safe levels of electrical stimulation for neural prostheses are based on a limited range of the stimulus parameters used clinically. Recent studies have reported particulate platinum (Pt) associated with long-term clinical use of these devices, highlighting the need for more carefully defined safety limits. We previously reported no adverse effects of Pt corrosion products in the cochleae of guinea pigs following 4 weeks of electrical stimulation using charge densities far greater than the published safe limits for cochlear implants. The present study examines the histopathological effects of Pt within the cochlea following continuous stimulation at a charge density well above the defined safe limits for periods up to 6 months. Approach. Six cats were bilaterally implanted with Pt electrode arrays and unilaterally stimulated using charge balanced current pulses at a charge density of 267 μC cm−2 phase−1 using a tripolar electrode configuration. Electrochemical measurements were made throughout the implant duration and evoked potentials recorded at the outset and on completion of the stimulation program. Cochleae were examined histologically for particulate Pt, tissue response, and auditory nerve survival; electrodes were examined for surface corrosion; and cochlea, brain, kidney, and liver tissue analysed for trace levels of Pt. Main results. Chronic stimulation resulted in both a significant increase in tissue response and particulate Pt within the tissue capsule surrounding the electrode array compared with implanted, unstimulated control cochleae. Importantly, there was no stimulus-induced loss of auditory neurons (ANs) or increase in evoked potential thresholds. Stimulated electrodes were significantly more corroded compared with unstimulated electrodes. Trace analysis revealed Pt in both stimulated and control cochleae although significantly greater levels were detected within stimulated cochleae. There was no evidence of Pt in brain or liver; however, trace levels of Pt were recorded in the kidneys of two animals. Finally, increased charge storage capacity and charge injection limit reflected the more extensive electrode corrosion associated with stimulated electrodes. Significance. Long-term electrical stimulation of Pt electrodes at a charge density well above existing safety limits and nearly an order of magnitude higher than levels used clinically, does not adversely affect the AN population or reduce neural function, despite a stimulus-induced tissue response and the accumulation of Pt corrosion product. The mechanism resulting in Pt within the unstimulated cochlea is unclear, while the level of Pt observed systemically following stimulation at these very high charge densities does not appear to be of clinical significance.

Neurotrophin therapy has potential to reverse some forms of hearing loss. However, cochlear pharmacokinetic studies are challenging due to small fluid volumes. Here a radioactive tracer was used to determine neurotrophin-3 retention, distribution and clearance after intracochlear administration. 125I-neurotrophin-3 was injected into guinea pig cochleae using a sealed injection technique comparing dosing volumes, rates and concentrations up to 750 μg/mL. Retention was measured by whole-cochlear gamma counts at five time points while distribution and clearance were assessed by autoradiography. Smaller injection volumes and higher concentrations correlated with higher retention of neurotrophin-3. Distribution of neurotrophin-3 was widespread throughout the cochlear tissue, decreasing in concentration from base to apex. Tissue distribution was non-uniform, with greatest density in cells lining the scala tympani and lower density in neural target tissue. The time constant for clearance of neurotrophin-3 from cochlear tissues was 38 h but neurotrophin-3 remained detectable for at least 2 weeks. Neurotrophin-3 was evident in the semi-circular canals with minor spread to the contralateral cochlea. This study is the first comprehensive evaluation of the disposition profile for a protein therapy in the cochlea. The findings and methods in this study will provide valuable guidance for the development of protein therapies for hearing loss.

Introduction: To evaluate the use of mycobacterial blood cultures (MBC) in diagnosing tuberculosis (TB) in patients with prolonged fever admitted to a Vietnamese referral hospital. Results: MBCs from 94 patients (66% male; median age 33 years; 75% HIV positive) were evaluated: 14 were mycobacterium positive (all HIV positive), and MBC was the only positive specimen in 9 cases (41%). Three positive cases were identified as Mycobacterium avium and the remaining M. tuberculosis (one case could not be identified). Conclusion: MBC can be a valuable additional method to diagnose TB, particularly in immunosuppressed HIV patients when sputum cannot be collected.

An automated capillary DNA sequencer with laser-induced fluorescence detection can be utilised for DNA fragment analysis. The precise mobilities of DNA fragments with different chemical termini are especially important in the determination of the sequence specificity of DNA damaging agents. The aim of this study was to examine the electrophoretic mobility profile of DNA fragments with different 3'-termini. The nature of the 3'-teminal residue was found to have a major effect on the electrophoretic mobility of the DNA fragment, especially for 3'-phosphoglycolate termini that migrated anomalously by 3-6 nucleotides. Using the automated capillary sequencer, the electrophoretic mobilities of DNA fragments with different 3'-termini including 3'-hydrogen, 3'-hydroxyl, 3'-phosphate, and 3'-phosphoglycolate were extensively quantified and compared relative to each other. The 3'-hydrogen termini were generated by dideoxy sequencing; 3'-hydroxyl ends by minus sequencing; 3'-phosphate by Maxam-Gilbert chemical sequencing; and 3'-phosphoglycolate by bleomycin cleavage. The mobilities of these DNA fragments with different 3'-termini were found to be: (slowest) 3'-hydroxyl

While intron retention (IR) is considered a widely conserved and distinct mechanism of gene expression control, its regulation is poorly understood. Here we show that DNA methylation directly regulates IR. We also find reduced occupancy of MeCP2 near the splice junctions of retained introns, mirroring the reduced DNA methylation at these sites. Accordingly, MeCP2 depletion in tissues and cells enhances IR. By analysing the MeCP2 interactome using mass spectrometry and RNA co-precipitation, we demonstrate that decreased MeCP2 binding near splice junctions facilitates IR via reduced recruitment of splicing factors, including Tra2b, and increased RNA polymerase II stalling. These results suggest an association between IR and a slower rate of transcription elongation, which reflects inefficient splicing factor recruitment. In summary, our results reinforce the interdependency between alternative splicing involving IR and epigenetic controls of gene expression., Intron retention is a conserved mechanism that controls gene expression but its regulation is poorly understood. Here, the authors provide evidence that DNA methylation regulates intron retention and find reduced MeCP2 occupancy and splicing factor recruitment near affected splice junctions.

The rapid progress seen in the development of quantum coherent devices for information processing has motivated serious consideration of quantum computer architecture and organization. One topic which remains open for investigation and optimization relates to the design of the classical-quantum interface, where control operations on individual qubits are applied according to higher-level algorithms; accommodating competing demands on performance and scalability remains a major outstanding challenge. In this work we present a resource-efficient, scalable framework for the implementation of embedded physical-layer classical controllers for quantum information systems. Design drivers and key functionalities are introduced, leading to the selection of Walsh functions as an effective functional basis for both programming and controller hardware implementation. This approach leverages the simplicity of real-time Walsh-function generation in classical digital hardware, and the fact that a wide variety of physical-layer controls such as dynamic error suppression are known to fall within the Walsh family. We experimentally implement a real-time FPGA-based Walsh controller producing Walsh timing signals and Walsh-synthesized analog waveforms appropriate for critical tasks in error-resistant quantum control and noise characterization. These demonstrations represent the first step towards a unified framework for the realization of physical-layer controls compatible with large-scale quantum information processing., Updated structure and background. Related manuscripts available from http://www.physics.usyd.edu.au/~mbiercuk/Publications.html

International audience; Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 degrees C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 degrees C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

In this review, the use of automated DNA sequencing techniques to determine the sequence specificity of compounds that interact with DNA is discussed. The sequence specificity of a DNA-damaging agent is an essential element in determining the cellular mechanism of action of a drug. A number of DNA-damaging compounds are mutagenic, carcinogenic, as well as being widely used as cancer chemotherapeutic agents. The distribution of lesions in a sequence of DNA can give vital clues in the determination of the precise mechanism of interaction of the agent with DNA. The DNA sequence specificity of a number of DNA-damaging agents has been delineated using automated DNA sequencing technology, and these studies are discussed in this review. The current state-of-the-art methodology involves capillary electrophoresis with laser-induced fluorescence detection usually on an Applied Biosystems ABI 3730 capillary sequencer. This current technique has higher resolution, greater sensitivity, higher precision, more rapid separation times, is safer and easier to perform than previous methods. The two main methods to determine the DNA sequence selectivity of compounds that interact with DNA are described: end labelling and the polymerase stop assay. The interaction of the antitumour drug, bleomycin, with DNA is utilized to illustrate the recent technological advances.

The DNA sequence specificity of the cancer chemotherapeutic agent bleomycin was examined in a human telomeric DNA sequence and compared with that of non-telomeric sequences. The target DNA sequence contained 17 repeats of the human telomeric sequence and other primary sites of bleomycin cleavage. The 377-base-pair target DNA was fluorescently labelled at the 3'-end, damaged with bleomycin and electrophoresed in an ABI 3730 automated capillary sequencer to determine the intensity and sequence specificity of bleomycin damage. The results revealed that bleomycin cleaved primarily at 5'-GT in the telomeric sequence 5'-GGGTTA. Maxam-Gilbert chemical sequencing reactions were utilised as DNA size markers to determine the precise sites of bleomycin cleavage. The telomeric region contained strong sites of bleomycin cleavage and constituted 57% of the 30 most intense bleomycin damage sites in the DNA sequence examined. These data indicated that telomeric DNA sequences are a major site for bleomycin damage.

Properties of enteric neurons are transformed by inflammation and protein kinase C (PKC) isoforms are involved both in long-term changes in enteric neurons, and in transducing the effects of substances released during inflammation. We investigated roles of PKCepsilon in submucosal neurons by studying translocation in response to inflammatory mediators, effects on neuron excitability, and the changes in PKCepsilon distribution in a trinitrobenzene sulphonate model of ileitis.Immunohistochemical detection and analysis of association with membrane and cytosolic fractions, and Western blot analysis of cytosolic and particulate fractions were used to quantify translocation. Electrophysiology methods were used to measure effects on neuron excitability.All submucosal neurons were immunoreactive for the novel PKC, PKCepsilon, and direct PKC activators, phorbol 12,13-dibutyrate, ingenol 3,20-dibenzoate, and the PKCepsilon-specific activator, transactivator of transduction-Psiepsilon receptor for activated C kinase, all caused PKCepsilon translocation from cytoplasm to surfaces of the neurons. Electrophysiologic studies showed that the stimulant of novel PKCs, ingenol (1 micromol/L), increased excitability of all neurons. Stimulation of protease-activated receptors caused PKCepsilon translocation selectively in vasoactive intestinal peptide secretomotor neurons, whereas a neurokinin 3 tachykinin receptor agonist caused translocation in neuropeptide Y and calretinin neurons. In all cases translocation was reduced significantly by a PKCepsilon-specific translocation inhibitor peptide. Increased PKCepsilon at the plasma membrane occurred in all neurons 6-7 days after an inflammatory stimulus.Major targets for PKCepsilon include ion channels near the plasma membrane. PKCepsilon is likely to have a significant role in controlling the excitability of submucosal neurons and is probably an intermediate in causing hyperexcitability after inflammation.

To asses the impact of locally developed antimicrobial treatment guidelines in the initial empiric treatment of ICU patients with severe hospital-acquired pneumonia (HAP).Observational cohort study with pre-guideline and post-guideline data collection.A total of 48 pre-guideline patients with 56 episodes of severe HAP defined by the National Nosocomial Infections Surveillance (NNIS) compared with 58 guideline-treated (GUIDE) patients with 61 episodes of severe HAP.The two groups were similar in terms of mean (+/- SD) age (NNIS group, 67.7 +/- 9.6 years; GUIDE group, 68.0 +/- 11.5 years) and simplified acute physiology score (NNIS group, 12.9 +/- 3.9; GUIDE group, 12.6 +/- 3.1) at the HAP diagnosis, and the proportion of the most frequent isolates (ie, Pseudomonas and methicillin-resistant Staphylococcus aureus). There was wide variation in initial antibiotic use in NNIS-treated patients, with cefotaxime, ceftazidime, and piperacillin being the most common agents compared with all of the GUIDE patients who received an imipenem-cilastin-based regimen. Vancomycin use was similar in both groups. The GUIDE patients had a higher percentage of adequately treated patients (81% vs 46%, respectively; p0.01) with a lower mortality rate at 14 days (8% vs 23%, respectively; p = 0.03). A lower mortality rate was also noted at the end of 30 days and the end of hospitalization but was not statistically significant. Appropriate imipenem use (as defined by the guidelines) occurred in 74% of the cases, and there was no increase in the number of imipenem-resistant organisms isolated during the course of the study.These guidelines represent a successful implementation of a "deescalation" approach, because the recommended empiric therapy with broad-spectrum antibiotics was switched to therapy with narrower spectrum agents after 3 days. Based on our experience, this approach improves the adequacy of antibiotic treatment, with improvement in short-term survival and without increasing the emergence of resistant organisms.

This paper discusses the application of adaptive sectorisation in the capacity enhancement of a CDMA cellular system dealing with non-uniform traffic. It considers the particular case where the traffic distribution consists of isolated areas of congested traffic called Hot Spots (HS). It is envisaged that the traffic density inside a HS is many more times that outside the HS. A more even traffic distribution among sectors is attempted by re-adjustment of sector boundaries using finite antenna beam switching. System capacity is estimated on the basis of tolerable interference in a sector taking into account of both intrasector and intersector interference in a multicell environment. Interference is evaluated under the assumption of perfect power control in the uplink. Normal propagation environment is assumed in the cell that allows path losses and shadowing losses in the mobile radio signal. Variation in the antenna gain is also accommodated by taking appropriate radiation pattern into account. It is shown that successive sector adjustment with increasing HS traffic intensity can deter call blocking in the HS sector. It is also shown that a significant improvement in system capacity could be obtained with adaptive sectorisation particularly when the HS user density is several orders of magnitude higher than that outside the HS.

Low-frequency stimulation of synaptic inputs to after-hyperpolarising (AH) neurons in the guinea-pig small intestine causes sustained increases in excitability that far outlast the stimulus period. This excitation has been called sustained, slow, post-synaptic excitation (SSPE). Intracellular microelectrodes were used to record the effects of the protein kinase C (PKC) stimulant, phorbol dibutyrate (PDBu), and compare these with changes seen during the SSPE, in AH neurons of the small intestine of the guinea-pig. PDBu (1 nM–1 µM) increased excitability, depolarised the membrane and increased input resistance concentration dependently, mimicking the effects of low-frequency stimulation of pre-synaptic inputs. These changes developed slowly after the start of infusion and were only slowly reversible after wash out. PDBu suppressed a late after-hyperpolarising potential (AHP) that depends on Ca2+ entry via voltage-gated Ca2+ channels during the action potential. The effects of PDBu (10 nM) on the late AHP were indistinguishable from those observed during the SSPE. PDBu, at a concentration that inhibited the AHP, had no effect on the action potential half-width or the slope of its first repolarisation phase (the early phase of repolarisation is slowed by the Ca2+ influx of the action potential). Thus PDBu inhibited K+ channel opening underlying the late AHP, but did not suppress Ca2+ entry during the action potential. The hyperpolarisation-activated cation current (I h) in intrinsic primary afferent neurons (IPANs) was not affected by PDBu. We conclude that PDBu mimics the sustained excitation caused by low-frequency stimulation of synaptic inputs to IPANs by closing IK channels responsible for the AHP or restricting their opening by Ca2+ and by reducing the current carried by K+ channels that are active at rest. IK channels, the opening of which results in the AHP, have consensus sites for PKC and are likely targets for phosphorylation during the SSPE.

The mechanical engineering department at Washington State University has just completed a 6 year program in offering a modern sequence of design/manufacturing electives. Using theoretical foundations, site visits, guest lectures, IT based labs, and hands-on manufacturing exercises students learn about the role of information technology and electronic data in modern manufacturing enterprise systems, design for manufacture concepts, design automation, and also hands-on machining using advanced programming concepts of CNC code and CNC machines that are integrated with CAD/CAM systems. In this paper, two key courses of this sequence will be discussed and the innovations that they have enabled in engineering design education of the students will be presented. What is significant is that thru these courses we have achieved a dynamic interplay between the new and the old — we have been able to leapfrog into concepts of modern industry where IT and modern computing tools play a critical role in the manufacturing enterprise system and bring about considerable design and manufacturing automation; at the same time we have also retained and enhanced traditional concepts from before where design engineers had considerable hands-on knowledge with machining and manufacturing processes.

Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown.The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a).Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptor agonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84.Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin.

The electrophoretic mobility of DNA fragments that differ by a single 3′-terminal nucleotide was assessed by capillary electrophoresis. This was accomplished using dideoxy sequencing with a 5′-fluorescently labelled primer to generate DNA fragments with 3′-hydrogen ends. The resulting DNA fragments were electrophoresed on the ABI 3730 automated capillary sequencer, and the data were analysed with the GeneMapper software to determine the electrophoretic mobility differences on addition of a 3′-terminal nucleotide. It was found that the 3′-terminal nucleotide gave rise to different electrophoretic mobility profiles depending on the identity of the terminal nucleotide. The apparent electrophoretic mobility was (faster) –C > −A > −T > −G (slower). The C-terminated fragments were the fastest and the G-terminated fragments the slowest, relative to other nucleotides. It was proposed that the terminal nucleotide effect was due to changes in partial net charges on the nucleotides that resulted in alterations in the electrophoretic mobility of the DNA fragments in the automated capillary DNA sequencer. Other alternative explanations are also discussed. Copyright © 2012 John Wiley & Sons, Ltd.

The electrophoretic mobility of DNA fragments that differ by a single 3'-terminal nucleotide was assessed by capillary electrophoresis. This was accomplished using dideoxy sequencing with a 5'-fluorescently labelled primer to generate DNA fragments with 3'-hydrogen ends. The resulting DNA fragments were electrophoresed on the ABI 3730 automated capillary sequencer, and the data were analysed with the GeneMapper software to determine the electrophoretic mobility differences on addition of a 3'-terminal nucleotide. It was found that the 3'-terminal nucleotide gave rise to different electrophoretic mobility profiles depending on the identity of the terminal nucleotide. The apparent electrophoretic mobility was (faster) -C-A-T-G (slower). The C-terminated fragments were the fastest and the G-terminated fragments the slowest, relative to other nucleotides. It was proposed that the terminal nucleotide effect was due to changes in partial net charges on the nucleotides that resulted in alterations in the electrophoretic mobility of the DNA fragments in the automated capillary DNA sequencer. Other alternative explanations are also discussed.

In this review, the use of automated DNA sequencing techniques to determine the sequence specificity of compounds that interact with DNA is discussed. The sequence specificity of a DNA-damaging agent is an essential element in determining the cellular mechanism of action of a drug. A number of DNA-damaging compounds are mutagenic, carcinogenic, as well as being widely used as cancer chemotherapeutic agents. The distribution of lesions in a sequence of DNA can give vital clues in the determination of the precise mechanism of interaction of the agent with DNA. The DNA sequence specificity of a number of DNA-damaging agents has been delineated using automated DNA sequencing technology, and these studies are discussed in this review. The current state-of-the-art methodology involves capillary electrophoresis with laser-induced fluorescence detection usually on an Applied Biosystems ABI 3730 capillary sequencer. This current technique has higher resolution, greater sensitivity, higher precision, more rapid separation times, is safer and easier to perform than previous methods. The two main methods to determine the DNA sequence selectivity of compounds that interact with DNA are described: end labelling and the polymerase stop assay. The interaction of the antitumour drug, bleomycin, with DNA is utilized to illustrate the recent technological advances.

Background Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses. Methods and Findings Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found. Conclusion In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.

We investigated the responses of morphologically identified myenteric neurons of the guinea-pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin-resistant component, and a prolonged after-hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca(2+)-dependent K(+) channel blocker TRAM-34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin-resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current (I(h)) antagonist Cs(+). It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.

OBJECTIVES: To determine prospectively the causative pathogens of central nervous system (CNS) infections in patients admitted to a tertiary referral hospital in Hanoi, Vietnam. METHODS: From May 2007 to December 2008, cerebrospinal fluid (CSF) samples from 352 adults with suspected meningitis or encephalitis underwent routine testing, staining (Gram, Ziehl-Nielsen, India ink), bacterial culture and polymerase chain reaction targeting Neisseria meningitidis, Streptococcus pneumoniae, S. suis, Haemophilus influenzae type b, Herpes simplex virus (HSV), Varicella Zoster virus (VZV), enterovirus, and 16S ribosomal RNA. Blood cultures and clinically indicated radiology were also performed. Patients were classified as having confirmed or suspected bacterial (BM), tuberculous (TBM), cryptococcal (CRM), eosinophilic (EOM) meningitis, aseptic encephalitis/meningitis (AEM), neurocysticercosis and others. RESULTS: 352 (male: 66%) patients were recruited: median age 34 years (range 13-85). 95/352 (27.3%) diagnoses were laboratory confirmed and one by cranial radiology: BM (n = 62), TBM (n = 9), AEM (n = 19), CRM (n = 5), and neurocysticercosis (n = 1, cranial radiology). S. suis predominated as the cause of BM [48/62 (77.4%)]; Listeria monocytogenese (n = 1), S. pasteurianus (n = 1) and N. meningitidis (n = 2) were infrequent. AEM viruses were: HSV (n = 12), VZV (n = 5) and enterovirus (n = 2). 5 patients had EOM. Of 262/352 (74.4%) patients with full clinical data, 209 (79.8%) were hospital referrals and 186 (71%) had been on antimicrobials. 21 (8%) patients died: TBM (15.2%), AEM (10%), and BM (2.8%). CONCLUSIONS: Most infections lacked microbiological confirmation. S. suis was the most common cause of BM in this setting. Improved diagnostics are needed for meningoencephalitic syndromes to inform treatment and prevention strategies.

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